# Patent

## Title
Compounds and methods for modulating gene expression

## Abstract

The present disclosure describes short antisense compounds, including such compounds comprising chemically-modified high-affinity monomers 8-16 monomers in length. Certain such short antisense compound are useful for the reduction of target nucleic acids and/or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing.

## Background
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0061US7C1SEQ_ST25.txt, created on Apr. 27, 2015 which is 732 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND
Targeting disease-causing gene sequences was first suggested nearly 40 years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate expression of specific disease-causing genes.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and effects modulation of gene expression activity or function, such as transcription, translation or splicing. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi is a form of antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. Sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as research tools for identifying and characterizing nucleases and as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of any one of a variety of diseases.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
Despite the expansion of knowledge since the discovery of antisense technology, there remains an unmet need for antisense compounds with greater efficacy, reduced toxicity and lower cost. Until the present disclosure, high-affinity modifications have not been employed in the design of short antisense compounds for reducing target RNA in vivo. This is because of concerns regarding the degree of target specificity that a sequence 15 nucleotides or shorter would have when employed to reduce target in a living system. Previous studies have described that greater specificity, and therefore greater potential for potency, is achieved by antisense compounds between 16 and 20 nucleobases in length.
The present disclosure describes incorporation of chemically-modified high-affinity nucleotides into antisense compounds allows for short antisense compounds about 8-16 nucleobases in length useful in the reduction of target RNAs in animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment.
SUMMARY
Disclosed herein are short antisense compounds and methods of using said compounds to reduce target RNA expression in cells or tissues. In certain embodiments, provided herein is a method of reducing expression of a target in an animal, comprising administering to the animal a short antisense compound targeted to a nucleic acid of such target. In certain embodiments, shorts antisense compounds are oligonucleotide compounds. In certain embodiments short antisense oligonucleotides are about 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length and comprises a gap region flanked on each side by a wing, wherein each wing independently consists of 1 to 3 nucleotides. Preferred motifs include but are not limited to wing-deoxy gap-wing motifs selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. In a preferred embodiment, the short antisense oligonucleotide comprise at least one high-affinity modification. In a further embodiment, the high-affinity modification includes chemically-modified high-affinity nucleotides. In a preferred embodiment, each wing independently consists of 1 to 3 high-affinity modified nucleotides. In one embodiment the high affinity modified nucleotides are sugar-modified nucleotides.
In certain embodiments short antisense compounds exhibit greater uptake in the gut as compared to antisense compounds of greater length. Thus, also provided herein are methods of reducing a target in an animal, comprising orally administering the short antisense compounds of the present invention.
In certain embodiments, short antisense compounds are targeted to a nucleic acid encoding a protein selected from ApoB, SGLT2, PCSK9, SOD1, CRP, GCCR, GCGR, DGAT2, PTP1B and PTEN.
Further provided are methods of treating a metabolic disorder in an animal, comprising administering to an animal in need of such therapy a short antisense compound targeted to a nucleic acid involved in regulating glucose metabolism or clearance, lipid metabolism, cholesterol metabolism, or insulin signaling.
Also provided are methods of increasing insulin sensitivity, decreasing blood glucose or decreasing HbA1c in an animal, comprising administering to said animal a short antisense compound targeted to a nucleic acid encoding a target involved in regulating glucose metabolism or clearance, lipid metabolism, cholesterol metabolism, or insulin signaling.
Further provided are methods of decreasing total serum cholesterol, serum LDL, serum VLDL, serum HDL, serum triglycerides, serum apolipoprotein(a) or free fatty acids in an animal, comprising administering to said animal a short antisense compound targeted to a nucleic acid encoding a target that is involved in regulating glucose metabolism or clearance, lipid metabolism, cholesterol metabolism, or insulin signaling, wherein said short antisense compound is 8 to 16 nucleotides in length and comprises a gap region flanked on each side by a wing, wherein each wing independently consists of 1 to 3 high-affinity modified nucleotides.
Certain targets involved in regulating glucose metabolism or clearance, lipid metabolism, cholesterol metabolism, or insulin signaling include, but are not limited to, GCGR and ApoB-100. Thus, provided are short antisense compounds targeting nucleic acids encoding GCGR and ApoB-100 and methods of reducing expression of said targets and/or target nucleic acids in animal. In addition, provided is the use of short antisense compounds targeting nucleic acids encoding GCGR, and ApoB-100 for the treatment of a metabolic or cardiovascular disease or condition.
In certain embodiments, short antisense compounds further comprise a conjugate group. Conjugate groups include, but are not limited to, C16 and cholesterol.
In certain embodiments short antisense compounds comprise at least one modified nucleobase, internucleoside linkage or sugar moiety. In certain embodiments, such modified internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, short antisense compounds comprise at least one high affinity modification. In certain such embodiments, the high-affinity modification is a chemically-modified high-affinity nucleotide. In certain embodiments, chemically-modified high affinity nucleotides are sugar-modified nucleotides. In certain embodiments, at least one of the sugar-modified nucleotides comprises a bridge between the 4′ and the 2′ position of the sugar. Each of the sugar-modified nucleotides is, independently, in the β-D or α-L sugar conformation. In certain embodiments, each of said high-affinity modified nucleotides confers a ΔTm of at least 1 to 4 degrees per nucleotide. In certain embodiments, each of said sugar-modified nucleotides comprises a 2′-substituent group that is other than H or OH. Such sugar-modified nucleotides include those having a 4′ to 2′ bridged bicyclic sugar moiety. In certain embodiments, each of the 2′-substituent groups is, independently, alkoxy, substituted alkoxy, or halogen. In certain embodiments, each of the 2′-substituent groups is OCH2CH2OCH3 (2′-MOE).
In certain embodiments, short antisense compounds have one or more sugar-modified nucleotides comprising a bridge between the 4′ and 2′ position of the sugar, wherein each of said bridges independently comprises from 2 to 4 linked groups independently selected from —[C(R1)(R2)]n—, —C(R1)═C(R2)—, —C(R1)═N—, —C(═NR1)—, —C(═O)—, —C(═S)—, —O—, —Si(R1)2—, —S(═O)x— and —N(R1)—;
wherein

    
    
        x is 0, 1, or 2;
        n is 1, 2, 3, or 4;
        
            each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2 The-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
            each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
        
        
    
    


In one aspect, each of said bridges is, independently, —[C(R1)(R2)]n—, —[C(R1)(R2)]n—O—, —C(R1R2)—N(R1)—O— or —C(R1R2)—O—N(R1)—. In another aspect, each of said bridges is, independently, 4′-(CH2)3-2′, 4′-(CH2)2-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2—N(R1)—O-2′— wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.
In certain embodiments, provided herein are short antisense compounds useful in the reduction of targets and/or target RNAs associated with disease states in animals. In certain embodiments, provided are methods of using the short antisense compounds for reducing expression of a target RNA in an animal. In certain embodiments, provided herein is the use of a short antisense compound in the preparation of a medicament for the treatment of a metabolic disorder in an animal. In certain embodiments, provided herein is the use of a short antisense compound in the preparation of a medicament for increasing insulin sensitivity, decreasing blood glucose or decreasing HbA1c in an animal. Also provided is the use of a short antisense compound in the preparation of a medicament for decreasing total serum cholesterol, serum LDL, serum VLDL, serum HDL, serum triglycerides, serum apolipoprotein(a) or free fatty acids in an animal.
In certain embodiments, short antisense compounds provided herein exhibit equal or increased potency with regard to target RNA knockdown as compared to longer parent antisense oligonucleotide at least 20 nucleotides in length. In certain embodiments, short antisense compounds exhibit a faster onset of action (target RNA reduction) as compared to the parent antisense oligonucleotide. In certain embodiments, increased potency is in the kidney. In certain embodiments, target RNA is predominately expressed in the kidney. In certain embodiments, increased potency is in the liver. In certain embodiments, target RNA is predominately expressed in the liver.


DETAILED DESCRIPTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. U.S. patent application Ser. Nos. 10/712,795 and 10/200,710 are hereby expressly incorporated by reference in their entirety for any purpose.
A. DEFINITIONS
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and which is hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and sequences from GenBank and other data bases referred to throughout in the disclosure herein are incorporated by reference in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, the term “nucleoside” means a glycosylamine comprising a nucleobase and a sugar. Nucleosides includes, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups.
As used herein, the term “nucleotide” refers to a glycosomine comprising a nucleobase and a sugar having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.
As used herein, the term “nucleobase” refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.
As used herein, the term “heterocyclic base moiety” refers to a nucleobase comprising a heterocycle.
As used herein, the term “deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
As used herein, the term “ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.
As used herein, the term “oligomeric compound” refers to a polymeric structure comprising two or more sub-structures and capable of hybridizing to a region of a nucleic acid molecule. In certain embodiments, oligomeric compounds are oligonucleosides. In certain embodiments, oligomeric compounds are oligonucleotides. In certain embodiments, oligomeric compounds are antisense compounds. In certain embodiments, oligomeric compounds are antisense oligonucleotides. In certain embodiments, oligomeric compounds are short antisense compounds. In certain embodiments, oligomeric compounds are short antisense oligonucleotides. In certain embodiments, oligomeric compounds are chimeric oligonucleotides.
As used herein, the term “monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.
As used herein “oligonucleoside” refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
As used herein, the term “oligonucleotide” refers to an oligomeric compound comprising a plurality of linked nucleotides. In certain embodiment, one or more nucleotides of an oligonucleotide is modified. In certain embodiments, an oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In certain embodiments, oligonucleotides are composed of naturally- and/or non-naturally-occurring nucleobases, sugars and covalent internucleotide linkages, and may further include non-nucleic acid conjugates.
As used herein “internucleotide linkage” refers to a covalent linkage between adjacent nucleotides.
As used herein, the term “monomeric linkage” refers to a covalent linkage between two monomers. Monomeric linkages include, but are not limited to internucleotide linkages and internucleoside linkages.
As used herein “naturally occurring internucleotide linkage” refers to a 3′ to 5′ phosphodiester linkage.
As used herein, the term “antisense compound” refers to an oligomeric compound that is at least partially complementary to a target nucleic acid molecule to which it hybridizes. In certain embodiments, an antisense compound modulates (increases or decreases) expression of a target nucleic acid. Antisense compounds include, but are not limited to, compounds that are oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. Consequently, while all antisense compounds are oligomeric compounds, not all oligomeric compounds are antisense compounds.
As used herein, the term “antisense oligonucleotide” refers to an antisense compound that is an oligonucleotide.
As used herein, the term “parent antisense oligonucleotide” refers to an oligonucleotide 20 nucleotides in length having a deoxy gap region having ten 2′-deoxyribonucleotides, flanked by a first and a second wing region each having five 2′-O-(2-methoxyethyl) ribonucleotides (a 5-10-5 MOE gapmer) and comprising the sequence of the corresponding short antisense compound to which it is a parent.
As used herein, the term “short antisense compound” refers to an antisense compound about 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers in length. In certain embodiments, a short antisense compound has at least one high-affinity modification.
As used herein, the term “short antisense oligonucleotide” or refers to an antisense oligonucleotide about 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides in length. In certain embodiments, a short antisense oligonucleotide has at least one high-affinity modification.
As used herein, the term “short gapmer” refers to a short antisense oligonucleotide having a first and a second wing region each independently 1 to 3 nucleotides in length and a gap region 2 to 14 nucleobase in length.
As used herein, the term “motif” refers to the pattern of unmodified and modified nucleotides in a short antisense compound
As used herein, the term “chimeric antisense oligomer” refers to an antisense oligomeric compound, having at least one sugar, nucleobase or internucleoside linkage that is differentially modified as compared to at least on other sugar, nucleobase or internucleoside linkage within the same antisense oligomeric compound. The remainder of the sugars, nucleobases and internucleoside linkages can be independently modified or unmodified, the same or different.
As used herein, the term “chimeric antisense oligonucleotide” refers to an antisense oligonucleotide, having at least one sugar, nucleobase or internucleoside linkage that is differentially modified as compared to at least on other sugar, nucleobase or internucleoside linkage within the same antisense oligonucleotide. The remainder of the sugars, nucleobases and internucleoside linkages can be independently modified or unmodified, the same or different.
As used herein, the term “mixed-backbone antisense oligonucleotide” refers to an antisense oligonucleotide wherein at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleotide linkage of the antisense oligonucleotide.
As used herein, the term “target” refers to a protein, the modulation of which is desired.
As used herein, the term “target gene” refers to a gene encoding a target.
As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding a target” refer to any nucleic acid molecule the expression or activity of which is capable of being modulated by an antisense compound. Target nucleic acids include, but are not limited to, RNA (including, but not limited to pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding a target, and also cDNA derived from such RNA, and miRNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent.
As used herein, the term “targeting” or “targeted to” refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule.
As used herein, the term “5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.
As used herein, the term “3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.
As used herein, the term “target region,” refers to a portion of a target nucleic acid to which one or more antisense compounds is complementary.
As used herein, the term “target segment” refers to a smaller or sub-portions of a region within a target nucleic acid.
As used herein, the term “nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
As used herein, the term “non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
As used herein, the term “complementary” refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity. In certain embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). Preferably the antisense compounds contain no more than about 15%, more preferably not more than about 10%, most preferably not more than 5% or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target nucleic acid.
As used herein, the term “mismatch” refers to a non-complementary nucleobase within a complementary oligomeric compound.
As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.
As used herein, the term “specifically hybridizes” refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
As used herein, “designing” or “designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
As used herein, the term “modulation” refers to a perturbation of function or activity when compared to the level of the function or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As further example, modulation of expression can include perturbing splice site selection of pre-mRNA processing.
As used herein, the term “expression” refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
As used herein, “variant” refers to an alternative RNA transcript that can be produced from the same genomic region of DNA. Variants include, but are not limited to “pre-mRNA variants” which are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Variants also include, but are not limited to, those with alternate splice junctions, or alternate initiation and termination codons.
As used herein, “high-affinity modified monomer” refers to a monomer having at least one modified nucleobase, internucleoside linkage or sugar moiety, when compared to naturally occurring monomers, such that the modification increases the affinity of an antisense compound comprising the high-affinity modified monomer to its target nucleic acid. High-affinity modifications include, but are not limited to, monomers (e.g., nucleosides and nucleotides) comprising 2′-modified sugars.
As used herein, the term “2′-modified” or “2′-substituted” means a sugar comprising substituent at the 2′ position other than H or OH. 2′-modified monomers, include, but are not limited to, BNA's and monomers (e.g., nucleosides and nucleotides) with 2′-substituents, such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rm), or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, short antisense compounds comprise a 2′-modified monomer that does not have the formula 2′-O(CH2)nH, wherein n is one to six. In certain embodiments, short antisense compounds comprise a 2′-modified monomer that does not have the formula 2′-OCH3. In certain embodiments, short antisense compounds comprise a 2′-modified monomer that does not have the formula or, in the alternative, 2′-O(CH2)2OCH3.
As used herein, the term “bicyclic nucleic acid” or “BNA” or “bicyclic nucleoside” or “bicyclic nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
As used herein, unless otherwise indicated, the term “methyleneoxy BNA” alone refers to β-D-methyleneoxy BNA.
As used herein, the term “MOE” refers to a 2′-methoxyethyl substituent.
As used herein, the term “gapmer” refers to a chimeric oligomeric compound comprising a central region (a “gap”) and a region on either side of the central region (the “wings”), wherein the gap comprises at least one modification that is different from that of each wing. Such modifications include nucleobase, monomeric linkage, and sugar modifications as well as the absence of modification (unmodified). Thus, in certain embodiments, the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap. In certain embodiments, each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification. In certain embodiments the nucleotides in the gap and the nucleotides in the wings all comprise high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in the wings. In certain embodiments, the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other. In certain embodiments, nucleotides in the gap are unmodified and nucleotides in the wings are modified. In certain embodiments, the modification(s) in each wing are the same. In certain embodiments, the modification(s) in one wing are different from the modification(s) in the other wing. In certain embodiments, short antisense compounds are gapmers having 2′-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
As used herein, the term “prodrug” refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
As used herein, the term “pharmaceutically acceptable salts” refers to salts of active compounds that retain the desired biological activity of the active compound and do not impart undesired toxicological effects thereto.
As used herein, the term “cap structure” or “terminal cap moiety” refers to chemical modifications, which have been incorporated at either terminus of an antisense compound.
As used herein, the term “prevention” refers to delaying or forestalling the onset or development of a condition or disease for a period of time from hours to days, preferably weeks to months.
As used herein, the term “amelioration” refers to a lessening of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
As used herein, the term “treatment” refers to administering a composition of the invention to effect an alteration or improvement of the disease or condition. Prevention, amelioration, and/or treatment may require administration of multiple doses at regular intervals, or prior to onset of the disease or condition to alter the course of the disease or condition. Moreover, a single agent may be used in a single individual for each prevention, amelioration, and treatment of a condition or disease sequentially, or concurrently.
As used herein, the term “pharmaceutical agent” refers to a substance provides a therapeutic benefit when administered to a subject.
As used herein, the term “therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.
As used herein, “administering” means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
As used herein, the term “co-administration” refers to administration of two or more pharmaceutical agents to an animal. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.
As used herein, the term “pharmaceutical composition” refers to a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
As used herein, the term “individual” refers to a human or non-human animal selected for treatment or therapy.
As used herein, the term “animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
As used herein, the term “subject” refers to an animal, including, but not limited to a human, to whom a pharmaceutical composition is administered.
As used herein, the term “duration” refers to the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.
As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein.
As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
As used herein, the term “pharmaceutical agent” refers to a substance provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide is a pharmaceutical agent.
As used herein, the term “active pharmaceutical ingredient” refers to the substance in a pharmaceutical composition that provides a desired effect.
As used herein, the term “therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. In certain embodiments, a therapeutically effective amount of an antisense compound is the amount that needs to be administered to result in an observable benefit.
As used herein, the term “hypercholesterolemia” refers to a condition characterized by elevated serum cholesterol.
As used herein, the term “hyperlipidemia” refers to a condition characterized by elevated serum lipids.
As used herein, the term “hypertriglyceridemia” refers to a condition characterized by elevated triglyceride levels.
As used herein, the term “non-familial hypercholesterolemia” refers to a condition characterized by elevated cholesterol that is not the result of a single inherited gene mutation.
As used herein, the term “polygenic hypercholesterolemia” refers to a condition characterized by elevated cholesterol that results from the influence of a variety of genetic factors. In certain embodiments, polygenic hypercholesterolemia may be exacerbated by dietary intake of lipids.
As used herein, the term “familial hypercholesterolemia (FH)” refers to an autosomal dominant metabolic disorder characterized by a mutation in the LDL-receptor (LDL-R) gene, markedly elevated LDL-C and premature onset of atherosclerosis. A diagnosis of familial hypercholesterolemia is made when a individual meets one or more of the following criteria: genetic testing confirming 2 mutated LDL-receptor genes; genetic testing confirming one mutated LDL-receptor gene; document history of untreated serum LDL-cholesterol greater than 500 mg/dL; tendinous and/or cutaneous xanthoma prior to age 10 years; or, both parents have documented elevated serum LDL-cholesterol prior to lipid-lowering therapy consistent with heterozygous familial hypercholesterolemia.
As used herein, the term “homozygous familial hypercholesterolemia” or “HoFH” refers to a condition characterized by a mutation in both maternal and paternal LDL-R genes.
As used herein, the term “heterozygous familial hypercholesterolemia” or “HeFH” refers to a condition characterized by a mutation in either the maternal or paternal LDL-R gene.
As used herein, the term “mixed dyslipidemia” refers to a condition characterized by elevated serum cholesterol and elevated serum triglycerides.
As used herein, the term “diabetic dyslipidemia” or “Type II diabetes with dyslipidemia” refers to a condition characterized by Type II diabetes, reduced HDL-C, elevated serum triglycerides, and elevated small, dense LDL particles.
As used herein, the term “CHD risk equivalents,” refers to indicators of clinical atherosclerotic disease that confer a high risk for coronary heart disease. For example, in certain embodiments, CHD risk equivalents include, without limitation, clinical coronary heart disease, symptomatic carotid artery disease, peripheral arterial disease, and/or abdominal aortic aneurysm.
As used herein, the term “non-alcoholic fatty liver disease (NAFLD)” refers to a condition characterized by fatty inflammation of the liver that is not due to excessive alcohol use (for example, alcohol consumption of over 20 g/day). In certain embodiments, NAFLD is related to insulin resistance and the metabolic syndrome.
As used herein, the term “non-alcoholic steatohepatitis (NASH)” refers to a condition characterized by inflammation and the accumulation of fat and fibrous tissue in the liver, that is not due to excessive alcohol use. NASH is an extreme form of NAFLD.
As used herein, the term “major risk factors” refers to factors that contribute to a high risk for a particular disease or condition. In certain embodiments, major risk factors for coronary heart disease include, without limitation, cigarette smoking, hypertension, low HDL-C, family history of coronary heart disease, and age.
As used herein, the term “CHD risk factors” refers to CHD risk equivalents and major risk factors.
As used herein, the term “coronary heart disease (CHD)” refers to a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.
As used herein, the term “reduced coronary heart disease risk” refers to a reduction in the likelihood that a individual will develop coronary heart disease. In certain embodiments, a reduction in coronary heart disease risk is measured by an improvement in one or more CHD risk factors, for example, a decrease in LDL-C levels.
As used herein, the term “atherosclerosis” refers to a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called “atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.
As used herein, the term “history of coronary heart disease” refers to the occurrence of clinically evident coronary heart disease in the medical history of a individual or a individual's family member.
As used herein, the term “Early onset coronary heart disease” refers to a diagnosis of coronary heart disease prior to age 50.
As used herein, the term “statin intolerant individual” refers to a individual who as a result of statin therapy experiences one or more of creatine kinase increases, liver function test abnormalities, muscle aches, or central nervous system side effects.
As used herein, the term “efficacy” refers to the ability to produce a desired effect. For example, efficacy of a lipid-lowering therapy may be reduction in the concentration of one or more of LDL-C, VLDL-C, IDL-C, non-HDL-C, ApoB, lipoprotein(a), or triglycerides.
As used herein, the term “acceptable safety profile” refers to a pattern of side effects that is within clinically acceptable limits.
As used herein, the term “side effects” refers to physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
As used herein, the term “injection site reaction” refers to inflammation or abnormal redness of skin at a site of injection in an individual.
As used herein, the term “individual compliance” refers to adherence to a recommended or prescribed therapy by an individual.
As used herein, the term “lipid-lowering therapy” refers to a therapeutic regimen provided to a individual to reduce one or more lipids in a individual. In certain embodiments, a lipid-lowering therapy is provide to reduce one or more of ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in an individual.
As used herein, the term “lipid-lowering agent” refers to a pharmaceutical agent provided to a individual to achieve a lowering of lipids in the individual. For example, in certain embodiments, a lipid-lowering agent is provided to an individual to reduce one or more of ApoB, LDL-C, total cholesterol, and triglycerides.
As used herein, the term “LDL-C target” refers to an LDL-C level that is desired following lipid-lowering therapy.
As used herein, the term “comply” refers to the adherence with a recommended therapy by an individual.
As used herein, the term “recommended therapy” refers to a therapeutic regimen recommended by a medical professional for the treatment, amelioration, or prevention of a disease.
As used herein, the term “low LDL-receptor activity” refers to LDL-receptor activity that is not sufficiently high to maintain clinically acceptable levels of LDL-C in the bloodstream.
As used herein, the term “cardiovascular outcome” refers to the occurrence of major adverse cardiovascular events.
As used herein, the term “improved cardiovascular outcome” refers to a reduction in the occurrence of major adverse cardiovascular events, or the risk thereof. Examples of major adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.
As used herein, the term “surrogate markers of cardiovascular outcome” refers to indirect indicators of cardiovascular events, or the risk thereof. For example, surrogate markers of cardiovascular outcome include carotid intimal media thickness (CIMT). Another example of a surrogate marker of cardiovascular outcome includes atheroma size. Atheroma size may be determined by intravascular ultrasound (IVUS).
As used herein, the term “increased HDL-C” refers to an increase in serum HDL-C in an individual over time.
As used herein, the term “lipid-lowering” refers to a reduction in one or more serum lipids in an individual over time.
As used herein, the term “metabolic disorder” refers to a condition characterized by an alteration or disturbance in metabolic function. “Metabolic” and “metabolism” are terms well know in the art and generally include the whole range of biochemical processes that occur within a living organism. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance and metabolic syndrome.
As used herein, the term “metabolic syndrome” refers to a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. It has been closely linked to the generalized metabolic disorder known as insulin resistance. The National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) established criteria for diagnosis of metabolic syndrome when three or more of five risk determinants are present. The five risk determinants are abdominal obesity defined as waist circumference of greater than 102 cm for men or greater than 88 cm for women, triglyceride levels greater than or equal to 150 mg/dL, HDL cholesterol levels of less than 40 mg/dL for men and less than 50 mg/dL for women, blood pressure greater than or equal to 130/85 mm Hg and fasting glucose levels greater than or equal to 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
The term “alkyl,” as used herein, refers to a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred. The term “lower alkyl” as used herein includes from 1 to about 6 carbon atoms. Alkyl groups as used herein may optionally include one or more further substituent groups.
The term “alkenyl,” as used herein, refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
The term “alkynyl,” as used herein, refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substitutent groups.
The term “aminoalkyl” as used herein, refers to an amino substituted alkyl radical. This term is meant to include C1-C12 alkyl groups having an amino substituent at any position and wherein the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and/or amino portions of the aminoalkyl group can be further substituted with substituent groups.
The term “aliphatic,” as used herein, refers to a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines Aliphatic groups as used herein may optionally include further substitutent groups.
The term “alicyclic” or “alicyclyl” refers to a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substitutent groups.
The term “alkoxy,” as used herein, refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substitutent groups.
The terms “halo” and “halogen,” as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.
The terms “aryl” and “aromatic,” as used herein, refer to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substitutent groups.
The terms “aralkyl” and “arylalkyl,” as used herein, refer to a radical formed between an alkyl group and an aryl group wherein the alkyl group is used to attach the aralkyl group to a parent molecule. Examples include, but are not limited to, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substitutent groups attached to the alkyl, the aryl or both groups that form the radical group.
The term “heterocyclic radical” as used herein, refers to a radical mono-, or poly-cyclic ring system that includes at least one heteroatom and is unsaturated, partially saturated or fully saturated, thereby including heteroaryl groups. Heterocyclic is also meant to include fused ring systems wherein one or more of the fused rings contain at least one heteroatom and the other rings can contain one or more heteroatoms or optionally contain no heteroatoms. A heterocyclic group typically includes at least one atom selected from sulfur, nitrogen or oxygen. Examples of heterocyclic groups include, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as used herein may optionally include further substitutent groups.
The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatom. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substitutent groups.
The term “heteroarylalkyl,” as used herein, refers to a heteroaryl group as previously defined having an alky radical that can attach the heteroarylalkyl group to a parent molecule. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, napthyridinylpropyl and the like. Heteroarylalkyl groups as used herein may optionally include further substitutent groups on one or both of the heteroaryl or alkyl portions.
The term “mono or poly cyclic structure” as used in the present invention includes all ring systems that are single or polycyclic having rings that are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic, heteroarylalkyl. Such mono and poly cyclic structures can contain rings that are uniform or have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or poly cyclic structures can be further substituted with substituent groups such as for example phthalimide which has two ═O groups attached to one of the rings. In another aspect, mono or poly cyclic structures can be attached to a parent molecule directly through a ring atom, through a substituent group or a bifunctional linking moiety.
The term “acyl,” as used herein, refers to a radical formed by removal of a hydroxyl group from an organic acid an d has the general formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substitutent groups.
The term “hydrocarbyl” includes groups comprising C, O and H. Included are straight, branched and cyclic groups having any degree of saturation. Such hydrocarbyl groups can include one or more heteroatoms selected from N, O and S and can be further mono or poly substituted with one or more substituent groups.
The terms “substituent” and “substituent group,” as used herein, include groups that are typically added to other groups or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be added to one available site or to many available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound. Such groups include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxo (—O—Raa), aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino (—NRbbR—), imino(═NRbb), amido (—C(O)NRbbRcc or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)NRbbRcc or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)NRbbRcc), thioureido (—N(Rbb)C(S)NRbbRcc), guanidinyl (—N(Rbb)C(═NRbb)NRbbRcc), amidinyl (—C(═NRbb)NRbbRcc or —N(Rbb)C(NRbb)Raa), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb), sulfonamidyl (—S(O)2NRbbRcc or —N(Rbb)S(O)2Rbb) and conjugate groups. Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation H, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl.
B. CERTAIN OLIGOMERIC COMPOUNDS
In certain embodiments, it is desirable to chemically modify oligomeric compounds, compared to naturally occurring oligomers, such as DNA or RNA. Certain such modifications alter the activity of the oligomeric compound. Certain such chemical modifications can alter activity by, for example: increasing affinity of an antisense compound for its target nucleic acid, increasing its resistance to one or more nucleases, and/or altering the pharmacokinetics or tissue distribution of the oligomeric compound. In certain instances, the use of chemistries that increase the affinity of an oligomeric compound for its target can allow for the use of shorter oligomeric compounds.
1. Certain Monomers
In certain embodiment, oligomeric compounds comprise one or more modified monomer. In certain such embodiments, oligomeric compounds comprise one or more high affinity monomer. In certain embodiments, such high-affinity monomer is selected from monomers (e.g., nucleosides and nucleotides) comprising 2′-modified sugars, including, but not limited to: BNA's and monomers (e.g., nucleosides and nucleotides) with 2′-substituents such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Ra), or O—CH2—C(═O)—N(Rm)(Ra), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
In certain embodiments, the oligomeric compounds including, but no limited to short antisense compounds of the present invention, comprise one or more high affinity monomers provided that the oligomeric compound does not comprise a nucleotide comprising a 2′-O(CH2)nH, wherein n is one to six.
In certain embodiments, the oligomeric compounds including, but no limited to short antisense compounds of the present invention, comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a nucleotide comprising a 2′-OCH3 or a 2′-O(CH2)2OCH3.
In certain embodiments, the oligomeric compounds including, but no limited to short antisense compounds of the present invention, comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a α-L-Methyleneoxy (4′-CH2—O-2′) BNA.
In certain embodiments, the oligomeric compounds including, but no limited to short antisense compounds of the present invention, comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a β-D-Methyleneoxy (4′-CH2—O-2′) BNA.
In certain embodiments, the oligomeric compounds including, but no limited to short antisense compounds of the present invention, comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a α-L-Methyleneoxy (4′-CH2—O-2′) BNA or a β-D-Methyleneoxy (4′-CH2—O-2′) BNA.
a. Certain Nucleobases
The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. For those nucleosides that include a pentofuranosyl sugar, a phosphate group can be linked to the 2′,3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleotide backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and of DNA is a 3′ to 5′ phosphodiester linkage.
In addition to “unmodified” or “natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein. In certain embodiments, a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp.
In certain embodiments, nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
b. Certain Sugars
Oligomeric compounds provided herein may comprise one or more monomer, including a nucleoside or nucleotide, having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA).
In certain embodiments, oligomeric compounds comprise one or more monomers that is a BNA. In certain such embodiments, BNA s include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA, (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA and (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, as depicted in Figure 1.




In certain embodiments, BNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R1)(R2)]n—, —C(R1)═C(R2)—, —C(R1)═N—, —C(═NR1)—, —C(═O)—, —C(═S)—, —O—, —Si(R1)27, —S(═O)x— and —N(R1)—;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
In one embodiment, each of the bridges of the BNA compounds is, independently, —[C(R1)(R2)]n—, —[C(R1)(R2)]n—O—, —C(R1R2)—N(R1)—O— or —C(R1R2)—O—N(R1)—. In another embodiment, each of said bridges is, independently, 4′-CH2-2′,4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2—N(R1)—O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.
Certain BNA's have been prepared and disclosed in the patent literature as well as in scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO 2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Examples of issued US patents and published applications that disclose BNA s include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.
Also provided herein are BNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is linked to the 4′ carbon atom of the sugar ring thereby forming a methyleneoxy (4′-CH2—O-2′) linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (—CH2—) group bridging the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH2—O-2′) BNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ethyleneoxy (4′-CH2CH2—O-2′) BNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy (4′-CH2—O-2′) BNA and other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638).
An isomer of methyleneoxy (4′-CH2—O-2′) BNA that has also been discussed is alpha-L-methyleneoxy (4′-CH2—O-2′) BNA which has been shown to have superior stability against a 3′-exonuclease. The alpha-L-methyleneoxy (4′-CH2—O-2′) BNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
The synthesis and preparation of the methyleneoxy (4′-CH2—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4′-CH2—O-2′) BNA, phosphorothioate-methyleneoxy (4′-CH2—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars (BNA's), including methyleneoxy (4′-CH2—O-2′) BNA and ethyleneoxy (4′-(CH2)2—O-2′ bridge) BNA; substituted sugars, especially 2′-substituted sugars having a 2′-F, 2′-OCH3 or a 2′-O(CH2)2—OCH3 substituent group; and 4′-thio modified sugars. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and 6,600,032; and WO 2005/121371.
In certain embodiments, BNA's include bicyclic nucleoside having the formula:




wherein:

Bx is a heterocyclic base moiety;
T1 is H or a hydroxyl protecting group;
T2 is H, a hydroxyl protecting group or a reactive phosphorus group;
Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.
In certain such embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJ1.
In certain embodiments, the Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—), substituted alkoxy or azido.
In certain embodiments, the Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2, 5J1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is —CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.
In certain such embodiments, the Z group is in the (R)-configuration:




In certain such embodiments, the Z group is in the (S)-configuration:




In certain embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is T1 is 4,4′-dimethoxytrityl.
In certain embodiments, T2 is a reactive phosphorus group wherein preferred reactive phosphorus groups include diisopropylcyanoethoxy phosphoramidite and H-phosphonate. In certain embodiments T1 is 4,4′-dimethoxytrityl and T2 is diisopropylcyanoethoxy phosphoramidite.
In certain embodiments, oligomeric compounds have at least one monomer of the formula:




or of the formula:





or of the formula:





wherein

Bx is a heterocyclic base moiety;
T3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;
T4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;
wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; and
Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O or NJ1.
In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is, independently, C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is, independently, C1-C6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is, independently, substituted C1-C6 alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.
In certain embodiments, at least one substituent group is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In another embodiment, each substituent group is, independently, C1-C6 alkoxy (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more C1-C6 alkoxy).
In certain embodiments, at least one C1-C6 alkoxy substituent group is CH3O— (e.g., at least one Z is CH3OCH2—). In another embodiment, each C1-C6 alkoxy substituent group is CH3O— (e.g., each Z is CH3OCH2—).
In certain embodiments, at least one substituent group is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogen). In certain embodiments, each substituent group is, independently, halogen (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more halogen). In certain embodiments, at least one halogen substituent group is fluoro (e.g., at least one Z is CH2FCH2—, CHF2CH2— or CF3CH2—). In certain embodiments, each halo substituent group is fluoro (e.g., each Z is, independently, CH2FCH2—, CHF2CH2— or CF3CH2—).
In certain embodiments, at least one substituent group is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent group is, independently, hydroxyl (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2—. In another embodiment, each Z is HOCH2—.
In certain embodiments, at least one Z is CH3—, CH3CH2—, CH2OCH3—, CH2F— or HOCH2—. In certain embodiments, each Z is, independently, CH3—, CH3CH2—, CH2OCH3—, CH2F— or HOCH2—.
In certain embodiments, at least one Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, at least one Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.
In certain embodiments, each Z group is, independently, C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is, independently, C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.
In certain embodiments, at least one Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In certain embodiments, at least one Z group is —CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.
In certain embodiments, each Z group is, independently, —CH2Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is, independently, —CH2Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.
In certain embodiments, at least one Z is CH3—. In another embodiment, each Z is, CH3—.
In certain embodiments, the Z group of at least one monomer is in the (R)-configuration represented by the formula:




or the formula:





or the formula:





In certain embodiments, the Z group of each monomer of the formula is in the (R)-configuration.
In certain embodiments, the Z group of at least one monomer is in the (S)-configuration represented by the formula:




or the formula:





or the formula:





In certain embodiments, the Z group of each monomer of the formula is in the (S)-configuration.
In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In a further embodiment T3 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.
In certain embodiments, oligomeric compounds have at least one region of at least two contiguous monomers of the formula:




or of the formula:





or of the formula:





In certain embodiments, the oligomeric compound comprises at least two regions of at least two contiguous monomers of the above formula. In certain embodiments, the oligomeric compound comprises a gapped oligomeric compound. In certain embodiments, the oligmeric compound comprises at least one region of from about 8 to about 14 contiguous β-D-2′-deoxyribofuranosyl nucleosides. In certain embodiments, the oligomeric compound comprises at least one region of from about 9 to about 12 contiguous β-D-2′-deoxyribofuranosyl nucleosides.
In certain embodiments, monomers include sugar mimetics. In certain such embodiments, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of a sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.
3. Monomeric Linkages
Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound. The two main classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Oligomeric compounds having non-phosphorus linking groups are referred to as oligonucleosides. Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, linkages having a chiral atom can be prepared a racemic mixtures, as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art.
The oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or 13 such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
4. Oligomeric Compounds
In certain embodiments, provided herein are oligomeric compounds having reactive phosphorus groups useful for forming linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Methods of preparation and/or purification of precursors or oligomeric compounds are not a limitation of the compositions or methods provided herein. Methods for synthesis and purification of oligomeric compounds including DNA, RNA, oligonucleotides, oligonucleosides, and antisense compounds are well known to those skilled in the art.
Generally, oligomeric compounds comprise a plurality of monomeric subunits linked together by linking groups. Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs. As such, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
In certain embodiments, the present invention provides chimeric oligomeric compounds. In certain such embodiments, chimeric oligomeric compounds are chimeric oligonucleotides. In certain such embodiments, the chimeric oligonucleotides comprise differently modified nucleotides. In certain embodiments, chimeric oligonucleotides are mixed-backbone antisense oligonucleotides.
In general a chimeric oligomeric compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Any combination of modifications and/or mimetic groups can comprise a chimeric oligomeric compound as described herein.
In certain embodiments, chimeric oligomeric compounds typically comprise at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. In certain embodiments, an additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligomeric compounds when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain embodiments, chimeric oligomeric compounds are gapmers. In certain embodiments, chimeric compounds are short antisense compounds. In certain embodiments, short antisense compounds are gapmers. In certain such embodiments, a mixed-backbone antisense oligomer has one type of internucleotide linkages in one or both wings and a different type of internucleotide linkages in the gap. In certain such embodiments, the mixed-backbone antisense oligonucleotide has phosphodiester linkages in the wings and phosphorothioate linkages in the gap. In certain embodiments in which the internucleotide linkages in a wing is different from the internucleotide linkages in the gap, the internucleotide linkage bridging that wing and the gap is the same as the internucleotide linkage in the wing. In certain embodiments in which the internucleotide linkages in a wing is different from the internucleotide linkages in the gap, the internucleotide linkage bridging that wing and the gap is the same as the internucleotide linkage in the gap.
C. CERTAIN SHORT ANTISENSE COMPOUNDS
Disclosed herein are short antisense compounds 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds comprise one or more chemical modifications. In certain such embodiments, short antisense compounds comprise at least one modified nucleotide. In certain embodiments short antisense compounds comprise at least two or more modified nucleotides. In certain embodiments, short antisense compounds comprise at least one modified internucleotide linkage. In certain embodiments, short antisense compounds are mixed-backbone oligonucleotides. In certain embodiments, short antisense compounds are chimeric oligonucleotides. In certain embodiments, short antisense oligonucleotides are uniformly modified. In certain embodiments, short antisense oligonucleotides comprise modifications independently selected at each nucleobase and at each linkage.
In certain embodiments, short antisense compounds are short gapmers. In certain such embodiments, short gapmers comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds comprise 1 to 3 high-affinity modifications in each wing. In certain embodiments, high affinity modifications of the short antisense compounds allow for a target affinity similar to, or even greater than, the target affinity of longer antisense compounds. In certain embodiments, the high-affinity modified nucleotides are sugar modified nucleotides. Such sugar modified nucleotides include those comprising a bridge between the 4′ and 2′ position of the sugar. Exemplary high affinity sugar modifications include, but are not limited to, BNA s and other 2′-modifications such as 2′-MOE. In an alternate embodiment of the invention, the high affinity modification is not a 2′-O—(CH2)nH (n=1-6) sugar-modified nucleotide. In an additional alternate embodiment, the high affinity modified nucleotide is not a 2′-OCH3 or a 2′-OCH2CH2OCH3 nucleotide. In certain embodiments, the high-affinity modified nucleotides confer a ΔTm of at least 1, at least 1.5, at least 2, at least 2.5, at least 3.0, at least 3.5 or at least 4.0 degrees per nucleotide. Some high-affinity nucleotide modifications are known in the art to increase toxicity. As shown herein, short antisense compounds having a limited number (generally 2 to 6) of high affinity modifications exhibit little to no increase in toxicity but retain or increase affinity for the target RNA, while also significantly reducing expression of the RNA target. Short antisense compounds of the invention may optionally comprise a conjugate group, such as, for example, cholesterol or C16.
1. Certain Wings
In certain embodiments, the short antisense compounds comprise a 5′ wing and/or a 3′ wing. In such embodiments, the features of the 3′ wing and the features of the 5′ wing are selected independently. Thus, in such embodiments, the number of monomers in the 5′ wing and the number of monomers (length) in the 3′ wing may be the same or may be different; the modifications, if any, in the 5′ wing may be the same as the modifications, if any, in the 3′ wing or such modifications, if any, may be different; and the monomeric linkages in the 5′ wing and the monomeric linkages in the 3′ wing may be the same or they may be different.
In certain embodiments a wing comprises one, two or three monomers (i.e. has a length of 1, 2, or 3). In certain embodiments, the monomers of a wing are modified. In certain such embodiments, the monomers of the wing are modified to increase affinity of the antisense compound for its target nucleic acid. In certain embodiments, the monomers of a wing are nucleosides or nucleotides. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers (nucleosides or nucleotides) of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, the monomeric linkages in a wing are naturally occurring internucleotide linkages. In certain embodiments, the monomeric linkages in a wing are non-naturally occurring internucleotide or internucleoside linkages. In certain such embodiments, the monomeric linkages in the wing are more resistant to one or more nucleases than naturally occurring internucleotide linkages. In certain such embodiments, the monomeric linkages in the wing are phosphorothioate linkages (P═S). In certain embodiments where a wing has more than one monomeric linkage, the monomeric linkages are the same as one another. In certain embodiments where a wing has more than one monomers linkage, the monomers linkages are different from each other.
One of ordinary skill in the art will recognize that the features and modifications discussed above may be used in any combination to prepare a wing. The table below provides non-limiting examples showing how one might prepare a wing by selecting a certain number of monomers, monomeric modifications (if any), and monomeric linkages both within the wing.













Monomer type/
monomeric linkages within


Length
modifications
wing







1
2′ MOE
None


1
BNA
None


1
Methyleneoxy
None



BNA


1
ENA
None


2
2′ MOE
P═S


2
BNA
P═S


2
Methyleneoxy
P═S



BNA


2
ENA
P═S


2
2′ MOE
P═O


2
BNA
P═O


2
Methyleneoxy
P═O



BNA


2
ENA
P═O


3
2′ MOE
P═S


3
BNA
P═S


3
Methyleneoxy
P═S



BNA


3
ENA
P═S


3
2′ MOE
P═O


3
BNA
P═O


3
Methyleneoxy
P═O



BNA


3
ENA
P═O









In certain embodiments in which a wing comprises two, three or four monomers, those two, three or four monomers all comprise the same modifications, if any. In certain embodiments in which a wing comprises two, three or four monomers, one or more of those two, three or four nucleobases comprises one or more modifications that is different from one or more of the modifications of one or more of the remaining monomers.
2. Certain Gaps
In certain embodiments, the short antisense compounds comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, the monomeric linkages in the gap are naturally occurring internucleotide linkages. In certain embodiments, the monomeric linkages in the gap are non-naturally occurring linkages. In certain such embodiments, the monomeric linkages in the gap are more resistant to one or more nuclease than naturally occurring internucleotide linkages. In certain such embodiments, the monomeric linkages in the gap are phosphorothioate linkages (P═S). In certain embodiments, the monomeric linkages in the gap are all the same as one another. In certain embodiments, the monomeric linkages within the gap are not all the same.
One of ordinary skill in the art will recognize that the features and modifications discussed above may be used in any combination to prepare a gap. The table below provides non-limiting examples showing how one might prepare a gap by selecting a certain number of monomers, monomeric modifications (if any), and monomeric linkages within the gap region.













Monomer type/
Monomeric linkages within


Length
modifications
gap

















5
DNA
P═S


6
DNA
P═S


7
DNA
P═S


8
DNA
P═S


9
DNA
P═S


10
DNA
P═S


11
DNA
P═S


12
DNA
P═S


13
DNA
P═S


14
DNA
P═S


6
DNA
P═O


7
DNA
P═O


8
DNA
P═O


9
DNA
P═O


10
DNA
P═O


11
DNA
P═O


12
DNA
P═O


8
RNA
P═S


9
RNA
P═S


10
RNA
P═S


11
RNA
P═S


12
RNA
P═S









3. Certain Gapped Antisense Oligomeric Compounds
One of ordinary skill in the art will recognize that the wings and the gaps discussed above may be selected and then combined in a variety of combinations to generate gapped oligomeric compounds, including, but not limited to, gapped antisense oligomeric compounds, and gapped antisense oligonucleotides. The features (length, modifications, linkages) of the 5′ wing and the 3′ wing may be selected independently of one another. The features of the gap include at least one difference in modification compared to the features of the 5′ wing and at least one difference compared to the features of the 3′ wing (i.e., there must be at least one difference in modification between neighboring regions to distinguish those neighboring regions from one another). The features of the gap may otherwise be selected independently.
In certain embodiments, the monomeric linkages within a wing and the monomeric linkages within the gap are the same. In certain embodiments, the monomeric linkages within a wing and the monomeric linkages within the gap are different. In certain such embodiments, the monomeric linkage bridging the wing and the gap are the same as the monomeric linkages in the wing. In certain embodiments, the monomeric linkage bridging the wing and the gap are the same as the monomeric linkages in the gap. In certain embodiments, short antisense compounds have uniform linkages throughout the compound. In certain such embodiments, all of the linkages are phosphorothioate (P═S) linkages.
One of ordinary skill in the art will recognize that the 3′ wings, 5′ wings, gaps, and linkages discussed above may be used in any combination to prepare a gapmer. The table below provides non-limiting examples showing how one might prepare a gapmer by selecting a certain 5′ wing, a gap, a 3′ wing and certain linkages bridging the gap and each wing.














5′ Wing
5′ Bridge
Gap
3′ Bridge
3′ Wing

















Length
Monomer
Link
Link
Length
Monomer
Link
Link 
Length
Monomer
Link




















2
MOE
P═S
P═S
6
DNA
P═S
P═S
2
MOE
P═S


2
BNA
P═S
P═O
8
DNA
P═O
P═S
3
BNA
P═S


1
MOE
None
P═S
10
DNA
P═S
P═S
1
MOE
P═S


2
MOE
P═S
P═S
8
RNA
P═S
P═S
2
MOE
P═S


3
Methyleneoxy
P═S
P═S
8
RNA
P═S
P═S
3
MOE
P═S



BNA











3
DNA
P═O
P═O
10
RNA
P═S
P═O
3
2′OH
P═O


2
2-F
P═S
P═S
5
RNA
P═S
P═S
2
2′-F
P═S


1
MOE
P═O
P═S
5
DNA
P═O
P═S
4
MOE
P═S









In certain embodiments, the oligomeric compounds disclosed herein may comprise from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleobases. In certain embodiments, oligomeric compounds are antisense compounds.
In certain embodiments, short antisense compounds are 8 nucleobases in length.
In certain embodiments, short antisense compounds are 9 nucleobases in length.
In certain embodiments, short antisense compounds are 10 nucleobases in length.
In certain embodiments, short antisense compounds are 11 nucleobases in length.
In certain embodiments, short antisense compounds are 12 nucleobases in length.
In certain embodiments, short antisense compounds are 13 nucleobases in length.
In certain embodiments, short antisense compounds are 14 nucleobases in length.
In certain embodiments, short antisense compounds are 15 nucleobases in length.
In certain embodiments, short antisense compounds are 16 nucleobases in length.
In certain embodiments, short antisense compounds are 8 monomers in length. In certain embodiments, short antisense compounds are 9 monomers in length. In certain embodiments, short antisense compounds are 10 monomers in length. In certain embodiments, short antisense compounds are 11 monomers in length. In certain embodiments, short antisense compounds are monomers in length. In certain embodiments, short antisense compounds are 13 monomers in length. In certain embodiments, short antisense compounds are 14 monomers in length. In certain embodiments, short antisense compounds are 15 monomers in length. In certain embodiments, short antisense compounds are 16 monomers in length. In certain embodiments, short antisense compounds comprise 9 to 15 monomers. In certain embodiments, short antisense compounds comprise 10 to 15 monomers. In certain embodiments, short antisense compounds comprise 12 to 14 monomers. In certain embodiments, short antisense compounds comprise 12 to 14 nucleotides or nucleosides.
One having skill in the art and informed by the short antisense compounds illustrated herein will be able, without undue experimentation, to identify further short antisense compounds.
In certain embodiments, short antisense compounds comprise a gap flanked by more than one wing on either or both sides. Thus, in certain embodiments, a short antisense compound comprises two or more 5′ wings and two or more 3′ wings. In certain embodiments, a short antisense compound comprises one 5′ wing and two or more 3′ wings. In certain embodiments, a short antisense compound comprises one 3′ wing and two or more 5′ wings. Certain such embodiments comprise, for example, the following regions: a first 5′ wing-a bridge-a second 5′ wing-a bridge-a gap-a bridge-a second 3′ wing-a bridge-a first 3′-wing. In such embodiments, each region has at least one difference in modification when compared to its neighboring region. Thus, in such embodiments, the second 5′ wing and the second 3′ wing each independently comprises one or more differences in modification compared to the gap and compared to the first 5′ wing and the first 3′ wing. In such embodiments, the modifications of the first 3′ wing and first 5′ wing may either or both be the same or different from the modifications of the gap, if any.
4. Certain Conjugate Groups
In one aspect, oligomeric compounds are modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound. A preferred list of conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
Preferred conjugate groups amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
Linking groups or bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Linking groups are useful for attachment of chemical functional groups, conjugate groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like. Some nonlimiting examples of bifunctional linking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
5. Synthesis, Purification and Analysis
Oligomerization of modified and unmodified nucleosides and nucleotides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
Oligomeric compounds provided herein can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The invention is not limited by the method of antisense compound synthesis.
Methods of purification and analysis of oligomeric compounds are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The method of the invention is not limited by the method of oligomer purification.
D. ANTISENSE
Antisense mechanisms are all those involving the hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
One type of antisense mechanism involving target degradation includes an RNase H. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
In certain embodiments, chemically-modified antisense compounds have a higher affinity for target RNAs than does non-modified DNA. In certain such embodiments, that higher affinity in turn provides increased potency allowing for the administration of lower doses of such compounds, reduced potential for toxicity and improvement in therapeutic index and decreased overall cost of therapy.
The present disclosure demonstrates that the incorporation of chemically-modified high-affinity nucleotides and nucleosides into antisense compounds allows for the design of short antisense compounds 8-16 nucleobases in length useful for the reduction of target RNAs and/or target proteins in cells, tissues, and animals, including, but not limited to, humans with increased potency and improved therapeutic index. Thus, in certain embodiments, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Certain such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, certain short antisense compounds have greater potential for oral dosing.
To address the need for more potent antisense compounds, provided herein are short antisense compounds (8-16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length) with increased activity in vivo relative to longer compounds. Certain short antisense compounds are gapmer compounds comprising high-affinity chemically-modified nucleotides on the 3′ and 5′ ends (wings) of the compound. In certain embodiments, the addition of high-affinity modified nucleotides allows antisense compounds to be active against, and specific for, their intended target RNA in vivo despite being shorter in length. Contemplated herein are short antisense compounds wherein each of the wings independently comprises 1 to 3 high-affinity modified nucleotides. In certain embodiments, the high-affinity modifications are sugar modifications. High-affinity modified nucleotides include, but are not limited to, BNA s or other 2′-modified nucleotides, such as 2′-MOE nucleotides. Also contemplated are short antisense compounds having at least one modified internucleotide linkage, such as a phosphorothioate internucleotide linkage. In certain embodiments, the short antisense compounds of the present invention can have all phosphorothioate internucleoside linkages. The short antisense compounds optionally comprise a conjugate group. As shown herein, short antisense compounds have greater affinity for target RNA than they have for DNA and are significantly more potent in vivo as shown by reduction of target mRNA as well as by amelioration of a variety of disease indications.
As used herein, an RNA which is involved in regulating glucose metabolism or clearance, lipid metabolism, cholesterol metabolism or insulin metabolism is any RNA involved in the biochemical pathways that regulate these processes. Such RNAs are well known in the art. Examples of target genes include, but are not limited to, ApoB-100 (also known as APOB; Ag(x) antigen; apoB-48; apolipoprotein B; apolipoprotein B-100; apolipoprotein B-48) and GCGR (also known as glucagon receptor; GR), CRP, DGAT2, GCCR, PCSK9, PTEN, PTP1B, SGLT2, and SOD1.
1. Modulation of Target Expression
In certain embodiments, a target is identified and antisense oligonucleotides are designed to modulate that target or its expression. In certain embodiments, designing an oligomeric compound to a target nucleic acid molecule can be a multistep process. Typically the process begins with the identification of a target protein, the activity of which is to be modulated, and then identifying the nucleic acid the expression of which yields the target protein. In certain embodiments, designing of an antisense compound results in an antisense compound that is hybridizable to the targeted nucleic acid molecule. In certain embodiments, the antisense compound is an antisense oligonucleotide or antisense oligonucleoside. In certain embodiments, an antisense compound and a target nucleic acid are complementary to one another. In certain such embodiments, an antisense compound is perfectly complementary to a target nucleic acid. In certain embodiments, an antisense compound includes one mismatch. In certain embodiments, an antisense compound includes two mismatches. In certain embodiments, an antisense compound includes three or more mismatches.
Modulation of expression of a target nucleic acid can be achieved through alteration of any number of nucleic acid functions. In certain embodiments, the functions of RNA to be modulated include, but are not limited to, translocation functions, which include, but are not limited to, translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, and translation of protein from the RNA. RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3′ maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level. Thus, in one embodiment modulation of expression can mean increase or decrease in target RNA or protein levels. In another embodiment modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.
In certain embodiments, expression of a target gene is modulated using an oligomeric compound comprising from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of a target gene using one or more antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleobases.
In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 8 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 9 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 8 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 10 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 10 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 11 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 12 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 13 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 14 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 15 nucleobases in length. In certain embodiments, methods of modulating a target gene comprises use of a short antisense compound that is 16 nucleobases in length.
In certain embodiments, methods of modulating expression of a target gene comprises use of a short antisense compound comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of a target gene comprises use of a short antisense compound comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of a target gene comprises use of a short antisense compound comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of a target gene comprises use of a short antisense compound comprising 12 or 14 nucleotides or nucleosides.
2. Hybridization
In certain embodiments, antisense compounds specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
As used herein, “stringent hybridization conditions” or “stringent conditions” refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions” under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated.
3. Complementarity
It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, such as by determining melting temperature (Tm). Tm or ΔTm can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
4. Identity
Antisense compounds, or a portion thereof, may have a defined percent identity to a SEQ ID NO, or a compound having a specific Isis number. As used herein, a sequence is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, an RNA which contains uracil in place of thymidine in the disclosed sequences of the compounds described herein would be considered identical as they both pair with adenine. This identity may be over the entire length of the oligomeric compound, or in a portion of the antisense compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO. It is understood by those skilled in the art that an antisense compound need not have an identical sequence to those described herein to function similarly to the antisense compound described herein. Shortened versions of antisense compounds taught herein, or non-identical versions of the antisense compounds taught herein, are also provided herein. Non-identical versions are those wherein each base does not have the same pairing activity as the antisense compounds disclosed herein. Bases do not have the same pairing activity by being shorter or having at least one abasic site. Alternatively, a non-identical version can include at least one base replaced with a different base with different pairing activity (e.g., G can be replaced by C, A, or T). Percent identity is calculated according to the number of bases that have identical base pairing corresponding to the SEQ ID NO or antisense compound to which it is being compared. The non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both.
For example, a 16-mer having the same sequence as nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer. Alternatively, a 20-mer containing four nucleobases not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art.
The percent identity is based on the percent of nucleobases in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase antisense compound comprising the full sequence of the complement of a 20 nucleobase active target segment would have a portion of 100% identity with the complement of the 20 nucleobase active target segment, while further comprising an additional 10 nucleobase portion. In the context of the instant description, the complement of an active target segment may constitute a single portion. In preferred embodiments, the oligonucleotides provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to at least a portion of the complement of the active target segments presented herein.
E. TARGET NUCLEIC ACIDS, REGIONS AND SEGMENTS
In certain embodiments, short antisense compounds may be designed to target any target nucleic acid. In certain embodiments, the target nucleic acid encodes a target that is clinically relevant. In such embodiments, modulation of the target nucleic acid results in clinical benefit. Certain target nucleic acids include, but are not limited to, the target nucleic acids illustrated in Table 1.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding ApoB. Nucleic acid molecules that encode ApoB include, without limitation, SEQ ID NO: 1 and SEQ ID NO: 2.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding SGLT2. Nucleic acid molecules that encode SGLT2 include, without limitation, SEQ ID NO: 3.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding PCSK9. Nucleic acid molecules that encode PCSK9 include, without limitation, SEQ ID NO: 4.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding SOD1. Nucleic acid molecules that encode SOD1 include, without limitation, SEQ ID NO: 5.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding CRP. Nucleic acid molecules that encode CRP include, without limitation, SEQ ID NO: 6.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding GCCR. Nucleic acid molecules that encode GCCR include, without limitation, SEQ ID NO: 7 and SEQ ID NO: 8.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding GCGR. Nucleic acid molecules that encode GCGR include, without limitation, SEQ ID NO: 9.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding DGAT2. Nucleic acid molecules that encode DGAT2 include, without limitation, SEQ ID NO: 10.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding PTP1B. Nucleic acid molecules that encode PTP1B include, without limitation, SEQ ID NO: 11 and SEQ ID NO: 12.
In certain embodiments, a target nucleic acid is a nucleic acid molecule encoding PTEN. Nucleic acid molecules that encode PTEN include, without limitation, SEQ ID NO: 14 or SEQ ID NO: 15.







TABLE 1







Certain Target Nucleic Acids













SEQ ID


Target
Species
GENBANK ® Accession Number
NO













ApoB
Human
NM_000384.1
1


ApoB
Mouse
XM_137955.5
2


SGLT2
Human
NM_003041.1
3


PCSK9
Human
NM_174936.2
4


SOD1
Human
X02317.1
5


CRP
Human
NM_000567.1
6


GCCR
Mouse
BC031885.1
7


GCCR
Human
Nucleotides 1 to 10600 of AC012634
8


GCGR
Human
NM_000160.1
9


DGAT2
Human
NM_032564.2
10


PTP1B
Human
NM_002827.2
11


PTP1B
Human
Nucleotides 1417800 to 1425600 of
12




NT_011362.9


PTEN
Mouse
U92437.1
13


PTEN
Human
NM_000314.4
14


PTEN
Human
Nucleotides 8063255 to 8167140 of
15




NT_033890.3









The targeting process usually includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect will result.
In certain embodiments, the 5′-most nucleotide of a target region is the 5′ target site of a short antisense compound and the 3′-most nucleotide of a target region is the 3′ target site of the same short antisense compound. In certain embodiments, the 5′-most nucleotide of a target region is the 5′ target site of a short antisense compound and the 3′-most nucleotide of a target region is the 3′ target site of a different short antisense compound. In certain embodiments, a target region comprises a nucleotide sequence within 10, 15, or 20 nucleotides of a 5′ target site or a 3′ target site.
In certain embodiments, a target region is a structurally defined region of the nucleic acid. For example, in certain such embodiments, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region.
The locations on the target nucleic acid defined by having one or more active short antisense compounds targeted thereto are referred to as “active target segments.” In certain embodiments, the target nucleic acid having one or more active short antisense compounds targeted thereto is a target RNA. When an active target segment is defined by multiple short antisense compounds, the compounds are preferably separated by no more than about 10 nucleotides on the target sequence, more preferably no more than about 5 nucleotides on the target sequence, even more preferably the short antisense compounds are contiguous, most preferably the short antisense compounds are overlapping. There may be substantial variation in activity (e.g., as defined by percent inhibition) of the short antisense compounds within an active target segment. Active short antisense compounds are those that modulate the expression of their target nucleic acid, including but not limited to a target RNA. Active short antisense compounds inhibit expression of their target RNA at least 10%, preferably 20%. In a preferred embodiment, at least about 50%, preferably about 70% of the short antisense compounds targeted to the active target segment modulate expression of their target RNA at least 40%. In a more preferred embodiment, the level of inhibition required to define an active short antisense compound is defined based on the results from the screen used to define the active target segments.
A suitable target segment is at least about an 8-nucleobase portion of a target region to which an active short antisense compound is targeted. Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA comprises about 8 to about 16 nucleobases). Target segments are also represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA comprises about 8 to about 16 nucleobases). It is also understood that antisense target segments may be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative target segment, and may extend in either or both directions until the short antisense compound comprises about 8 to about 16 nucleobases. One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further target segments.
Once one or more target regions, segments or sites have been identified, short antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
The short antisense compounds may also be targeted to regions of the target nucleobase sequence comprising any consecutive nucleobases 8 to 16 nucleobases in length along the target nucleic acid molecule.
Target segments 8-16 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative target segments are considered to be suitable for targeting as well. Thus, the short antisense compounds may also encompass 8-16 nucleobases within those segments identified herein as beginning at a particular 5′ target site. Any segment of 8, 9, 10, 11, or more preferably 12, 13, 14, 15 or 16 contiguous nucleobases in a 50, preferably 25, more preferably 16 nucleobase perimeter around these regions are also considered to be suitable for targeting.
In a further embodiment, the “suitable target segments” identified herein may be employed in a screen for additional short antisense compounds that modulate the expression of a target nucleic acid. “Modulators” are those compounds that decrease or increase the expression of a target nucleic acid and which comprise at least an 8-nucleobase portion which is complementary to a target segment. The screening method comprises the steps of contacting a target segment of a nucleic acid with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a target nucleic acid. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a target nucleic acid, the modulator may then be employed in further investigative studies of the function of the target, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
For all short antisense compounds discussed herein, sequence, monomer, monomeric modification, and monomeric linkage may each be selected independently. In certain embodiments, short antisense compounds are described by a motif. In such embodiments, any motif may be used with any sequence, whether or not the sequence and/or the motif is specifically disclosed herein. In certain embodiments, short antisense compounds comprise modifications that are not amenable to description by motif (for example, short antisense compounds comprising several different modifications and/or linkages at various positions throughout the compound). Such combinations may be incorporated for any sequence, whether or not it is disclosed herein. The sequence listing accompanying this filing provides certain nucleic acid sequences independent of chemical modification. Though that listing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications and/or motifs.
In certain embodiments, short antisense compounds comprise at least one high-affinity modified monomer. In certain embodiments, provided are short antisense compounds targeted to nucleic acid molecules encoding targets including, but not limited to, ApoB-100 (also known as APOB; Ag(x) antigen; apoB-48; apolipoprotein B; apolipoprotein B-100; apolipoprotein B-48), GCGR (also known as glucagon receptor; GR), CRP, DGAT2, GCCR, PCSK9, PTEN, PTP1B, SGLT2, and SOD1. In certain such embodiments, such short antisense compounds are targeted to a nucleic acid molecule encoding any of those targets.
F. CERTAIN TARGETS
In certain embodiments, short antisense compounds may be designed to modulate any target. In certain embodiments, the target is clinically relevant. In such embodiments, modulation of the target results in clinical benefit. Certain targets are preferentially expressed in the kidney. Certain targets are preferentially expressed in the liver. Certain targets are associated with a metabolic disorder. Certain targets are associated to a cardiovascular disorder. In certain embodiments, a target is selected from: ApoB, SGLT2, PCSK9, SOD1, CRP, GCCR, GCGR, DGAT2, PTP1B, and PTEN. In certain embodiments, a target is selected from: ApoB, SGLT2, PCSK9, SOD1, CRP, GCCR, GCGR, DGAT2, and PTP1B. In certain embodiments, a target is any protein other than SGLT2.
In certain embodiments, short antisense compounds exhibit liver and kidney-specific target RNA reduction in vivo. Such property renders those short antisense compounds particularly useful for inhibition of many target RNAs involved in metabolic and cardiovascular diseases. Thus, provided herein are methods of treating cardiovascular or metabolic disorders by contacting said kidney or liver tissues with short antisense compounds targeted to RNAs associated with said disorders. Thus, also provided are methods for ameliorating any of a variety of metabolic or cardiovascular disease indications with the short antisense compounds of the present invention.
1. ApoB
ApoB (also known as apolipoprotein B-100; ApoB-100, apolipoprotein B-48; ApoB-48 and Ag(x) antigen), is a large glycoprotein that serves an indispensable role in the assembly and secretion of lipids and in the transport and receptor-mediated uptake and delivery of distinct classes of lipoproteins. ApoB performs a variety of activities, from the absorption and processing of dietary lipids to the regulation of circulating lipoprotein levels (Davidson and Shelness, Annu. Rev. Nutr., 2000, 20, 169-193). This latter property underlies its relevance in terms of atherosclerosis susceptibility, which is highly correlated with the ambient concentration of ApoB-containing lipoproteins (Davidson and Shelness, Annu. Rev. Nutr., 2000, 20, 169-193). ApoB-100 is the major protein component of LDL-C and contains the domain required for interaction of this lipoprotein species with the LDL receptor. Elevated levels of LDL-C are a risk factor for cardiovascular disease, including atherosclerosis.
Definitions
“ApoB” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound.
“ApoB nucleic acid” means any nucleic acid encoding ApoB. For example, in certain embodiments, a ApoB nucleic acid includes, without limitation, a DNA sequence encoding ApoB, an RNA sequence transcribed from DNA encoding ApoB, and an mRNA sequence encoding ApoB.
“ApoB mRNA” means an mRNA encoding ApoB.
ApoB Therapeutic Indications
In certain embodiments, the invention provides methods of modulating the expression of ApoB in an individual comprising administering a short antisense compound targeted to an ApoB nucleic acid. In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions comprising a short antisense compound targeted to an ApoB nucleic acid. In certain embodiments, the individual has hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.
Guidelines for lipid-lowering therapy were established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). The guidelines include obtaining a complete lipoprotein profile, typically after a 9 to 12 hour fast, for determination of LDL-C, total cholesterol, and HDL-C levels. According to the most recently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189 mg/dL, and greater than or equal to 190 mg/dL are considered borderline high, high, and very high, respectively. Total cholesterol levels of 200-239 and greater than or equal to 240 mg/dL are considered borderline high and high, respectively. HDL-C levels of less than 40 mg/dL are considered low.
In certain embodiments, the individual has been identified as in need of lipid-lowering therapy. In certain such embodiments, the individual has been identified as in need of lipid-lowering therapy according to the guidelines established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 190 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 160 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy has LDL-C above 100 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 160 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certain such embodiments the individual should maintain LDL-C below 70 mg/dL.
In certain embodiments the invention provides methods for reducing ApoB in an individual. In certain embodiments the invention provides methods for reducing ApoB-containing lipoprotein in an individual. In certain embodiments the invention provides methods for reducing LDL-C in an individual. In certain embodiments the invention provides methods for reducing VLDL-C in an individual. In certain embodiments the invention provides methods for reducing IDL-C in an individual. In certain embodiments the invention provides methods for reducing non-HDL-C in an individual. In certain embodiments the invention provides methods for reducing Lp(a) in an individual. In certain embodiments the invention provides methods for reducing serum triglyceride in an individual. In certain embodiments the invention provides methods for reducing liver triglyceride in an individual. In certain embodiments the invention provides methods for reducing Ox-LDL-C in an individual. In certain embodiments the invention provides methods for reducing small LDL particles in an individual. In certain embodiments the invention provides methods for reducing small VLDL particles in an individual. In certain embodiments the invention provides methods for reducing phospholipids in an individual. In certain embodiments the invention provides methods for reducing oxidized phospholipids in an individual.
In certain embodiments the invention provides methods for reducing Ox-LDL-C concentration in a subject. In certain such embodiments, the reduction in ApoB, LDL-C, VLDL-C, IDL-C, total cholesterol, non-HDL-C, Lp(a), triglyerides, or Ox-LDL-C is, independently, selected from at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 100%. In certain such embodiments, the reduction in ApoB, LDL-C, VLDL-C, IDL-C, total cholesterol, non-HDL-C, Lp(a), triglyerides, or Ox-LDL-C is, independently, selected from at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 70%. In certain such embodiments, the reduction in ApoB, LDL-C, VLDL-C, IDL-C, total cholesterol, non-HDL-C, Lp(a), triglyerides, or Ox-LDL-C is, independently, selected from at least 40%, at least 50%, at least 60%, and at least 70%.
In certain embodiments, the invention provides method for raising HDL-C concentration in a subject.
In certain embodiments, the methods provided by the present invention do not lower HDL-C. In certain embodiments, the methods provided by the present invention do not result in accumulation of lipids in the liver. In certain embodiments, the methods provided by the present invention do not cause hepatic steatosis.
In certain embodiments, the invention provides methods for lowering ApoB concentration in a subject while reducing side effects associated with treatment. In certain such embodiments, a side effect is liver toxicity. In certain such embodiments, a side effect is abnormal liver function. In certain such embodiments, a side effect is elevated alanine aminotransferase (ALT). In certain such embodiments, a side effect is elevated aspartate aminotransferase (AST).
In certain embodiments, the invention provides methods for lowering ApoB concentration in a subject who is not reaching target LDL-C levels as a result of lipid-lowering therapy. In certain such embodiments, a short antisense compound targeted to an ApoB nucleic acid is the only lipid-lowering agent administered to the subject. In certain such embodiments, the subject has not complied with recommended lipid-lowering therapy. In certain such embodiments, a pharmaceutical composition of the invention is co-administered with an additional different lipid-lowering therapy. In certain such embodiments, an additional lipid-lowering therapy is LDL-apheresis. In certain such embodiments, an additional lipid-lowering therapy is a statin. In certain such embodiments, an additional lipid-lowering therapy is ezetimibe.
In certain embodiments, the invention provides methods for lowering ApoB concentration in a statin-intolerant subject. In certain such embodiments, the subject has creatine kinase concentration increases as a result of statin administration. In certain such embodiments, the subject has liver function abnormalities as a result of statin administration. In certain such embodiments the subject has muscle aches as a result of statin administration. In certain such embodiments the subject has central nervous system side effects as a result of statin administration. In certain embodiments, the subject has not complied with recommended statin administration.
In certain embodiments, the invention provides methods for lowering liver triglycerides in a subject. In certain such embodiments, the subject has elevated liver triglycerides. In certain such embodiments, the subject has steatohepatitis. In certain such embodiments, the subject has steatosis. In certain such embodiments, liver triglyceride levels are measured by magnetic resonance imaging.
In certain embodiments, the invention provides methods for reducing coronary heart disease risk in a subject. In certain embodiments the invention provides methods for slowing the progression of atherosclerosis in a subject. In certain such embodiments the invention provides methods for stopping the progression of atherosclerosis in a subject. In certain such embodiments the invention provides methods for reducing the size and/or prevalence of atherosclerotic plaques in a subject. In certain embodiments the methods provided reduce a subject's risk of developing atherosclerosis.
In certain embodiments the methods provided improve the cardiovascular outcome in a subject. In certain such embodiments improved cardiovascular outcome is the reduction of the risk of developing coronary heart disease. In certain such embodiments, improved cardiovascular outcome is a reduction in the occurrence of one or more major cardiovascular events, which include, but are not limited to, death, myocardial infarction, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia. In certain such embodiments, the improved cardiovascular outcome is evidenced by improved carotid intimal media thickness. In certain such embodiments, improved carotid intimal media thickness is a decrease in thickness. In certain such embodiments, improved carotid intimal media thickness is a prevention an increase of intimal media thickness.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid is for use in therapy. In certain embodiments, the therapy is the reduction of LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments, the therapy is the treatment of hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease. In additional embodiments, the therapy is the reduction of CHD risk. In certain the therapy is prevention of atherosclerosis. In certain embodiments, the therapy is the prevention of coronary heart disease.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid is used for the preparation of a medicament for reducing LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid is used for the preparation of a medicament for reducing coronary heart disease risk. In certain embodiments a short antisense compound targeted to an ApoB nucleic acid is used for the preparation of a medicament for the treatment of hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.
ApoB Combination Therapies
In certain embodiments, one or more pharmaceutical compositions comprising a short antisense compound targeted to an ApoB nucleic acid are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain such embodiments, the one or more pharmaceutical agents are lipid-lowering agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid include lipid-lowering agents. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.
In certain embodiments, a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments the statin is selected from atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.
In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe.
In certain embodiments, a co-administered lipid-lowering agent is a co-formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe/simvastatin.
In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).
In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.
In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.
In certain embodiments, a pharmaceutical composition comprising a short antisense compound targeted to an ApoB nucleic acid may be administered in conjunction with a lipid-lowering therapy. In certain such embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain such embodiments, a lipid-lowering therapy is LDL apheresis.
In one embodiment, the antisense compounds provided herein can be used to lower the level of apolipoprotein B-containing lipoproteins in a human subject. As used herein, “apolipoprotein B-containing lipoprotein” refers to any lipoprotein that has apolipoprotein B as its protein component, and is understood to include LDL, VLDL, IDL, and lipoprotein(a). LDL, VLDL, IDL and lipoprotein(a) each contain one molecule of apolipoprotein B, thus a serum apolipoprotein B measurement reflects the total number of these lipoproteins. As is known in the art, each of the aforementioned lipoproteins is atherogenic. Thus, lowering one or more apolipoprotein B-containing lipoproteins in serum may provide a therapeutic benefit to a human subject. Small LDL particles are considered to be particularly atherogenic relative to large LDL particles, thus lowering small LDL particles can provide a therapeutic benefit to a human subject. Additional lipid parameters can also be determined in a subject. Reduction of total cholesterol:HDL ratio or LDL:HDL ratio is a clinically desirable improvement in cholesterol ratio. Similarly, it is clinically desirable to reduce serum triglycerides in humans who exhibit elevated lipid levels.
Other indications of cardiovascular disease that can be measured in a subject include serum LDL particle size; serum LDL cholesteryl ester concentration; serum LDL cholesteryl ester composition; the extent of polyunsaturation of serum LDL cholesteryl esters; and serum HDL cholesterol levels. As used herein, “serum LDL particle size” refers to the classification of serum LDL particle size, which may be very small, small, medium, or large, and is typically expressed in g/μmol. In the context of the present invention, “serum LDL cholesteryl ester concentration” means the amount of cholesteryl ester present in LDL particles, and is typically measured as mg/dL. In the context of the present invention, “serum LDL cholesteryl ester composition” is a measurement of the percentage of saturated, monounsaturated and polyunsaturated cholesteryl ester fatty acids present in serum LDL particles. “Polyunsaturation of serum LDL cholesteryl esters” means the percentage of polyunsaturated cholesteryl ester fatty acids in serum LDL particles.
Methods of obtaining serum or plasma samples for analysis and methods of preparation of the serum samples to allow for analysis are well known to those skilled in the art. With regard to measurements of lipoproteins, cholesterol, triglyceride and cholesteryl esters, the terms “serum” and “plasma” are herein used interchangeably.
In another embodiment, the antisense compounds provided herein can be used to treat metabolic disorders. A variety of biomarkers can be used for evaluating metabolic disease. For example, blood glucose levels can be determined by a physician or even by the patient using a commonly available test kit or glucometer (for example, the Ascensia ELITETM kit, Ascensia (Bayer), Tarrytown N.Y., or Accucheck, Roche Diagnostics). Glycated hemoglobin (HbA1c) can also be measured. HbA1c is a stable minor hemoglobin variant formed in vivo via posttranslational modification by glucose, and it contains predominantly glycated NH2-terminal β-chains. There is a strong correlation between levels of HbA1c and the average blood glucose levels over the previous 3 months. Thus HbA1c is often viewed as the “gold standard” for measuring sustained blood glucose control (Bunn, H. F. et al., 1978, Science. 200, 21-7). HbA1c can be measured by ion-exchange HPLC or immunoassay; home blood collection and mailing kits for HbA1c measurement are now widely available. Serum fructosamine is another measure of stable glucose control and can be measured by a colorimetric method (Cobas Integra, Roche Diagnostics).
Certain Short Antisense Compounds Targeted to an ApoB Nucleic Acid
In certain embodiments, short antisense compounds are targeted to an ApoB nucleic acid having the sequence of GENBANK® Accession No. NM_000384.1, incorporated herein as SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 1 is at least 90% complementary to SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 1 is at least 95% complementary to SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 1 is 100% complementary to SEQ ID NO: 1. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 1 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 2 and Table 3.
The nucleotide sequence set forth in each SEQ ID NO in Tables 2 and 3 is independent of any modification to a sugar moiety, a monomeric linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Tables 2 and 3 illustrate examples of short antisense compounds targeted to SEQ ID NO: 1. Table 2 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 1. Table 3 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 1. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 2







Short Antisense Compounds targeted to SEQ ID NO: 1













5′
3′





ISIS
Target
Target


SEQ


No
Site
Site
Sequence (5′-3′)
Gapmer Motif
ID NO















372816
263
278
CCGGAGGTGCTTGAAT
3-10-3 MOE
16


 


372894
264
277
CGGAGGTGCTTGAA
2-10-2 MOE
17


 


372817
428
443
GAAGCCATACACCTCT
3-10-3 MOE
18


 


372895
429
442
AAGCCATACACCTC
2-10-2 MOE
19


 


372818
431
446
GTTGAAGCCATACACC
3-10-3 MOE
20


 


372896
432
445
TTGAAGCCATACAC
2-10-2 MOE
21


 


372819
438
453
CCTCAGGGTTGAAGCC
3-10-3 MOE
22


 


372897
439
452
CTCAGGGTTGAAGC
2-10-2 MOE
23


 


372820
443
458
TTTGCCCTCAGGGTTG
3-10-3 MOE
24


 


372898
444
457
TTGCCCTCAGGGTT
2-10-2 MOE
25


 


372821
468
483
AGTTCTTGGTTTTCTT
3-10-3 MOE
26


 


372899
469
482
GTTCTTGGTTTTCT
2-10-2 MOE
27


 


372822
587
602
CCTCTTGATGTTCAGG
3-10-3 MOE
28


 


372900
588
601
CTCTTGATGTTCAG
2-10-2 MOE
29


 


372823
592
607
ATGCCCCTCTTGATGT
3-10-3 MOE
30


 


372901
593
606
TGCCCCTCTTGATG
2-10-2 MOE
31


 


346583
715
728
TGCCACATTGCCCT
3-8-3 MOE
32


 


346584
716
729
TTGCCACATTGCCC
3-8-3 MOE
33


 


346585
717
730
GTTGCCACATTGCC
3-8-3 MOE
34


 


346586
718
731
TGTTGCCACATTGC
3-8-3 MOE
35


 


346587
719
732
CTGTTGCCACATTG
3-8-3 MOE
36


 


346588
720
733
TCTGTTGCCACATT
3-8-3 MOE
37


 


346589
721
734
TTCTGTTGCCACAT
3-8-3 MOE
38


 


346590
722
735
TTTCTGTTGCCACA
3-8-3 MOE
39


 


346591
723
736
ATTTCTGTTGCCAC
3-8-3 MOE
40


 


372824
929
944
GTAGGAGAAAGGCAGG
3-10-3 MOE
41


 


372902
930
943
TAGGAGAAAGGCAG
2-10-2 MOE
42


 


372825
1256
1271
GGCTTGTAAAGTGATG
3-10-3 MOE
43


 


372903
1257
1270
GCTTGTAAAGTGAT
2-10-2 MOE
44


 


372826
1304
1319
CCACTGGAGGATGTGA
3-10-3 MOE
45


 


372904
1305
1318
CACTGGAGGATGTG
2-10-2 MOE
46


 


372829
2135
2150
TTTCAGCATGCTTTCT
3-10-3 MOE
47


 


372907
2136
2149
TTCAGCATGCTTTC
2-10-2 MOE
48


 


372832
2774
2789
CATATTTGTCACAAAC
3-10-3 MOE
49


 


372910
2775
2788
ATATTTGTCACAAA
2-10-2 MOE
50


 


372833
2779
2794
ATGCCCATATTTGTCA
3-10-3 MOE
51


 


372911
2780
2793
TGCCCATATTTGTC
2-10-2 MOE
52


 


372835
2961
2976
TTTTGGTGGTAGAGAC
3-10-3 MOE
53


 


372913
2962
2975
TTTGGTGGTAGAGA
2-10-2 MOE
54


 


346592
3248
3261
TCTGCTTCGCACCT
3-8-3 MOE
55


 


346593
3249
3262
GTCTGCTTCGCACC
3-8-3 MOE
56


 


346594
3250
3263
AGTCTGCTTCGCAC
3-8-3 MOE
57


 


346595
3251
3264
CAGTCTGCTTCGCA
3-8-3 MOE
58


 


346596
3252
3265
TCAGTCTGCTTCGC
3-8-3 MOE
59


 


346597
3253
3266
CTCAGTCTGCTTCG
3-8-3 MOE
60


 


346598
3254
3267
CCTCAGTCTGCTTC
3-8-3 MOE
61


 


346599
3255
3268
GCCTCAGTCTGCTT
3-8-3 MOE
62


 


346600
3256
3269
AGCCTCAGTCTGCT
3-8-3 MOE
63


 


372836
3350
3365
AACTCTGAGGATTGTT
3-10-3 MOE
64


 


372914
3351
3364
ACTCTGAGGATTGT
2-10-2 MOE
65


 


372837
3355
3370
TCATTAACTCTGAGGA
3-10-3 MOE
66


 


372915
3356
3369
CATTAACTCTGAGG
2-10-2 MOE
67


 


372838
3360
3375
ATTCATCATTAACTCT
3-10-3 MOE
68


 


372916
3361
3374
TTCATCATTAACTC
2-10-2 MOE
69


 


372839
3409
3424
TTGTTCTGAATGTCCA
3-10-3 MOE
70


 


387461
3409
3424
TTGTTCTGAATGTCCA
3-10-3 Methyleneoxy
70






BNA Unmodified






cytosines in gap


 


380147
3409
3424
TTGTTCTGAATGTCCA
3-10-3 Methyleneoxy
70






BNA


 


372917
3410
3423
TGTTCTGAATGTCC
2-10-2 MOE
73


 


372840
3573
3588
CAGATGAGTCCATTTG
3-10-3 MOE
74


 


372918
3574
3587
AGATGAGTCCATTT
2-10-2 MOE
75


 


372841
3701
3716
ATCCACAGGGAAATTG
3-10-3 MOE
76


 


372919
3702
3715
TCCACAGGGAAATT
2-10-2 MOE
77


 


372843
4219
4234
CAGTTGTACAAGTTGC
3-10-3 MOE
78


 


372921
4220
4233
AGTTGTACAAGTTG
2-10-2 MOE
79


 


372844
4301
4316
CACAGAGTCAGCCTTC
3-10-3 MOE
80


 


372922
4302
4315
ACAGAGTCAGCCTT
2-10-2 MOE
81


 


372845
4308
4323
GGTCAACCACAGAGTC
3-10-3 MOE
82


 


372923
4309
4322
GTCAACCACAGAGT
2-10-2 MOE
83


 


346601
5588
5601
CAGCCACATGCAGC
3-8-3 MOE
84


 


346602
5589
5602
CCAGCCACATGCAG
3-8-3 MOE
85


 


346603
5590
5603
ACCAGCCACATGCA
3-8-3 MOE
86


 


346604
5591
5604
TACCAGCCACATGC
3-8-3 MOE
87


 


346605
5592
5605
TTACCAGCCACATG
3-8-3 MOE
88


 


346606
5593
5606
GTTACCAGCCACAT
3-8-3 MOE
89


 


346607
5594
5607
GGTTACCAGCCACA
3-8-3 MOE
90


 


346608
5595
5608
AGGTTACCAGCCAC
3-8-3 MOE
91


 


346609
5596
5609
TAGGTTACCAGCCA
3-8-3 MOE
92


 


372851
5924
5939
AGGTTCTGCTTTCAAC
3-10-3 MOE
93


 


372929
5925
5938
GGTTCTGCTTTCAA
2-10-2 MOE
94


 


372854
6664
6679
TACTGATCAAATTGTA
3-10-3 MOE
95


 


372932
6665
6678
ACTGATCAAATTGT
2-10-2 MOE
96


 


372855
6908
6923
TTTTTCTTGTATCTGG
3-10-3 MOE
97


 


372933
6909
6922
TTTTCTTGTATCTG
2-10-2 MOE
98


 


372856
7190
7205
ATCCATTAAAACCTGG
3-10-3 MOE
99


 


372934
7191
7204
TCCATTAAAACCTG
2-10-2 MOE
100


 


372858
7817
7832
ATATTGCTCTGCAAAG
3-10-3 MOE
101


 


372936
7818
7831
TATTGCTCTGCAAA
2-10-2 MOE
102


 


346610
7818
7831
TATTGCTCTGCAAA
3-8-3 MOE
102


 


346611
7819
7832
ATATTGCTCTGCAA
3-8-3 MOE
104


 


346612
7820
7833
AATATTGCTCTGCA
3-8-3 MOE
105


 


346613
7821
7834
GAATATTGCTCTGC
3-8-3 MOE
106


 


346614
7822
7835
AGAATATTGCTCTG
3-8-3 MOE
107


 


346615
7823
7836
TAGAATATTGCTCT
3-8-3 MOE
108


 


346616
7824
7837
ATAGAATATTGCTC
3-8-3 MOE
109


 


346617
7825
7838
GATAGAATATTGCT
3-8-3 MOE
110


 


346618
7826
7839
GGATAGAATATTGC
3-8-3 MOE
111


 


372859
7995
8010
ATGGAATCCTCAAATC
3-10-3 MOE
112


 


372937
7996
8009
TGGAATCCTCAAAT
2-10-2 MOE
113


 


372861
8336
8351
GAATTCTGGTATGTGA
3-10-3 MOE
114


 


372939
8337
8350
AATTCTGGTATGTG
2-10-2 MOE
115


 


372862
8341
8356
AGCTGGAATTCTGGTA
3-10-3 MOE
116


 


372940
8342
8355
GCTGGAATTCTGGT
2-10-2 MOE
117


 


372863
8539
8554
TGAAAATCAAAATTGA
3-10-3 MOE
118


 


372941
8540
8553
GAAAATCAAAATTG
2-10-2 MOE
119


 


372871
9344
9359
AAACAGTGCATAGTTA
3-10-3 MOE
120


 


372949
9345
9358
AACAGTGCATAGTT
2-10-2 MOE
121


 


372872
9515
9530
TTCAGGAATTGTTAAA
3-10-3 MOE
122


 


372950
9516
9529
TCAGGAATTGTTAA
2-10-2 MOE
123


 


372875
9794
9809
TTTTGTTTCATTATAG
3-10-3 MOE
124


 


372953
9795
9808
TTTGTTTCATTATA
2-10-2 MOE
125


 


372877
10157
10172
GATGACACTTGATTTA
3-10-3 MOE
126


 


372955
10158
10171
ATGACACTTGATTT
2-10-2 MOE
127


 


372878
10161
10176
GTGTGATGACACTTGA
3-10-3 MOE
128


 


372956
10162
10175
TGTGATGACACTTG
2-10-2 MOE
129


 


372879
10167
10182
TATTCAGTGTGATGAC
3-10-3 MOE
130


 


372957
10168
10181
ATTCAGTGTGATGA
2-10-2 MOE
131


 


372880
10172
10187
ATTGGTATTCAGTGTG
3-10-3 MOE
132


 


372958
10173
10186
TTGGTATTCAGTGT
2-10-2 MOE
133


 


346619
10838
10851
CCTCTAGCTGTAAG
3-8-3 MOE
134


 


346620
10839
10852
CCCTCTAGCTGTAA
3-8-3 MOE
135


 


346621
10840
10853
GCCCTCTAGCTGTA
3-8-3 MOE
136


 


346622
10841
10854
GGCCCTCTAGCTGT
3-8-3 MOE
137


 


346623
10842
10855
AGGCCCTCTAGCTG
3-8-3 MOE
138


 


346624
10843
10856
GAGGCCCTCTAGCT
3-8-3 MOE
139


 


346625
10844
10857
AGAGGCCCTCTAGC
3-8-3 MOE
140


 


346626
10845
10858
AAGAGGCCCTCTAG
3-8-3 MOE
141


 


346627
10846
10859
AAAGAGGCCCTCTA
3-8-3 MOE
142


 


372890
13689
13704
GAATGGACAGGTCAAT
3-10-3 MOE
143


 


372968
13690
13703
AATGGACAGGTCAA
2-10-2 MOE
144


 


372891
13694
13709
GTTTTGAATGGACAGG
3-10-3 MOE
145


 


372969
13695
13708
TTTTGAATGGACAG
2-10-2 MOE
146


 


372892
13699
13714
TGGTAGTTTTGAATGG
3-10-3 MOE
147


 


372970
13700
13713
GGTAGTTTTGAATG
2-10-2 MOE
148


 


346628
13907
13920
TCACTGTATGGTTT
3-8-3 MOE
149


 


346629
13908
13921
CTCACTGTATGGTT
3-8-3 MOE
150


 


346630
13909
13922
GCTCACTGTATGGT
3-8-3 MOE
151


 


346631
13910
13923
GGCTCACTGTATGG
3-8-3 MOE
152


 


346632
13911
13924
TGGCTCACTGTATG
3-8-3 MOE
153


 


346633
13912
13925
CTGGCTCACTGTAT
3-8-3 MOE
154


 


346634
13913
13926
GCTGGCTCACTGTA
3-8-3 MOE
155


 


346635
13914
13927
GGCTGGCTCACTGT
3-8-3 MOE
156


 


346636
13915
13928
AGGCTGGCTCACTG
3-8-3 MOE
157


 


346637
13963
13976
CAGGTCCAGTTCAT
3-8-3 MOE
158


 


346638
13964
13977
GCAGGTCCAGTTCA
3-8-3 MOE
159


 


346639
13965
13978
TGCAGGTCCAGTTC
3-8-3 MOE
160


 


346640
13966
13979
GTGCAGGTCCAGTT
3-8-3 MOE
161


 


346641
13967
13980
GGTGCAGGTCCAGT
3-8-3 MOE
162


 


346642
13968
13981
TGGTGCAGGTCCAG
3-8-3 MOE
163


 


346643
13969
13982
TTGGTGCAGGTCCA
3-8-3 MOE
164


 


346644
13970
13983
TTTGGTGCAGGTCC
3-8-3 MOE
165


 


346645
13971
13984
CTTTGGTGCAGGTC
3-8-3 MOE
166


 


346646
14051
14064
TAACTCAGATCCTG
3-8-3 MOE
167


 


346647
14052
14065
ATAACTCAGATCCT
3-8-3 MOE
168


 


346648
14053
14066
AATAACTCAGATCC
3-8-3 MOE
169


 


346649
14054
14067
AAATAACTCAGATC
3-8-3 MOE
170


 


346650
14055
14068
AAAATAACTCAGAT
3-8-3 MOE
171


 


346651
14056
14069
CAAAATAACTCAGA
3-8-3 MOE
172


 


346652
14057
14070
GCAAAATAACTCAG
3-8-3 MOE
173


 


346653
14058
14071
AGCAAAATAACTCA
3-8-3 MOE
174


 


346654
14059
14072
TAGCAAAATAACTC
3-8-3 MOE
175
















TABLE 3







Short antisense compounds targeted to SEQ


ID NO: 1 and having 1 or 2 mismatches













5′
3′


SEQ


Isis
Target
Target


ID


NO.
Site
Site
Sequence (5′-3′)
Gapmer Motif
NO















372894
771
784
CGGAGGTGCTTGAA
2-10-2 MOE
17


 


372905
1111
1124
CAGGGCCTGGAGAG
2-10-2 MOE
176


 


346628
1493
1506
TCACTGTATGGTTT
3-8-3 MOE
149


 


372828
2006
2021
TCTGAAGTCCATGATC
3-10-3 MOE
177


 


372906
2007
2020
CTGAAGTCCATGAT
2-10-2 MOE
178


 


372830
2382
2397
TGGGCATGATTCCATT
3-10-3 MOE
179


 


372908
2383
2396
GGGCATGATTCCAT
2-10-2 MOE
180


 


346616
3162
3175
ATAGAATATTGCTC
3-8-3 MOE
109


 


346617
3163
3176
GATAGAATATTGCT
3-8-3 MOE
110


 


372929
3513
3526
GGTTCTGCTTTCAA
2-10-2 MOE
94


 


372946
3800
3813
TGGAGCCCACGTGC
2-10-2 MOE
181


 


372904
4040
4053
CACTGGAGGATGTG
2-10-2 MOE
46


 


372842
4084
4099
TTGAAGTTGAGGGCTG
3-10-3 MOE
182


 


372920
4085
4098
TGAAGTTGAGGGCT
2-10-2 MOE
183


 


346586
4778
4791
TGTTGCCACATTGC
3-8-3 MOE
35


 


372847
5030
5045
ACCAGTATTAATTTTG
3-10-3 MOE
184


 


372925
5031
5044
CCAGTATTAATTTT
2-10-2 MOE
185


 


372848
5192
5207
GTGTTCTTTGAAGCGG
3-10-3 MOE
186


 


372926
5193
5206
TGTTCTTTGAAGCG
2-10-2 MOE
187


 


372953
5625
5638
TTTGTTTCATTATA
2-10-2 MOE
125


 


372935
7585
7598
AGTTACTTTGGTGT
2-10-2 MOE
188


 


372860
8255
8270
TGGTACATGGAAGTCT
3-10-3 MOE
189


 


372938
8256
8269
GGTACATGGAAGTC
2-10-2 MOE
190


 


391260
8256
8269
GGTACATGGAAGTC
2-10-2 MOE
190


 


392068
8256
8269
GGTACATGGAAGTC
2-10-2 MOE
190


 


387462
8256
8269
GGTACATGGAAGTC
2-10-2 Methyleneoxy
190






BNA


 


391872
8256
8269
GGTACATGGAAGTC
1-1-10-2 2′-
190






(butylacetomido) -






palmitamide






Methyleneoxy






BNA/Methyleneoxy






BNA






Unmodified cytosines in






gap


 


380148
8256
8269
GGTACATGGAAGTC
2-10-2 Methyleneoxy
190






BNA


 


391871
8256
8269
GGTACATGGAAGTC
1-1-10-2 2′-
190






(butylacetomido) -






palmitamide/MOE/MOE






Unmodified cytosines in






gap


 


391755
8256
8269
GGTACATGGAAGTC
2-10-2 ENA
190






mC in wing only


 


398296
8256
8269
GGTACATGGAAGTC
2-10-2 (6′S)-6′-methyl-
190






Methyleneoxy BNA






Unmodified Cytosines


 


372942
8455
8468
TCCATGCCATATGT
2-10-2 MOE
200


 


372865
8888
8903
CCCTGAAGAAGTCCAT
3-10-3 MOE
201


 


372943
8889
8902
CCTGAAGAAGTCCA
2-10-2 MOE
202


 


372866
8908
8923
GCCCAGTTCCATGACC
3-10-3 MOE
203


 


372944
8909
8922
CCCAGTTCCATGAC
2-10-2 MOE
204


 


372867
9058
9073
TTGAGGAAGCCAGATT
3-10-3 MOE
205


 


372945
9059
9072
TGAGGAAGCCAGAT
2-10-2 MOE
206


 


372870
9261
9276
TGGATGCAGTAATCTC
3-10-3 MOE
207


 


372948
9262
9275
GGATGCAGTAATCT
2-10-2 MOE
208


 


372881
10185
10200
TATAAAGTCCAGCATT
3-10-3 MOE
209


 


372959
10186
10199
ATAAAGTCCAGCAT
2-10-2 MOE
210


 


372882
10445
10460
AAGTTCCTGCTTGAAG
3-10-3 MOE
211


 


372960
10446
10459
AGTTCCTGCTTGAA
2-10-2 MOE
212


 


372964
11451
11464
AATGGTGAAGTACT
2-10-2 MOE
213


 


346612
13459
13472
AATATTGCTCTGCA
3-8-3 MOE
105


 


346613
13460
13473
GAATATTGCTCTGC
3-8-3 MOE
106









In certain embodiments, a target region is nucleotides 263-278 of SEQ ID NO: 1. In certain such embodiments, short antisense compounds targeted to nucleotides 263-278 of SEQ ID NO: 1 comprise a nucleotide sequence selected from SEQ ID NO: 16 or 17. In certain such embodiments, a short antisense compound targeted to nucleotides 263-278 of SEQ ID NO: 1 is selected from Isis NO. 372816 or 372894.
In certain embodiments, a target region is nucleotides 428-483 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 428-483 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27. In certain such embodiments, a short antisense compound targeted to nucleotides 428-483 of SEQ ID NO: 1 is selected from Isis NO. 372817, 372895, 372818, 372896, 372819, 372897, 372820, 372898, 372821, or 372899.
In certain embodiments, a target region is nucleotides 428-458 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 428-458 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 18, 19, 20, 21, 22, 23, 24, or 25. In certain such embodiments, a short antisense compound targeted to nucleotides 428-458 of SEQ ID NO: 1 is selected from Isis NO. 372817, 372895, 372818, 372896, 372819, 372897, 372820, or 372898.
In certain embodiments, a target region is nucleotides 468-483 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 468-483 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 26 or 27. In certain such embodiments, a short antisense compound targeted to nucleotides 468-483 of SEQ ID NO: 1 is selected from Isis NO. 372821 or 372899.
In certain embodiments, a target region is nucleotides 587-607 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 587-607 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 28, 29, 30, or 31. In certain such embodiments, a short antisense compound targeted to nucleotides 587-607 of SEQ ID NO: 1 is selected from ISIS NO. 372822, 372900, 372823, or 372901.
In certain embodiments, a target region is nucleotides 715-736 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 715-736 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39, or 40. In certain such embodiments, a short antisense compound targeted to nucleotides 715-736 of SEQ ID NO: 1 is selected from Isis NO. 346583, 346584, 346585, 346586, 346587, 346588, 346589, 346590, or 346591.
In certain embodiments, a target region is nucleotides 929-944 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 929-944 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 41 or 42. In certain such embodiments, a short antisense compound targeted to nucleotides 929-944 of SEQ ID NO: 1 is selected from Isis NO. 372824 or 372902.
In certain embodiments, a target region is nucleotides 1256-1319 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 1256-1319 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 43, 44, 45, or 46. In certain such embodiments, a short antisense compound targeted to nucleotides 1256-1319 of SEQ ID NO: 1 is selected from Isis NO. 372825, 372903, 372826, or 372904.
In certain embodiments, a target region is nucleotides 1256-1271 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 1256-1271 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 43 or 44. In certain such embodiments, a short antisense compound targeted to nucleotides 1256-1271 of SEQ ID NO: 1 is selected from Isis NO. 372825 or 372903.
In certain embodiments, a target region is nucleotides 1304-1319 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 1304-1319 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 45 or 46. In certain such embodiments, a short antisense compound targeted to nucleotides 1304-1319 of SEQ ID NO: 1 is selected from Isis NO. 372826 or 372904.
In certain embodiments, a target region is nucleotides 2135-2150 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 2135-2150 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 47 or 48. In certain such embodiments, a short antisense compound targeted to nucleotides 2135-2150 of SEQ ID NO: 1 is selected from ISIS NO. 372829 or 372907.
In certain embodiments, a target region is nucleotides 2774-2794 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 2774-2794 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 49, 50, 51, or 52. In certain such embodiments, a short antisense compound targeted to nucleotides 2774-2794 of SEQ ID NO: 1 is selected from ISIS NO. 372832, 372910, 372833, or 372911.
In certain embodiments, a target region is nucleotides 2961-2976 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 2961-2976 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 53 or 54. In certain such embodiments, a short antisense compound targeted to nucleotides 2961-2976 of SEQ ID NO: 1 is selected from ISIS NO. 372835 or 372913.
In certain embodiments, a target region is nucleotides 3248-3269 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 3248-3269 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 55, 56, 57, 58, 59, 60, 61, 62, or 63. In certain such embodiments, a short antisense compound targeted to nucleotides 3248-3269 of SEQ ID NO: 1 is selected from ISIS NO. 346592, 346593, 346594, 346595, 346596, 346597, 346598, 346599, or 346600.
In certain embodiments, a target region is nucleotides 3350-3375 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 3350-3375 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 64, 65, 66, 67, 68, or 69. In certain such embodiments, a short antisense compound targeted to nucleotides 3350-3375 of SEQ ID NO: 1 is selected from ISIS NO. 372836, 372914, 372837, 372915, 372838, or 372916.
In certain embodiments, a target region is nucleotides 3409-3424 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 3409-3424 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 70 or 73. In certain such embodiments, a short antisense compound targeted to nucleotides 3409-3424 of SEQ ID NO: 1 is selected from ISIS NO. 372839, 387461, 380147, or 372917.
In certain embodiments, a target region is nucleotides 3573-3588 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 3573-3588 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 74 or 75. In certain such embodiments, a short antisense compound targeted to nucleotides 3573-3588 of SEQ ID NO: 1 is selected from ISIS NO. 372840 or 372918.
In certain embodiments, a target region is nucleotides 3701-3716 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 3701-3716 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 76 or 77. In certain such embodiments, a short antisense compound targeted to nucleotides 3701-3716 of SEQ ID NO: 1 is selected from ISIS NO. 372841 or 372919.
In certain embodiments, a target region is nucleotides 4219-4234 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 4219-4234 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 78 or 79. In certain such embodiments, a short antisense compound targeted to nucleotides 4219-4234 of SEQ ID NO: 1 is selected from ISIS NO. 372843 or 372921.
In certain embodiments, a target region is nucleotides 4301-4323 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 4301-4323 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 80, 81, 82, or 83. In certain embodiments, a short antisense compound targeted to nucleotides 4301-4323 of SEQ ID NO: 1 is selected from ISIS NO. 372844, 372922, 372845, or 372923.
In certain embodiments, a target region is nucleotides 5588-5609 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 5588-5609 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, or 92. In certain such embodiments, a short antisense compound targeted to nucleotides 5588-5609 of SEQ ID NO: 1 is selected from ISIS NO. 346601, 346602, 346603, 346604, 346605, 346606, 346607, 346608, or 346609.
In certain embodiments, a target region is nucleotides 5924-5939 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 5924-5939 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 93 or 94. In certain such embodiments, a short antisense compound targeted to nucleotides 5924-5939 of SEQ ID NO: 1 is selected from ISIS NO. 372851 or 372929.
In certain embodiments, a target region is nucleotides 6664-6679 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 6664-6679 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 95 or 96. In certain such embodiments, a short antisense compound targeted to nucleotides 6664-6679 of SEQ ID NO: 1 is selected from ISIS NO. 372854 or 372932.
In certain embodiments, a target region is nucleotides 6908-6923 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 6908-6923 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 97 or 98. In certain such embodiments, a short antisense compound targeted to nucleotides 6908-6923 of SEQ ID NO: 1 is selected from ISIS NO. 372855 or 372933.
In certain embodiments, a target region is nucleotides 7190-7205 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 7190-7205 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 99 or 100. In certain such embodiments, a short antisense compound targeted to nucleotides 7190-7205 of SEQ ID NO: 1 is selected from ISIS NO. 372856 or 372934.
In certain embodiments, a target region is nucleotides 7817-7839 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 7817-7839 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 101, 102, 104, 105, 106, 107, 108, 109, 110, or 111. In certain such embodiments, a short antisense compound targeted to nucleotides 7817-7839 of SEQ ID NO: 1 is selected from ISIS NO. 372858, 372936, 346610, 346611, 346612, 346613, 346614, 346615, 346616, 346617, or 346618.
In certain embodiments, a target region is nucleotides 7995-8010 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 7995-8010 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 112 or 113. In certain such embodiments, a short antisense compound targeted to nucleotides 7995-8010 of SEQ ID NO: 1 is selected from ISIS NO. 372859 or 372937.
In certain embodiments, a target region is nucleotides 8336-8356 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 8336-8356 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 114, 115, 116, or 117. In certain such embodiments, a short antisense compound targeted to nucleotides 8336-8356 of SEQ ID NO: 1 is selected from ISIS NO. 372861, 372939, 372862, or 372940.
In certain embodiments, a target region is nucleotides 8539-8554 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 8539-8554 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 118 or 119. In certain such embodiments, a short antisense compound targeted to nucleotides 8539-8554 of SEQ ID NO: 1 is selected from ISIS NO. 372863 or 372941.
In certain embodiments, a target region is nucleotides 9344-9359 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 9344-9359 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 120 or 121. In certain such embodiments, a short antisense compound targeted to nucleotides 9344-9359 of SEQ ID NO: 1 is selected from ISIS NO. 372871 or 372949.
In certain embodiments, a target region is nucleotides 9515-9530 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 9515-9530 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 122 or 123. In certain such embodiments, a short antisense compound targeted to nucleotides 9515-9530 of SEQ ID NO: 1 is selected from ISIS NO. 372872 or 372950.
In certain embodiments, a target region is nucleotides 9794-9809 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 9794-9809 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 124 or 125. In certain such embodiments, a short antisense compound targeted to nucleotides 9794-9809 of SEQ ID NO: 1 is selected from ISIS NO. 372875 or 372953.
In certain embodiments, a target region is nucleotides 10157-10187 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 10157-10187 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 126, 127, 128, 129, 130, 131, 132, or 133. In certain such embodiments, a short antisense compound targeted to nucleotides 10157-10187 of SEQ ID NO: 1 is selected from ISIS NO. 372877, 372955, 372878, 372956, 372879, 372957, 372880, or 372958.
In certain embodiments, a target region is nucleotides 10838-10859 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 10838-10859 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 134, 135, 136, 137, 138, 139, 140, 141, or 142. In certain such embodiments, a short antisense compound targeted to nucleotides 10838-10859 of SEQ ID NO: 1 is selected from ISIS NO. 346619, 346620, 346621, 346622, 346623, 346624, 346625, 346626, or 346627.
In certain embodiments, a target region is nucleotides 13689-13714 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 13689-13714 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 143, 144, 145, 146, 147, or 148. In certain such embodiments, a short antisense compound targeted to nucleotides 13689-13714 of SEQ ID NO: 1 is selected from ISIS NO. 372890, 372968, 372891, 372969, 372892, or 372970.
In certain embodiments, a target region is nucleotides 13907-13928 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 13907-13928 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 149, 150, 151, 152, 153, 154, 155, 156, or 157. In certain such embodiments, a short antisense compound targeted to nucleotides 13907-13928 of SEQ ID NO: 1 is selected from ISIS NO. 346628, 346629, 346630, 346631, 346632, 346633, 346634, 346635, or 346636.
In certain embodiments, a target region is nucleotides 13963-13984 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 13963-13984 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 158, 159, 160, 161, 162, 163, 164, 165, or 166. In certain such embodiments, a short antisense compound targeted to nucleotides 13963-13984 of SEQ ID NO: 1 is selected from ISIS NO. 346637, 346638, 346639, 346640, 346641, 346642, 346643, 346644, or 346645.
In certain embodiments, a target region is nucleotides 14051-14072 of SEQ ID NO: 1. In certain such embodiments, a short antisense compound targeted to nucleotides 14051-14072 of SEQ ID NO: 1 comprises a nucleotide sequence selected from SEQ ID NO 167, 168, 169, 170, 171, 172, 173, 174, or 175. In certain such embodiments, a short antisense compound targeted to nucleotides 14051-14072 of SEQ ID NO: 1 is selected from ISIS NO. 346646, 346647, 346648, 346649, 346650, 346651, 346652, 346653, or 346654.
In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to an ApoB nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to an ApoB nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of ApoB. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to an ApoB nucleic acid, wherein the short antisense compound targeted to an ApoB nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of ApoB using one or more short antisense compounds targeted to an ApoB nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of ApoB comprise use of a short antisense compound targeted to an ApoB nucleic acid comprising 12 or 14 nucleotides or nucleosides.
In certain embodiments, short antisense compounds targeting a ApoB nucleic acid may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a ApoB nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.
2. SGLT-2
Sodium dependent glucose transporter 2 (SGLT-2) is expressed in the kidney proximal tubule epithelial cells, and functions to reabsorb glucose preventing glucose loss in the urine. For the human genome SGLT-2 is a member of an 11-membered family of sodium substrate co-transporters. Many of these family members share sequence homology, for example SGLT-1 shares about 59% sequence identity with SGLT-2 and about 70% sequence identity with SGLT-3. SGLT-1 is a glucose transporter found in the heart and the CNS. SGLT-3 is a glucose sensing sodium channel in the small intestine. The separate localization patterns for these SGLTs is one point of distinction between the homologous family members. (Handlon, A. L., Expert Opin. Ther. Patents (2005) 15(11):1532-1540; Kanai et al., J. Clin. Invest., 1994, 93, 397-404; Wells et al., Am. J. Physiol. Endocrinol. Metab., 1992, 263, F459-465).
Studies of human SGLT2 injected into Xenopus oocytes demonstrated that this protein mediates sodium-dependent transport of D-glucose and .alpha.-methyl-D-glucopyranoside (.alpha.-MeGlc; a glucose analog) with a Km value of 1.6 mM for .alpha.-MeGlc and a sodium to glucose coupling ratio of 1:1 (Kanai et al., J. Clin. Invest., 1994, 93, 397-404; You et al., J. Biol. Chem., 1995, 270, 29365-29371). This transport activity was suppressed by phlorizin, a plant glycoside that binds to the glucose site of the SGLTs but is not transported and thus inhibits SGLT action (You et al., J. Biol. Chem., 1995, 270, 29365-29371).
Diabetes is a disorder characterized by hyperglycemia due to deficient insulin action. Chronic hyperglycemia is a major risk factor for diabetes-associated complications, including heart disease, retinopathy, nephropathy and neuropathy. As the kidneys play a major role in the regulation of plasma glucose levels, renal glucose transporters are becoming attractive drug targets (Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18). Diabetic nephropathy is the most common cause of end-stage renal disease that develops in many patients with diabetes. Glucotoxicity, which results from long-term hyperglycemia, induces tissue-dependent insulin resistance in diabetic patients (Nawano et al., Am. J. Physiol. Endocrinol. Metab., 2000, 278, E535-543).
Definitions
“Sodium dependent glucose transporter 2” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound. Sodium dependent glucose transporter 2 is generally referred to as SGLT2 but may also be referred to as SLC5A2; sodium-glucose transporter 2; sodium-glucose cotransporter, kidney low affinity; sodium-glucose cotransporter, renal; solute carrier family 5 (sodium/glucose cotransporter), member 2; SL52.
“SGLT2 nucleic acid” means any nucleic acid encoding SGLT2. For example, in certain embodiments, a SGLT2 nucleic acid includes, without limitation, a DNA sequence encoding SGLT2, an RNA sequence transcribed from DNA encoding SGLT2, and an mRNA sequence encoding SGLT2. “SGLT2 mRNA” means an mRNA encoding a SGLT2 protein.
Therapeutic Indications
In certain embodiments, short antisense compounds are used to modulate expression of SGLT-2 and related proteins. In certain embodiments, such modulation is accomplished by providing short antisense compounds that hybridize with one or more target nucleic acid molecules encoding SGLT-2, including, but is not limited to, SGLT2, SL52, SLC5A2, Sodium-Glucose Co-Transporter, Kidney Low Affinity Sodium-Glucose Co-Transporter, Renal Sodium-Glucose Co-Transporter 2 and Solute Carrier Family 5 Sodium/Glucose Co-Transporter Member 2. Also provided are methods of treating metabolic and/or cardiovascular disease and disorders as described herein. In particular embodiments, short antisense compounds that inhibit the expression of SGLT2 are used in methods of lowering blood glucose levels in an animal and methods of delaying or preventing the onset of type 2 diabetes. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds of the invention to the animal, which may be in need of treatment. The one or more compounds can be a short antisense compound targeting a nucleic acid encoding SGLT2. Provided herein are methods of enhancing inhibition of expression of SGLT2 in kidney cells or kidney tissues, comprising contacting the cells or tissues with one or more of the compounds of the invention, such as short antisense compounds targeting a nucleic acid encoding SGLT2.
While certain compounds, compositions and methods have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the same.
In certain embodiments, short antisense compounds are chimeric oligomeric compounds having mixed phosphorothioate and phosphodiester backbones. Certain mixed backbone short antisense compounds have a central gap comprising at least 5 contiguous 2′-deoxy nucleosides flanked by two wings each of which comprises at least one 2′-O-methoxyethyl nucleoside. In certain embodiments, the internucleoside linkages of the mixed backbone compounds are phosphorothioate linkages in the gap and phosphodiester linkages in the two wings. In certain embodiments, mixed backbone compounds have phosphorothioate linkages in the wings, except for one phosphodiester linkage at one or both of the extreme 5′ and 3′ ends of the oligonucleotide. In certain embodiments short antisense compounds targeted to SGLT2 have a motif (wing-deoxy gap-wing) selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 2-8-2, 1-9-2, 1-8-1, 3-6-3 or 1-6-1.
In certain embodiments short antisense compounds targeted to SGLT2 have a motif (wing-deoxy gap-wing) selected from 1-10-1, 1-10-2, 2-8-2, 1-9-2, 1-8-1, 3-6-3 or 1-6-1.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid and having a mixed backbone are efficiently delivered to the kidney. In certain embodiments, administration of short antisense compounds targeted to an SGLT2 nucleic acid and having a mixed backbone results in modulation of target gene expression in the kidney. In certain such embodiments, there is little or no liver or kidney toxicity. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid and having a mixed backbone are more potent for reducing SGLT-2 mRNA and have a faster onset compared with a short antisense compound that does not have a mixed back-bone, but is otherwise identical. In certain such embodiments, such increase potency and/or reduced toxicity is in mouse and/or rat. In certain such embodiments, such increase potency and/or reduced toxicity is in a human.
By way of example, and only for illustrative purposes, ISIS 145733, which comprises uniform phosphorothioate linkages and ISIS 257016 which comprises phosphodiester linkage in the wings and phosphorothioate linkages in the gap, are otherwise identical. Both comprise the sequence GAAGTAGCCACCAACTGTGC (SEQ ID NO. 1572). Both of the oligonucleotides further comprise a gap consisting of ten 2′-deoxynucleotides, flanked on each side by five-nucleotide “2”-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The mixed back-bone compound, ISIS 257016, was about 50 times more potent for reducing SGLT-2 mRNA compared to the non-mixed parent compound, ISIS 145733 (see EXAMPLE 9).
Pharmacokinetic studies of certain mixed backbone compound ISIS 257016 indicate that in certain embodiments, the compound acts as a prodrug that is metabolized to a 12 nucleobase pharmacophore. Studies with ISIS 370717, a 12 nucleobase short antisense compound corresponding to ISIS 257016, show that the compound has a similar pharmacological profile to ISIS 257016 but with a faster onset of action. ISIS 370717 is a 12 nucleobase antisense oligonucleotide targeted to SGLT-2 comprising the sequence TAGCCACCAACT (SEQ ID NO. 1554), further comprising a gap consisting of ten 2′-deoxynucleotides, flanked on both sides by one-nucleotide wings. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The internucleoside linkages are phosphorothioate (P═S) throughout the oligonucleotide. The similarity in pharmacological activity of ISIS 257016 and ISIS 370717 supports the pharmacokinetic studies indicating ISIS 257016 was a prodrug having a 12 nucleotide pharmacophore (see EXAMPLE 10). Further, studies with stabilized (end-capped) versions of ISIS 257016 show dramatic loss of activity.
In certain embodiments, short antisense compounds comprising 2′ MOE monomers in the wings are efficiently delivered to the kidney and treatment with such compounds results in efficient modulation of target gene expression in the kidney without liver or kidney toxicity. It is further shown herein that in certain embodiments, short antisense compounds are more potent for reducing SGLT-2 mRNA and have a faster onset compared with parent oligonucleotides targeted to SGLT-2 mRNA in mouse and rat. 2′ MOE gap shortmers are shown herein to improve potency and bioavailability over parent compounds.
By way of example, and only for illustrative purposes studies with ISIS 370717 reveal significantly higher accumulation of the short antisense compound in the kidney tissue (approximately 500 micro grams per gram of tissue) compared to the longer parent. Moreover, SGLT-2 mRNA was reduced by more than 80% over the controls (see EXAMPLE 11). ISIS 370717 1-10-1 gapmer was used as a template to make sequence related oligos with varying motifs. Studies evaluating wing, gap and total length variations around the ISIS 370717 12 mer oligonucleotide can be seen in EXAMPLE 12. Certain motifs evaluated included 1-10-1, 2-8-2, 1-8-1, 3-6-3, and 1-6-1 (see Table 60 in EXAMPLE 12). The compounds were analyzed for their effect on SGLT2 mRNA levels. All the motifs inhibited the expression of SGLT2 in vivo in a dose-dependent manner. The 1-10-1, 2-8-2 and 1-8-1 gapmers were found to be particularly potent. SGLT-2 mRNA was reduced by more than 80% over the controls using these motifs.
In certain embodiments, the invention provides short antisense compounds targeted to an SGLT2 nucleic acid and having a motif selected from: 1-10-1 and 1-10-2 MOE gapmer. (see Table 62 in EXAMPLE 13). Certain such compounds were analyzed for their effect on rat SGLT2 mRNA. Results in Table 63 illustrate that both the 1-10-1 and 1-10-2 MOE gapmers inhibit the expression of SGLT2 in vivo in a dose-dependent manner and over 80% reduction of SGLT-2 mRNA could be achieved.
Certain additional 1-10-1 and 2-8-2 MOE gapmers were evaluated in both mouse and rat in vivo models (see, e.g., EXAMPLE 14 and 15). Greater than 80% reduction in SGLT-2 mRNA was achieved with many of the 1-10-1 and 2-8-2 MOE gapmers at relatively low concentrations of oligo and in the absence of any toxicity effects.
In another non-limiting example, the effect of ISIS 388625 on dog SGLT2 mRNA levels was also analyzed. Dog studies illustrate that greater than 80% inhibition of the expression of SGLT2 can be achieved at a 1 mg/kg/wk dose. Even greater inhibition can be achieved at slightly higher doses. Administration of ISIS 388625 in dog was also shown to improved glucose tolerance. Peak plasma glucose levels were decreased by over 50% on average and the subsequent drop in glucose was lessened compared to saline controls in a standard glucose tolerance test (See EXAMPLE 17). Also, in a rat model of diabetes, short antisense compounds were shown to significantly decrease plasma glucose levels and HbA1C over time compared to PBS and control treated animals (See Example 16).
The animals in all studies were further evaluated for toxicity. For example, total body weight, liver, spleen and kidney weight were evaluated. Significant changes in spleen, liver or body weight can indicate that a particular compound causes toxic effects. All changes were found to be within the margin of error. No significant changes in body weight were observed during the treatment or at study termination. No significant changes in liver or spleen weights were observed.
Certain Short Antisense Compounds Targeted to an SGLT2 Nucleic Acid
In certain embodiments, short antisense compounds are targeted to an SGLT2 nucleic acid having the sequence of GENBANK® Accession No. NM_003041.1, incorporated herein as SEQ ID NO: 2. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 3 is at least 90% complementary to SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 3 is at least 95% complementary to SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 3 is 100% complementary to SEQ ID NO: 1. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 3 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 4 and 5.
The nucleotide sequence set forth in each SEQ ID NO set forth in Tables 4 and 5 is independent of any modification to a sugar moiety, a monomeric linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Tables 4 and 5 illustrate examples of short antisense compounds targeted to SEQ ID NO: 3. Table 4 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 3. Table 5 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 3. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 4







Short Antisense Compounds Targeted to SEQ ID NO: 3













5′
3′





ISIS
Target
Target


SEQ ID


No
Site
Site
Sequence (5′-3′)
Gapmer Motif
NO















379684
84
95
TGTCAGCAGGAT
1-10-1 MOE
214


 


405193
113
124
CAGCAGGAAATA
2-8-2 MOE
215


 


405194
114
125
CCAGCAGGAAAT
2-8-2 MOE
216


 


405195
115
126
ACCAGCAGGAAA
2-8-2 MOE
217


 


405196
116
127
GACCAGCAGGAA
2-8-2 MOE
218


 


405197
117
128
TGACCAGCAGGA
2-8-2 MOE
219


 


379685
117
128
TGACCAGCAGGA
1-10-1 MOE
219


 


405198
118
129
ATGACCAGCAGG
2-8-2 MOE
221


 


405199
119
130
AATGACCAGCAG
2-8-2 MOE
222


 


405200
120
131
CAATGACCAGCA
2-8-2 MOE
223


 


405201
121
132
CCAATGACCAGC
2-8-2 MOE
224


 


379686
135
146
ACCACAAGCCAA
1-10-1 MOE
225


 


379711
172
183
TAGCCGCCCACA
1-10-1 MOE
226


 


388628
172
183
TAGCCGCCCACA
2-8-2 MOE
226


 


405202
207
218
CCGGCCACCACA
2-8-2 MOE
228


 


405203
208
219
ACCGGCCACCAC
2-8-2 MOE
229


 


405204
236
247
GATGTTGCTGGC
2-8-2 MOE
230


 


379687
236
247
GATGTTGCTGGC
1-10-1 MOE
230


 


405205
237
248
CGATGTTGCTGG
2-8-2 MOE
232


 


405206
238
249
CCGATGTTGCTG
2-8-2 MOE
233


 


405207
239
250
GCCGATGTTGCT
2-8-2 MOE
234


 


405208
240
251
TGCCGATGTTGC
2-8-2 MOE
235


 


405209
241
252
CTGCCGATGTTG
2-8-2 MOE
236


 


405210
260
271
CAGGCCCACAAA
2-8-2 MOE
237


 


405211
261
272
CCAGGCCCACAA
2-8-2 MOE
238


 


405212
262
273
GCCAGGCCCACA
2-8-2 MOE
239


 


379688
288
299
CCAAGCCACTTG
1-10-1 MOE
240


 


379689
318
329
AGAGCGCATTCC
1-10-1 MOE
241


 


379690
435
446
ACAGGTAGAGGC
1-10-1 MOE
242


 


405248
474
485
AGATCTTGGTGA
2-8-2 MOE
243


 


379691
474
485
AGATCTTGGTGA
1-10-1 MOE
243


 


382676
527
539
TGTTCCAGCCCAG
1-10-2 MOE
245


 


388625
528
539
TGTTCCAGCCCA
2-8-2 MOE
246


 


389780
528
539
TGTTCCAGCCCA
1-9-2 MOE
246


 


379692
528
539
TGTTCCAGCCCA
1-10-1 MOE
246


 


392170
528
539
TGTTCCAGCCCA
1-10-1
246






Methyleneoxy






BNA


 


392173
528
539
TGTTCCAGCCCA
2-8-2
246






Methyleneoxy






BNA


 


405213
529
540
ATGTTCCAGCCC
2-8-2 MOE
251


 


405214
564
575
TGGTGATGCCCA
2-8-2 MOE
252


 


405215
565
576
ATGGTGATGCCC
2-8-2 MOE
253


 


405216
566
577
CATGGTGATGCC
2-8-2 MOE
254


 


379693
566
577
CATGGTGATGCC
1-10-1 MOE
254


 


405217
567
578
TCATGGTGATGC
2-8-2 MOE
256


 


405218
568
579
ATCATGGTGATG
2-8-2 MOE
257


 


405219
587
598
CCCTCCTGTCAC
2-8-2 MOE
258


 


405220
588
599
GCCCTCCTGTCA
2-8-2 MOE
259


 


405221
589
600
AGCCCTCCTGTC
2-8-2 MOE
260


 


405222
590
601
CAGCCCTCCTGT
2-8-2 MOE
261


 


405223
591
602
CCAGCCCTCCTG
2-8-2 MOE
262


 


405224
592
603
GCCAGCCCTCCT
2-8-2 MOE
263


 


379694
629
640
GACGAAGGTCTG
1-10-1 MOE
264


 


405225
707
718
GTATTTGTCGAA
2-8-2 MOE
265


 


379695
737
748
GGACACCGTCAG
1-10-1 MOE
266


 


379696
974
985
CAGCTTCAGGTA
1-10-1 MOE
267


 


405226
998
1009
CATGACCATGAG
2-8-2 MOE
268


 


405227
999
1010
GCATGACCATGA
2-8-2 MOE
269


 


405228
1000
1011
GGCATGACCATG
2-8-2 MOE
270


 


405229
1001
1012
TGGCATGACCAT
2-8-2 MOE
271


 


405230
1002
1013
CTGGCATGACCA
2-8-2 MOE
272


 


379697
1002
1013
CTGGCATGACCA
1-10-1 MOE
272


 


405231
1003
1014
CCTGGCATGACC
2-8-2 MOE
274


 


379698
1091
1102
GCAGCCCACCTC
1-10-1 MOE
275


 


405232
1092
1103
AGCAGCCCACCT
2-8-2 MOE
276


 


405233
1093
1104
GAGCAGCCCACC
2-8-2 MOE
277


 


405234
1130
1141
CATGAGCTTCAC
2-8-2 MOE
278


 


405235
1131
1142
GCATGAGCTTCA
2-8-2 MOE
279


 


382677
1131
1143
GGCATGAGCTTCA
1-10-2 MOE
280


 


388626
1132
1143
GGCATGAGCTTC
2-8-2 MOE
281


 


379699
1132
1143
GGCATGAGCTTC
1-10-1 MOE
281


 


405236
1133
1144
GGGCATGAGCTT
2-8-2 MOE
283


 


405237
1157
1168
CAGCATGAGTCC
2-8-2 MOE
284


 


405238
1158
1169
CCAGCATGAGTC
2-8-2 MOE
285


 


379700
1158
1169
CCAGCATGAGTC
1-10-1 MOE
285


 


405239
1159
1170
GCCAGCATGAGT
2-8-2 MOE
287


 


379701
1230
1241
CCATGGTGAAGA
1-10-1 MOE
288


 


405240
1542
1553
CACAGCTGCCCG
2-8-2 MOE
289


 


405241
1543
1554
ACACAGCTGCCC
2-8-2 MOE
290


 


405242
1544
1555
CACACAGCTGCC
2-8-2 MOE
291


 


382678
1544
1556
GCACACAGCTGCC
1-10-2 MOE
292


 


388627
1545
1556
GCACACAGCTGC
2-8-2 MOE
293


 


379702
1545
1556
GCACACAGCTGC
1-10-1 MOE
293


 


379703
1701
1712
GCCGGAGACTGA
1-10-1 MOE
295


 


405243
1976
1987
ATTGAGGTTGAC
2-8-2 MOE
296


 


405244
1977
1988
CATTGAGGTTGA
2-8-2 MOE
297


 


405245
1978
1989
GCATTGAGGTTG
2-8-2 MOE
298


 


405246
1979
1990
GGCATTGAGGTT
2-8-2 MOE
299


 


405247
1980
1991
GGGCATTGAGGT
2-8-2 MOE
300
















TABLE 5







Short antisense compounds targeted to


SEQ ID NO: 3 and having 1 or 2 mismatches













5′
3′





ISIS
Target
Target
Sequence
Gapmer
SEQ ID


No
Site
Site
(5′-3′)
Motif
NO















405200
96
107
CAATGACCAGCA
2-8-2 MOE
223


 


405215
382
393
ATGGTGATGCCC
2-8-2 MOE
253


 


405216
383
394
CATGGTGATGCC
2-8-2 MOE
254


 


379693
383
394
CATGGTGATGCC
1-10-1 MOE
254


 


379701
471
482
CCATGGTGAAGA
1-10-1 MOE
288


 


405218
472
483
ATCATGGTGATG
2-8-2 MOE
257


 


405246
536
547
GGCATTGAGGTT
2-8-2 MOE
299


 


405248
570
581
AGATCTTGGTGA
2-8-2 MOE
243


 


379691
570
581
AGATCTTGGTGA
1-10-1 MOE
243


 


379698
683
694
GCAGCCCACCTC
1-10-1 MOE
275


 


405232
684
695
AGCAGCCCACCT
2-8-2 MOE
276


 


379711
685
696
TAGCCGCCCACA
1-10-1 MOE
226


 


388628
685
696
TAGCCGCCCACA
2-8-2 MOE
226


 


379698
950
961
GCAGCCCACCTC
1-10-1 MOE
275


 


405232
951
962
AGCAGCCCACCT
2-8-2 MOE
276


 


405235
978
989
GCATGAGCTTCA
2-8-2 MOE
279


 


382677
978
990
GGCATGAGCTTCA
1-10-2 MOE
280


 


388626
979
990
GGCATGAGCTTC
2-8-2 MOE
281


 


379699
979
990
GGCATGAGCTTC
1-10-1 MOE
281


 


405236
980
991
GGGCATGAGCTT
2-8-2 MOE
283


 


379698
1043
1054
GCAGCCCACCTC
1-10-1 MOE
275


 


405239
1171
1182
GCCAGCATGAGT
2-8-2 MOE
287


 


405209
1213
1224
CTGCCGATGTTG
2-8-2 MOE
236


 


405233
1364
1375
GAGCAGCCCACC
2-8-2 MOE
277


 


405240
1366
1377
CACAGCTGCCCG
2-8-2 MOE
289


 


405211
1500
1511
CCAGGCCCACAA
2-8-2 MOE
238


 


405212
1501
1512
GCCAGGCCCACA
2-8-2 MOE
239


 


379695
1643
1654
GGACACCGTCAG
1-10-1 MOE
266


 


379698
1875
1886
GCAGCCCACCTC
1-10-1 MOE
275


 


405239
1993
2004
GCCAGCATGAGT
2-8-2 MOE
287


 


405211
2210
2221
CCAGGCCCACAA
2-8-2 MOE
238


 


405212
2211
2222
GCCAGGCCCACA
2-8-2 MOE
239









In certain embodiments, a target region is nucleotides 85-184 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 85-184 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 85-184 comprises a nucleotide sequence selected from SEQ ID NO 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, or 227. In certain such embodiments, a short antisense compound targeted to nucleotides 85-184 of SEQ ID NO: 3 is selected from Isis No 379684, 405193, 405194, 405195, 405196, 405197, 379685, 405198, 405199, 405200, 405201, 379686, 379711 or 388628.
In certain embodiments, a target region is nucleotides 113-132 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 113-132 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 113-132 comprises a nucleotide sequence selected from SEQ ID NO 215, 216, 217, 218, 219, 221, 222, 223, or 224. In certain such embodiments, a short antisense compound targeted to nucleotides 113-132 of SEQ ID NO: 3 is selected from Isis No 405193, 405194, 405195, 405196, 405197, 379685, 405198, 405199, 405200, or 405201.
In certain embodiments, a target region is nucleotides 207-329 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 207-329 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 207-329 comprises a nucleotide sequence selected from SEQ ID NO 228, 229, 230, 232, 233, 234, 235, 236, 237, 238, 239, 240, or 241. In certain such embodiments, a short antisense compound targeted to nucleotides 207-329 of SEQ ID NO: 3 is selected from Isis No 405202, 405203, 405204, 379687, 405205, 405206, 405207, 405208, 405209, 405210, 405211, 405212, 379688, or 379689.
In certain embodiments, a target region is nucleotides 207-273 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 207-273 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 207-273 comprises a nucleotide sequence selected from SEQ ID NO 228, 229, 230, 232, 233, 234, 235, 236, 237, 238, or 239. In certain such embodiments, a short antisense compound targeted to nucleotides 207-273 of SEQ ID NO: 3 is selected from Isis No 405202, 405203, 405204, 379687, 405205, 405206, 405207, 405208, 405209, 405210, 405211, or 405212.
In certain embodiments, a target region is nucleotides 207-219 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 207-219 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 207-219 comprises a nucleotide sequence selected from SEQ ID NO 228 or 229. In certain such embodiments, a short antisense compound targeted to nucleotides 207-219 of SEQ ID NO: 3 is selected from Isis NO. 405202 or 405203.
In certain embodiments, a target region is nucleotides 236-252 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 236-252 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 236-252 comprises a nucleotide sequence selected from SEQ ID NO 230, 232, 233, 234, 235, or 236. In certain such embodiments, a short antisense compound targeted to nucleotides 236-252 of SEQ ID NO: 3 is selected from Isis NO. 405204, 379687, 405205, 405206, 405207, 405208, or 405209.
In certain embodiments, a target region is nucleotides 260-273 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 260-273 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 260-273 comprises a nucleotide sequence selected from SEQ ID NO 237, 238, or 239. In certain such embodiments, a short antisense compound targeted to nucleotides 260-273 of SEQ ID NO: 3 is selected from Isis NO. 405210, 405211, or 405212.
In certain embodiments, a target region is nucleotides 435-640 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 435-640 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 435-640 comprises a nucleotide sequence selected from SEQ ID NO 242, 243, 245, 246, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, or 264. In certain such embodiments, a short antisense compound targeted to nucleotides 435-640 of SEQ ID NO: 3 is selected from Isis NO. 379690, 405248, 379691, 389780, 379692, 382676, 388625, 392170, 392173, 405213, 405214, 405215, 405216, 379693, 405217, 405218, 405219, 405220, 405221, 405222, 405223, 405224, or 379694.
In certain embodiments, a target region is nucleotides 527-540 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 527-540 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 527-540 comprises a nucleotide sequence selected from SEQ ID NO 245, 246, or 251. In certain such embodiments, a short antisense compound targeted to nucleotides 527-540 of SEQ ID NO: 3 is selected from Isis NO. 389780, 379692, 382676, 388626, 392170, 392173, or 405213.
In certain embodiments, a target region is nucleotides 564-603 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 564-603 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 564-603 comprises a nucleotide sequence selected from SEQ ID NO 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, or 263. In certain such embodiments, a short antisense compound targeted to nucleotides 564-603 of SEQ ID NO: 3 is selected from Isis NO. 405214, 405215, 405216, 379693, 405217, 405218, 405219, 405220, 405221, 405222, 405223, or 405224.
In certain embodiments, a target region is nucleotides 564-579 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 564-579 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 564-579 comprises a nucleotide sequence selected from SEQ ID NO 252, 253, 254, 256, or 257. In certain such embodiments, a short antisense compound targeted to nucleotides 564-579 of SEQ ID NO: 3 is selected from Isis NO. 405214, 405215, 405216, 379693, 405217, or 405218.
In certain embodiments, a target region is nucleotides 587-603 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 587-603 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 587-603 comprises a nucleotide sequence selected from SEQ ID NO 258, 259, 260, 261, 262, or 263. In certain such embodiments, a short antisense compound targeted to nucleotides 587-603 of SEQ ID NO: 3 is selected from Isis NO. 405219, 405220, 405221, 405222, 405223, or 405224.
In certain embodiments, a target region is nucleotides 974-1014 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 974-1014 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 974-1014 comprises a nucleotide sequence selected from SEQ ID NO 267, 268, 269, 270, 271, 272, or 274. In certain such embodiments, a short antisense compound targeted to nucleotides 974-1014 of SEQ ID NO: 3 is selected from Isis NO. 379696, 405226, 405227, 405228, 405229, 405230, 379697, or 405231.
In certain embodiments, a target region is nucleotides 998-1014 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 998-1014 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 998-1014 comprises a nucleotide sequence selected from SEQ ID NO 268, 269, 270, 271, 272, or 274. In certain such embodiments, a short antisense compound targeted to nucleotides 998-1014 of SEQ ID NO: 3 is selected from Isis NO. 405226, 405227, 405228, 405229, 405230, 379697, or 405231.
In certain embodiments, a target region is nucleotides 1091-1170 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1091-1170 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1091-1170 comprises a nucleotide sequence selected from SEQ ID NO 275, 276, 277, 278, 279, 280, 281, 283, 284, 285, 286, or 287. In certain such embodiments, a short antisense compound targeted to nucleotides 1091-1170 of SEQ ID NO: 3 is selected from Isis NO. 379698, 405232, 405233, 405234, 405235, 388626, 379699, 382677, 405236, 405237, 405238, 379700, or 405239.
In certain embodiments, a target region is nucleotides 1091-1104 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1091-1104 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1091-1104 comprises a nucleotide sequence selected from SEQ ID NO 275, 276, or 277. In certain such embodiments, an short antisense compound targeted to nucleotides 1091-1104 of SEQ ID NO: 3 is selected from Isis NO. 379698, 405232, or 405233.
In certain embodiments, a target region is nucleotides 1130-1144 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1130-1144 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1130-1144 comprises a nucleotide sequence selected from SEQ ID NO 278, 279, 280, 281, or 283. In certain such embodiments, a short antisense compound targeted to nucleotides 1130-1144 of SEQ ID NO: 3 is selected from Isis NO. 405234, 405235, 388626, 379699, 382677, or 405236.
In certain embodiments, a target region is nucleotides 1157-1170 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1157-1170 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1157-1170 comprises a nucleotide sequence selected from SEQ ID NO 284, 285, or 287. In certain such embodiments, a short antisense compound targeted to nucleotides 1157-1170 of SEQ ID NO: 3 is selected from Isis NO. 405237, 405238, 379700, or 405239.
In certain embodiments, a target region is nucleotides 1542-1556 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1542-1556 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1542-1556 comprises a nucleotide sequence selected from SEQ ID NO 289, 290, 291, 292, or 293. In certain such embodiments, a short antisense compound targeted to nucleotides 1542-1556 of SEQ ID NO: 3 is selected from Isis NO. 405240, 405241, 405242, 388629, 379702, or 382678.
In certain embodiments, a target region is nucleotides 1976-1991 of SEQ ID NO: 3. In certain embodiments, a short antisense compound is targeted to nucleotides 1976-1991 of SEQ ID NO: 3. In certain such embodiments, a short antisense compound targeted to nucleotides 1976-1991 comprises a nucleotide sequence selected from SEQ ID NO 296, 297, 298, 299, or 300. In certain such embodiments, a short antisense compound targeted to nucleotides 1976-1991 of SEQ ID NO: 3 is selected from Isis NO. 405243, 405244, 405245, 405246, or 405247.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to an SGLT2 nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to an SGLT2 nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of SGLT2. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to an SGLT2 nucleic acid, wherein the short antisense compound targeted to an SGLT2 nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of SGLT2 using one or more short antisense compounds targeted to an SGLT2 nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of SGLT2 comprise use of a short antisense compound targeted to an SGLT2 nucleic acid comprising 12 or 14 nucleotides or nucleosides.
3. PCSK9
In individuals with autosomal dominant hypercholesterolemia (ADH), elevated LDL-C levels have been linked to mutations in the genes encoding LDL-receptor (LDL-R), apolipoprotein B (apoB), or proprotein convertase subtilisin/kexin type 9 (PCSK9) (Abifadel et al., Nat. Genet., 2003, 34:154-156). PCSK9 was identified as a third locus associated with ADH when gain-of-function mutations in PCSK9 were found to be linked to elevated LDL-C levels. ApoB participates in the intracellular assembly and secretion of triglyceride-rich lipoproteins and is a ligand for the LDL-R. PCSK9 is proposed to reduce LDL-R expression levels in the liver. Reduced LDL-R expression results in reduced hepatic uptake of circulating ApoB-containing lipoproteins, which in turn leads to elevated cholesterol.
Definitions
“PCSK9” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound.
“PCSK9 nucleic acid” means any nucleic acid encoding PCSK9. For example, in certain embodiments, a PCSK9nucleic acid includes, without limitation, a DNA sequence encoding PCSK9, an RNA sequence transcribed from DNA encoding PCSK9, and an mRNA sequence encoding PCSK9.
“PCSK9 mRNA” means an mRNA encoding PCSK9.
PCSK9 Therapeutic Indications
In certain embodiments, the invention provides methods of modulating the expression of PCSK9 in an individual comprising administering a short antisense compound targeted to a PCSK9 nucleic acid. In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.
Guidelines for lipid-lowering therapy were established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). The guidelines include obtaining a complete lipoprotein profile, typically after a 9 to 12 hour fast, for determination of LDL-C, total cholesterol, and HDL-C levels. According to the most recently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189 mg/dL, and greater than or equal to 190 mg/dL are considered borderline high, high, and very high, respectively. Total cholesterol levels of 200-239 and greater than or equal to 240 mg/dL are considered borderline high and high, respectively. HDL-C levels of less than 40 mg/dL are considered low.
In certain embodiments, the individual has been identified as in need of lipid-lowering therapy. In certain such embodiments, the individual has been identified as in need of lipid-lowering therapy according to the guidelines established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 190 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 160 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy has LDL-C above 100 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 160 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certain such embodiments the individual should maintain LDL-C below 70 mg/dL.
In certain embodiments the invention provides methods for reducing ApoB in an individual. In certain embodiments the invention provides methods for reducing ApoB-containing lipoprotein in an individual. In certain embodiments the invention provides methods for reducing LDL-C in an individual. In certain embodiments the invention provides methods for reducing VLDL-C in an individual. In certain embodiments the invention provides methods for reducing IDL-C in an individual. In certain embodiments the invention provides methods for reducing non-HDL-C in an individual. In certain embodiments the invention provides methods for reducing Lp(a) in an individual. In certain embodiments the invention provides methods for reducing serum triglyceride in an individual. In certain embodiments the invention provides methods for reducing liver triglyceride in an individual. In certain embodiments the invention provides methods for reducing Ox-LDL-C in an individual. In certain embodiments the invention provides methods for reducing small LDL particles in an individual. In certain embodiments the invention provides methods for reducing small VLDL particles in an individual. In certain embodiments the invention provides methods for reducing phospholipids in an individual. In certain embodiments the invention provides methods for reducing oxidized phospholipids in an individual.
In certain embodiments, the methods provided by the present invention do not lower HDL-C. In certain embodiments, the methods provided by the present invention do not result in accumulation of lipids in the liver.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to a PCSK9 nucleic acid is for use in therapy. In certain embodiments, the therapy is the reduction of LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments, the therapy is the treatment of hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease. In additional embodiments, the therapy is the reduction of CHD risk. In certain the therapy is prevention of atherosclerosis. In certain embodiments, the therapy is the prevention of coronary heart disease.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to a PCSK9 nucleic acid is used for the preparation of a medicament for reducing LDL-C, ApoB, VLDL-C, IDL-C, non-HDL-C, Lp(a), serum triglyceride, liver triglyceride, Ox-LDL-C, small LDL particles, small VLDL, phospholipids, or oxidized phospholipids in an individual. In certain embodiments pharmaceutical composition comprising a short antisense compound targeted to PCKS9 is used for the preparation of a medicament for reducing coronary heart disease risk. In certain embodiments a short antisense compound targeted to a PCSK9 nucleic acid is used for the preparation of a medicament for the treatment of hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease, type II diabetes, type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.
PCSK9 Combination Therapies
In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include lipid-lowering agents. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.
In certain embodiments, a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments the statin is selected from atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.
In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe.
In certain embodiments, a co-administered lipid-lowering agent is a co-formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe/simvastatin.
In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).
In certain embodiments, a co-administered lipid-lowering agent is an oligonucleotide targeted to an ApoB nucleic acid.
In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.
In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.
In certain embodiments, the pharmaceutical compositions of the present invention may be administered in conjunction with a lipid-lowering therapy. In certain such embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain such embodiments, a lipid-lowering therapy is LDL apheresis.
Certain Short Antisense Compounds Targeted to a PCSK9 Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a PCSK9 nucleic acid having the sequence of GENBANK® Accession No. NM_174936.2, incorporated herein as SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 4 is at least 90% complementary to SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 4 is at least 95% complementary to SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 4 is 100% complementary to SEQ ID NO: 4. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 4 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 6 or Table 7.
The nucleotide sequence set forth in each SEQ ID NO in Tables 6 and 7 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Short antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Tables 6 and 7 illustrate examples of short antisense compounds targeted to SEQ ID NO: 4. Table 6 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 4. Table 7 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 4. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 6







Short Antisense Compounds targeted to SEQ ID NO: 4













5′
3′


SEQ


ISIS
Target
Target
Sequence

ID


NO.
Site
Site
(5′-3′)
Gapmer Motif
NO















400297
695
708
ATGGGGCAACTTCA
2-10-2 MOE
329


 


400298
696
709
CATGGGGCAACTTC
2-10-2 MOE
330


 


400299
697
710
ACATGGGGCAACTT
2-10-2 MOE
331


 


400300
742
755
GGGATGCTCTGGGC
2-10-2 MOE
332


 


400301
757
770
CGCTCCAGGTTCCA
2-10-2 MOE
333


 


400302
828
841
GATACACCTCCACC
2-10-2 MOE
334


 


400303
829
842
AGATACACCTCCAC
2-10-2 MOE
335


 


400304
830
843
GAGATACACCTCCA
2-10-2 MOE
336


 


400305
937
950
GCCTGTCTGTGGAA
2-10-2 MOE
337


 


400306
952
965
CTGTCACACTTGCT
2-10-2 MOE
338


 


400307
988
1001
CGGCCGCTGACCAC
2-10-2 MOE
339


 


400308
989
1002
CCGGCCGCTGACCA
2-10-2 MOE
340


 


400309
990
1003
CCCGGCCGCTGACC
2-10-2 MOE
341


 


400310
991
1004
TCCCGGCCGCTGAC
2-10-2 MOE
342


 


400311
992
1005
ATCCCGGCCGCTGA
2-10-2 MOE
343


 


400312
993
1006
CATCCCGGCCGCTG
2-10-2 MOE
344


 


400313
994
1007
GCATCCCGGCCGCT
2-10-2 MOE
345


 


400314
1057
1070
GTGCCCTTCCCTTG
2-10-2 MOE
346


 


400315
1075
1088
ATGAGGGTGCCGCT
2-10-2 MOE
347


 


400316
1076
1089
TATGAGGGTGCCGC
2-10-2 MOE
348


 


400317
1077
1090
CTATGAGGGTGCCG
2-10-2 MOE
349


 


400318
1078
1091
CCTATGAGGGTGCC
2-10-2 MOE
350


 


400319
1093
1106
CGAATAAACTCCAG
2-10-2 MOE
351


 


400320
1094
1107
CCGAATAAACTCCA
2-10-2 MOE
352


 


400321
1095
1108
TCCGAATAAACTCC
2-10-2 MOE
353


 


400322
1096
1109
TTCCGAATAAACTC
2-10-2 MOE
354


 


400323
1147
1160
GCCAGGGGCAGCAG
2-10-2 MOE
355


 


400324
1255
1268
GAGTAGAGGCAGGC
2-10-2 MOE
356


 


400325
1334
1347
CCCCAAAGTCCCCA
2-10-2 MOE
357


 


400326
1335
1348
TCCCCAAAGTCCCC
2-10-2 MOE
358


 


400327
1336
1349
GTCCCCAAAGTCCC
2-10-2 MOE
359


 


400328
1453
1466
ACGTGGGCAGCAGC
2-10-2 MOE
360


 


400329
1454
1467
CACGTGGGCAGCAG
2-10-2 MOE
361


 


400330
1455
1468
CCACGTGGGCAGCA
2-10-2 MOE
362


 


400331
1456
1469
GCCACGTGGGCAGC
2-10-2 MOE
363


 


400332
1569
1582
CAGGGAACCAGGCC
2-10-2 MOE
364


 


400333
1570
1583
TCAGGGAACCAGGC
2-10-2 MOE
365


 


400334
1571
1584
CTCAGGGAACCAGG
2-10-2 MOE
366


 


400335
1572
1585
CCTCAGGGAACCAG
2-10-2 MOE
367


 


400336
1573
1586
TCCTCAGGGAACCA
2-10-2 MOE
368


 


400337
1574
1587
GTCCTCAGGGAACC
2-10-2 MOE
369


 


400338
1575
1588
GGTCCTCAGGGAAC
2-10-2 MOE
370


 


400339
1576
1589
TGGTCCTCAGGGAA
2-10-2 MOE
371


 


400340
1577
1590
CTGGTCCTCAGGGA
2-10-2 MOE
372


 


400341
1578
1591
GCTGGTCCTCAGGG
2-10-2 MOE
373


 


400342
1621
1634
GTGCTGGGGGGCAG
2-10-2 MOE
374


 


400343
1622
1635
GGTGCTGGGGGGCA
2-10-2 MOE
375


 


400344
1623
1636
GGGTGCTGGGGGGC
2-10-2 MOE
376


 


400345
1624
1637
TGGGTGCTGGGGGG
2-10-2 MOE
377


 


400346
1738
1751
GAGCAGCTCAGCAG
2-10-2 MOE
378


 


400347
1739
1752
GGAGCAGCTCAGCA
2-10-2 MOE
379


 


400348
1740
1753
TGGAGCAGCTCAGC
2-10-2 MOE
380


 


400349
1741
1754
CTGGAGCAGCTCAG
2-10-2 MOE
381


 


400350
1834
1847
CCCTCACCCCCAAA
2-10-2 MOE
382


 


400351
1835
1848
ACCCTCACCCCCAA
2-10-2 MOE
383


 


400352
1836
1849
CACCCTCACCCCCA
2-10-2 MOE
384


 


400353
1837
1850
ACACCCTCACCCCC
2-10-2 MOE
385


 


400354
1838
1851
GACACCCTCACCCC
2-10-2 MOE
386


 


400355
1839
1852
AGACACCCTCACCC
2-10-2 MOE
387


 


400356
1840
1853
TAGACACCCTCACC
2-10-2 MOE
388


 


400357
2083
2096
TGGCAGCAGGAAGC
2-10-2 MOE
389


 


400358
2084
2097
ATGGCAGCAGGAAG
2-10-2 MOE
390


 


400359
2085
2098
CATGGCAGCAGGAA
2-10-2 MOE
391


 


400360
2086
2099
GCATGGCAGCAGGA
2-10-2 MOE
392


 


400361
2316
2329
GGCAGCAGATGGCA
2-10-2 MOE
393


 


400362
2317
2330
CGGCAGCAGATGGC
2-10-2 MOE
394


 


400363
2318
2331
CCGGCAGCAGATGG
2-10-2 MOE
395


 


400364
2319
2332
TCCGGCAGCAGATG
2-10-2 MOE
396


 


400365
2320
2333
CTCCGGCAGCAGAT
2-10-2 MOE
397


 


400366
2321
2334
GCTCCGGCAGCAGA
2-10-2 MOE
398


 


400367
2322
2335
GGCTCCGGCAGCAG
2-10-2 MOE
399


 


400368
2323
2336
CGGCTCCGGCAGCA
2-10-2 MOE
400


 


400369
2324
2337
CCGGCTCCGGCAGC
2-10-2 MOE
401


 


400370
2325
2338
GCCGGCTCCGGCAG
2-10-2 MOE
402


 


400371
3543
3556
AGTTACAAAAGCAA
2-10-2 MOE
403


 


403739
988
1001
CGGCCGCTGACCAC
2-10-2
339






(6′S)-6′-






methyl-






Methyleneoxy






BNA


 


403740
1455
1468
CCACGTGGGCAGCA
2-10-2
362






(6′S)-6′-






methyl-






Methyleneoxy






BNA
















TABLE 7







Short antisense compounds targeted to


SEQ ID NO: 4 and having 1 or 2 mismatches













5′
3′


SEQ


ISIS
Target
Target
Sequence

ID


NO.
Site
Site
(5′-3′)
Gapmer Motif
NO















400323
349
362
GCCAGGGGCAGCAG
2-10-2 MOE
355


 


400370
679
692
GCCGGCTCCGGCAG
2-10-2 MOE
402


 


400361
1860
1873
GGCAGCAGATGGCA
2-10-2 MOE
393


 


400323
1873
1886
GCCAGGGGCAGCAG
2-10-2 MOE
355


 


400310
2257
2270
TCCCGGCCGCTGAC
2-10-2 MOE
342


 


400361
2653
2666
GGCAGCAGATGGCA
2-10-2 MOE
393


 


400350
2811
2824
CCCTCACCCCCAAA
2-10-2 MOE
382


 


400351
2812
2825
ACCCTCACCCCCAA
2-10-2 MOE
383


 


400352
2813
2826
CACCCTCACCCCCA
2-10-2 MOE
384


 


400353
2814
2827
ACACCCTCACCCCC
2-10-2 MOE
385


 


400334
2966
2979
CTCAGGGAACCAGG
2-10-2 MOE
366


 


400332
3379
3392
CAGGGAACCAGGCC
2-10-2 MOE
364


 


400340
3448
3461
CTGGTCCTCAGGGA
2-10-2 MOE
372


 


400341
3449
3462
GCTGGTCCTCAGGG
2-10-2 MOE
373









In certain embodiments, a target region is nucleotides 695-710 of SEQ ID NO: 4. In certain such embodiments, short antisense compounds targeted to nucleotides 695-710 of SEQ ID NO: 4 comprise a nucleotide sequence selected from SEQ ID NO: 329, 330, or 331. In certain such embodiments, a short antisense compound targeted to nucleotides 695-710 of SEQ ID NO: 4 is selected from Isis NO. 400297, 400298, or 400299.
In certain embodiments, a target region is nucleotides 742-770 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 742-770 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 332 or 333. In certain such embodiments, a short antisense compound targeted to nucleotides 742-770 of SEQ ID NO: 4 is selected from Isis NO. 400300 or 400301.
In certain embodiments, a target region is nucleotides 828-843 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 828-843 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 334, 335, or 336. In certain such embodiments, a short antisense compound targeted to nucleotides 828-843 of SEQ ID NO: 4 is selected from ISIS No. 400302, 400303, or 400304.
In certain embodiments, a target region is nucleotides 937-1007 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 937-1007 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 337, 338, 339, 340, 341, 342, 343, 344, or 345. In certain such embodiments, a short antisense compound targeted to nucleotides 937-1007 of SEQ ID NO: 4 is selected from Isis NO. 400305, 400306, 400307, 400308, 400309, 400310, 400311, 400312, 400313, or 403739.
In certain embodiments, a target region is nucleotides 937-965 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 937-965 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 337 or 338. In certain such embodiments, a short antisense compound targeted to nucleotides 937-965 of SEQ ID NO: 4 is selected from Isis NO. 400305 or 400306.
In certain embodiments, a target region is nucleotides 988-1007 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 988-1007 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 339, 340, 341, 342, 343, 344, or 345. In certain such embodiments, a short antisense compound targeted to nucleotides 937-1007 of SEQ ID NO: 4 is selected from Isis NO. 400307, 400308, 400309, 400310, 400311, 400312, 4003313, or 403739.
In certain embodiments, a target region is nucleotides 1057-1160 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1160 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 346, 347, 348, 349, 350, 351, 352, 353, 354, or 355. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1160 of SEQ ID NO: 4 is selected from ISIS NO. 400314, 400315, 400316, 400317, 400318, 400319, 400320, 400321, 400322, or 400323.
In certain embodiments, a target region is nucleotides 1057-1109 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1109 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 346, 347, 348, 349, 350, 351, 352, 353, or 354. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1109 of SEQ ID NO: 4 is selected from ISIS NO. 400314, 400315, 400316, 400317, 400318, 400319, 400320, 400321, or 400322.
In certain embodiments, a target region is nucleotides 1057-1091 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1091 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 346, 347, 348, 349, or 350. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1091 of SEQ ID NO: 4 is selected from ISIS NO. 400314, 400315, 400316, 400317, or 400318.
In certain embodiments, a target region is nucleotides 1093-1109 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1093-1109 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 351, 352, 353, or 354. In certain such embodiments, a short antisense compound targeted to nucleotides 1057-1109 of SEQ ID NO: 4 is selected from ISIS NO. 400319, 400320, 400321, or 400322.
In certain embodiments, a target region is nucleotides 1334-1349 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1334-1349 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 357, 358, or 359. In certain such embodiments, a short antisense compound targeted to nucleotides 1334-1349 of SEQ ID NO: 4 is selected from ISIS NO 400325, 400326, or 400327.
In certain embodiments, a target region is nucleotides 1453-1469 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1453-1469 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 360, 361, 362, or 363. In certain such embodiments, a short antisense compound targeted to nucleotides 1453-1469 of SEQ ID NO: 4 is selected from ISIS NO 400328, 400329, 400330, 400331, or 403470.
In certain embodiments, a target region is nucleotides 1569-1591 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1569-1591 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 364, 365, 366, 367, 368, 369, 370, 371, 372, or 373. In certain such embodiments, a short antisense compound targeted to nucleotides 1569-1591 of SEQ ID NO: 4 is selected from ISIS NO 400332, 400333, 400334, 400335, 400336, 400337, 400338, 400339, 400340, or 400341.
In certain embodiments, a target region is nucleotides 1621-1637 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1621-1637 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 374, 375, 376, or 377. In certain such embodiments, a short antisense compound targeted to nucleotides 1621-1637 of SEQ ID NO: 4 is selected from ISIS NO 400342, 400343, 400344, or 400345.
In certain embodiments, a target region is nucleotides 1738-1754 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1738-1754 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 378, 379, 380, or 381. In certain such embodiments, a short antisense compound targeted to nucleotides 1738-1754 of SEQ ID NO: 4 is selected from ISIS NO 400346, 400347, 400348, or 400349.
In certain embodiments, a target region is nucleotides 1834-1853 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 1834-1853 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 382, 383, 384, 385, 386, 387, or 388. In certain embodiments, a short antisense compound targeted to nucleotides 1834-1853 of SEQ ID NO: 4 is selected from ISIS NO 400350, 400351, 400352, 400353, 400354, 400355, or 400356.
In certain embodiments, a target region is nucleotides 2083-2099 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 2083-2099 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 389, 390, 391, or 392. In certain such embodiments, a short antisense compound targeted to nucleotides 2083-2099 of SEQ ID NO: 4 is selected from ISIS NO 400357, 400358, 400359, or 400360.
In certain embodiments, a target region is nucleotides 2316-2338 of SEQ ID NO: 4. In certain such embodiments, a short antisense compound targeted to nucleotides 2316-2338 of SEQ ID NO: 4 comprises a nucleotide sequence selected from SEQ ID NO 393, 394, 395, 396, 397, 398, 399, 400, 401, or 402. In certain such embodiments, a short antisense compound targeted to nucleotides 2316-2338 of SEQ ID NO: 4 is selected from ISIS NO 400361, 400362, 400363, 400364, 400365, 400366, 400367, 400368, 400369, or 400370.
In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a PCSK9 nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeting a PCSK9 nucleic acid may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a PCSK9 nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.
In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a PCSK9 nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of PCSK9. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a PCSK9 nucleic acid, wherein the short antisense compound targeted to a PCSK9 nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of PCSK9 using one or more short antisense compounds targeted to a PCSK9 nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of PCSK9 comprise use of a short antisense compound targeted to a PCSK9 nucleic acid comprising 12 or 14 nucleotides or nucleosides.
4. Superoxide Dismutase 1 Enzyme (SOD1) The enzymes known as the superoxide dismutases (SODs) provide defense against oxidative damage of biomolecules by catalyzing the dismutation of superoxide to hydrogen peroxide (H2O2) (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112). Two major classes of superoxide dismutases exist. One consists of a group of enzymes with active sites containing copper and zinc while the other class has either manganese or iron at the active site (Fridovich, Annu. Rev. Biochem., 1995, 64, 97-112).
Mutations in the superoxide dismutase 1 gene are associated with a dominantly-inherited form of amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) a disorder characterized by a selective degeneration of upper and lower motor neurons (Cleveland and Liu, Nat. Med., 2000, 6, 1320-1321). The deleterious effects of various mutations on superoxide dismutase 1 are most likely mediated through a gain of toxic function rather than a loss of superoxide dismutase 1 activity, as the complete absence of superoxide dismutase 1 in mice neither diminishes life nor provokes overt disease (Al-Chalabi and Leigh, Curr. Opin. Neurol., 2000, 13, 397-405; Alisky and Davidson, Hum. Gene Ther., 2000, 11, 2315-2329).
Over 100 mutations of the human SOD1 gene have been identified, and altogether account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. Some mutations, such as the A4V mutation most commonly found in the United States, are highly lethal and result in survival only nine months from the onset of disease symptoms. Other mutations of SOD1 manifest in a slower disease course.
Definitions
“SOD1” means the gene product or protein of which expression is to be modulated by administration of a short antisense compound.
“SOD1 nucleic acid” means any nucleic acid encoding SOD1. For example, in certain embodiments, a SOD1 nucleic acid includes, without limitations, a DNA sequence encoding SOD1, an RNA sequence transcribed from DNA encoding SOD1, and an mRNA sequence encoding SOD1.
“SOD1 mRNA” means an mRNA encoding SOD1.
SOD1 Therapeutic Indications
It has been discovered that antisense inhibition of superoxide dismutase 1 (SOD1) in an animal model of familial ALS reduces both SOD1 mRNA and protein, and further results in a slowing of disease progression and, importantly, increased survival time. Accordingly, in certain embodiments, the invention provides methods for the slowing of disease progression in an individual suffering from familial ALS by administering to such an individual a short antisense compound targeted to an SOD1 nucleic acid. In certain such embodiments, a short antisense compound targeted to SOD1 are delivered directly to the cerebrospinal fluid of the individual. In certain such embodiments, methods further comprise increasing survival time of an individual suffering from familial ALS. Slowing of disease progression is indicated by an improvement in one or more indicators of ALS disease progression, including, without limitation, the revised ALS functional rating scale, pulmonary function tests, and muscle strength measurements.
SOD1 Combination Therapies
In certain embodiments, one or more pharmaceutical compositions comprising a short antisense compound targeted to an SOD1 nucleic acid is co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to SOD1 include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.
Certain Short Antisense Compounds Targeted to a SOD1 Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a SOD1 nucleic acid having the sequence of GENBANK® Accession No. NM_X02317.1, incorporated herein as SEQ ID NO: 5. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 5 is at least 90% complementary to SEQ ID NO: 5. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 5 is at least 95% complementary to SEQ ID NO: 5. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 5 is 100% complementary to SEQ ID NO: 5. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 5 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 8 or Table 9.
The nucleotide sequence set forth in each SEQ ID NO in Tables 8 and 9 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Short antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Table 8 illustrates examples of short antisense compounds targeted to SEQ ID NO: 5. Table 8 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 5. The column labeled ‘gapmer motif indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2’-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.
In certain embodiments, short antisense compounds targeting a SOD1 nucleic acid may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a SOD1 nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.







TABLE 8







Short Antisense Compounds targeted to SEQ ID NO: 5













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO















387541
85
100
GTCGCCCTTCAGCACG
3-10-3 MOE
406


 


387540
86
99
TCGCCCTTCAGCAC
2-10-2 MOE
407


 


387539
87
98
CGCCCTTCAGCA
1-10-1 MOE
408









In certain embodiments, a target region is nucleotides 85-100 of SEQ ID NO: 5. In certain such embodiments, short antisense compounds targeted to nucleotides 85-100 of SEQ ID NO: 5 comprise a nucleotide sequence selected from SEQ ID NO: 406, 407, or 408. In certain such embodiments, a short antisense compound targeted to nucleotides 85-100 of SEQ ID NO: 5 is selected from Isis No. 387541, 387540, or 387539.
In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a SOD1 nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a SOD1 nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of SOD1. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a SOD1 nucleic acid, wherein the short antisense compound targeted to a SOD1 nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of SOD1 using one or more short antisense compounds targeted to a SOD1 nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of SOD1 comprise use of a short antisense compound targeted to a SOD1 nucleic acid comprising 12 or 14 nucleotides or nucleosides.
5. CRP
CRP (also known as C-reactive protein and PTX1) is an essential human acute-phase reactant produced in the liver in response to a variety of inflammatory cytokines. The protein, first identified in 1930, is highly conserved and considered to be an early indicator of infectious or inflammatory conditions. Plasma CRP levels increase 1,000-fold in response to infection, ischemia, trauma, burns, and inflammatory conditions. In clinical trials where patients receive lipid-lowering therapy, such as statin therapy, it has been demonstrated that patients having reductions in both LDL-C and CRP have a reduced risk of future coronary events relative to patients experiencing only reductions in LDL-C.
Definitions
“CRP” means the gene product or protein of which expression is to be modulated by a short antisense compound.
“CRP nucleic acid” means any nucleic acid encoding CRP. For example, in certain embodiments, a CRP nucleic acid includes, without limitations, a DNA sequence encoding CRP, an RNA sequence transcribed from DNA encoding CRP, and an mRNA sequence encoding CRP.
“CRP mRNA” means an mRNA encoding CRP.
CRP Therapeutic Indications
In certain embodiments, the invention provides methods of modulating CRP expression in an individual comprising administering to the individual a short antisense compound targeted to a CRP nucleic acid. In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions comprising a short antisense compound targeted to a CRP nucleic acid. In certain embodiments, the individual has hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, one or more risk factors for coronary heart disease. In certain embodiments, the individual has acute coronary syndrome, vascular injury, arterial occlusion, unstable angina, post peripheral vascular disease, post myocardial infarction (MI), thrombosis, deep vein thrombus, end-stage renal disease (ESRD), chronic renal failure, complement activation, congestive heart failure, or systemic vasculitis. In certain embodiments, the individual has had a stroke.
In certain embodiments, the individual has undergone a procedure selected from elective stent placement, angioplasty, post percutaneous transluminal angioplasty (PTCA), cardiac transplantation, renal dialysis or cardiopulmonary bypass.
In certain embodiments, the individual has an inflammatory disease. In certain such embodiments, the inflammatory disease is selected from inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, or osteoarthritis.
Guidelines for lipid-lowering therapy were established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). The guidelines include obtaining a complete lipoprotein profile, typically after a 9 to 12 hour fast, for determination of LDL-C, total cholesterol, and HDL-C levels. According to the most recently established guidelines, LDL-C levels of 130-159 mg/dL, 160-189 mg/dL, and greater than or equal to 190 mg/dL are considered borderline high, high, and very high, respectively. Total cholesterol levels of 200-239 and greater than or equal to 240 mg/dL are considered borderline high and high, respectively. HDL-C levels of less than 40 mg/dL are considered low.
In certain embodiments, the individual has been identified as in need of lipid-lowering therapy. In certain such embodiments, the individual has been identified as in need of lipid-lowering therapy according to the guidelines established in 2001 by Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), and updated in 2004 (Grundy et al., Circulation, 2004, 110, 227-239). In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 190 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 160 mg/dL. In certain such embodiments, the individual in need of lipid-lowering therapy has LDL-C above 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy has LDL-C above 100 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 160 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 130 mg/dL. In certain such embodiments the individual in need of lipid-lowering therapy should maintain LDL-C below 100 mg/dL. In certain such embodiments the individual should maintain LDL-C below 70 mg/dL.
In certain embodiments the invention provides methods for reducing CRP in an individual. In certain such embodiments, the reduction in CRP is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 100%.
In certain embodiments, the methods provided by the present invention do not lower HDL-C. In certain embodiments, the methods provided by the present invention do not result in accumulation of lipids in the liver. In certain embodiments, the methods provided by the present invention do not cause hepatic steatosis.
In certain embodiments, the invention provides methods for lowering CRP concentration in a subject while reducing side effects associated with treatment. In certain such embodiments, a side effect is liver toxicity. In certain such embodiments, a side effect is abnormal liver function. In certain such embodiments, a side effect is elevated alanine aminotransferase (ALT). In certain such embodiments, a side effect is elevated aspartate aminotransferase (AST).
In certain embodiments, the invention provides methods for lowering CRP concentration in a subject who is not reaching target LDL-C levels as a result of lipid-lowering therapy. In certain such embodiments, a short antisense compound targeted to a CRP nucleic acid is the only pharmaceutical agent administered to the subject. In certain such embodiments, the subject has not complied with recommended lipid-lowering therapy. In certain such embodiments, a pharmaceutical composition of the invention is co-administered with an additional different lipid-lowering therapy. In certain such embodiments, an additional lipid-lowering therapy is LDL-apheresis. In certain such embodiments, an additional lipid-lowering therapy is a statin. In certain such embodiments, an additional lipid-lowering therapy is ezetimibe.
In certain embodiments, the invention provides methods for lowering CRP concentration in a statin-intolerant subject. In certain such embodiments, the subject has creatine kinase concentration increases as a result of statin administration. In certain such embodiments, the subject has liver function abnormalities as a result of statin administration. In certain such embodiments the subject has muscle aches as a result of statin administration. In certain such embodiments the subject has central nervous system side effects as a result of statin administration. In certain embodiments, the subject has not complied with recommended statin administration.
In certain embodiments, the invention provides methods for reducing coronary heart disease risk in a subject. In certain embodiments the invention provides methods for slowing the progression of atherosclerosis in a subject. In certain such embodiments the invention provides methods for stopping the progression of atherosclerosis in a subject. In certain such embodiments the invention provides methods for reducing the size and/or prevalence of atherosclerotic plaques in a subject. In certain embodiments the methods provided reduce a subject's risk of developing atherosclerosis.
In certain embodiments the methods provided improve the cardiovascular outcome in a subject. In certain such embodiments improved cardiovascular outcome is the reduction of the risk of developing coronary heart disease. In certain such embodiments, improved cardiovascular outcome is a reduction in the occurrence of one or more major cardiovascular events, which include, but are not limited to, death, myocardial infarction, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia. In certain such embodiments, the improved cardiovascular outcome is evidenced by improved carotid intimal media thickness. In certain such embodiments, improved carotid intimal media thickness is a decrease in thickness. In certain such embodiments, improved carotid intimal media thickness is a prevention an increase of intimal media thickness.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid is for use in therapy. In certain embodiments, the therapy is the reduction of CRP in an individual. In certain embodiments, the therapy is the treatment of hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, or early onset coronary heart disease. In additional embodiments, the therapy is the reduction of CHD risk. In certain the therapy is prevention of atherosclerosis. In certain embodiments, the therapy is the prevention of coronary heart disease. In certain embodiments, the therapy is the treatment of acute coronary syndrome, chronic renal failure, vascular injury, arterial occlusion, atherothrombosis, unstable angina, post peripheral vascular disease, post myocardial infarction (MI), thrombosis, deep vein thrombus, end-stage renal disease (ESRD), complement activation, congestive heart failure, or systemic vasculitis. In certain embodiments the therapy is the treatment of an individual who has undergone a procedure selected from elective stent placement, angioplasty, post percutaneous transluminal angioplasty (PTCA), cardiac transplantation, renal dialysis or cardiopulmonary bypass. In certain embodiments, the therapy is the treatment of an inflammatory disorder.
In certain embodiments a pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for reducing CRP in an individual. In certain embodiments pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for reducing coronary heart disease risk. In certain embodiments a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment of hypercholesterolemia, non-familial hypercholesterolemia, familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk of developing atherosclerosis, coronary heart disease, a history of coronary heart disease, early onset coronary heart disease, or one or more risk factors for coronary heart disease.
In certain embodiments, a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment of acute coronary syndrome, chronic renal failure, vascular injury, arterial occlusion, atherothrombosis, unstable angina, post peripheral vascular disease, post myocardial infarction (MI), thrombosis, deep vein thrombus, end-stage renal disease (ESRD), complement activation, congestive heart failure, or systemic vasculitis. In certain embodiments, a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment of an individual who has had a stroke.
In certain embodiments, a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment in an individual who has undergone a procedure selected from elective stent placement, angioplasty, post percutaneous transluminal angioplasty (PTCA), cardiac transplantation, renal dialysis or cardiopulmonary bypass.
In certain embodiments, a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment of an inflammatory disease. In certain such embodiments, a short antisense compound targeted to a CRP nucleic acid is used for the preparation of a medicament for the treatment of inflammatory bowel disease, ulcerative colitis, rheumatoid arthritis, or osteoarthritis.
CRP Combination Therapies
In certain embodiments, one or more pharmaceutical compositions comprising a short antisense compound targeted to a CRP nucleic acid are co-administered with one or more other pharmaceutical agents. In certain embodiments, the one or more other pharmaceutical agents is a lipid-lowering agent. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid include lipid-lowering agents. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.
In certain embodiments, a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments the statin is selected from atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.
In certain embodiments, a co-administered lipid-lowering agent is ISIS 301012.
In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe.
In certain embodiments, a co-administered lipid-lowering agent is a co-formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe/simvastatin.
In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).
In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.
In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.
In certain embodiments, a pharmaceutical composition comprising a short antisense compound targeted to a CRP nucleic acid may be administered in conjunction with a lipid-lowering therapy. In certain such embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain such embodiments, a lipid-lowering therapy is LDL apheresis.
Certain Short Antisense Compounds Targeted to a CRP Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a CRP nucleic acid having the sequence of GENBANK® Accession No. NM_000567.1, incorporated herein as SEQ ID NO: 6. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 6 is at least 90% complementary to SEQ ID NO: 6. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 6 is at least 95% complementary to SEQ ID NO: 6. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 6 is 100% complementary to SEQ ID NO: 6. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 6 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 9.
The nucleotide sequence set forth in each SEQ ID NO in Table 9 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Short antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Table 9 illustrates examples of short antisense compounds targeted to SEQ ID NO: 6. Table 9 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 6. The column labeled ‘gapmer motif indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2’-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.
In certain embodiments, short antisense compounds targeting a CRP nucleic acid may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a CRP nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.







TABLE 9







Short Antisense Compounds targeted to SEQ ID NO: 6













5′
3′


Seq


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO





353506
1257
1272
ACTCTGGACCCAAACC
3-10-3 MOE
409


 


353507
1258
1271
CTCTGGACCCAAAC
2-10-2 MOE
410


 


353484
1305
1320
CCATTTCAGGAGACCT
3-10-3 MOE
411


 


353485
1306
1319
CATTTCAGGAGACC
2-10-2 MOE
412









In certain embodiments, a target region is nucleotides 1305-1320 of NM_000567.1. In certain such embodiments, short antisense compounds targeted to nucleotides 1305-1320 of NM_000567.1 comprise a nucleotide sequence selected from SEQ ID NO: 1305 or 1306. In certain such embodiments, a short antisense compound targeted to nucleotides 263-278 of NM_000567.1 is selected from Isis NO. 353484 or 353485.
In certain embodiments, a target region is nucleotides 1257-1272 of NM_000567.1. In certain such embodiments, a short antisense compound targeted to nucleotides 1257-1272 of NM_000567.1 comprises a nucleotide sequence selected from SEQ ID NO 1257 or 1258. In certain such embodiments, a short antisense compound targeted to nucleotides 428-483 of NM_000567.1 is selected from Isis NO. 353506 or 353507.
In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a CRP nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a CRP nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a CRP nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of CRP. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a CRP nucleic acid, wherein the short antisense compound targeted to a CRP nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of CRP using one or more short antisense compounds targeted to a CRP nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating CRP comprise use of a short antisense compound targeted to a CRP nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of CRP comprise use of a short antisense compound targeted to a CRP nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of CRP comprise use of a short antisense compound targeted to a CRP nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of CRP comprise use of a short antisense compound targeted to a CRP nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of CRP comprise use of a short antisense compound targeted to a CRP nucleic acid comprising 12 or 14 nucleotides or nucleosides.
6. Glucocorticoid Receptor (GCCR)
Glucocorticoids were among the first steroid hormones to be identified and are responsible for a multitude of physiological functions, including the stimulation of gluconeogenesis, decreased glucose uptake and utilization in peripheral tissues, increased glycogen deposition, suppression of immune and inflammatory responses, inhibition of cytokine synthesis and acceleration of various developmental events. Glucocorticoids are also especially important for combating stress. Stress-induced elevation of glucocorticoid synthesis and release leads to, among other responses, increased ventricular workload, inhibition of inflammatory mediators, inhibition of cytokine synthesis and increased glucose production (Karin, Cell, 1998, 93, 487-490).
Both natural glucocorticoids and their synthetic derivatives exert their action through the glucocorticoid receptor, a ubiquitously expressed cytoplasmic member of the nuclear hormone superfamily of receptors. Human glucocorticoid receptor is also known as nuclear receptor subfamily 3, group C, member 1; NR3C1; GCCR; GCR; GRL; Glucocorticoid receptor, lymphocyte. The gene is located on human chromosome 5q11-q13 and consists of 9 exons (Encio and Detera-Wadleigh, J Biol Chem, 1991, 266, 7182-7188; Gehring et al., Proc Natl Acad Sci USA, 1985, 82, 3751-3755). Multiple forms of human glucocorticoid receptor mRNA exist: a 5.5 kb human glucocorticoid receptor α cDNA containing exons 1-8 and exon 9α; a 4.3 kb human glucocorticoid receptor β cDNA containing exons 1-8 and exon 9β; and a 7.0 kb human glucocorticoid receptor α cDNA containing exons 1-8 and the entire exon 9, which includes exon 9α, exon 9β and the ‘J region’, which is flanked by exons 9α and 9β (Hollenberg et al., Nature, 1985, 318, 635-641; Oakley et al., J Biol Chem, 1996, 271, 9550-9559). Human glucocorticoid receptor α is the predominant isoform of the receptor and the one that exhibits steroid binding activity (Hollenberg et al., Nature, 1985, 318, 635-641). Additionally, through usage of three different promoters three different exon 1 variants can be transcribed, and alternative splicing of one exon 1 variant can result in three different versions of this exon. Thus, human glucocorticoid receptor mRNA may contain 5 different versions of exon 1 (Breslin et al., Mol Endocrinol, 2001, 15, 1381-1395).
Examination of the expression patterns of the α and β isoforms of human glucocorticoid receptor mRNA reveals that the α isoform is more abundantly expressed. Both isoforms are expressed in similar tissues and cell types, including lung, kidney, heart, liver, skeletal muscle, macrophages, neutrophils and peripheral blood mononuclear cells. Only human glucocorticoid receptor α is expressed in colon. At the level of protein, while the α isoform is detected in all tissues examined, the β isoform is undetectable, suggesting that under physiological conditions, the default splicing pathway is the one that produces the α isoform (Pujols et al., Am J Physiol Cell Physiol, 2002, 283, C1324-1331). The β isoform of glucocorticoid receptor binds neither a glucocorticoid agonist nor an antagonist. Furthermore, the β isoform is localized primarily in the nucleus in transfected cells, independent of hormone stimulation. When both isoforms are expressed in the same cell, the glucocorticoid receptor β inhibits the hormone-induced, glucocorticoid receptor α-mediated stimulation of gene expression, suggesting that the β isoform functions as an inhibitor of glucocorticoid receptor α activity (Oakley et al., J Biol Chem, 1996, 271, 9550-9559). Unless otherwise noted, the human glucocorticoid receptor described herein is defined as the ubiquitous product(s) of the gene located on chromosome 5q11-q13.
Cell lines transfected with a complementary glucocorticoid receptor antisense RNA strand exhibited a reduction in glucocorticoid receptor mRNA levels and a decreased response to the glucocorticoid receptor agonist dexamethasone (Pepin and Barden, Mol Cell Biol, 1991, 11, 1647-1653). Transgenic mice bearing an antisense glucocorticoid receptor gene construct were used to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis (Pepin et al., Nature, 1992, 355, 725-728). In another study of similarly genetically engineered mice, energy intake and expenditure, heart and vastus lateralis muscle lipoprotein lipase activity, and heart and brown adipose tissue norepinephrine were lower than in control animals. Conversely, fat content and total body energy were significantly higher than in control animals. These results suggest that a defective glucocorticoid receptor system may affect energy balance through increasing energetic efficiency, and they emphasize the modulatory effects of hypothalamic-pituitary-adrenal axis changes on muscle lipoprotein lipase activity (Richard et al., Am J Physiol, 1993, 265, R146-150).
Behavioral effects of glucocorticoid receptor antagonists have been measured in animal models designed to assess anxiety, learning and memory. Reduced expression of glucocorticoid receptor in rats long-term intracerebroventricularly infused with antisense oligodeoxynucleotides targeting glucocorticoid receptor mRNA did not interfere with spatial navigation in the Morris water maze test (Engelmann et al., Eur J Pharmacol, 1998, 361, 17-26). Bilateral infusion of an antisense oligodeoxynucleotide targeting the glucocorticoid receptor mRNA into the dentate gyms of the rat hippocampus reduced the immobility of rats in the Porsolt forced swim test (Korte et al., Eur J Pharmacol, 1996, 301, 19-25).
Glucocorticoids are frequently used for their immunosuppressive, anti-inflammatory effects in the treatment of diseases such as allergies, asthma, rheumatoid arthritis, AIDS, systemic lupus erythematosus and degenerative osteoarthritis. Negative regulation of gene expression, such as that caused by the interaction of glucocorticoid receptor with NF-kB, is proposed to be at least partly responsible for the anti-inflammatory action of glucocorticoids in vivo. Interleukin-6, tumor necrosis factor α and interleukin-1 are the three cytokines that account for most of the hypothalamic-pituitary-adrenal (HPA) axis stimulation during the stress of inflammation. The HPA axis and the systemic sympathetic and adrenomedullary system are the peripheral components of the stress system, responsible for maintaining basal and stress-related homeostasis. Glucocorticoids, the end products of the HPA axis, inhibit the production of all three inflammatory cytokines and also inhibit their effects on target tissues, with the exception of interleukin-6, which acts synergistically with glucocorticoids to stimulate the production of acute-phase reactants. Glucocorticoid treatment decreases the activity of the HPA axis (Chrousos, N Engl J Med, 1995, 332, 1351-1362).
In some cases, patients are refractory to glucocorticoid treatment. One reason for this resistance to steroids lies with mutations or polymorphisms present in the glucocorticoid receptor gene. A total of 15 missense, three nonsense, three frameshift, one splice site, and two alternative spliced mutations, as well as 16 polymorphisms, have been reported in the NR3C1 gene in association with glucocorticoid resistance (Bray and Cotton, Hum Mutat, 2003, 21, 557-568). Additional studies in humans have suggested a positive association between metabolic syndrome incidence and progression, with alleles at the glucocorticoid receptor (GR) gene (Rosmond, Obes Res, 2002, 10, 1078-1086).
Other cases of glucocorticoid insensitivity are associated with altered expression of glucocorticoid receptor isoforms. A study of human glucocorticoid receptor β isoform mRNA expression in glucocorticoid-resistant ulcerative colitis patients revealed the presence of this mRNA was significantly higher than in the glucocorticoid-sensitive patients, suggesting that the expression of human glucocorticoid receptor 13 mRNA in the peripheral blood mononuclear cells may serve as a predictor of glucocorticoid response in ulcerative colitis (Honda et al., Gastroenterology, 2000, 118, 859-866). Increased expression of glucocorticoid receptor β is also observed in a significantly high number of glucocorticoid-insensitive asthmatics. Additionally, cytokine-induced abnormalities in the DNA binding capacity of the glucocorticoid receptor were found in peripheral blood mononuclear cells from glucocorticoid-insensitive patients transfection, and HepG2 cells with the glucocorticoid receptor β gene resulted in a significant reduction of glucocorticoid receptor α DNA-binding capacity (Leung et al., J Exp Med, 1997, 186, 1567-1574). Dexamethasone binding studies demonstrate that human glucocorticoid receptor 13 does not alter the affinity of glucocorticoid receptor α for hormonal ligands, but rather its ability to bind to the GRE (Bamberger et al., J Clin Invest, 1995, 95, 2435-2441). Taken together, these results illustrate that glucocorticoid receptor β, through competition with glucocorticoid receptor α for GRE target sites, may function as a physiologically and pathophysiologically relevant endogenous inhibitor of glucocorticoid action.
In the liver, glucocorticoid agonists increase hepatic glucose production by activating the glucocorticoid receptor, which subsequently leads to increased expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase. Through gluconeogenesis, glucose is formed through non-hexose precursors, such as lactate, pyruvate and alanine (Link, Curr Opin Investig Drugs, 2003, 4, 421-429). Steroidal glucocorticoid receptor antagonists such as RU 486 have been tested in rodent models of diabetes. Mice deficient in the leptin receptor gene, termed db/db mice, are genetically obese, diabetic and hyperinsulinemic. Treatment of hyperglycemic db/db mice with RU 486 decreased blood glucose levels by approximately 49%, without affecting plasma insulin levels. Additionally, RU 486 treatment reduced the expression of glucocorticoid receptor responsive genes PEPCK, glucose-6-phosphatase, glucose transporter type 2 and tyrosine aminotransferase in db/db mice as compared to untreated animals (Friedman et al., J Biol Chem, 1997, 272, 31475-31481). RU 486 also ameliorates diabetes in the ob/ob mouse model of diabetes, obesity and hyperinsulinemia, through a reduction in serum insulin and blood glucose levels (Gettys et al., Int J Obes Relat Metab Disord, 1997, 21, 865-873).
As increased gluconeogenesis is considered to be the major source of increased glucose production in diabetes, a number of therapeutic targets for the inhibition of hepatic glucose production have been investigated. Due to the ability of antagonists of the glucocorticoid receptor to ameliorate diabetes in animal models, such compounds are among the potential therapies being explored. However, there are detrimental systemic effects of glucocorticoid receptor antagonists, including activation of the HPA axis (Link, Curr Opin Investig Drugs, 2003, 4, 421-429). Increased HPA axis activity is associated with suppression of immune-related inflammatory action, which can increase susceptibility to infectious agents and neoplasms. Conditions associated with suppression of immune-mediated inflammation through defects in the HPA axis, or its target tissues, include Cushing's syndrome, chronic stress, chronic alcoholism and melancholic depression (Chrousos, N Engl J Med, 1995, 332, 1351-1362). Thus, it is of great value to develop liver-specific glucocorticoid receptor antagonists. Steroidal glucocorticoid receptor antagonists have been conjugated to bile acids for the purpose of targeting them to the liver (Apelqvist et al., 2000). Currently, there are no known therapeutic agents that target the glucocorticoid receptor without undesired peripheral effects (Link, Curr Opin Investig Drugs, 2003, 4, 421-429). Consequently, there remains a long felt need for agents capable of effectively inhibiting hepatic glucocorticoid receptor.
Definitions
“Glucocorticoid receptor” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound. Glucocorticoid receptor is generally referred to as GCCR.
“GCCR nucleic acid” means any nucleic acid encoding GCCR. For example, in certain embodiments, a GCCR nucleic acid includes, without limitation, a DNA sequence encoding GCCR, an RNA sequence transcribed from DNA encoding GCCR, and an mRNA sequence encoding GCCR. “GCCR mRNA” means an mRNA encoding GCCR.
Therapeutic Indications
Antisense technology is an effective means of reducing the expression of specific gene products and therefore is useful in a number of therapeutic, diagnostic and research applications for the modulation of glucocorticoid receptor expression. Furthermore, in certain embodiments, liver is one of the tissues in which the highest concentrations of antisense oligonucleotides are found following administration (Geary et al., Curr. Opin. Investig. Drugs, 2001, 2, 562-573). Therefore, in such embodiments, antisense technology represents an attractive method for the liver-specific inhibition of glucocorticoid receptor.
In certain embodiments, short antisense compounds targeted to a nucleic acid encoding glucocorticoid receptor are preferentially distributed to the liver. In certain embodiments, short antisense compounds have increased potency in the liver when compared to a longer parent compound. In certain embodiments, target RNA is predominantly expressed in the liver.
For therapeutics, a subject, suspected of having a disease or disorder which can be treated by modulating the expression of GCCR is treated by administering one or more short antisense compound. In a non-limiting example, the methods comprise the step of administering to an animal a therapeutically effective amount of a short antisense compound. Certain short antisense compounds inhibit the activity of GCCR and/or inhibit expression of GCCR. In certain embodiments, the activity or expression of GCCR in a subject is inhibited by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%. In certain embodiments, the activity or expression of GCCR in a subject is inhibited by at least 30%. In certain embodiments, the activity or expression of GCCR in a subject is inhibited by at least 50% or more.
The reduction of the expression of GCCR may be measured, for example, in blood, plasma, serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. In certain embodiments, cells contained within such fluids, tissues or organs being analyzed comprise nucleic acids encoding GCCR and/or they contain the GCCR protein itself.
Certain pharmaceutical and other compositions comprising short antisense compounds are also provided. In certain embodiments, short antisense compounds are be utilized in pharmaceutical compositions by adding to them an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
In certain embodiments, short antisense compounds targeting a GCCR nucleic acid have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a GCCR nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. In certain embodiments, short antisense compounds targeting a GCCR nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-10-1, 2-10-2, 3-10-3, and 1-9-2. In certain embodiments, short antisense compounds targeting a GCCR nucleic acid have a motif (wing-deoxy gap-wing) selected from 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 2-10-2 and 2-8-2.
In certain embodiments, provided herein are methods of treating an individual by administering one or more short antisense compound targeted to a GCCR nucleic acid or a pharmaceutical composition comprising such compound. Further provided are methods of treating a subject having a disease or conditions associated with GCCR activity by administering a short antisense compound targeted to a GCCR nucleic acid. In addition to diabetes, particularly type 2 diabetes, diseases and conditions associated with GCCR include but are not limited to, obesity, Metabolic syndrome X, Cushing's Syndrome, Addison's disease, inflammatory diseases such as asthma, rhinitis and arthritis, allergy, autoimmune disease, immunodeficiency, anorexia, cachexia, bone loss or bone frailty, and wound healing. Metabolic syndrome, metabolic syndrome X or simply Syndrome X refers to a cluster of risk factors that include obesity, dyslipidemia, particularly high blood triglycerides, glucose intolerance, high blood sugar and high blood pressure. In certain embodiments, short antisense compounds targeted to GCCR are used for amelioration of hyperglycemia induced by systemic steroid therapy. Moreover, antisense technology provides a means of inhibiting the expression of the glucocorticoid receptor β isoform, demonstrated to be overexpressed in patients refractory to glucocorticoid treatment.
In certain embodiments, the invention provides short antisense compounds targeted to a nucleic acid encoding GCGR, and which modulate the expression of glucocorticoid receptor. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of glucocorticoid receptor and methods of modulating the expression of glucocorticoid receptor in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of glucocorticoid receptor are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.
Certain Short Antisense Compounds Targeted to a GCCR Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a GCCR nucleic acid having the sequence of nucleotides 1 to 106000 of GENBANK® Accession No. AC012634, incorporated herein as SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 8 is at least 90% complementary to SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 8 is at least 95% complementary to SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 8 is 100% complementary to SEQ ID NO: 8. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 8 includes a nucleotide sequence selected from the nucleotide sequences set forth in Tables 10 and 11.
The nucleotide sequence set forth in each SEQ ID NO in Tables 10 and 11 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Short antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise a gapmer motif. In certain embodiments, a short antisense compound targeted to a GCCR nucleic acid comprises a 2-10-2 gapmer motif.
Tables 10 and 11 illustrate examples of short antisense compounds targeted to SEQ ID NO: 8. Table 10 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 8. Table 11 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 8. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 10







Short Antisense Compounds targeted to SEQ ID NO: 8













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO





371644
88142
88155
TTTGGGAGGTGGTC
2-10-2 MOE
413


 


371645
88156
88169
CACACCAGGCAGAG
2-10-2 MOE
414


 


371649
88212
88225
CTTTACAGCTTCCA
2-10-2 MOE
415


 


371651
88242
88255
CACTACCTTCCACT
2-10-2 MOE
416


 


371652
88248
88261
AACACACACTACCT
2-10-2 MOE
417


 


371653
88256
88269
CTCTTCAAAACACA
2-10-2 MOE
418


 


371665
92037
92050
GTAATTGTGCTGTC
2-10-2 MOE
419


 


371669
92086
92099
TTTTTCTTCGAATT
2-10-2 MOE
420


 


371671
92114
92127
CATTTTCGATAGCG
2-10-2 MOE
421


 


371673
92142
92155
ACCTTCCAGGTTCA
2-10-2 MOE
422
















TABLE 11







Short antisense compounds targeted to


SEQ ID NO: 8 and having 1 or 2 mismatches













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO
Site
Site
Sequence (5′-3′)
Motif
NO















371638
2039
2052
ATAGGAAGCATAAA
2-10-2 MOE
423


 


371650
4949
4962
TCTTTTAAAGAAGA
2-10-2 MOE
424


 


371673
10187
10200
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371660
13465
13478
AAGGATATTTTAAA
2-10-2 MOE
425


 


371660
14428
14441
AAGGATATTTTAAA
2-10-2 MOE
425


 


371654
15486
15499
GAACAAAAATTAAA
2-10-2 MOE
427


 


371661
16638
16651
TTCCACAGATCTGT
2-10-2 MOE
428


 


371653
17892
17905
CTCTTCAAAACACA
2-10-2 MOE
418


 


371679
18444
18457
TTTATAAAGTAAAG
2-10-2 MOE
429


 


371645
19816
19829
CACACCAGGCAGAG
2-10-2 MOE
414


 


371638
21555
21568
ATAGGAAGCATAAA
2-10-2 MOE
423


 


371650
21775
21788
TCTTTTAAAGAAGA
2-10-2 MOE
424


 


371679
21902
21915
TTTATAAAGTAAAG
2-10-2 MOE
429


 


371655
22507
22520
TACTGTGAGAAATA
2-10-2 MOE
433


 


371655
22722
22735
TACTGTGAGAAATA
2-10-2 MOE
433


 


371672
25662
25675
TTCCAGCTTGAAGA
2-10-2 MOE
435


 


371678
25926
25939
GATCAGTTCTCATG
2-10-2 MOE
436


 


371655
26041
26054
TACTGTGAGAAATA
2-10-2 MOE
433


 


371638
29770
29783
ATAGGAAGCATAAA
2-10-2 MOE
423


 


371668
30551
30564
TTATCAATGATGCA
2-10-2 MOE
439


 


371670
40584
40597
GCATGCTGGACAGT
2-10-2 MOE
440


 


371654
43331
43344
GAACAAAAATTAAA
2-10-2 MOE
427


 


371650
46024
46037
TCTTTTAAAGAAGA
2-10-2 MOE
424


 


371659
50372
50385
TTGCACCTGAACTA
2-10-2 MOE
443


 


371634
50565
50578
CAGAATATATTTCT
2-10-2 MOE
444


 


371673
56942
56955
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371654
62372
62385
GAACAAAAATTAAA
2-10-2 MOE
427


 


371679
63537
63550
TTTATAAAGTAAAG
2-10-2 MOE
429


 


371654
64908
64921
GAACAAAAATTAAA
2-10-2 MOE
427


 


371661
65795
65808
TTCCACAGATCTGT
2-10-2 MOE
428


 


371645
70997
71010
CACACCAGGCAGAG
2-10-2 MOE
414


 


371661
77400
77413
TTCCACAGATCTGT
2-10-2 MOE
428


 


371663
82329
82342
ATAAGAGATTAAAA
2-10-2 MOE
450


 


371633
83426
83439
TCCCCCTTCTCATT
2-10-2 MOE
451


 


371662
85873
85886
GGGCATTGTTAAAA
2-10-2 MOE
452


 


371654
86476
86489
GAACAAAAATTAAA
2-10-2 MOE
427


 


371679
86516
86529
TTTATAAAGTAAAG
2-10-2 MOE
429


 


371641
88097
88110
AGAACTCACATCTG
2-10-2 MOE
455


 


371642
88111
88124
GAGCTGGACGGAGG
2-10-2 MOE
456


 


371646
88170
88183
AAGCTTCATCGGAG
2-10-2 MOE
457


 


371647
88184
88197
ATAATGGCATCCCG
2-10-2 MOE
458


 


371650
88226
88239
TCTTTTAAAGAAGA
2-10-2 MOE
424


 


371673
91493
91506
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371664
92030
92043
TGCTGTCCTATAAG
2-10-2 MOE
460


 


371666
92044
92057
CACAAAGGTAATTG
2-10-2 MOE
461


 


371667
92058
92071
ATCATTTCTTCCAG
2-10-2 MOE
462


 


371668
92072
92085
TTATCAATGATGCA
2-10-2 MOE
463


 


371670
92100
92113
GCATGCTGGACAGT
2-10-2 MOE
440


 


371672
92128
92141
TTCCAGCTTGAAGA
2-10-2 MOE
435


 


371674
92147
92160
CCATTACCTTCCAG
2-10-2 MOE
466


 


371637
92983
92996
GCATAAACAGGGTT
2-10-2 MOE
467


 


371654
93928
93941
GAACAAAAATTAAA
2-10-2 MOE
427


 


371641
99772
99785
AGAACTCACATCTG
2-10-2 MOE
455


 


371679
99883
99896
TTTATAAAGTAAAG
2-10-2 MOE
429


 


371660
99933
99946
AAGGATATTTTAAA
2-10-2 MOE
425


 


371635
105004
105017
TATGAAAGGAATGT
2-10-2 MOE
472


 


371654
105028
105041
GAACAAAAATTAAA
2-10-2 MOE
427


 


371676
106482
106495
TTCCTTAAGCTTCC
2-10-2 MOE
474


 


371650
107838
107851
TCTTTTAAAGAAGA
2-10-2 MOE
424


 


371673
110922
110935
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371673
111580
111593
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371634
114608
114621
CAGAATATATTTCT
2-10-2 MOE
444


 


371638
115040
115053
ATAGGAAGCATAAA
2-10-2 MOE
423


 


371660
116244
116257
AAGGATATTTTAAA
2-10-2 MOE
425


 


371663
116657
116670
ATAAGAGATTAAAA
2-10-2 MOE
450


 


371673
118068
118081
ACCTTCCAGGTTCA
2-10-2 MOE
422


 


371666
118834
118847
CACAAAGGTAATTG
2-10-2 MOE
461


 


371660
119858
119871
AAGGATATTTTAAA
2-10-2 MOE
425


 


371660
120210
120223
AAGGATATTTTAAA
2-10-2 MOE
425


 


371662
120876
120889
GGGCATTGTTAAAA
2-10-2 MOE
452


 


371655
124004
124017
TACTGTGAGAAATA
2-10-2 MOE
433


 


371656
124170
124183
GAACAGTTAAACAT
2-10-2 MOE
485









In certain embodiments, a target region is nucleotides 88142-88269 of SEQ ID NO: 8. In certain embodiments, a short antisense compound is targeted to nucleotides 88142-88269 of SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to nucleotides 88142-88269 comprises a nucleotide sequence selected from SEQ ID NO 413, 414, 415, 416, 417, or 418. In certain such embodiments, an antisense compound targeted to nucleotides 88142-88269 of SEQ ID NO: 8 is selected from Isis NO. 371644, 371645, 371649, 371651, 371652, or 371653.
In certain embodiments, a target region is nucleotides 88142-88169 of SEQ ID NO: 8. In certain embodiments, a short antisense compound is targeted to nucleotides 88142-88169 of SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to nucleotides 88142-88169 comprises a nucleotide sequence selected from SEQ ID NO 413 or 414. In certain such embodiments, an antisense compound targeted to nucleotides 88142-88169 of SEQ ID NO: 8 is selected from Isis NO. 371644 or 371645.
In certain embodiments, a target region is nucleotides 88242-88269 of SEQ ID NO: 8. In certain embodiments, a short antisense compound is targeted to nucleotides 88242-88269 of SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to nucleotides 88242-88269 comprises a nucleotide sequence selected from SEQ ID NO 416, 417, or 418. In certain such embodiments, an antisense compound targeted to nucleotides 88242-88269 of SEQ ID NO: 8 is selected from Isis NO. 371651, 371652, or 371653.
In certain embodiments, a target region is nucleotides 92037-92155 of SEQ ID NO: 8. In certain embodiments, a short antisense compound is targeted to nucleotides 92037-92155 of SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to nucleotides 92037-92155 comprises a nucleotide sequence selected from SEQ ID NO 419, 420, 421, or 422. In certain such embodiments, an antisense compound targeted to nucleotides 92037-92155 of SEQ ID NO: 8 is selected from Isis NO. 371665, 371669, 371671, or 171673.
In certain embodiments, a target region is nucleotides 92114-92155 of SEQ ID NO: 8. In certain embodiments, a short antisense compound is targeted to nucleotides 92114-92155 of SEQ ID NO: 8. In certain such embodiments, a short antisense compound targeted to nucleotides 92114-92155 comprises a nucleotide sequence selected from SEQ ID NO 421 or 422. In certain such embodiments, an antisense compound targeted to nucleotides 92114-92155 of SEQ ID NO: 8 is selected from Isis NO. 371671 or 171673.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a GCCR nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of GCCR. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a GCCR nucleic acid, wherein the short antisense compound targeted to a GCCR nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of GCCR using one or more short antisense compounds targeted to a GCCR nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of GCCR comprise use of a short antisense compound targeted to a GCCR nucleic acid comprising 12 or 14 nucleotides or nucleosides.
7. Glucagon Receptor (GCGR)
The maintenance of normal glycemia is a carefully regulated metabolic event. Glucagon, the 29-amino acid peptide responsible for maintaining blood glucose levels in the postabsorbative state, increases glucose release from the liver by activating hepatic glycogenolysis, gluconeogenesis, stimulating lipolysis in adipose tissue, and stimulating insulin secretion. During high blood glucose levels, insulin reverses the glucagon-mediated enhancement of glycogenolysis and gluconeogenesis. In patients with diabetes, insulin is either not available or not fully effective. While treatment for diabetes has traditionally focused on increasing insulin levels, antagonism of glucagon function has been considered as an alternative therapy. As glucagon exerts its physiological effects by signaling through the glucagon receptor, the glucagon receptor has been proposed as a potential therapeutic target for diabetes (Madsen et al., Curr. Pharm. Des., 1999, 5, 683-691).
Glucagon receptor is belongs to the superfamily of G-protein-coupled receptors having seven transmembrane domains. It is also a member of the smaller sub-family of homologous receptors which bind peptides that are structurally similar to glucagon. The gene encoding human glucagon receptor was cloned in 1994 and analysis of the genomic sequence revealed multiple introns and an 82% identity to the rat glucagon receptor gene (Lok et al., Gene, 1994, 140, 203-209; MacNeil et al., Biochem. Biophys. Res. Commun., 1994, 198, 328-334). Cloning of the rat glucagon receptor gene also led to the description of multiple alternative splice variants (Maget et al., FEBS Lett., 1994, 351, 271-275). The human glucagon receptor gene is localized to chromosome 17q25 (Menzel et al., Genomics, 1994, 20, 327-328). A missense mutation of Gly to Ser at codon 40 in the glucagon receptor gene leads to a 3-fold lower affinity for glucagon (Fujisawa et al., Diabetologia, 1995, 38, 983-985) and this mutation has been linked to several disease states, including non-insulin-dependent diabetes mellitus (Fujisawa et al., Diabetologia, 1995, 38, 983-985), hypertension (Chambers and Morris, Nat. Genet., 1996, 12, 122), and central adiposity (Siani et al., Obes. Res., 2001, 9, 722-726).
Definitions
“Glucagon receptor” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound. Glucagon receptor is generally referred to as GCGR but may also be referred to as GR, GGR, MGC138246, MGC93090.
“GCGR nucleic acid” means any nucleic acid encoding GCGR. For example, in certain embodiments, a GCGR nucleic acid includes, without limitation, a GCGR sequence encoding GCGR, an RNA sequence transcribed from DNA encoding GCGR, and an mRNA sequence encoding GCGR. “GCGR mRNA” means an mRNA encoding a GCGR protein.
Therapeutic Indications
Antisense technology is an effective means for reducing glucagon receptor (GCGR) expression and has proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications. As such, in certain embodiments, the present invention provides short antisense compounds targeted to a nucleic acid encoding glucagon receptor, and which modulate the expression of glucagon receptor. Further provided herein are short antisense compounds capable of inhibiting GCGR expression. Also provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions comprising a short antisense compound targeted to a GCGR nucleic acid. In certain embodiments, because short antisense compounds targeted to a GCGR nucleic acid inhibit GCGR expression, provided herein are methods of treating a subject having a disease or condition associated with GCGR activity by administering one or more pharmaceutical compositions comprising a short antisense compound targeted to a GCGR nucleic acid. For example, provided herein are methods of treating a subject having high blood glucose, hyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolic syndrome, obesity and/or insulin resistance.
Also contemplated herein are pharmaceutical composition comprising one or more short antisense compounds targeted to GCGR and optionally a pharmaceutically acceptable carrier, diluent, enhancer or excipient. Certain compounds of the invention can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to glucagon effects mediated by GCGR.
Certain embodiments of the present invention include methods of reducing the expression of GCGR in tissues or cells comprising contacting said cells or tissues with a short antisense compound targeted to a nucleic acid encoding GCGR or pharmaceutical composition comprising such a short antisense compound. In certain such embodiments, the invention provides methods of decreasing blood glucose levels, blood triglyceride levels, or blood cholesterol levels in a subject comprising administering to the subject a short antisense compound or a pharmaceutical composition. Blood levels may be plasma levels or serum levels. Also contemplated are methods of improving insulin sensitivity, methods of increasing GLP-1 levels and methods of inhibiting hepatic glucose output in an animal comprising administering to said animal an antisense oligonucleotide or a pharmaceutical composition of the invention. An improvement in insulin sensitivity may be indicated by a reduction in circulating insulin levels.
In certain embodiments, the invention provides methods of treating a subject having a disease or condition associated with glucagon activity via GCGR comprising administering to the subject a therapeutically or prophylactically effective amount of a short antisense compound or a pharmaceutical composition. In certain embodiments, such disease or condition may be a metabolic disease or condition. In certain embodiments, the metabolic disease or condition is diabetes, hyperglycemia, hyperlipidemia, metabolic syndrome X, obesity, primary hyperglucagonemia, insulin deficiency, or insulin resistance. In some embodiments, the diabetes is Type 2 diabetes. In some embodiments the obesity is diet-induced. In some embodiments, hyperlipidemia is associated with elevated blood lipid levels. Lipids include cholesterol and triglycerides. In one embodiment, the condition is liver steatosis. In some embodiments, the steatosis is steatohepatitis or non-alcoholic steatohepatitis.
In certain embodiments, the invention provides methods of preventing or delaying the onset of elevated blood glucose levels in an animal as well as methods of preserving beta-cell function in an animal using the oligomeric compounds delineated herein.
Certain short antisense compounds targeted to GCGR can be used to modulate the expression of GCGR in a subject in need thereof, such as an animal, including, but not limited to, a human In certain embodiments, such methods comprise the step of administering to said animal an effective amount of a short antisense compound that reduces expression of GCGR RNA. In certain embodiments, short antisense compounds effectively reduce the levels or function of GCGR RNA. Because reduction in GCGR mRNA levels can lead to alteration in GCGR protein products of expression as well, such resultant alterations can also be measured. Certain antisense compounds that effectively reduce the levels or function of GCGR RNA or protein products of expression is considered an active antisense compound. In certain embodiments, short antisense compounds reduce the expression of GCGR causing a reduction of RNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.
Further provided are methods of screening for modulators of glucagon receptor and methods of modulating the expression of glucagon receptor in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more short antisense compounds targeted to GCGR or with compositions comprising such compounds. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of glucagon receptor are also set forth herein. Certain such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.
The reduction of the expression of glucagon receptor may be measured, for example, in blood, plasma, serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding glucagon receptor protein and/or the glucagon receptor protein itself.
Pharmaceutical and other compositions comprising short antisense compounds are also provided. In certain embodiments short antisense compounds targeted to a nucleic acid encoding GCGR are utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
The short antisense compounds targeting a GCGR nucleic acid may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a GCGR nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. In certain embodiments, short antisense compounds targeting a GCGR nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 2-10-2, 3-10-3, 3-8-3, 1-1-10-2.
Certain Short Antisense Compounds Targeted to a GCGR Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a GCGR nucleic acid having the sequence GENBANK® Accession No. NM_000160.1, incorporated herein as SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 9 is at least 90% complementary to SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 9 is at least 95% complementary to SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 9 is 100% complementary to SEQ ID NO: 9. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 9 includes a nucleotide sequence selected from the nucleotide sequences set forth in Tables 12 and 13.
The nucleotide sequences set forth in each SEQ ID NO in Tables 12 and 13 are independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Short antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise a gapmer motif. In certain embodiments, a short antisense compound targeted to a GCCR nucleic acid comprises a 3-10-3 gapmer motif. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise a gapmer motif. In certain embodiments, a short antisense compound targeted to a GCCR nucleic acid comprises a 3-8-3 gapmer motif. In certain embodiments, short antisense compounds targeted to a GCCR nucleic acid comprise a gapmer motif. In certain embodiments, a short antisense compound targeted to a GCCR nucleic acid comprises a 2-10-2 gapmer motif.
Tables 12 and 13 illustrate examples of short antisense compounds targeted to SEQ ID NO: 9. Table 12 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 9. Table 13 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 9. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 12







Short Antisense Compounds targeted to SEQ ID NO: 9













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO















338463
378
393
TAGAGCTTCCACTTCT
3-10-3 MOE
486


 


338534
378
391
GAGCTTCCACTTCT
3-8-3 MOE
487


 


327130
499
512
TGTTGGCCGTGGTA
3-8-3 MOE
488


 


327131
500
513
ATGTTGGCCGTGGT
3-8-3 MOE
489


 


327132
501
514
GATGTTGGCCGTGG
3-8-3 MOE
490


 


327133
502
515
AGATGTTGGCCGTG
3-8-3 MOE
491


 


327134
503
516
GAGATGTTGGCCGT
3-8-3 MOE
492


 


327135
504
517
GGAGATGTTGGCCG
3-8-3 MOE
493


 


327136
505
518
AGGAGATGTTGGCC
3-8-3 MOE
494


 


327137
506
519
CAGGAGATGTTGGC
3-8-3 MOE
495


 


327138
507
520
GCAGGAGATGTTGG
3-8-3 MOE
496


 


327139
508
521
GGCAGGAGATGTTG
3-8-3 MOE
497


 


327140
531
544
GTGGTGCCAAGGCA
3-8-3 MOE
498


 


327141
532
545
TGTGGTGCCAAGGC
3-8-3 MOE
499


 


327142
533
546
TTGTGGTGCCAAGG
3-8-3 MOE
500


 


327143
534
547
TTTGTGGTGCCAAG
3-8-3 MOE
501


 


327144
535
548
CTTTGTGGTGCCAA
3-8-3 MOE
502


 


327145
536
549
ACTTTGTGGTGCCA
3-8-3 MOE
503


 


327146
537
550
CACTTTGTGGTGCC
3-8-3 MOE
504


 


327147
538
551
GCACTTTGTGGTGC
3-8-3 MOE
505


 


327148
539
552
TGCACTTTGTGGTG
3-8-3 MOE
506


 


327149
540
553
TTGCACTTTGTGGT
3-8-3 MOE
507


 


327150
545
558
CGGTGTTGCACTTT
3-8-3 MOE
508


 


327151
546
559
GCGGTGTTGCACTT
3-8-3 MOE
509


 


327152
547
560
AGCGGTGTTGCACT
3-8-3 MOE
510


 


327153
548
561
AAGCGGTGTTGCAC
3-8-3 MOE
511


 


327154
549
562
GAAGCGGTGTTGCA
3-8-3 MOE
512


 


327155
550
563
CGAAGCGGTGTTGC
3-8-3 MOE
513


 


327156
551
564
ACGAAGCGGTGTTG
3-8-3 MOE
514


 


327157
552
565
CACGAAGCGGTGTT
3-8-3 MOE
515


 


327158
553
566
ACACGAAGCGGTGT
3-8-3 MOE
516


 


327159
554
567
AACACGAAGCGGTG
3-8-3 MOE
517


 


345897
684
697
GCTGCTGTACATCT
2-10-2 MOE
518


 


327160
684
697
GCTGCTGTACATCT
3-8-3 MOE
518


 


327161
685
698
AGCTGCTGTACATC
3-8-3 MOE
520


 


327162
686
699
AAGCTGCTGTACAT
3-8-3 MOE
521


 


327163
687
700
GAAGCTGCTGTACA
3-8-3 MOE
522


 


327164
688
701
GGAAGCTGCTGTAC
3-8-3 MOE
523


 


327165
689
702
TGGAAGCTGCTGTA
3-8-3 MOE
524


 


327166
690
703
CTGGAAGCTGCTGT
3-8-3 MOE
525


 


327167
691
704
CCTGGAAGCTGCTG
3-8-3 MOE
526


 


327168
692
705
ACCTGGAAGCTGCT
3-8-3 MOE
527


 


327169
693
706
CACCTGGAAGCTGC
3-8-3 MOE
528


 


327170
694
707
TCACCTGGAAGCTG
3-8-3 MOE
529


 


327171
695
708
ATCACCTGGAAGCT
3-8-3 MOE
530


 


327172
696
709
CATCACCTGGAAGC
3-8-3 MOE
531


 


327173
697
710
ACATCACCTGGAAG
3-8-3 MOE
532


 


327174
698
711
TACATCACCTGGAA
3-8-3 MOE
533


 


327175
699
712
GTACATCACCTGGA
3-8-3 MOE
534


 


327176
700
713
TGTACATCACCTGG
3-8-3 MOE
535


 


327177
701
714
GTGTACATCACCTG
3-8-3 MOE
536


 


327178
869
882
TAGCGGGTCCTGAG
3-8-3 MOE
537


 


327179
870
883
GTAGCGGGTCCTGA
3-8-3 MOE
538


 


327180
871
884
TGTAGCGGGTCCTG
3-8-3 MOE
539


 


327181
872
885
CTGTAGCGGGTCCT
3-8-3 MOE
540


 


327182
873
886
GCTGTAGCGGGTCC
3-8-3 MOE
541


 


327183
874
887
GGCTGTAGCGGGTC
3-8-3 MOE
542


 


327184
875
888
TGGCTGTAGCGGGT
3-8-3 MOE
543


 


327185
876
889
CTGGCTGTAGCGGG
3-8-3 MOE
544


 


327186
877
890
TCTGGCTGTAGCGG
3-8-3 MOE
545


 


327187
878
891
TTCTGGCTGTAGCG
3-8-3 MOE
546


 


327188
955
968
TGAACACCGCGGCC
3-8-3 MOE
547


 


327189
956
969
ATGAACACCGCGGC
3-8-3 MOE
548


 


327190
957
970
CATGAACACCGCGG
3-8-3 MOE
549


 


327191
958
971
GCATGAACACCGCG
3-8-3 MOE
550


 


327192
959
972
TGCATGAACACCGC
3-8-3 MOE
551


 


327193
960
973
TTGCATGAACACCG
3-8-3 MOE
552


 


327194
961
974
ATTGCATGAACACC
3-8-3 MOE
553


 


327195
962
975
TATTGCATGAACAC
3-8-3 MOE
554


 


327196
963
976
ATATTGCATGAACA
3-8-3 MOE
555


 


327197
964
977
CATATTGCATGAAC
3-8-3 MOE
556


 


327198
1019
1032
AGGTTGTGCAGGTA
3-8-3 MOE
557


 


327199
1020
1033
CAGGTTGTGCAGGT
3-8-3 MOE
558


 


327200
1021
1034
GCAGGTTGTGCAGG
3-8-3 MOE
559


 


327201
1022
1035
AGCAGGTTGTGCAG
3-8-3 MOE
560


 


327202
1023
1036
CAGCAGGTTGTGCA
3-8-3 MOE
561


 


327203
1024
1037
CCAGCAGGTTGTGC
3-8-3 MOE
562


 


327204
1025
1038
CCCAGCAGGTTGTG
3-8-3 MOE
563


 


327205
1026
1039
GCCCAGCAGGTTGT
3-8-3 MOE
564


 


327206
1027
1040
GGCCCAGCAGGTTG
3-8-3 MOE
565


 


327207
1028
1041
AGGCCCAGCAGGTT
3-8-3 MOE
566


 


338491
1160
1175
TGTCATTGCTGGTCCA
3-10-3 MOE
567


 


338562
1160
1173
TCATTGCTGGTCCA
3-8-3 MOE
568


 


338498
1307
1322
TGGCCAGCCGGAACTT
3-10-3 MOE
569


 


338569
1307
1320
GCCAGCCGGAACTT
3-8-3 MOE
570


 


338499
1329
1344
GGGATGAGGGTCAGCG
3-10-3 MOE
571


 


338570
1329
1342
GATGAGGGTCAGCG
3-8-3 MOE
572


 


385067
1364
1377
AAGGCAAAGACCAC
3-8-3 MOE
573


 


338573
1401
1414
GGAGCGCAGGGTGC
3-8-3 MOE
574


 


338580
1487
1500
TGCACCTCCTTGTT
3-8-3 MOE
575
















TABLE 13







Short antisense compounds targeted to


SEQ ID NO: 1 and having 1 or 2 mismatches













5′
3′





ISIS
Target
Target


SEQ


NO.
Site
Site
Sequence (5′-3′)
Gapmer Motif
ID NO















338577
158
171
CAGCAGACCCTGGA
3-8-3 MOE
576


 


338458
237
252
ACATCTGGCAGAGGTT
3-10-3 MOE
577


 


338529
237
250
ATCTGGCAGAGGTT
3-8-3 MOE
578


 


338466
318
333
CAGGCCAGCAGGAGTA
3-10-3 MOE
579


 


338537
318
331
GGCCAGCAGGAGTA
3-8-3 MOE
580


 


338533
364
377
CAAACAAAAAGTCC
3-8-3 MOE
582


 


338462
364
379
CTCAAACAAAAAGTCC
3-10-3 MOE
581


 


338535
397
410
GGTGACATTGGTCA
3-8-3 MOE
584


 


338464
397
412
GTGGTGACATTGGTCA
3-10-3 MOE
583


 


338466
470
485
CAGGCCAGCAGGAGTA
3-10-3 MOE
579


 


338537
470
483
GGCCAGCAGGAGTA
3-8-3 MOE
580


 


385048
497
510
TTGGCAGTGGTGTT
3-8-3 MOE
587


 


385049
500
513
ATGTTGGCAGTGGT
3-8-3 MOE
588


 


338467
503
518
AGGAAATGTTGGCAGT
3-10-3 MOE
589


 


338538
503
516
GAAATGTTGGCAGT
3-8-3 MOE
590


 


385050
506
519
CAGGAAATGTTGGC
3-8-3 MOE
591


 


385051
509
522
GGGCAGGAAATGTT
3-8-3 MOE
592


 


385052
523
536
AAGGTAGGTACCAG
3-8-3 MOE
593


 


385053
526
539
ACCAAGGTAGGTAC
3-8-3 MOE
594


 


385056
535
548
CTTTGTGGCACCAA
3-8-3 MOE
595


 


385057
538
551
GCACTTTGTGGCAC
3-8-3 MOE
596


 


338539
539
552
TGCACTTTGTGGCA
3-8-3 MOE
597


 


385058
541
554
GCTGCACTTTGTGG
3-8-3 MOE
598


 


385059
544
557
GGTGCTGCACTTTG
3-8-3 MOE
599


 


385060
547
560
GGCGGTGCTGCACT
3-8-3 MOE
600


 


385063
556
569
TGAACACTAGGCGG
3-8-3 MOE
601


 


385064
559
572
TCTTGAACACTAGG
3-8-3 MOE
602


 


338469
561
576
CACCTCTTGAACACTA
3-10-3 MOE
603


 


338540
561
574
CCTCTTGAACACTA
3-8-3 MOE
604


 


385065
562
575
ACCTCTTGAACACT
3-8-3 MOE
605


 


385066
565
578
CACACCTCTTGAAC
3-8-3 MOE
606


 


338541
590
603
CCTCGAACCCACTG
3-8-3 MOE
607


 


338473
658
673
CTTCTGGACCTCGATC
3-10-3 MOE
608


 


338544
658
671
TCTGGACCTCGATC
3-8-3 MOE
609


 


338474
681
696
CTGCTATACATCTTGG
3-10-3 MOE
610


 


338545
681
694
GCTATACATCTTGG
3-8-3 MOE
611


 


338475
703
718
CACGGTGTACATCACC
3-10-3 MOE
612


 


338546
703
716
CGGTGTACATCACC
3-8-3 MOE
613


 


338547
718
731
ACAGACTGTAGCCC
3-8-3 MOE
615


 


338476
718
733
GGACAGACTGTAGCCC
3-10-3 MOE
614


 


338550
889
902
CATCGCCAATCTTC
3-8-3 MOE
617


 


338479
889
904
GTCATCGCCAATCTTC
3-10-3 MOE
616


 


338551
899
912
ACACTGAGGTCATC
3-8-3 MOE
619


 


338480
899
914
TCACACTGAGGTCATC
3-10-3 MOE
618


 


338552
924
937
CGCCCCGTCACTGA
3-8-3 MOE
620


 


338555
992
1005
AGCAACCAGCAATA
3-8-3 MOE
622


 


338484
992
1007
CCAGCAACCAGCAATA
3-10-3 MOE
621


 


338485
1018
1033
CAGGCTGTACAGGTAC
3-10-3 MOE
623


 


338556
1018
1031
GGCTGTACAGGTAC
3-8-3 MOE
624


 


338558
1051
1064
AGCTCCTCTCAGAG
3-8-3 MOE
626


 


338487
1051
1066
GAAGCTCCTCTCAGAG
3-10-3 MOE
625


 


338559
1079
1092
CAGCCAATGCCCAG
3-8-3 MOE
628


 


338488
1079
1094
CCCAGCCAATGCCCAG
3-10-3 MOE
627


 


338560
1131
1144
AAACAGACACTTGA
3-8-3 MOE
630


 


338489
1131
1146
TCAAACAGACACTTGA
3-10-3 MOE
629


 


338490
1145
1160
AGCACTGAACATTCTC
3-10-3 MOE
631


 


338561
1145
1158
CACTGAACATTCTC
3-8-3 MOE
632


 


338563
1181
1194
ATCCACCAGAATCC
3-8-3 MOE
634


 


338492
1181
1196
GGATCCACCAGAATCC
3-10-3 MOE
633


 


338564
1216
1229
TGATCAGTAAGGCC
3-8-3 MOE
635


 


338565
1232
1245
ACAAAGATGAAAAA
3-8-3 MOE
637


 


338494
1232
1247
GGACAAAGATGAAAAA
3-10-3 MOE
636


 


338566
1267
1280
CACGCAGCTTGGCC
3-8-3 MOE
639


 


338495
1267
1282
GGCACGCAGCTTGGCC
3-10-3 MOE
638


 


338571
1344
1357
GACCCCCAGCAGAG
3-8-3 MOE
641


 


338500
1344
1359
TGGACCCCCAGCAGAG
3-10-3 MOE
640


 


385068
1366
1379
CAAAGGCAAAGACC
3-8-3 MOE
642


 


385069
1369
1382
TCACAAAGGCAAAG
3-8-3 MOE
643


 


385070
1372
1385
CAGTCACAAAGGCA
3-8-3 MOE
644


 


385071
1375
1388
CGTCAGTCACAAAG
3-8-3 MOE
645


 


385072
1378
1391
GCTCGTCAGTCACA
3-8-3 MOE
646


 


385073
1381
1394
CATGCTCGTCAGTC
3-8-3 MOE
647


 


386608
1384
1397
GGGCATGCTCGTCA
1-12-1 MOE
648


 


386593
1384
1397
GGGCATGCTCGTCA
2-10-2 MOE
648


 


396146
1384
1397
GGGCATGCTCGTCA
2-10-2 MOE
648


 


338572
1384
1397
GGGCATGCTCGTCA
3-8-3 MOE
648


 


396149
1384
1397
GGGCATGCTCGTCA
1-1-10-2 2′-
648






(butylacetamido)-






palmitamide/OMe/






OMe


 


386627
1384
1397
GGGCATGCTCGTCA
2-10-2
648






Methyleneoxy






BNA


 


386610
1387
1400
CTTGGGCATGCTCG
1-12-1 MOE
654


 


386595
1387
1400
CTTGGGCATGCTCG
2-10-2 MOE
654


 


385074
1387
1400
CTTGGGCATGCTCG
3-8-3 MOE
654


 


385075
1390
1403
TGCCTTGGGCATGC
3-8-3 MOE
657


 


385076
1393
1406
GGGTGCCTTGGGCA
3-8-3 MOE
648


 


385077
1396
1409
GCAGGGTGCCTTGG
3-8-3 MOE
659


 


385078
1399
1412
AGCGCAGGGTGCCT
3-8-3 MOE
660


 


338502
1401
1416
GTGGAGCGCAGGGTGC
3-10-3 MOE
661


 


385079
1402
1415
TGGAGCGCAGGGTG
3-8-3 MOE
662


 


385080
1405
1418
TGGTGGAGCGCAGG
3-8-3 MOE
663


 


385081
1408
1421
GCTTGGTGGAGCGC
3-8-3 MOE
664


 


385082
1411
1424
AGAGCTTGGTGGAG
3-8-3 MOE
665


 


338503
1412
1427
AAAAGAGCTTGGTGGA
3-10-3 MOE
666


 


338574
1412
1425
AAGAGCTTGGTGGA
3-8-3 MOE
667


 


385083
1414
1427
AAAAGAGCTTGGTG
3-8-3 MOE
668


 


385084
1417
1430
CAAAAAAGAGCTTG
3-8-3 MOE
669


 


338504
1434
1449
AAGGAGCTGAGGAACA
3-10-3 MOE
670


 


338575
1434
1447
GGAGCTGAGGAACA
3-8-3 MOE
671


 


327167
1441
1454
CCTGGAAGCTGCTG
3-8-3 MOE
526


 


338576
1445
1458
AGACCCTGGAAGGA
3-8-3 MOE
673


 


338505
1445
1460
GCAGACCCTGGAAGGA
3-10-3 MOE
672


 


338506
1449
1464
ACCAGCAGACCCTGGA
3-10-3 MOE
674


 


338577
1449
1462
CAGCAGACCCTGGA
3-8-3 MOE
576


 


338507
1464
1479
CAGTAGAGAACAGCCA
3-10-3 MOE
676


 


338578
1464
1477
GTAGAGAACAGCCA
3-8-3 MOE
677


 


338508
1475
1490
TGTTGAGGAAACAGTA
3-10-3 MOE
678


 


338579
1475
1488
TTGAGGAAACAGTA
3-8-3 MOE
679


 


338509
1487
1502
CCTGCACCTCCTTGTT
3-10-3 MOE
680


 


338580
1610
1623
TGCACCTCCTTGTT
3-8-3 MOE
575









In certain embodiments, a target region is nucleotides 378-391 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 378-391 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 378-391 comprises a nucleotide sequence selected from SEQ ID NO 486 or 487. In certain such embodiments, a short antisense compound targeted to nucleotides 378-391 of SEQ ID NO: 9 is selected from Isis No 338463 or 338534.
In certain embodiments, a target region is nucleotides 499-521 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 499-521 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 499-521 comprises a nucleotide sequence selected from SEQ ID NO 488, 489, 490, 491, 492, 493, 494, 495, 496, or 497. In certain such embodiments, a short antisense compound targeted to nucleotides 499-521 of SEQ ID NO: 9 is selected from Isis No 327130, 327131, 327132, 327133, 327134, 327135, 327136, 327137, 327138, or 327139.
In certain embodiments, a target region is nucleotides 531-553 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 531-553 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 531-553 comprises a nucleotide sequence selected from SEQ ID NO 498, 499, 500, 501, 502, 503, 504, 505, 506, or 507. In certain such embodiments, a short antisense compound targeted to nucleotides 531-553 of SEQ ID NO: 9 is selected from Isis No 327140, 327141, 327142, 327143, 327144, 327145, 327146, 327147, 327148, or 327149.
In certain embodiments, a target region is nucleotides 545-567 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 545-567 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 545-567 comprises a nucleotide sequence selected from SEQ ID NO 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517. In certain such embodiments, a short antisense compound targeted to nucleotides 545-567 of SEQ ID NO: 9 is selected from Isis No 327150, 327151, 327152, 327153, 327154, 327155, 327156, 327157, 327158, or 327159.
In certain embodiments, a target region is nucleotides 531-567 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 531-567 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 531-567 comprises a nucleotide sequence selected from SEQ ID NO 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517. In certain such embodiments, a short antisense compound targeted to nucleotides 531-567 of SEQ ID NO: 9 is selected from Isis No 327140, 327141, 327142, 327143, 327144, 327145, 327146, 327147, 327148, 327149, 327150, 327151, 327152, 327153, 327154, 327155, 327156, 327157, 327158, or 327159.
In certain embodiments, a target region is nucleotides 684-714 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 684-714 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 684-714 comprises a nucleotide sequence selected from SEQ ID NO 518, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, or 536. In certain such embodiments, a short antisense compound targeted to nucleotides 684-714 of SEQ ID NO: 9 is selected from Isis No 345897, 327160, 327161, 327162, 327163, 327164, 327165, 327166, 327167, 327168, 327169, 327170, 327171, 327172, 327173, 327174, 327175, 327176, or 327177.
In certain embodiments, a target region is nucleotides 869-891 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 869-891 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 869-891 comprises a nucleotide sequence selected from SEQ ID NO 537, 538, 539, 540, 541, 542, 543, 544, 545, or 546. In certain such embodiments, a short antisense compound targeted to nucleotides 869-891 of SEQ ID NO: 9 is selected from Isis No 327178, 327179, 327180, 327181, 327182, 327183, 327184, 327185, 327186, or 327187.
In certain embodiments, a target region is nucleotides 955-977 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 955-977 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 955-977 comprises a nucleotide sequence selected from SEQ ID NO 547, 548, 549, 550, 551, 552, 553, 554, 555, or 556. In certain such embodiments, a short antisense compound targeted to nucleotides 955-977 of SEQ ID NO: 9 is selected from Isis No 327188, 327189, 327190, 327191, 327192, 327193, 327194, 327195, 327196, or 327197.
In certain embodiments, a target region is nucleotides 1019-1041 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 1019-1041 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 1019-1041 comprises a nucleotide sequence selected from SEQ ID NO 557, 558, 559, 560, 561, 562, 563, 564, 565, or 566. In certain such embodiments, a short antisense compound targeted to nucleotides 1019-1041 of SEQ ID NO: 9 is selected from Isis No 327198, 327199, 327200, 327201, 327202, 327203, 327204, 327205, 327206, or 327207.
In certain embodiments, a target region is nucleotides 1160-1175 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 1160-1175 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 1160-1175 comprises a nucleotide sequence selected from SEQ ID NO 567 or 568. In certain such embodiments, a short antisense compound targeted to nucleotides 1160-1175 of SEQ ID NO: 9 is selected from Isis No 338491 or 338562.
In certain embodiments, a target region is nucleotides 1307-1377 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 1307-1377 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 1307-1377 comprises a nucleotide sequence selected from SEQ ID NO 569, 570, 571, 572, or 573. In certain such embodiments, a short antisense compound targeted to nucleotides 1307-1377 of SEQ ID NO: 9 is selected from Isis No 338498, 338569, 338499, 338570, or 385067.
In certain embodiments, a target region is nucleotides 1307-1414 of SEQ ID NO: 9. In certain embodiments, a short antisense compound is targeted to nucleotides 1307-1414 of SEQ ID NO: 9. In certain such embodiments, a short antisense compound targeted to nucleotides 1307-1414 comprises a nucleotide sequence selected from SEQ ID NO 569, 570, 571, 572, 573, or 574. In certain such embodiments, a short antisense compound targeted to nucleotides 1307-1414 of SEQ ID NO: 9 is selected from Isis No 338498, 338569, 338499, 338570, 385067, or 338573.
In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a GCGR nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a GCGR nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of GCGR. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a GCGR nucleic acid, wherein the short antisense compound targeted to a GCGR nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of GCGR using one or more short antisense compounds targeted to a GCGR nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 14 monomers in length.
In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of GCGR comprise use of a short antisense compound targeted to a GCGR nucleic acid comprising 12 or 14 nucleotides or nucleosides.
8. DGAT2
Diacylglycerol transferase 2 (also known as DGAT2, diacylglycerol 0-transferase 2, acyl-CoA:diacylglycerol acyltransferase 2), Diacylglycerol transferase 2 has been shown to be implicated in the absorption process of triglycerides (also called triacylglycerols) from food.
The absorption of triglycerides from food is a very efficient process which occurs by a series of steps wherein the dietary triacylglycerols are hydrolyzed in the intestinal lumen and then resynthesized within enterocytes. The resynthesis of triacylglycerols can occur via the monoacylglycerol pathwaywhich commences with monoacylglycerol acyltransferase (MGAT) catalyzing the synthesis of diacylglycerol from monoacylglycerol and fatty acyl-CoA. An alternative synthesis of diacylglycerols is provided by the glycerol-phosphate pathway which describes the coupling of two molecules of fatty acyl-CoA to glycerol-3-phosphate. In either case, diacylglycerol is then acylated with another molecule of fatty acyl-CoA in a reaction catalyzed by one of two diacylglycerol acyltransferase enzymes to form the triglyceride (Farese et al., Curr. Opin. Lipidol., 2000, 11, 229-234).
The reaction catalyzed by diacylglycerol acyltransferase is the final and only committed step in triglyceride synthesis. As such, diacylglycerol acyltransferase is involved in intestinal fat absorption, lipoprotein assembly, regulating plasma triglyceride concentrations, and fat storage in adipocytes. The first diacylglycerol acyltransferase, diacylglycerol transferase 1, was identified in 1960 and the human and mouse genes encoding this protein were isolated in 1998 (Cases et al., Proc. Natl. Acad. Sci. U.S.A, 1998, 95, 13018-13023; Oelkers et al., J. Biol. Chem., 1998, 273, 26765-26771). Mice lacking diacylglycerol acyltransferase 1 are viable and can still synthesize triglycerides through other biological routes, suggesting the existence of multiple mechanisms for triglyceride synthesis (Smith et al., Nat. Genet., 2000, 25, 87-90).
A second diacylglycerol transferase, diacylglycerol transferase 2 (also known as DGAT2, diacylglycerol 0-transferase 2, acyl-CoA:diacylglycerol acyltransferase 2), was subsequently identified in the fungus Mortierella, humans and mice (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876; Lardizabal et al., J. Biol. Chem., 2001, 276, 38862-38869). Enzymatic assays indicate that this recently identified protein does possess diacylglycerol transferase activity that utilizes a broad range of long chain fatty acyl-CoA substrates (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876).
Diacylglycerol transferase 2 is a member of a family of genes whose sequences are unrelated to diacylglycerol transferase 1. In addition to differing in sequence compared to diacylglycerol transferase 1, in vitro assays illustrate that diacylglycerol transferase 2 has higher activity at lower concentrations of magnesium chloride and oleoyl-CoA (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). The predicted protein sequence of diacylglycerol transferase 2 contains at least one putative transmembrane domain, three potential N-linked glycosylation sites, six potential protein kinase C phosphorylation consensus sites, as well as sequences in common with a putative glycerol phosphorylation site found in acyltransferase enzymes (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). The International Radiation Hybrid Mapping Consortium has mapped human diacylglycerol transferase 2 to chromosome 11q13.3.
In human tissues, the highest levels of diacylglycerol transferase 2 are detected in liver and white adipose tissues, with lower levels found in mammary gland, testis and peripheral blood leukocytes (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876). Two mRNA species of 2.4 and 1.8 kilobases are detected in human tissues, whereas the major diacylglycerol transferase 2 mRNA species in mouse tissues is 2.4 kilobases. In addition to liver and white adipose tissues, diacylglycerol transferase 2 is expressed in all segments of the small intestine in mice, with higher expression in the proximal intestine and lower expression in the distal intestine (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876).
Diacylglycerol transferase activity exhibits distinct patterns during postnatal development of the rat liver. As there is no correlation between the mRNA expression and activity patterns, post-translational modifications may participate in the regulation of diacylglycerol transferase 2 activity during rat development (Waterman et al., J. Lipid. Res., 2002, 43, 1555-1562).
Diacylglycerol transferase 2 mRNA is preferentially upregulated by insulin treatment, as shown by in vitro assays measuring the diacylglycerol activity from the membrane fraction of cultured mouse adipocytes (Meegalla et al., Biochem. Biophys. Res. Commun., 2002, 298, 317-323). In fasting mice, diacylglycerol transferase 2 expression is greatly reduced, and dramatically increases upon refeeding. The expression patterns of two enzymes that participate in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase, respond to fasting and refeeding in a similar fashion. These results, combined with the observation that diacylglycerol transferase 2 is abundantly expressed in liver, suggest that diacylglycerol transferase 2 is tightly linked to the endogenous fatty acid synthesis pathway (Meegalla et al., Biochem. Biophys. Res. Commun., 2002, 298, 317-323).
Studies of mice harboring a disruption in the diacylglycerol acyltransferase 1 gene provide evidence that diacylglycerol acyltransferase 2 contributes to triglyceride synthesis. Levels of diacylglycerol transferase 2 mRNA expression are similar in intestinal segments from both wild type and diacylglycerol transferase 1-deficient mice (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479). Using magnesium chloride to distinguish between diacylglycerol transferase 1 and 2 activity, Buhman, et al. observed that, in diacylglycerol transferase 1-deficient mice, diacylglycerol transferase activity is reduced to 50% in the proximal intestine and to 10-15% in the distal intestine (Buhman et al., J. Biol. Chem., 2002, 277, 25474-25479).
Additionally, diacylglycerol transferase 2 mRNA levels are not up-regulated the liver or adipose tissues of diacylglycerol transferase 1-deficient mice, even after weeks of high-fat diet (Cases et al., J. Biol. Chem., 2001, 276, 38870-38876; Chen et al., J. Clin. Invest., 2002, 109, 1049-1055). However, in ob/ob mice, which have a mutation in the leptin gene that results in obesity, diacylglycerol transferase 2 is more highly expressed than in wild type mice, suggesting that diacylglycerol transferase 2 may be partly responsible for the highly accumulated fat mass seen in these mice. Furthermore, the combined mutations of leptin and diacylglycerol transferase 1 leads to a three-fold elevation in diacylglycerol transferase 2 expression in white adipose tissue, compared to the levels in the same tissue from diacylglycerol transferase 1-deficient mice (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055). Diacylglycerol transferase 2 mRNA is also upregulated in the skin of these mice (Chen et al., J. Clin. Invest., 2002, 109, 175-181). These data suggest leptin normally downregulates diacylglycerol transferase 2 expression, and that the upregulation of diacylglycerol transferase 2 in white adipose tissue in these mice may provide an alternate pathway for the triglyceride synthesis that still occurs in leptin deficient/diacylglycerol transferase 1-deficient mice (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055).
Diacylglycerol acyltransferase 1 knockout mice exhibit interesting phenotypes in that they are lean, resistant to diet-induce obesity, have decreased levels of tissue triglycerides and increased sensitivity to insulin and leptin (Chen et al., J. Clin. Invest., 2002, 109, 1049-1055; Smith et al., Nat. Genet., 2000, 25, 87-90). As diacylglycerol transferase 2 also participates in triglyceride synthesis, interfering with diacylglycerol transferase 2 may similarly lead to reduced body fat content.
Definitions
“DGAT2” means the gene product or protein of which expression is to be modulated by administration of a short antisense compound.
“DGAT2 nucleic acid” means any nucleic acid encoding DGAT2. For example, in certain embodiments, a DGAT2 nucleic acid includes, without limitation, a DNA sequence encoding DGAT2, an RNA sequence transcribed from DNA encoding DGAT2, and an mRNA sequence encoding DGAT2.
“DGAT2 mRNA” means an mRNA encoding DGAT2.
Therapeutic Indications
Antisense technology is an effective means for reducing DGAT2 expression and has proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications. As such, in certain embodiments, the present invention provides compounds targeted to nucleic acid encoding DGAT2, which modulate the expression of DGAT2. Further provided herein are short antisense compounds capable of effectively inhibiting DGAT2 expression.
In certain embodiments, a subject, suspected of having a disease or associated with DGAT2 is treated by administering one or more short antisense compounds targeted to a nucleic acid encoding DGAT2. For example, in a non-limiting embodiment, such methods comprise the step of administering to an animal a therapeutically effective amount of a short antisense compound. In certain such embodiments, short antisense compounds effectively inhibit the activity of DGAT2 or inhibit the expression of DGAT2. In one embodiment, the activity or expression of DGAT2 in a subject is inhibited by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%. In certain embodiments, the activity or expression of DGAT2 in a subject is inhibited by about 30%. More preferably, the activity or expression of DGAT2 in a subject is inhibited by 50% or more.
The reduction of the expression of DGAT2 may be measured, for example, in blood, plasma, serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding DGAT2 and/or the DGAT2 protein itself.
In certain embodiments, pharmaceutical and other compositions comprising the compounds of the invention are also provided. For example, short antisense compounds targeted to a DGAT2 nucleic acid can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
Certain short antisense compounds targeting DGAT2 may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a DGAT2 nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1. In certain embodiments, short antisense compounds targeting a DGAT2 nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-10-1, 2-10-2 and 3-10-3.
Provided herein are methods of treating an individual by administering one or more short antisense compound targeted to a DGAT2 nucleic acid or a pharmaceutical composition comprising such compound. Further provided are methods of treating a subject having a disease or conditions associated with DGAT2 activity by administering a short antisense compound targeted to a DGAT2 nucleic acid. Diseases and conditions associated with DGAT2 include, but are not limited to, cardiovascular disorders, obesity, diabetes, cholesterolemia, and liver steatosis.
Certain Short Antisense Compounds Targeted to a DGAT2 Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a DGAT2 nucleic acid having the sequence of GENBANK® Accession No. NM_032564.2, incorporated herein as SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 10 is at least 90% complementary to SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 10 is at least 95% complementary to SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 10 is 100% complementary to SEQ ID NO: 10. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 10 includes a nucleotide sequence selected from the nucleotide sequences set forth in Tables 14 and 15.
Each nucleotide sequence set forth in each Tables 14 and 15 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds comprising a nucleotide sequence as set forth in Tables 14 and 15 may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Tables 14 and 15 illustrate examples of short antisense compounds targeted to SEQ ID NO: 10. Table 14 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 10. Table 15 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 10. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 14







Short Antisense Compounds targeted


to SEQ ID NO: 10













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO















372556
231
244
ATGAGGGTCTTCAT
2-10-2 MOE
681


 


372557
249
262
ACCCCGGAGTAGGC
2-10-2 MOE
682


 


382601
249
260
CCCGGAGTAGGC
1-10-1 MOE
683


 


372480
251
266
CAGGACCCCGGAGTAG
3-10-3 MOE
684


 


372481
252
267
GCAGGACCCCGGAGTA
3-10-3 MOE
685


 


372558
252
265
AGGACCCCGGAGTA
2-10-2 MOE
686


 


372559
253
266
CAGGACCCCGGAGT
2-10-2 MOE
687


 


382603
331
342
CAGACCCCTCGC
1-10-1 MOE
688


 


382604
361
372
AGAGGATGCTGG
1-10-1 MOE
689


 


372485
392
407
GAGCCAGGTGACAGAG
3-10-3 MOE
690


 


372563
393
406
AGCCAGGTGACAGA
2-10-2 MOE
691


 


382605
397
408
TGAGCCAGGTGA
1-10-1 MOE
692


 


372565
414
427
TTTTCCACCTTGGA
2-10-2 MOE
693


 


382606
482
493
CTGCAGGCCACT
1-10-1 MOE
694


 


372497
651
666
TCACCAGCTGGATGGG
3-10-3 MOE
695


 


372498
652
667
TTCACCAGCTGGATGG
3-10-3 MOE
696


 


372575
652
665
CACCAGCTGGATGG
2-10-2 MOE
697


 


372576
653
666
TCACCAGCTGGATG
2-10-2 MOE
698


 


382607
655
666
TCACCAGCTGGA
1-10-1 MOE
699


 


372499
656
671
TGTCTTCACCAGCTGG
3-10-3 MOE
700


 


372577
657
670
GTCTTCACCAGCTG
2-10-2 MOE
701


 


372500
659
674
GTGTGTCTTCACCAGC
3-10-3 MOE
702


 


372578
660
673
TGTGTCTTCACCAG
2-10-2 MOE
703


 


372501
661
676
TTGTGTGTCTTCACCA
3-10-3 MOE
704


 


372579
662
675
TGTGTGTCTTCACC
2-10-2 MOE
705


 


372502
664
679
AGGTTGTGTGTCTTCA
3-10-3 MOE
706


 


372580
665
678
GGTTGTGTGTCTTC
2-10-2 MOE
707


 


372503
666
681
GCAGGTTGTGTGTCTT
3-10-3 MOE
708


 


372581
667
680
CAGGTTGTGTGTCT
2-10-2 MOE
709


 


372504
669
684
TCAGCAGGTTGTGTGT
3-10-3 MOE
710


 


372582
670
683
CAGCAGGTTGTGTG
2-10-2 MOE
711


 


372505
671
686
GGTCAGCAGGTTGTGT
3-10-3 MOE
712


 


372506
672
687
TGGTCAGCAGGTTGTG
3-10-3 MOE
713


 


372583
672
685
GTCAGCAGGTTGTG
2-10-2 MOE
714


 


372584
673
686
GGTCAGCAGGTTGT
2-10-2 MOE
715


 


372507
676
691
CTGGTGGTCAGCAGGT
3-10-3 MOE
716


 


372585
677
690
TGGTGGTCAGCAGG
2-10-2 MOE
717


 


382608
680
691
CTGGTGGTCAGC
1-10-1 MOE
718


 


372508
681
696
AGTTCCTGGTGGTCAG
3-10-3 MOE
719


 


372586
682
695
GTTCCTGGTGGTCA
2-10-2 MOE
720


 


372509
684
699
TATAGTTCCTGGTGGT
3-10-3 MOE
721


 


372587
685
698
ATAGTTCCTGGTGG
2-10-2 MOE
722


 


372510
686
701
GATATAGTTCCTGGTG
3-10-3 MOE
723


 


372588
687
700
ATATAGTTCCTGGT
2-10-2 MOE
724


 


372511
691
706
CCAAAGATATAGTTCC
3-10-3 MOE
725


 


372512
692
707
TCCAAAGATATAGTTC
3-10-3 MOE
726


 


372589
692
705
CAAAGATATAGTTC
2-10-2 MOE
727


 


372590
693
706
CCAAAGATATAGTT
2-10-2 MOE
728


 


382609
724
735
CCAGGCCCATGA
1-10-1 MOE
729


 


372514
725
740
GGCACCCAGGCCCATG
3-10-3 MOE
730


 


372592
726
739
GCACCCAGGCCCAT
2-10-2 MOE
731


 


372515
730
745
CAGAAGGCACCCAGGC
3-10-3 MOE
732


 


372593
731
744
AGAAGGCACCCAGG
2-10-2 MOE
733


 


382610
851
862
CCAGACATCAGG
1-10-1 MOE
734


 


382611
867
878
GACAGGGCAGAT
1-10-1 MOE
735


 


382602
868
879
TGACAGGGCAGA
1-10-1 MOE
736


 


382612
911
922
CCACTCCCATTC
1-10-1 MOE
737


 


372524
965
980
GCCAGGCATGGAGCTC
3-10-3 MOE
738


 


372602
966
979
CCAGGCATGGAGCT
2-10-2 MOE
739


 


382613
968
979
CCAGGCATGGAG
1-10-1 MOE
740


 


382614
987
998
CAGGGTGACTGC
1-10-1 MOE
741


 


372525
989
1004
GTTCCGCAGGGTGACT
3-10-3 MOE
742


 


372603
990
1003
TTCCGCAGGGTGAC
2-10-2 MOE
743


 


372526
992
1007
GCGGTTCCGCAGGGTG
3-10-3 MOE
744


 


372604
993
1006
CGGTTCCGCAGGGT
2-10-2 MOE
745


 


372530
1106
1121
TCGGCCCCAGGAGCCC
3-10-3 MOE
746


 


372608
1107
1120
CGGCCCCAGGAGCC
2-10-2 MOE
747


 


372531
1109
1124
CCATCGGCCCCAGGAG
3-10-3 MOE
748


 


372609
1110
1123
CATCGGCCCCAGGA
2-10-2 MOE
749


 


372532
1112
1127
GACCCATCGGCCCCAG
3-10-3 MOE
750


 


372610
1113
1126
ACCCATCGGCCCCA
2-10-2 MOE
751


 


372533
1117
1132
TTCTGGACCCATCGGC
3-10-3 MOE
752


 


382615
1117
1128
GGACCCATCGGC
1-10-1 MOE
753


 


372611
1118
1131
TCTGGACCCATCGG
2-10-2 MOE
754


 


372536
1199
1214
CACCAGCCCCCAGGTG
3-10-3 MOE
755


 


372614
1200
1213
ACCAGCCCCCAGGT
2-10-2 MOE
756


 


372537
1204
1219
TAGGGCACCAGCCCCC
3-10-3 MOE
757


 


372615
1205
1218
AGGGCACCAGCCCC
2-10-2 MOE
758


 


372538
1209
1224
TGGAGTAGGGCACCAG
3-10-3 MOE
759


 


372616
1210
1223
GGAGTAGGGCACCA
2-10-2 MOE
760


 


382616
1215
1226
CTTGGAGTAGGG
1-10-1 MOE
761


 


372539
1218
1233
TGATGGGCTTGGAGTA
3-10-3 MOE
762


 


372617
1219
1232
GATGGGCTTGGAGT
2-10-2 MOE
763


 


372540
1293
1308
TGTGGTACAGGTCGAT
3-10-3 MOE
764


 


372618
1294
1307
GTGGTACAGGTCGA
2-10-2 MOE
765


 


382617
1294
1305
GGTACAGGTCGA
1-10-1 MOE
766


 


372541
1295
1310
GGTGTGGTACAGGTCG
3-10-3 MOE
767


 


372619
1296
1309
GTGTGGTACAGGTC
2-10-2 MOE
768


 


372542
1298
1313
CATGGTGTGGTACAGG
3-10-3 MOE
769


 


372620
1299
1312
ATGGTGTGGTACAG
2-10-2 MOE
770


 


372543
1300
1315
TACATGGTGTGGTACA
3-10-3 MOE
771


 


372621
1301
1314
ACATGGTGTGGTAC
2-10-2 MOE
772


 


372544
1303
1318
ATGTACATGGTGTGGT
3-10-3 MOE
773


 


372622
1304
1317
TGTACATGGTGTGG
2-10-2 MOE
774


 


382618
1313
1324
GCCTCCATGTAC
1-10-1 MOE
775


 


382619
1325
1336
AGCTTCACCAGG
1-10-1 MOE
776


 


382620
1383
1394
GTTCACCTCCAG
1-10-1 MOE
777
















TABLE 15







Short antisense compounds targeted to


SEQ ID NO: 10 and having 1 or 2 mismatches













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO
Site
Site
Sequence (5′-3′)
Motif
NO















372608
151
164
CGGCCCCAGGAGCC
2-10-2 MOE
747


 


372474
156
171
CATGCCCCAGCCGCCG
3-10-3 MOE
778


 


372552
157
170
ATGCCCCAGCCGCC
2-10-2 MOE
779


 


382609
167
178
CCAGGCCCATGA
1-10-1 MOE
729


 


372478
230
245
GATGAGGGTCTTCATG
3-10-3 MOE
780


 


372479
248
263
GACCCCGGAGTAGGCA
3-10-3 MOE
781


 


382611
317
328
GACAGGGCAGAT
1-10-1 MOE
735


 


372483
352
367
ATGCTGGAGCCAGTGC
3-10-3 MOE
782


 


372561
353
366
TGCTGGAGCCAGTG
2-10-2 MOE
783


 


372562
373
386
GTCTTGGAGGGCCG
2-10-2 MOE
784


 


382602
388
399
TGACAGGGCAGA
1-10-1 MOE
736


 


372613
392
405
CCCAGGTGTCAGAG
2-10-2 MOE
785


 


372486
412
427
TTTTCCACCTTGGATC
3-10-3 MOE
786


 


372564
413
426
TTTCCACCTTGGAT
2-10-2 MOE
787


 


372487
413
428
TTTTTCCACCTTGGAT
3-10-3 MOE
788


 


372488
418
433
AGGTGTTTTTCCACCT
3-10-3 MOE
789


 


372566
419
432
GGTGTTTTTCCACC
2-10-2 MOE
790


 


372489
459
474
CCAGGAAGGATAGGAC
3-10-3 MOE
791


 


372567
460
473
CAGGAAGGATAGGA
2-10-2 MOE
792


 


382612
475
486
CCACTCCCATTC
1-10-1 MOE
737


 


372490
483
498
TGACACTGCAGGCCAC
3-10-3 MOE
793


 


372568
484
497
GACACTGCAGGCCA
2-10-2 MOE
794


 


372491
492
507
ACATGAGGATGACACT
3-10-3 MOE
795


 


372569
493
506
CATGAGGATGACAC
2-10-2 MOE
796


 


372492
503
518
GCAGAAGGTGTACATG
3-10-3 MOE
797


 


372570
504
517
CAGAAGGTGTACAT
2-10-2 MOE
798


 


372493
512
527
GCAGTCAGTGCAGAAG
3-10-3 MOE
799


 


372571
513
526
CAGTCAGTGCAGAA
2-10-2 MOE
800


 


372496
612
627
ACACGGCCCAGTTTCG
3-10-3 MOE
801


 


372574
613
626
CACGGCCCAGTTTC
2-10-2 MOE
802


 


372513
717
732
GGCCCATGATGCCATG
3-10-3 MOE
803


 


372591
718
731
GCCCATGATGCCAT
2-10-2 MOE
804


 


372516
732
747
TACAGAAGGCACCCAG
3-10-3 MOE
805


 


372594
733
746
ACAGAAGGCACCCA
2-10-2 MOE
806


 


372518
812
827
GAAGTTGCCAGCCAAT
3-10-3 MOE
807


 


372596
813
826
AAGTTGCCAGCCAA
2-10-2 MOE
808


 


372560
863
876
CAGGGCAGATCCTT
2-10-2 MOE
809


 


372519
887
902
CAAGTAGTCTATGGTG
3-10-3 MOE
810


 


372597
888
901
AAGTAGTCTATGGT
2-10-2 MOE
811


 


372520
894
909
TGGAAAGCAAGTAGTC
3-10-3 MOE
812


 


372598
895
908
GGAAAGCAAGTAGT
2-10-2 MOE
813


 


372527
1013
1028
GGCCAGCTTTACAAAG
3-10-3 MOE
814


 


372605
1014
1027
GCCAGCTTTACAAA
2-10-2 MOE
815


 


372606
1020
1033
CGCAGGGCCAGCTT
2-10-2 MOE
816


 


372529
1052
1067
AAAGGAATAGGTGGGA
3-10-3 MOE
817


 


372607
1053
1066
AAGGAATAGGTGGG
2-10-2 MOE
818


 


372534
1144
1159
GCGAAACCAATATACT
3-10-3 MOE
819


 


372612
1145
1158
CGAAACCAATATAC
2-10-2 MOE
820


 


372535
1192
1207
CCCCAGGTGTCAGAGG
3-10-3 MOE
821


 


372613
1193
1206
CCCAGGTGTCAGAG
2-10-2 MOE
822


 


372545
1332
1347
GATTGTCAAAGAGCTT
3-10-3 MOE
823


 


372623
1333
1346
ATTGTCAAAGAGCT
2-10-2 MOE
824


 


372546
1342
1357
TTGGTCTTGTGATTGT
3-10-3 MOE
825


 


372624
1343
1356
TGGTCTTGTGATTG
2-10-2 MOE
826


 


372547
1352
1367
AAGGCCGAATTTGGTC
3-10-3 MOE
827


 


372625
1353
1366
AGGCCGAATTTGGT
2-10-2 MOE
828


 


382601
1617
1628
CCCGGAGTAGGC
1-10-1 MOE
683


 


382606
1971
1982
CTGCAGGCCACT
1-10-1 MOE
694


 


382612
1988
1999
CCACTCCCATTC
1-10-1 MOE
737









In certain embodiments, a target region is nucleotides 231-267 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 231-267 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 231-267 comprises a nucleotide sequence selected from SEQ ID NO 681, 682, 683, 684, 685, 686, or 687. In certain such embodiments, a short antisense compound targeted to nucleotides 231-267 of SEQ ID NO: 10 is selected from Isis No 372556, 372557, 382601, 372480, 372481, 372558, or 372559.
In certain embodiments, a target region is nucleotides 249-267 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 249-267 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 249-267 comprises a nucleotide sequence selected from SEQ ID NO 683, 684, 685, 686, or 687. In certain such embodiments, a short antisense compound targeted to nucleotides 249-267 of SEQ ID NO: 10 is selected from Isis No 382601, 372480, 372481, 372558, or 372559.
In certain embodiments, a target region is nucleotides 331-493 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 331-493 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 331-493 comprises a nucleotide sequence selected from SEQ ID NO 688, 689, 690, 691, 692, 693, or 694. In certain such embodiments, a short antisense compound targeted to nucleotides 331-493 of SEQ ID NO: 10 is selected from Isis No 382603, 382604, 372485, 372563, 382605, 372565, or 382606.
In certain embodiments, a target region is nucleotides 331-427 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 331-427 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 331-427 comprises a nucleotide sequence selected from SEQ ID NO 688, 689, 690, 691, 692, or 693. In certain such embodiments, a short antisense compound targeted to nucleotides 331-427 of SEQ ID NO: 10 is selected from Isis No 382603, 382604, 372485, 372563, 382605, or 372565.
In certain embodiments, a target region is nucleotides 392-408 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 392-408 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 392-408 comprises a nucleotide sequence selected from SEQ ID NO 690, 691, or 692. In certain such embodiments, a short antisense compound targeted to nucleotides 392-408 of SEQ ID NO: 10 is selected from Isis No 372485, 372563, or 382605.
In certain embodiments, a target region is nucleotides 651-707 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 651-707 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 651-707 comprises a nucleotide sequence selected from SEQ ID NO 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, or 728. In certain such embodiments, a short antisense compound targeted to nucleotides 651-707 of SEQ ID NO: 10 is selected from Isis No 372497, 372498, 372575, 372576, 382607, 372499, 372577, 372500, 372578, 372501, 372579, 372502, 372580, 372503, 372581, 372504, 372582, 372505, 372506, 372583, 372584, 372507, 372585, 382608, 372508, 372586, 372509, 372587, 372510, 372588, 372511, 372512, 372589, or 372590.
In certain embodiments, a target region is nucleotides 724-745 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 724-745 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 724-745 comprises a nucleotide sequence selected from SEQ ID NO 729, 730, 731, 732, or 733. In certain such embodiments, a short antisense compound targeted to nucleotides 724-745 of SEQ ID NO: 10 is selected from Isis No 382609, 372514, 372592, 372515, or 372593.
In certain embodiments, a target region is nucleotides 651-745 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 651-745 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 651-745 comprises a nucleotide sequence selected from SEQ ID NO 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, or 733. In certain such embodiments, a short antisense compound targeted to nucleotides 651-745 of SEQ ID NO: 10 is selected from Isis No 372497, 372498, 372575, 372576, 382607, 372499, 372577, 372500, 372578, 372501, 372579, 372502, 372580, 372503, 372581, 372504, 372582, 372505, 372506, 372583, 372584, 372507, 372585, 382608, 372508, 372586, 372509, 372587, 372510, 372588, 372511, 372512, 372589, 372590, 382609, 372514, 372592, 372515, or 372593.
In certain embodiments, a target region is nucleotides 851-922 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 851-922 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 851-922 comprises a nucleotide sequence selected from SEQ ID NO 734, 735, 736, or 737. In certain such embodiments, a short antisense compound targeted to nucleotides 851-922 of SEQ ID NO: 10 is selected from Isis No 382610, 382611, 382602, or 382612.
In certain embodiments, a target region is nucleotides 851-879 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 851-879 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 851-879 comprises a nucleotide sequence selected from SEQ ID NO 734, 735, or 736. In certain such embodiments, a short antisense compound targeted to nucleotides 851-879 of SEQ ID NO: 10 is selected from Isis No 382610, 382611, or 382602.
In certain embodiments, a target region is nucleotides 965-1007 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 965-1007 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 965-1007 comprises a nucleotide sequence selected from SEQ ID NO 738, 739, 740, 741, 742, 743, 744, or 745. In certain such embodiments, a short antisense compound targeted to nucleotides 965-1007 of SEQ ID NO: 10 is selected from Isis No 372524, 372602, 382613, 382614, 372525, 372603, 372526, or 372604.
In certain embodiments, a target region is nucleotides 965-979 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 965-979 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 965-979 comprises a nucleotide sequence selected from SEQ ID NO 738, 739, or 740. In certain such embodiments, a short antisense compound targeted to nucleotides 965-979 of SEQ ID NO: 10 is selected from Isis No 372524, 372602, or 382613.
In certain embodiments, a target region is nucleotides 987-1007 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 987-1007 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 987-1007 comprises a nucleotide sequence selected from SEQ ID NO 741, 742, 743, 744, or 745. In certain such embodiments, a short antisense compound targeted to nucleotides 987-1007 of SEQ ID NO: 10 is selected from Isis No 382614, 372525, 372603, 372526, or 372604.
In certain embodiments, a target region is nucleotides 1106-1132 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 1106-1132 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 1106-1132 comprises a nucleotide sequence selected from SEQ ID NO 746, 747, 748, 749, 750, 751, 752, 753, or 754. In certain such embodiments, a short antisense compound targeted to nucleotides 1106-1132 of SEQ ID NO: 10 is selected from Isis No 372530, 372608, 372531, 372609, 372532, 372610, 372533, 382615, or 372611.
In certain embodiments, a target region is nucleotides 1199-1233 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 1199-1233 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 1199-1233 comprises a nucleotide sequence selected from SEQ ID NO 755, 756, 757, 758, 759, 760, 761, 762, or 763. In certain such embodiments, a short antisense compound targeted to nucleotides 1199-1233 of SEQ ID NO: 10 is selected from Isis No 372536, 372614, 372537, 372615, 372538, 372616, 382616, 372539, or 372617.
In certain embodiments, a target region is nucleotides 1293-1394 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 1293-1394 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1394 comprises a nucleotide sequence selected from SEQ ID NO 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, or 777. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1394 of SEQ ID NO: 10 is selected from Isis No 372540, 372618, 382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544, 372622, 382618, 382619, or 382620.
In certain embodiments, a target region is nucleotides 1293-1336 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 1293-1336 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1336 comprises a nucleotide sequence selected from SEQ ID NO 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, or 776. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1336 of SEQ ID NO: 10 is selected from Isis No 372540, 372618, 382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544, 372622, 382618, or 382619.
In certain embodiments, a target region is nucleotides 1293-1324 of SEQ ID NO: 10. In certain embodiments, a short antisense compound is targeted to nucleotides 1293-1324 of SEQ ID NO: 10. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1324 comprises a nucleotide sequence selected from SEQ ID NO 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, or 775. In certain such embodiments, a short antisense compound targeted to nucleotides 1293-1324 of SEQ ID NO: 10 is selected from Isis No 372540, 372618, 382617, 372541, 372619, 372542, 372620, 372543, 372621, 372544, 372622, or 382618.
In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a DGAT2 nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) and O—CH2—C(═O)—N(Rm)(Rm), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in a short antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a DGAT2 nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of DGAT2. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a DGAT2 nucleic acid, wherein the short antisense compound targeted to a DGAT2 nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of DGAT2 using one or more short antisense compounds targeted to a DGAT2 nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of DGAT2 comprise use of a short antisense compound targeted to a DGAT2 nucleic acid comprising 12 or 14 nucleotides or nucleosides.
9. PTP1B
PTP1B (also known as protein phosphatase 1B and PTPN1) is an endoplasmic reticulum (ER)-associated enzyme originally isolated as the major protein tyrosine phosphatase of the human placenta (Tonks et al., J. Biol. Chem., 1988, 263, 6731-6737; Tonks et al., J. Biol. Chem., 1988, 263, 6722-6730).
An essential regulatory role in signaling mediated by the insulin receptor has been established for PTP1B. In certain instances, PTP1B interacts with and dephosphorylates the activated insulin receptor both in vitro and in intact cells resulting in the downregulation of the signaling pathway (Goldstein et al., Mol. Cell. Biochem., 1998, 182, 91-99; Seely et al., Diabetes, 1996, 45, 1379-1385). In addition, PTP1B modulates the mitogenic actions of insulin (Goldstein et al., Mol. Cell. Biochem., 1998, 182, 91-99). In rat adipose cells overexpressing PTP1B, the translocation of the GLUT4 glucose transporter was inhibited, implicating PTP1B as a negative regulator of glucose transport as well (Chen et al., J. Biol. Chem., 1997, 272, 8026-8031).
Mouse knockout models lacking the PTP1B gene also point toward the negative regulation of insulin signaling by PTP1B. Mice harboring a disrupted PTP1B gene showed increased insulin sensitivity and increased phosphorylation of the insulin receptor. When placed on a high-fat diet, PTP1B −/− mice were resistant to weight gain and remained insulin sensitive (Elchebly et al., Science, 1999, 283, 1544-1548). These studies clearly establish PTP1B as a therapeutic target in the treatment of diabetes and obesity.
Diabetes and obesity (sometimes now collectively referred to as “diabesity”) are interrelated. Most human obesity is associated with insulin resistance and leptin resistance. In fact obesity may have an even greater impact on insulin action than does diabetes itself (Sindelka et al., Physiol Res., 2002, 51, 85-91). Syndrome X or metabolic syndrome is a new term for a cluster of conditions, that, when occurring together, may indicate a predisposition to diabetes and cardiovascular disease. These symptoms, including high blood pressure, high triglycerides, decreased HDL and obesity, tend to appear together in some individuals. Because of its role in both diabetes and obesity, PTP1B is believed to be a therapeutic target for a range of metabolic conditions, including diabetes, obesity and metabolic syndrome. By improving blood glucose control, inhibitors of PTP1B may also be useful in slowing, preventing, delaying or ameliorating the sequelae of diabetes, which include retinopathy, neuropathy, cardiovascular complications and nephropathy.
PTP1B, which is differentially regulated during the cell cycle (Schievella et al., Cell. Growth Differ., 1993, 4, 239-246), is expressed in insulin sensitive tissues as two different isoforms that arise from alternate splicing of the pre-mRNA (Shifrin and Neel, J. Biol. Chem., 1993, 268, 25376-25384). The ratio of the alternatively spliced products is affected by growth factors, such as insulin, and differs in various tissues examined (Sell and Reese, Mol. Genet. Metab., 1999, 66, 189-192). In these studies the levels of the variants correlated with the plasma insulin concentration and percentage body fat. These variants may therefore be used as a biomarker for patients with chronic hyperinsulinemia or type 2 diabetes.
Definitions
“Protein tyrosine phosphatase 1B” is the gene product or protein of which expression is to be modulated by administration of a short antisense compound. Protein tyrosine phosphatase 1B is generally referred to as PTP1B but may also be referred to as protein tyrosine phosphatase; PTPN1; RKPTP; protein tyrosine phosphatase, non-receptor type 1.
“PTP1B nucleic acid” means any nucleic acid encoding PTP1B. For example, in certain embodiments, a PTP1B nucleic acid includes, without limitation, a DNA sequence encoding PTP1B, an RNA sequence transcribed from DNA encoding PTP1B, and an mRNA sequence encoding PTP1B. “PTP1B mRNA” means an mRNA encoding a PTP1B protein.
Therapeutic Indications
Antisense technology is an effective means for reducing PTP1B expression and has proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications. As such, in certain embodiments, the present invention provides compounds targeted to a nucleic acid encoding PTP1B, which modulate the expression of PTP1B. Further provided herein are short antisense compounds capable of effectively inhibiting PTP1B expression.
In certain therapeutics, a subject, suspected of having a disease or disorder which can be treated by modulating the expression of PTP1B is treated by administering one or more short antisense compounds targeted to a nucleic acid encoding PTP1B. For example, in one non-limiting embodiment, the methods comprise the step of administering to an animal a therapeutically effective amount of a short antisense compound. The short antisense compounds of the present invention effectively inhibit the activity of PTP1B or inhibit the expression of PTP1B. In one embodiment, the activity or expression of PTP1B in a subject is inhibited by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%. In certain embodiments, activity or expression of PTP1B in a subject is inhibited by about 30%. In certain embodiments, the activity or expression of PTP1B in a subject is inhibited by 50% or more.
The reduction of the expression of PTP1B may be measured, for example, in blood, plasma, serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding PTP1B and/or the PTP1B protein itself.
Certain pharmaceutical and other compositions comprising the compounds of the invention are also provided. In certain embodiments short antisense compounds targeted to a PTP1B nucleic acid are utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
The short antisense compounds targeting PTP1B may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a PTP1B nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.
In certain embodiments provided herein are methods of treating an individual by administering one or more short antisense compound targeted to a PTP1B nucleic acid or a pharmaceutical composition comprising such compound. Further provided are methods of treating a subject having a disease or conditions associated with PTP1B activity by administering a short antisense compound targeted to a PTP1B nucleic acid. Diseases and conditions associated with PTP1B include but are not limited to high blood glucose or hyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolic syndrome, obesity and insulin resistance. Therefore, provided herein are methods of treating to high blood glucose or hyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolic syndrome, obesity and insulin resistance by administering a short antisense compound targeted to a PTP1B nucleic acid.
In certain embodiments the present invention provides compositions and methods for decreasing blood glucose levels in a subject or for preventing or delaying the onset of a rise in blood glucose levels in a subject, by administering to the subject a short antisense inhibitor of PTP1B expression.
In certain embodiments, the present invention provides compositions and methods for improving insulin sensitivity in a subject or for preventing or delaying the onset of insulin resistance in a subject, by administering to the subject a short antisense inhibitor of PTP1B expression.
In certain embodiments, the present invention provides compositions and methods for treating a metabolic condition in a subject or for preventing or delaying the onset of a metabolic condition in a subject, by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. Such metabolic condition may be any metabolic condition associated with PTP1B expression, including but not limited to diabetes and obesity. Also provided are methods of reducing adiposity. Also provided is a method of treating obesity wherein metabolic rate is increased.
In certain embodiments, the subject has Type 2 diabetes. In certain embodiments the subject exhibits elevated HbA1c levels In certain embodiments, HbA1c levels are at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10% or at least about 11%. In preferred embodiments, HbA1c levels are reduced to about 7% or below about 7%. In certain embodiments, the subject exhibits an elevated body mass index In certain embodiments, the elevated body mass index is greater than 25 kg/m2. In certain embodiments, the subject exhibits hyperglycemia or elevated blood glucose levels. In a particular embodiment, the blood glucose levels are fasting blood glucose levels. In certain embodiments, the elevated fasting blood glucose levels are at least 130 mg/dL. In certain embodiments, the subject exhibits hyperglycemia prior to the start of treatment or exhibits fasting blood glucose levels above about 130 mg/dL, baseline HbA1c levels of at least about 7%, or body mass index of greater than 25 kg/m2 or any combination thereof.
In certain embodiments a method of reducing one or more such levels by administering a short antisense compound targeted to a PTP1B nucleic acid is provided. For example, provided is a method of reducing fasting glucose levels, HbA1c levels or, body mass index levels or any combination thereof in a subject by administering to a subject a short antisense compound targeting PTP1B. Fasting glucose may be fasting blood glucose, fasting serum glucose, or fasting plasma glucose. In some embodiments, fasting plasma glucose levels are reduced by at least about 25 mg/dL or by at least about 10 mg/dL. In a certain embodiments, said subject does not achieve normal glucose levels on a therapeutic regimen of a glucose-lowering agent such as insulin, sulfonylurea, or metformin.
In certain embodiments the invention provides methods of altering lipid levels. Certain such methods reduce cholesterol, LDL and/or VLDL levels or any combination thereof in a subject by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. In certain embodiments HDL levels in a subject are increased by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. In certain embodiments, LDL:HDL ratio and/or total cholesterol:HDL ratio in a subject is reduced by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. In certain embodiments HDL:LDL ratio and/or HDL:total cholesterol ratio in a subject's increased by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. In certain embodiments lipid profile in a subject is improved by increasing HDL, lowering LDL, lowering VLDL, lowering triglycerides, lowering apolipoprotein B levels, or lowering total cholesterol levels, or a combination thereof, by administering to the subject a short antisense compound targeted to a PTP1B nucleic acid. In such embodiments, the subject is an animal, including a human.
Combination Therapy
In certain embodiments, one or more pharmaceutical compositions comprising a short antisense compound targeted to a PTP1B nucleic acid are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to a PTP1B nucleic acid include glucose-lowering agents and therapies. In some embodiments, the glucose-lowering agent is a PPAR agonist (gamma, dual, or pan), a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, a meglitinide, a thiazolidinedione, or a sulfonylurea.
In some embodiments, the glucose-lowering therapeutic is a GLP-1 analog. In some embodiments, the GLP-1 analog is exendin-4 or liraglutide.
In other embodiments, the glucose-lowering therapeutic is a sulfonylurea. In some embodiments, the sulfonylurea is acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a gliclazide.
In some embodiments, the glucose lowering drug is a biguanide. In some embodiments, the biguanide is metformin, and in some embodiments, blood glucose levels are decreased without increased lactic acidosis as compared to the lactic acidosis observed after treatment with metformin alone.
In some embodiments, the glucose lowering drug is a meglitinide. In some embodiments, the meglitinide is nateglinide or repaglinide.
In some embodiments, the glucose-lowering drug is a thiazolidinedione. In some embodiments, the thiazolidinedione is pioglitazone, rosiglitazone, or troglitazone. In some embodiments, blood glucose levels are decreased without greater weight gain than observed with rosiglitazone treatment alone.
In some embodiments, the glucose-lowering drug is an alpha-glucosidase inhibitor. In some embodiments, the alpha-glucosidase inhibitor is acarbose or miglitol.
In a certain embodiment, a co-administered glucose-lowering agent is ISIS 113715.
In a certain embodiment, glucose-lowering therapy is therapeutic lifestyle change.
In certain such embodiments, the glucose-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the glucose-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the glucose-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered glucose-lowering agent is the same as the dose that would be administered if the glucose-lowering agent was administered alone. In certain such embodiments the dose of a co-administered glucose-lowering agent is lower than the dose that would be administered if the glucose-lowering agent was administered alone. In certain such embodiments the dose of a co-administered glucose-lowering agent is greater than the dose that would be administered if the glucose-lowering agent was administered alone.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to a PTP1B nucleic acid include lipid-lowering agents. Such lipid lowering agents are discussed elsewhere in the application and are included here with respect to PTP1B. Such lipid lowering agents may be administered as described above for glucose lowering agents.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising a short antisense compound targeted to a PTP1B nucleic acid include anti-obesity agents therapeutics. Such anti-obesity agents therapeutics may be administered as described above for glucose lowering agents.
Further provided is a method of administering a short antisense compound targeted to a PTP1B nucleic acid via injection and further including administering a topical steroid at the injection site.
Medicaments
Also provided herein are uses of a short antisense compound which is targeted to a PTP1B nucleic acid for the preparation of a medicament for reducing blood glucose levels including fasting glucose levels, and HbA1c levels, body mass index levels or any combination thereof. The medicament can be administered during a loading period and a maintenance period. In some embodiments, the medicament is administered subcutaneously or intravenously. In other embodiments, the administration of said medicament occurs at least once daily, at least once weekly, or at least once monthly. In a particular embodiment the short antisense compound present in the medicament is administered in a dose lower than a short antisense compound with a longer sequence and particularly a sequence 20 or more nucleobases. The medicament may be administered to a subject that exhibits high blood glucose or hyperglycemia, prediabetes, diabetes, Type 2 diabetes, metabolic syndrome, obesity and insulin resistance.
Other aspects and advantages of short antisense compounds are provided herein. All aspect and advantages disclosed herein and specifically with regard to other targets is applicable with regard to compositions including short antisense compounds targeted to a PTP1B nucleic acid and methods of their use.
Certain Short Antisense Compounds Targeted to a PTP1B Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a PTP1B nucleic acid having the sequence of GENBANK® Accession No. NM_002827.2, incorporated herein as SEQ ID NO: 11 or the nucleotides 14178000 to 1425600 of the sequence of GENBANK® Accession No. NT_011362.9, incorporated herein as SEQ ID NO: 12. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 11 is at least 90% complementary to SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 11 is at least 95% complementary to SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 12 is 100% complementary to SEQ ID NO: 12. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 12 is at least 90% complementary to SEQ ID NO: 12. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 12 is at least 95% complementary to SEQ ID NO: 12. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 12 is 100% complementary to SEQ ID NO: 12.
In certain embodiments, a short antisense compound targeted to SEQ ID NO: 11 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Tables 16 and 17. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 12 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Tables 18 and 19.
Each nucleotide sequence set forth in each Tables 16, 17, 18, and 19 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds comprising a nucleotide sequence as set forth in Tables 16, 17, 18, and 19 may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Tables 16 and 17 illustrate examples of short antisense compounds targeted to SEQ ID NO: 11. Table 16 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 11. Table 17 illustrates short antisense compounds that have one or two mismatches with respect to SEQ ID NO: 11. Table 18 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 12. Table 19 illustrates short antisense compounds that have 1 or 2 mismatches with respect to SEQ ID NO: 12. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.







TABLE 16







Short Antisense Compounds targeted


to SEQ ID NO: 11













5′
3′


SEQ


ISIS
Target
Target

Gapmer
ID


NO.
Site
Site
Sequence (5′-3′)
Motif
NO















147022
177
188
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
178
189
CTTGTCGATCTC
1-10-1 MOE
859


 


147024
179
190
CCTTGTCGATCT
1-10-1 MOE
853


 


147019
195
206
TCGATCTCCTCG
1-10-1 MOE
877


 


147020
196
207
GTCGATCTCCTC
1-10-1 MOE
868


 


147021
197
208
TGTCGATCTCCT
1-10-1 MOE
882


 


147022
198
209
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
199
210
CTTGTCGATCTC
1-10-1 MOE
859


 


147024
200
211
CCTTGTCGATCT
1-10-1 MOE
853


 


147025
201
212
GCCTTGTCGATC
1-10-1 MOE
865


 


147026
202
213
AGCCTTGTCGAT
1-10-1 MOE
835


 


147027
203
214
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
204
215
CCAGCCTTGTCG
1-10-1 MOE
846


 


147073
204
215
CACTGATCCTGC
1-10-1 MOE
842


 


147029
205
216
CCCAGCCTTGTC
1-10-1 MOE
848


 


147030
206
217
TCCCAGCCTTGT
1-10-1 MOE
874


 


147036
212
223
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
213
224
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
214
225
CGCCCAGTTCCC
1-10-1 MOE
855


 


147039
215
226
CCGCCCAGTTCC
1-10-1 MOE
850


 


147040
216
227
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
217
228
AGCCGCCCAGTT
1-10-1 MOE
834


 


147073
311
322
CACTGATCCTGC
1-10-1 MOE
842


 


147042
323
334
GGTCAAAAGGGC
1-10-1 MOE
866


 


147043
324
335
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
325
336
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
326
337
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
327
338
CTGTGGTCAAAA
1-10-1 MOE
858


 


147047
328
339
ACTGTGGTCAAA
1-10-1 MOE
833


 


147051
332
343
TCCGACTGTGGT
1-10-1 MOE
875


 


147052
333
344
ATCCGACTGTGG
1-10-1 MOE
837


 


147053
334
345
AATCCGACTGTG
1-10-1 MOE
829


 


147054
335
346
TAATCCGACTGT
1-10-1 MOE
871


 


147055
336
347
TTAATCCGACTG
1-10-1 MOE
884


 


147056
337
348
TTTAATCCGACT
1-10-1 MOE
887


 


147057
338
349
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
339
350
AATTTAATCCGA
1-10-1 MOE
830


 


147059
340
351
CAATTTAATCCG
1-10-1 MOE
840


 


147060
341
352
GCAATTTAATCC
1-10-1 MOE
861


 


147061
342
353
TGCAATTTAATC
1-10-1 MOE
879


 


147045
679
690
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
680
691
CTGTGGTCAAAA
1-10-1 MOE
858


 


147045
787
798
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
788
799
CTGTGGTCAAAA
1-10-1 MOE
858


 


147066
816
827
CCTGCACTGACG
1-10-1 MOE
851


 


404131
992
1005
ACCTTCGATCACAG
2-10-2 MOE
831


 


147062
1024
1035
CACTGACGAGTC
1-10-1 MOE
841


 


147063
1025
1036
GCACTGACGAGT
1-10-1 MOE
862


 


147064
1026
1037
TGCACTGACGAG
1-10-1 MOE
880


 


147065
1027
1038
CTGCACTGACGA
1-10-1 MOE
857


 


147066
1028
1039
CCTGCACTGACG
1-10-1 MOE
851


 


147067
1029
1040
TCCTGCACTGAC
1-10-1 MOE
876


 


147068
1030
1041
ATCCTGCACTGA
1-10-1 MOE
838


 


147069
1031
1042
GATCCTGCACTG
1-10-1 MOE
860


 


147070
1032
1043
TGATCCTGCACT
1-10-1 MOE
878


 


147071
1033
1044
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
1034
1045
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
1035
1046
CACTGATCCTGC
1-10-1 MOE
842


 


147067
1199
1210
TCCTGCACTGAC
1-10-1 MOE
876


 


147040
1288
1299
GCCGCCCAGTTC
1-10-1 MOE
864


 


147040
1396
1407
GCCGCCCAGTTC
1-10-1 MOE
864


 


147022
1868
1879
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
1869
1880
CTTGTCGATCTC
1-10-1 MOE
859


 


147024
1870
1881
CCTTGTCGATCT
1-10-1 MOE
853


 


147019
1886
1897
TCGATCTCCTCG
1-10-1 MOE
877


 


147020
1887
1898
GTCGATCTCCTC
1-10-1 MOE
868


 


147021
1888
1899
TGTCGATCTCCT
1-10-1 MOE
882


 


147022
1889
1900
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
1890
1901
CTTGTCGATCTC
1-10-1 MOE
859


 


147025
1892
1903
GCCTTGTCGATC
1-10-1 MOE
865


 


147027
1894
1905
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
1895
1906
CCAGCCTTGTCG
1-10-1 MOE
846


 


147030
1897
1908
TCCCAGCCTTGT
1-10-1 MOE
874


 


147037
1904
1915
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
1905
1916
CGCCCAGTTCCC
1-10-1 MOE
855


 


147040
1907
1918
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
1908
1919
AGCCGCCCAGTT
1-10-1 MOE
834


 


147022
1976
1987
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
1977
1988
CTTGTCGATCTC
1-10-1 MOE
859


 


147024
1978
1989
CCTTGTCGATCT
1-10-1 MOE
853


 


147020
1995
2006
GTCGATCTCCTC
1-10-1 MOE
868


 


147021
1996
2007
TGTCGATCTCCT
1-10-1 MOE
882


 


147022
1997
2008
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
1998
2009
CTTGTCGATCTC
1-10-1 MOE
859


 


147024
1999
2010
CCTTGTCGATCT
1-10-1 MOE
853


 


147025
2000
2011
GCCTTGTCGATC
1-10-1 MOE
865


 


147026
2001
2012
AGCCTTGTCGAT
1-10-1 MOE
835


 


147027
2002
2013
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
2003
2014
CCAGCCTTGTCG
1-10-1 MOE
846


 


147029
2004
2015
CCCAGCCTTGTC
1-10-1 MOE
848


 


147030
2005
2016
TCCCAGCCTTGT
1-10-1 MOE
874


 


147036
2011
2022
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
2012
2023
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
2013
2024
CGCCCAGTTCCC
1-10-1 MOE
855


 


147039
2014
2025
CCGCCCAGTTCC
1-10-1 MOE
850


 


147040
2015
2026
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
2016
2027
AGCCGCCCAGTT
1-10-1 MOE
834


 


404199
2366
2379
GGTCATGCACAGGC
2-10-2 MOE
867


 


404134
2369
2382
TCAGGTCATGCACA
2-10-2 MOE
873


 


404132
2548
2561
CCTTGGAATGTCTG
2-10-2 MOE
852


 


147020
2613
2624
GTCGATCTCCTC
1-10-1 MOE
868


 


147020
2721
2732
GTCGATCTCCTC
1-10-1 MOE
868


 


404133
3289
3302
TATTCCATGGCCAT
2-10-2 MOE
872


 


147032
6220
6231
GTTCCCAGCCTT
1-10-1 MOE
870


 


147033
6221
6232
AGTTCCCAGCCT
1-10-1 MOE
836


 


147034
6222
6233
CAGTTCCCAGCC
1-10-1 MOE
844


 


147044
6288
6299
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
6289
6300
TGTGGTCAAAAG
1-10-1 MOE
883


 


147032
6329
6340
GTTCCCAGCCTT
1-10-1 MOE
870


 


147033
6330
6341
AGTTCCCAGCCT
1-10-1 MOE
836


 


147034
6331
6342
CAGTTCCCAGCC
1-10-1 MOE
844


 


147044
6397
6408
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
6398
6409
TGTGGTCAAAAG
1-10-1 MOE
883


 


147058
7057
7068
AATTTAATCCGA
1-10-1 MOE
830


 


147059
7058
7069
CAATTTAATCCG
1-10-1 MOE
840


 


147060
7059
7070
GCAATTTAATCC
1-10-1 MOE
861


 


147058
7166
7177
AATTTAATCCGA
1-10-1 MOE
830


 


147059
7167
7178
CAATTTAATCCG
1-10-1 MOE
840


 


147041
8084
8095
AGCCGCCCAGTT
1-10-1 MOE
834


 


147041
8192
8203
AGCCGCCCAGTT
1-10-1 MOE
834


 


147027
8630
8641
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
8631
8642
CCAGCCTTGTCG
1-10-1 MOE
846


 


147027
8738
8749
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
8739
8750
CCAGCCTTGTCG
1-10-1 MOE
846


 


147043
10957
10968
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
10958
10969
GTGGTCAAAAGG
1-10-1 MOE
869


 


147043
11065
11076
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
11066
11077
GTGGTCAAAAGG
1-10-1 MOE
869


 


147071
11605
11616
CTGATCCTGCAC
1-10-1 MOE
856


 


147070
11611
11622
TGATCCTGCACT
1-10-1 MOE
878


 


147071
11612
11623
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
12294
12305
ACTGATCCTGCA
1-10-1 MOE
832


 


147072
12299
12310
ACTGATCCTGCA
1-10-1 MOE
832


 


147030
12805
12816
TCCCAGCCTTGT
1-10-1 MOE
874


 


147031
12806
12817
TTCCCAGCCTTG
1-10-1 MOE
885


 


147053
12939
12950
AATCCGACTGTG
1-10-1 MOE
829


 


147030
12986
12997
TCCCAGCCTTGT
1-10-1 MOE
874


 


147031
12987
12998
TTCCCAGCCTTG
1-10-1 MOE
885


 


147053
13120
13131
AATCCGACTGTG
1-10-1 MOE
829


 


147051
13162
13173
TCCGACTGTGGT
1-10-1 MOE
875


 


147061
13316
13327
TGCAATTTAATC
1-10-1 MOE
879


 


147047
13339
13350
ACTGTGGTCAAA
1-10-1 MOE
833


 


147029
14058
14069
CCCAGCCTTGTC
1-10-1 MOE
848


 


147029
14239
14250
CCCAGCCTTGTC
1-10-1 MOE
848


 


147067
15560
15571
TCCTGCACTGAC
1-10-1 MOE
876


 


147068
15561
15572
ATCCTGCACTGA
1-10-1 MOE
838


 


147067
15742
15753
TCCTGCACTGAC
1-10-1 MOE
876


 


147069
15744
15755
GATCCTGCACTG
1-10-1 MOE
860


 


147042
16561
16572
GGTCAAAAGGGC
1-10-1 MOE
866


 


147042
16727
16738
GGTCAAAAGGGC
1-10-1 MOE
866


 


147030
17619
17630
TCCCAGCCTTGT
1-10-1 MOE
874


 


147064
17762
17773
TGCACTGACGAG
1-10-1 MOE
880


 


147030
17787
17798
TCCCAGCCTTGT
1-10-1 MOE
874


 


147064
17930
17941
TGCACTGACGAG
1-10-1 MOE
880


 


147042
19201
19212
GGTCAAAAGGGC
1-10-1 MOE
866


 


147042
19369
19380
GGTCAAAAGGGC
1-10-1 MOE
866


 


147027
21190
21201
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
21191
21202
CCAGCCTTGTCG
1-10-1 MOE
846


 


147027
21358
21369
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
21359
21370
CCAGCCTTGTCG
1-10-1 MOE
846


 


147070
22021
22032
TGATCCTGCACT
1-10-1 MOE
878


 


147070
22189
22200
TGATCCTGCACT
1-10-1 MOE
878


 


147047
22606
22617
ACTGTGGTCAAA
1-10-1 MOE
833


 


147043
24318
24329
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
24319
24330
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
24320
24331
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
24321
24332
CTGTGGTCAAAA
1-10-1 MOE
858


 


147043
24486
24497
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
24487
24498
GTGGTCAAAAGG
1-10-1 MOE
869


 


147046
24489
24500
CTGTGGTCAAAA
1-10-1 MOE
858


 


147047
24490
24501
ACTGTGGTCAAA
1-10-1 MOE
833


 


147040
25065
25076
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
25066
25077
AGCCGCCCAGTT
1-10-1 MOE
834


 


147046
25160
25171
CTGTGGTCAAAA
1-10-1 MOE
858


 


147039
25232
25243
CCGCCCAGTTCC
1-10-1 MOE
850


 


147040
25233
25244
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
25234
25245
AGCCGCCCAGTT
1-10-1 MOE
834


 


147046
25328
25339
CTGTGGTCAAAA
1-10-1 MOE
858


 


147057
25508
25519
ATTTAATCCGAC
1-10-1 MOE
839


 


147061
25512
25523
TGCAATTTAATC
1-10-1 MOE
879


 


147057
25676
25687
ATTTAATCCGAC
1-10-1 MOE
839


 


147069
28878
28889
GATCCTGCACTG
1-10-1 MOE
860


 


147070
28879
28890
TGATCCTGCACT
1-10-1 MOE
878


 


147053
30133
30144
AATCCGACTGTG
1-10-1 MOE
829


 


147053
30278
30289
AATCCGACTGTG
1-10-1 MOE
829


 


147054
30864
30875
TAATCCGACTGT
1-10-1 MOE
871


 


147043
30985
30996
TGGTCAAAAGGG
1-10-1 MOE
881


 


147054
31011
31022
TAATCCGACTGT
1-10-1 MOE
871


 


147043
31133
31144
TGGTCAAAAGGG
1-10-1 MOE
881


 


147036
32233
32244
CCCAGTTCCCAG
1-10-1 MOE
849


 


147072
32372
32383
ACTGATCCTGCA
1-10-1 MOE
832


 


147072
32520
32531
ACTGATCCTGCA
1-10-1 MOE
832


 


147069
33056
33067
GATCCTGCACTG
1-10-1 MOE
860


 


147070
33057
33068
TGATCCTGCACT
1-10-1 MOE
878


 


147071
33058
33069
CTGATCCTGCAC
1-10-1 MOE
856


 


147051
33126
33137
TCCGACTGTGGT
1-10-1 MOE
875


 


147070
33205
33216
TGATCCTGCACT
1-10-1 MOE
878


 


147071
33206
33217
CTGATCCTGCAC
1-10-1 MOE
856


 


147051
33274
33285
TCCGACTGTGGT
1-10-1 MOE
875


 


147046
33318
33329
CTGTGGTCAAAA
1-10-1 MOE
858


 


147049
33321
33332
CGACTGTGGTCA
1-10-1 MOE
854


 


147051
33323
33334
TCCGACTGTGGT
1-10-1 MOE
875


 


147046
33466
33477
CTGTGGTCAAAA
1-10-1 MOE
858


 


147047
33467
33478
ACTGTGGTCAAA
1-10-1 MOE
833


 


147051
33471
33482
TCCGACTGTGGT
1-10-1 MOE
875


 


147046
33640
33651
CTGTGGTCAAAA
1-10-1 MOE
858


 


147051
33645
33656
TCCGACTGTGGT
1-10-1 MOE
875


 


147046
33788
33799
CTGTGGTCAAAA
1-10-1 MOE
858


 


147051
33793
33804
TCCGACTGTGGT
1-10-1 MOE
875


 


147059
35437
35448
CAATTTAATCCG
1-10-1 MOE
840


 


147060
35438
35449
GCAATTTAATCC
1-10-1 MOE
861


 


147060
35586
35597
GCAATTTAATCC
1-10-1 MOE
861


 


147021
36093
36104
TGTCGATCTCCT
1-10-1 MOE
882


 


147061
36250
36261
TGCAATTTAATC
1-10-1 MOE
879


 


147061
36398
36409
TGCAATTTAATC
1-10-1 MOE
879


 


147073
37485
37496
CACTGATCCTGC
1-10-1 MOE
842


 


147073
37633
37644
CACTGATCCTGC
1-10-1 MOE
842


 


147043
40214
40225
TGGTCAAAAGGG
1-10-1 MOE
881


 


147061
40353
40364
TGCAATTTAATC
1-10-1 MOE
879


 


147043
40362
40373
TGGTCAAAAGGG
1-10-1 MOE
881


 


147061
40501
40512
TGCAATTTAATC
1-10-1 MOE
879


 


147031
42527
42538
TTCCCAGCCTTG
1-10-1 MOE
885


 


147032
42528
42539
GTTCCCAGCCTT
1-10-1 MOE
870


 


147034
42530
42541
CAGTTCCCAGCC
1-10-1 MOE
844


 


147031
42675
42686
TTCCCAGCCTTG
1-10-1 MOE
885


 


147032
42676
42687
GTTCCCAGCCTT
1-10-1 MOE
870


 


147033
42677
42688
AGTTCCCAGCCT
1-10-1 MOE
836


 


147034
42678
42689
CAGTTCCCAGCC
1-10-1 MOE
844


 


147074
43848
43859
CCACTGATCCTG
1-10-1 MOE
845


 


147074
43996
44007
CCACTGATCCTG
1-10-1 MOE
845


 


147051
45402
45413
TCCGACTGTGGT
1-10-1 MOE
875


 


147051
45550
45561
TCCGACTGTGGT
1-10-1 MOE
875


 


147074
46125
46136
CCACTGATCCTG
1-10-1 MOE
845


 


147057
46313
46324
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
46314
46325
AATTTAATCCGA
1-10-1 MOE
830


 


147059
46315
46326
CAATTTAATCCG
1-10-1 MOE
840


 


147061
46317
46328
TGCAATTTAATC
1-10-1 MOE
879


 


147057
46461
46472
ATTTAATCCGAC
1-10-1 MOE
839


 


147059
46463
46474
CAATTTAATCCG
1-10-1 MOE
840


 


147061
46465
46476
TGCAATTTAATC
1-10-1 MOE
879


 


147058
47413
47424
AATTTAATCCGA
1-10-1 MOE
830


 


147073
48221
48232
CACTGATCCTGC
1-10-1 MOE
842


 


147073
48369
48380
CACTGATCCTGC
1-10-1 MOE
842


 


147074
48370
48381
CCACTGATCCTG
1-10-1 MOE
845


 


147027
48566
48577
CAGCCTTGTCGA
1-10-1 MOE
843


 


147027
48714
48725
CAGCCTTGTCGA
1-10-1 MOE
843


 


147028
48715
48726
CCAGCCTTGTCG
1-10-1 MOE
846


 


147067
49050
49061
TCCTGCACTGAC
1-10-1 MOE
876


 


147068
49051
49062
ATCCTGCACTGA
1-10-1 MOE
838


 


147067
49198
49209
TCCTGCACTGAC
1-10-1 MOE
876


 


147073
49524
49535
CACTGATCCTGC
1-10-1 MOE
842


 


147073
49672
49683
CACTGATCCTGC
1-10-1 MOE
842


 


147074
49673
49684
CCACTGATCCTG
1-10-1 MOE
845


 


147036
50421
50432
CCCAGTTCCCAG
1-10-1 MOE
849


 


147036
52292
52303
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
52293
52304
GCCCAGTTCCCA
1-10-1 MOE
863


 


147036
52438
52449
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
52439
52450
GCCCAGTTCCCA
1-10-1 MOE
863


 


147034
53148
53159
CAGTTCCCAGCC
1-10-1 MOE
844


 


147034
53294
53305
CAGTTCCCAGCC
1-10-1 MOE
844


 


147042
53445
53456
GGTCAAAAGGGC
1-10-1 MOE
866


 


147043
53446
53457
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
53447
53458
GTGGTCAAAAGG
1-10-1 MOE
869


 


147042
53591
53602
GGTCAAAAGGGC
1-10-1 MOE
866


 


147030
53592
53603
TCCCAGCCTTGT
1-10-1 MOE
874


 


147043
53592
53603
TGGTCAAAAGGG
1-10-1 MOE
881


 


147031
53593
53604
TTCCCAGCCTTG
1-10-1 MOE
885


 


147044
53593
53604
GTGGTCAAAAGG
1-10-1 MOE
869


 


147030
53738
53749
TCCCAGCCTTGT
1-10-1 MOE
874


 


147031
53739
53750
TTCCCAGCCTTG
1-10-1 MOE
885


 


147040
53783
53794
GCCGCCCAGTTC
1-10-1 MOE
864


 


147041
53784
53795
AGCCGCCCAGTT
1-10-1 MOE
834


 


147041
53930
53941
AGCCGCCCAGTT
1-10-1 MOE
834


 


147042
55008
55019
GGTCAAAAGGGC
1-10-1 MOE
866


 


147043
55009
55020
TGGTCAAAAGGG
1-10-1 MOE
881


 


147042
55154
55165
GGTCAAAAGGGC
1-10-1 MOE
866


 


147043
55155
55166
TGGTCAAAAGGG
1-10-1 MOE
881


 


147058
55281
55292
AATTTAATCCGA
1-10-1 MOE
830


 


147058
55427
55438
AATTTAATCCGA
1-10-1 MOE
830


 


147019
55682
55693
TCGATCTCCTCG
1-10-1 MOE
877


 


147021
55684
55695
TGTCGATCTCCT
1-10-1 MOE
882


 


147021
55830
55841
TGTCGATCTCCT
1-10-1 MOE
882


 


147054
56275
56286
TAATCCGACTGT
1-10-1 MOE
871


 


147055
56276
56287
TTAATCCGACTG
1-10-1 MOE
884


 


147056
56277
56288
TTTAATCCGACT
1-10-1 MOE
887


 


147058
56279
56290
AATTTAATCCGA
1-10-1 MOE
830


 


147059
56280
56291
CAATTTAATCCG
1-10-1 MOE
840


 


147060
56281
56292
GCAATTTAATCC
1-10-1 MOE
861


 


147061
56282
56293
TGCAATTTAATC
1-10-1 MOE
879


 


147051
56418
56429
TCCGACTGTGGT
1-10-1 MOE
875


 


147053
56420
56431
AATCCGACTGTG
1-10-1 MOE
829


 


147054
56421
56432
TAATCCGACTGT
1-10-1 MOE
871


 


147055
56422
56433
TTAATCCGACTG
1-10-1 MOE
884


 


147056
56423
56434
TTTAATCCGACT
1-10-1 MOE
887


 


147057
56424
56435
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
56425
56436
AATTTAATCCGA
1-10-1 MOE
830


 


147061
56428
56439
TGCAATTTAATC
1-10-1 MOE
879


 


147045
57118
57129
TGTGGTCAAAAG
1-10-1 MOE
883


 


147045
57264
57275
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
57265
57276
CTGTGGTCAAAA
1-10-1 MOE
858


 


147071
58028
58039
CTGATCCTGCAC
1-10-1 MOE
856


 


147071
58174
58185
CTGATCCTGCAC
1-10-1 MOE
856


 


147043
61111
61122
TGGTCAAAAGGG
1-10-1 MOE
881


 


147071
61130
61141
CTGATCCTGCAC
1-10-1 MOE
856


 


147020
61226
61237
GTCGATCTCCTC
1-10-1 MOE
868


 


147043
61257
61268
TGGTCAAAAGGG
1-10-1 MOE
881


 


147071
61276
61287
CTGATCCTGCAC
1-10-1 MOE
856


 


147035
61277
61288
CCAGTTCCCAGC
1-10-1 MOE
847


 


147036
61278
61289
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
61279
61290
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
61280
61291
CGCCCAGTTCCC
1-10-1 MOE
855


 


147039
61281
61292
CCGCCCAGTTCC
1-10-1 MOE
850


 


147040
61282
61293
GCCGCCCAGTTC
1-10-1 MOE
864


 


147071
61309
61320
CTGATCCTGCAC
1-10-1 MOE
856


 


147020
61372
61383
GTCGATCTCCTC
1-10-1 MOE
868


 


147034
61422
61433
CAGTTCCCAGCC
1-10-1 MOE
844


 


147035
61423
61434
CCAGTTCCCAGC
1-10-1 MOE
847


 


147036
61424
61435
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
61425
61436
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
61426
61437
CGCCCAGTTCCC
1-10-1 MOE
855


 


147040
61428
61439
GCCGCCCAGTTC
1-10-1 MOE
864


 


147071
61455
61466
CTGATCCTGCAC
1-10-1 MOE
856


 


147073
62003
62014
CACTGATCCTGC
1-10-1 MOE
842


 


147073
62149
62160
CACTGATCCTGC
1-10-1 MOE
842


 


147066
63065
63076
CCTGCACTGACG
1-10-1 MOE
851


 


147068
63067
63078
ATCCTGCACTGA
1-10-1 MOE
838


 


147069
63146
63157
GATCCTGCACTG
1-10-1 MOE
860


 


147062
63207
63218
CACTGACGAGTC
1-10-1 MOE
841


 


147066
63211
63222
CCTGCACTGACG
1-10-1 MOE
851


 


147057
64054
64065
ATTTAATCCGAC
1-10-1 MOE
839


 


147036
64538
64549
CCCAGTTCCCAG
1-10-1 MOE
849


 


147037
64539
64550
GCCCAGTTCCCA
1-10-1 MOE
863


 


147037
64685
64696
GCCCAGTTCCCA
1-10-1 MOE
863


 


147066
64864
64875
CCTGCACTGACG
1-10-1 MOE
851


 


147067
64865
64876
TCCTGCACTGAC
1-10-1 MOE
876


 


147066
65010
65021
CCTGCACTGACG
1-10-1 MOE
851


 


147067
65011
65022
TCCTGCACTGAC
1-10-1 MOE
876


 


147045
65017
65028
TGTGGTCAAAAG
1-10-1 MOE
883


 


147045
65163
65174
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
65164
65175
CTGTGGTCAAAA
1-10-1 MOE
858


 


147068
65408
65419
ATCCTGCACTGA
1-10-1 MOE
838


 


147071
65411
65422
CTGATCCTGCAC
1-10-1 MOE
856


 


147069
65549
65560
GATCCTGCACTG
1-10-1 MOE
860


 


147068
65554
65565
ATCCTGCACTGA
1-10-1 MOE
838


 


147071
65557
65568
CTGATCCTGCAC
1-10-1 MOE
856


 


147029
67741
67752
CCCAGCCTTGTC
1-10-1 MOE
848


 


147030
67742
67753
TCCCAGCCTTGT
1-10-1 MOE
874


 


147031
67743
67754
TTCCCAGCCTTG
1-10-1 MOE
885


 


147028
67886
67897
CCAGCCTTGTCG
1-10-1 MOE
846


 


147029
67887
67898
CCCAGCCTTGTC
1-10-1 MOE
848


 


147030
67888
67899
TCCCAGCCTTGT
1-10-1 MOE
874


 


147031
67889
67900
TTCCCAGCCTTG
1-10-1 MOE
885


 


147043
68867
68878
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
68868
68879
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
68869
68880
TGTGGTCAAAAG
1-10-1 MOE
883


 


147043
69013
69024
TGGTCAAAAGGG
1-10-1 MOE
881


 


147044
69014
69025
GTGGTCAAAAGG
1-10-1 MOE
869


 


147045
69015
69026
TGTGGTCAAAAG
1-10-1 MOE
883


 


147046
69016
69027
CTGTGGTCAAAA
1-10-1 MOE
858


 


147071
69519
69530
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
69520
69531
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
69521
69532
CACTGATCCTGC
1-10-1 MOE
842


 


147071
69665
69676
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
69666
69677
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
69667
69678
CACTGATCCTGC
1-10-1 MOE
842


 


147074
69668
69679
CCACTGATCCTG
1-10-1 MOE
845


 


147066
69869
69880
CCTGCACTGACG
1-10-1 MOE
851


 


147066
70015
70026
CCTGCACTGACG
1-10-1 MOE
851


 


147023
70465
70476
CTTGTCGATCTC
1-10-1 MOE
859


 


147023
70611
70622
CTTGTCGATCTC
1-10-1 MOE
859


 


147062
70615
70626
CACTGACGAGTC
1-10-1 MOE
841


 


147063
70616
70627
GCACTGACGAGT
1-10-1 MOE
862


 


147064
70617
70628
TGCACTGACGAG
1-10-1 MOE
880


 


147065
70618
70629
CTGCACTGACGA
1-10-1 MOE
857


 


147066
70619
70630
CCTGCACTGACG
1-10-1 MOE
851


 


147063
70762
70773
GCACTGACGAGT
1-10-1 MOE
862


 


147064
70763
70774
TGCACTGACGAG
1-10-1 MOE
880


 


147065
70764
70775
CTGCACTGACGA
1-10-1 MOE
857


 


147066
70765
70776
CCTGCACTGACG
1-10-1 MOE
851


 


147072
70998
71009
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
70999
71010
CACTGATCCTGC
1-10-1 MOE
842


 


147072
71144
71155
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
71145
71156
CACTGATCCTGC
1-10-1 MOE
842


 


147074
71146
71157
CCACTGATCCTG
1-10-1 MOE
845


 


147037
71351
71362
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
71352
71363
CGCCCAGTTCCC
1-10-1 MOE
855


 


147039
71353
71364
CCGCCCAGTTCC
1-10-1 MOE
850


 


147037
71497
71508
GCCCAGTTCCCA
1-10-1 MOE
863


 


147038
71498
71509
CGCCCAGTTCCC
1-10-1 MOE
855


 


147039
71499
71510
CCGCCCAGTTCC
1-10-1 MOE
850


 


147061
71641
71652
TGCAATTTAATC
1-10-1 MOE
879


 


147061
71787
71798
TGCAATTTAATC
1-10-1 MOE
879
















TABLE 17







Short antisense compounds targeted to


SEQ ID NO: 11 and having 1 or 2 mismatches













5′
3′


SEQ 


ISIS
Target
Target
Sequence
Gapmer
ID


NO.
Site
Site
(5′-3′)
Motif
NO















147022
177
188
TTGTCGATCTCC
1-10-1 MOE
886


 


147023
178
189
CTTGTCGATCTC
1-10-1 MOE
859


 


147020
196
207
GTCGATCTCCTC
1-10-1 MOE
868


 


147022
198
209
TTGTCGATCTCC
1-10-1 MOE
886


 


147024
200
211
CCTTGTCGATCT
1-10-1 MOE
853


 


147026
202
213
AGCCTTGTCGAT
1-10-1 MOE
835


 


147028
204
215
CCAGCCTTGTCG
1-10-1 MOE
846


 


147029
205
216
CCCAGCCTTGTC
1-10-1 MOE
848


 


147030
206
217
TCCCAGCCTTGT
1-10-1 MOE
874


 


147036
212
223
CCCAGTTCCCAG
1-10-1 MOE
849


 


147073
311
322
CACTGATCCTGC
1-10-1 MOE
842


 


147046
327
338
CTGTGGTCAAAA
1-10-1 MOE
858


 


147047
328
339
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
329
340
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
330
341
CGACTGTGGTCA
1-10-1 MOE
854


 


147050
331
342
CCGACTGTGGTC
1-10-1 MOE
889


 


147051
332
343
TCCGACTGTGGT
1-10-1 MOE
875


 


147052
333
344
ATCCGACTGTGG
1-10-1 MOE
837


 


147053
334
345
AATCCGACTGTG
1-10-1 MOE
829


 


147054
335
346
TAATCCGACTGT
1-10-1 MOE
871


 


147055
336
347
TTAATCCGACTG
1-10-1 MOE
884


 


147056
337
348
TTTAATCCGACT
1-10-1 MOE
887


 


147057
338
349
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
339
350
AATTTAATCCGA
1-10-1 MOE
830


 


147060
341
352
GCAATTTAATCC
1-10-1 MOE
861


 


147061
342
353
TGCAATTTAATC
1-10-1 MOE
879


 


147062
1024
1035
CACTGACGAGTC
1-10-1 MOE
841


 


147063
1025
1036
GCACTGACGAGT
1-10-1 MOE
862


 


147068
1030
1041
ATCCTGCACTGA
1-10-1 MOE
838


 


147071
1033
1044
CTGATCCTGCAC
1-10-1 MOE
856


 


147073
1035
1046
CACTGATCCTGC
1-10-1 MOE
842


 


147074
1036
1047
CCACTGATCCTG
1-10-1 MOE
845


 


147067
1091
1102
TCCTGCACTGAC
1-10-1 MOE
876


 


147024
1891
1902
CCTTGTCGATCT
1-10-1 MOE
853


 


147026
1893
1904
AGCCTTGTCGAT
1-10-1 MOE
835


 


147029
1896
1907
CCCAGCCTTGTC
1-10-1 MOE
848


 


147036
1903
1914
CCCAGTTCCCAG
1-10-1 MOE
849


 


147039
1906
1917
CCGCCCAGTTCC
1-10-1 MOE
850


 


147019
1994
2005
TCGATCTCCTCG
1-10-1 MOE
877


 


401385
2815
2828
CCCAGTGGGTTTGA
2-10-2 MOE
890


 


147033
5265
5276
AGTTCCCAGCCT
1-10-1 MOE
836


 


147033
5373
5384
AGTTCCCAGCCT
1-10-1 MOE
836


 


147060
7168
7179
GCAATTTAATCC
1-10-1 MOE
861


 


147053
10527
10538
AATCCGACTGTG
1-10-1 MOE
829


 


147053
10635
10646
AATCCGACTGTG
1-10-1 MOE
829


 


147070
11604
11615
TGATCCTGCACT
1-10-1 MOE
878


 


147071
11612
11623
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
12294
12305
ACTGATCCTGCA
1-10-1 MOE
832


 


147072
12299
12310
ACTGATCCTGCA
1-10-1 MOE
832


 


147052
12938
12949
ATCCGACTGTGG
1-10-1 MOE
837


 


147052
13119
13130
ATCCGACTGTGG
1-10-1 MOE
837


 


147047
13158
13169
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
13159
13170
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
13160
13171
CGACTGTGGTCA
1-10-1 MOE
854


 


147048
13340
13351
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
13341
13352
CGACTGTGGTCA
1-10-1 MOE
854


 


147051
13343
13354
TCCGACTGTGGT
1-10-1 MOE
875


 


147061
13497
13508
TGCAATTTAATC
1-10-1 MOE
879


 


147069
15562
15573
GATCCTGCACTG
1-10-1 MOE
860


 


147068
15743
15754
ATCCTGCACTGA
1-10-1 MOE
838


 


147049
17181
17192
CGACTGTGGTCA
1-10-1 MOE
854


 


147049
17349
17360
CGACTGTGGTCA
1-10-1 MOE
854


 


147047
22438
22449
ACTGTGGTCAAA
1-10-1 MOE
833


 


147047
24322
24333
ACTGTGGTCAAA
1-10-1 MOE
833


 


147045
24488
24499
TGTGGTCAAAAG
1-10-1 MOE
883


 


147039
25064
25075
CCGCCCAGTTCC
1-10-1 MOE
850


 


147057
25508
25519
ATTTAATCCGAC
1-10-1 MOE
839


 


147057
25676
25687
ATTTAATCCGAC
1-10-1 MOE
839


 


147061
25680
25691
TGCAATTTAATC
1-10-1 MOE
879


 


147069
28731
28742
GATCCTGCACTG
1-10-1 MOE
860


 


147052
30132
30143
ATCCGACTGTGG
1-10-1 MOE
837


 


147052
30277
30288
ATCCGACTGTGG
1-10-1 MOE
837


 


147036
32085
32096
CCCAGTTCCCAG
1-10-1 MOE
849


 


147072
32520
32531
ACTGATCCTGCA
1-10-1 MOE
832


 


147071
33058
33069
CTGATCCTGCAC
1-10-1 MOE
856


 


147050
33125
33136
CCGACTGTGGTC
1-10-1 MOE
889


 


147069
33204
33215
GATCCTGCACTG
1-10-1 MOE
860


 


147050
33273
33284
CCGACTGTGGTC
1-10-1 MOE
889


 


147047
33319
33330
ACTGTGGTCAAA
1-10-1 MOE
833


 


147050
33322
33333
CCGACTGTGGTC
1-10-1 MOE
889


 


147052
33324
33335
ATCCGACTGTGG
1-10-1 MOE
837


 


147049
33469
33480
CGACTGTGGTCA
1-10-1 MOE
854


 


147050
33470
33481
CCGACTGTGGTC
1-10-1 MOE
889


 


147052
33472
33483
ATCCGACTGTGG
1-10-1 MOE
837


 


147047
33641
33652
ACTGTGGTCAAA
1-10-1 MOE
833


 


147047
33789
33800
ACTGTGGTCAAA
1-10-1 MOE
833


 


147059
35585
35596
CAATTTAATCCG
1-10-1 MOE
840


 


147021
36241
36252
TGTCGATCTCCT
1-10-1 MOE
882


 


147073
37633
37644
CACTGATCCTGC
1-10-1 MOE
842


 


147033
42529
42540
AGTTCCCAGCCT
1-10-1 MOE
836


 


147050
45401
45412
CCGACTGTGGTC
1-10-1 MOE
889


 


147050
45549
45560
CCGACTGTGGTC
1-10-1 MOE
889


 


147074
46125
46136
CCACTGATCCTG
1-10-1 MOE
845


 


147057
46313
46324
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
46462
46473
AATTTAATCCGA
1-10-1 MOE
830


 


147058
47413
47424
AATTTAATCCGA
1-10-1 MOE
830


 


147058
47561
47572
AATTTAATCCGA
1-10-1 MOE
830


 


147073
48221
48232
CACTGATCCTGC
1-10-1 MOE
842


 


147073
48369
48380
CACTGATCCTGC
1-10-1 MOE
842


 


147028
48567
48578
CCAGCCTTGTCG
1-10-1 MOE
846


 


147068
49199
49210
ATCCTGCACTGA
1-10-1 MOE
838


 


147036
50273
50284
CCCAGTTCCCAG
1-10-1 MOE
849


 


147040
53929
53940
GCCGCCCAGTTC
1-10-1 MOE
864


 


147047
54769
54780
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
54770
54781
GACTGTGGTCAA
1-10-1 MOE
888


 


147047
54915
54926
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
54916
54927
GACTGTGGTCAA
1-10-1 MOE
888


 


147019
55828
55839
TCGATCTCCTCG
1-10-1 MOE
877


 


147047
56268
56279
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
56269
56280
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
56270
56281
CGACTGTGGTCA
1-10-1 MOE
854


 


147050
56271
56282
CCGACTGTGGTC
1-10-1 MOE
889


 


147051
56272
56283
TCCGACTGTGGT
1-10-1 MOE
875


 


147052
56273
56284
ATCCGACTGTGG
1-10-1 MOE
837


 


147053
56274
56285
AATCCGACTGTG
1-10-1 MOE
829


 


147056
56277
56288
TTTAATCCGACT
1-10-1 MOE
887


 


147057
56278
56289
ATTTAATCCGAC
1-10-1 MOE
839


 


147047
56414
56425
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
56415
56426
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
56416
56427
CGACTGTGGTCA
1-10-1 MOE
854


 


147050
56417
56428
CCGACTGTGGTC
1-10-1 MOE
889


 


147052
56419
56430
ATCCGACTGTGG
1-10-1 MOE
837


 


147057
56424
56435
ATTTAATCCGAC
1-10-1 MOE
839


 


147058
56425
56436
AATTTAATCCGA
1-10-1 MOE
830


 


147059
56426
56437
CAATTTAATCCG
1-10-1 MOE
840


 


147060
56427
56438
GCAATTTAATCC
1-10-1 MOE
861


 


147046
57119
57130
CTGTGGTCAAAA
1-10-1 MOE
858


 


147071
58174
58185
CTGATCCTGCAC
1-10-1 MOE
856


 


147071
61130
61141
CTGATCCTGCAC
1-10-1 MOE
856


 


147034
61276
61287
CAGTTCCCAGCC
1-10-1 MOE
844


 


147071
61309
61320
CTGATCCTGCAC
1-10-1 MOE
856


 


147039
61427
61438
CCGCCCAGTTCC
1-10-1 MOE
850


 


147071
61455
61466
CTGATCCTGCAC
1-10-1 MOE
856


 


147073
62003
62014
CACTGATCCTGC
1-10-1 MOE
842


 


147062
63061
63072
CACTGACGAGTC
1-10-1 MOE
841


 


147068
63213
63224
ATCCTGCACTGA
1-10-1 MOE
838


 


147069
63292
63303
GATCCTGCACTG
1-10-1 MOE
860


 


147057
64054
64065
ATTTAATCCGAC
1-10-1 MOE
839


 


147057
64200
64211
ATTTAATCCGAC
1-10-1 MOE
839


 


147070
64427
64438
TGATCCTGCACT
1-10-1 MOE
878


 


147070
64573
64584
TGATCCTGCACT
1-10-1 MOE
878


 


147036
64684
64695
CCCAGTTCCCAG
1-10-1 MOE
849


 


147046
65018
65029
CTGTGGTCAAAA
1-10-1 MOE
858


 


147071
65557
65568
CTGATCCTGCAC
1-10-1 MOE
856


 


147069
65695
65706
GATCCTGCACTG
1-10-1 MOE
860


 


147047
66163
66174
ACTGTGGTCAAA
1-10-1 MOE
833


 


147047
66309
66320
ACTGTGGTCAAA
1-10-1 MOE
833


 


147028
67740
67751
CCAGCCTTGTCG
1-10-1 MOE
846


 


147046
68870
68881
CTGTGGTCAAAA
1-10-1 MOE
858


 


147047
68871
68882
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
68872
68883
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
68873
68884
CGACTGTGGTCA
1-10-1 MOE
854


 


147047
69017
69028
ACTGTGGTCAAA
1-10-1 MOE
833


 


147048
69018
69029
GACTGTGGTCAA
1-10-1 MOE
888


 


147049
69019
69030
CGACTGTGGTCA
1-10-1 MOE
854


 


147071
69519
69530
CTGATCCTGCAC
1-10-1 MOE
856


 


147073
69521
69532
CACTGATCCTGC
1-10-1 MOE
842


 


147071
69665
69676
CTGATCCTGCAC
1-10-1 MOE
856


 


147072
69666
69677
ACTGATCCTGCA
1-10-1 MOE
832


 


147024
70466
70477
CCTTGTCGATCT
1-10-1 MOE
853


 


147024
70612
70623
CCTTGTCGATCT
1-10-1 MOE
853


 


147062
70761
70772
CACTGACGAGTC
1-10-1 MOE
841


 


147072
70998
71009
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
70999
71010
CACTGATCCTGC
1-10-1 MOE
842


 


147072
71144
71155
ACTGATCCTGCA
1-10-1 MOE
832


 


147073
71145
71156
CACTGATCCTGC
1-10-1 MOE
842


 


147048
71366
71377
GACTGTGGTCAA
1-10-1 MOE
888


 


147048
71512
71523
GACTGTGGTCAA
1-10-1 MOE
888
















TABLE 18







Short Antisense Compounds targeted to SEQ ID NO: 12













5′
3′


Seq


ISIS
Target
Target


ID


NO.
Site
Site
Sequence (5′-3′)
Gapmer Motif
NO















398163
20
31
ATGTCAACCGGC
1-10-1 MOE
908


 


384545
23
34
CAAGTAGGATGT
1-10-1 MOE
951


 


147705
159
170
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147703
245
256
TGGCTTCATGTC
1-10-1 MOE
971


 


398090
283
296
TTGTTCTTAGGAAG
2-10-2 MOE
972


 


147704
285
296
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
291
302
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147709
311
322
CCATTTTTATCA
1-10-1 MOE
978


 


147733
349
360
TTCTTGATGTCC
1-10-1 MOE
891


 


147707
360
371
TAGTCATTATCT
1-10-1 MOE
977


 


147708
366
377
TTGATATAGTCA
1-10-1 MOE
997


 


390030
381
392
TTTATAAAACTG
1-10-1 MOE
1074


 


147709
386
397
CCATTTTTATCA
1-10-1 MOE
978


 


147081
393
404
GCTCCTTCCACT
1-10-1 MOE
1006


 


398091
393
406
GGGCTTCTTCCATT
2-10-2 MOE
979


 


398166
395
406
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147709
418
429
CCATTTTTATCA
1-10-1 MOE
978


 


147711
425
436
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147712
461
472
ACACCATCTCCC
1-10-1 MOE
1005


 


147713
466
477
CTCCCACACCAT
1-10-1 MOE
985


 


147714
471
482
TTCTGCTCCCAC
1-10-1 MOE
986


 


147715
496
507
GTTGAGCATGAC
1-10-1 MOE
1077


 


147716
521
532
TTAACGAGCCTT
1-10-1 MOE
949


 


147717
574
585
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
607
618
ATCTTCAGAGAT
1-10-1 MOE
996


 


147708
612
623
TTGATATAGTCA
1-10-1 MOE
997


 


147718
621
632
TAATATGACTTG
1-10-1 MOE
998


 


147746
625
636
TAAAAACAACAA
1-10-1 MOE
1073


 


398167
704
715
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
705
718
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


147723
715
726
GACTCCAAAGTC
1-10-1 MOE
892


 


398093
758
771
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
760
771
TCGGACTTTGAA
1-10-1 MOE
1008


 


147738
780
791
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398094
848
861
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398169
849
860
TCAGCCAGACAG
1-10-1 MOE
909


 


398164
873
884
TTGTCGATCTGC
1-10-1 MOE
1014


 


147735
973
984
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147737
984
995
ACAGCCAGGTAG
1-10-1 MOE
1067


 


368369
1025
1040
TCCTGCACTGACGAGT
3-10-3 MOE
893


 


368372
1031
1046
CACTGATCCTGCACTG
3-10-3 MOE
894


 


368353
1033
1046
CACTGATCCTGCAC
2-10-2 MOE
1007


 


368354
1035
1048
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368388
1035
1050
CTTCCACTGATCCTTA
3-10-3 MOE
895


 


368355
1036
1049
TTCCACTGATCCTG
2-10-2 MOE
1025


 


368356
1037
1050
CTTCCACTGATCCT
2-10-2 MOE
1027


 


368376
1037
1052
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


147076
1038
1049
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
1038
1051
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
1039
1050
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
1039
1052
TCCTTCCACTGATC
2-10-2 MOE
1031


 


368378
1039
1054
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


368359
1041
1054
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


147080
1042
1053
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
1043
1054
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
1043
1056
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


368380
1043
1058
GAAAGCTCCTTCCACT
3-10-3 MOE
896


 


147082
1044
1055
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368381
1045
1060
GGGAAAGCTCCTTCCA
3-10-3 MOE
1037


 


147739
1107
1118
CGTTTGGGTGGC
1-10-1 MOE
1023


 


147741
1165
1176
CACCCACTGGTG
1-10-1 MOE
1055


 


398097
1194
1207
GGCAGTCTTTATCC
2-10-2 MOE
897


 


147742
1273
1284
AACTTCAGTGTC
1-10-1 MOE
1041


 


147743
1388
1399
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147744
1392
1403
AGGAAGGGCTTC
1-10-1 MOE
1043


 


147745
1398
1409
TTGACCAGGAAG
1-10-1 MOE
1058


 


398157
1455
1468
GGAAACATACCCTG
2-10-2 MOE
1045


 


398167
1475
1486
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
1476
1489
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


368357
1596
1609
CCTTCCACTGATCC
2-10-2 MOE
1046


 


398160
1691
1704
GAATAGGTTAAGGC
2-10-2 MOE
1048


 


398163
1711
1722
ATGTCAACCGGC
1-10-1 MOE
908


 


147746
1750
1761
TAAAAACAACAA
1-10-1 MOE
1073


 


389949
1777
1788
GCGCGAGCCCGA
1-10-1 MOE
1061


 


398161
1790
1803
AACAATGTGTTGTA
2-10-2 MOE
1049


 


147746
1799
1810
TAAAAACAACAA
1-10-1 MOE
1073


 


398163
1819
1830
ATGTCAACCGGC
1-10-1 MOE
908


 


389950
1848
1859
CCCTGAAGGTTC
1-10-1 MOE
1063


 


398164
1889
1900
TTGTCGATCTGC
1-10-1 MOE
1014


 


147702
1917
1928
CTGGTAAATAGC
1-10-1 MOE
898


 


147088
1971
1982
CCCTCTACACCA
1-10-1 MOE
1050


 


398102
2003
2016
CTACCTGAGGATTT
2-10-2 MOE
899


 


398103
2010
2023
CCCAGTACTACCTG
2-10-2 MOE
900


 


147737
2386
2397
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398095
2407
2420
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398106
2441
2454
TGGAAAACTGCACC
2-10-2 MOE
1068


 


147745
2497
2508
TTGACCAGGAAG
1-10-1 MOE
1058


 


147712
2499
2510
ACACCATCTCCC
1-10-1 MOE
1005


 


147712
2607
2618
ACACCATCTCCC
1-10-1 MOE
1005


 


147745
2689
2700
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
2706
2717
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
2707
2720
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


398166
2966
2977
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147091
2992
3003
GTTCCCTCTACA
1-10-1 MOE
1004


 


147092
2993
3004
TGTTCCCTCTAC
1-10-1 MOE
901


 


389949
3008
3019
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147087
3149
3160
CCTCTACACCAG
1-10-1 MOE
982


 


147088
3150
3161
CCCTCTACACCA
1-10-1 MOE
1050


 


398113
3160
3173
AGGAGGTTAAACCA
2-10-2 MOE
905


 


147087
3257
3268
CCTCTACACCAG
1-10-1 MOE
982


 


147088
3258
3269
CCCTCTACACCA
1-10-1 MOE
1050


 


147737
3591
3602
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147737
3617
3628
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147079
3637
3648
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
3638
3649
CTCCTTCCACTG
1-10-1 MOE
1021


 


398095
3638
3651
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398106
3672
3685
TGGAAAACTGCACC
2-10-2 MOE
1068


 


398107
3678
3691
TATTCCTGGAAAAC
2-10-2 MOE
902


 


147691
3806
3817
GAGGTGGGAAAA
1-10-1 MOE
966


 


147683
3848
3859
GCTTACGATTGT
1-10-1 MOE
922


 


147738
3853
3864
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398167
3926
3937
CAGGCCATGTGG
1-10-1 MOE
1059


 


398109
3945
3958
CAAGAAGTGTGGTT
2-10-2 MOE
903


 


398167
4034
4045
CAGGCCATGTGG
1-10-1 MOE
1059


 


398110
4083
4096
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


398111
4168
4181
GTGAAAATGCTGGC
2-10-2 MOE
904


 


147706
4238
4249
GCTGACATCTCG
1-10-1 MOE
1071


 


398112
4282
4295
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


147746
4315
4326
TAAAAACAACAA
1-10-1 MOE
1073


 


398113
4391
4404
AGGAGGTTAAACCA
2-10-2 MOE
905


 


398115
4484
4497
AGTAAATATTGGCT
2-10-2 MOE
1076


 


390030
4491
4502
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
4537
4548
TTTATAAAACTG
1-10-1 MOE
1074


 


147703
5034
5045
TGGCTTCATGTC
1-10-1 MOE
971


 


147684
5035
5046
ACCCAGTCAGGG
1-10-1 MOE
964


 


398125
5075
5088
CAGTAAGGAATTTT
2-10-2 MOE
913


 


147696
5083
5094
TGGATGATTGGC
1-10-1 MOE
906


 


147684
5143
5154
ACCCAGTCAGGG
1-10-1 MOE
964


 


147712
5366
5377
ACACCATCTCCC
1-10-1 MOE
1005


 


147714
5416
5427
TTCTGCTCCCAC
1-10-1 MOE
986


 


398128
5443
5456
CTAAATTTAGTTCA
2-10-2 MOE
911


 


147712
5474
5485
ACACCATCTCCC
1-10-1 MOE
1005


 


147746
5498
5509
TAAAAACAACAA
1-10-1 MOE
1073


 


147714
5524
5535
TTCTGCTCCCAC
1-10-1 MOE
986


 


147736
5600
5611
AGGTAGGAGAAG
1-10-1 MOE
963


 


147085
5762
5773
TCTACACCAGGT
1-10-1 MOE
961


 


147679
5825
5836
CAAAAGGATCCC
1-10-1 MOE
907


 


390030
6803
6814
TTTATAAAACTG
1-10-1 MOE
1074


 


398142
6885
6898
CCAGCACACTGGAA
2-10-2 MOE
923


 


398142
6994
7007
CCAGCACACTGGAA
2-10-2 MOE
923


 


398166
7306
7317
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147684
7551
7562
ACCCAGTCAGGG
1-10-1 MOE
964


 


147085
8308
8319
TCTACACCAGGT
1-10-1 MOE
961


 


147085
8416
8427
TCTACACCAGGT
1-10-1 MOE
961


 


398163
8473
8484
ATGTCAACCGGC
1-10-1 MOE
908


 


147718
8523
8534
TAATATGACTTG
1-10-1 MOE
998


 


147718
8631
8642
TAATATGACTTG
1-10-1 MOE
998


 


147691
8806
8817
GAGGTGGGAAAA
1-10-1 MOE
966


 


147728
8835
8846
GCCAGACAGAAG
1-10-1 MOE
1013


 


147728
8943
8954
GCCAGACAGAAG
1-10-1 MOE
1013


 


398169
8946
8957
TCAGCCAGACAG
1-10-1 MOE
909


 


147742
9060
9071
AACTTCAGTGTC
1-10-1 MOE
1041


 


404136
9162
9175
TAAGTGTCCCTTTG
2-10-2 MOE
910


 


147746
9963
9974
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9966
9977
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9969
9980
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9991
10002
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10071
10082
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10074
10085
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10077
10088
TAAAAACAACAA
1-10-1 MOE
1073


 


390030
10170
10181
TTTATAAAACTG
1-10-1 MOE
1074


 


147084
10220
10231
CTACACCAGGTC
1-10-1 MOE
993


 


390030
10278
10289
TTTATAAAACTG
1-10-1 MOE
1074


 


147085
10329
10340
TCTACACCAGGT
1-10-1 MOE
961


 


147711
10684
10695
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
10792
10803
AAGGGCCCTGGG
1-10-1 MOE
1040


 


398128
11333
11346
CTAAATTTAGTTCA
2-10-2 MOE
911


 


147707
11960
11971
TAGTCATTATCT
1-10-1 MOE
977


 


147707
11965
11976
TAGTCATTATCT
1-10-1 MOE
977


 


147090
12013
12024
TTCCCTCTACAC
1-10-1 MOE
955


 


398096
12146
12159
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


398166
12214
12225
GGGCTTCTTCCA
1-10-1 MOE
1070


 


398135
12308
12321
GACTACATTTTACA
2-10-2 MOE
912


 


147741
12389
12400
CACCCACTGGTG
1-10-1 MOE
1055


 


398125
12431
12444
CAGTAAGGAATTTT
2-10-2 MOE
913


 


147714
12585
12596
TTCTGCTCCCAC
1-10-1 MOE
986


 


147718
12594
12605
TAATATGACTTG
1-10-1 MOE
998


 


398125
12612
12625
CAGTAAGGAATTTT
2-10-2 MOE
913


 


147737
12803
12814
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147746
12876
12887
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
12900
12911
GAGGTGGGAAAA
1-10-1 MOE
966


 


398137
13111
13124
TGTGTCCCTCAGTC
2-10-2 MOE
914


 


398138
13254
13267
AACATCAAGCTTGA
2-10-2 MOE
931


 


398137
13292
13305
TGTGTCCCTCAGTC
2-10-2 MOE
914


 


398138
13435
13448
AACATCAAGCTTGA
2-10-2 MOE
931


 


389764
14020
14031
CTGCAACATGAT
1-9-2 MOE
1018


 


389948
14067
14078
CCGTTGGACCCC
1-10-1 MOE
915


 


389948
14248
14259
CCGTTGGACCCC
1-10-1 MOE
915


 


147738
14279
14290
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147698
14572
14583
CCCGCCACCACC
1-10-1 MOE
928


 


147717
14750
14761
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
14932
14943
ATCTTCAGAGAT
1-10-1 MOE
996


 


398167
15374
15385
CAGGCCATGTGG
1-10-1 MOE
1059


 


147736
16444
16455
AGGTAGGAGAAG
1-10-1 MOE
963


 


147746
16510
16521
TAAAAACAACAA
1-10-1 MOE
1073


 


147738
16590
16601
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147746
16676
16687
TAAAAACAACAA
1-10-1 MOE
1073


 


398167
16797
16808
CAGGCCATGTGG
1-10-1 MOE
1059


 


398144
16911
16924
GACAGCTTCTATAA
2-10-2 MOE
916


 


389764
17096
17107
CTGCAACATGAT
1-9-2 MOE
1018


 


147709
17238
17249
CCATTTTTATCA
1-10-1 MOE
978


 


147709
17406
17417
CCATTTTTATCA
1-10-1 MOE
978


 


147695
17466
17477
TCATTCCCCACT
1-10-1 MOE
984


 


147746
17497
17508
TAAAAACAACAA
1-10-1 MOE
1073


 


147088
17539
17550
CCCTCTACACCA
1-10-1 MOE
1050


 


147711
17808
17819
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
17976
17987
AAGGGCCCTGGG
1-10-1 MOE
1040


 


398139
18049
18062
AGTGACTGACCACA
2-10-2 MOE
917


 


398139
18217
18230
AGTGACTGACCACA
2-10-2 MOE
917


 


398140
18596
18609
GTAGCATAGAGCCT
2-10-2 MOE
918


 


398140
18764
18777
GTAGCATAGAGCCT
2-10-2 MOE
918


 


398167
18927
18938
CAGGCCATGTGG
1-10-1 MOE
1059


 


398141
18947
18960
CAGATCTTGTCAAG
2-10-2 MOE
919


 


398167
19095
19106
CAGGCCATGTGG
1-10-1 MOE
1059


 


398141
19115
19128
CAGATCTTGTCAAG
2-10-2 MOE
919


 


147746
19207
19218
TAAAAACAACAA
1-10-1 MOE
1073


 


147711
19508
19519
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147729
19554
19565
GTAAGAGGCAGG
1-10-1 MOE
920


 


147718
19617
19628
TAATATGACTTG
1-10-1 MOE
998


 


390030
19618
19629
TTTATAAAACTG
1-10-1 MOE
1074


 


147701
19671
19682
CCATGGCGGGAC
1-10-1 MOE
921


 


147711
19676
19687
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147718
19785
19796
TAATATGACTTG
1-10-1 MOE
998


 


147079
20515
20526
TCCTTCCACTGA
1-10-1 MOE
1001


 


389764
20620
20631
CTGCAACATGAT
1-9-2 MOE
1018


 


398142
20653
20666
CCAGCACACTGGAA
2-10-2 MOE
923


 


147078
20682
20693
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
20683
20694
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
20704
20715
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
20705
20716
GCTCCTTCCACT
1-10-1 MOE
1006


 


389965
20788
20799
CTGCAACATGAT
1-10-1 MOE
1018


 


147746
20870
20881
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
21038
21049
TAAAAACAACAA
1-10-1 MOE
1073


 


147717
21080
21091
ATCTTCAGAGAT
1-10-1 MOE
996


 


147076
21222
21233
TTCCACTGATCC
1-10-1 MOE
1029


 


398094
21441
21454
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


147746
21633
21644
TAAAAACAACAA
1-10-1 MOE
1073


 


147738
21884
21895
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147683
21939
21950
GCTTACGATTGT
1-10-1 MOE
922


 


147743
22213
22224
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147736
22759
22770
AGGTAGGAGAAG
1-10-1 MOE
963


 


147736
22927
22938
AGGTAGGAGAAG
1-10-1 MOE
963


 


398142
23008
23021
CCAGCACACTGGAA
2-10-2 MOE
923


 


398147
23784
23797
CTACAGGACAATAC
2-10-2 MOE
957


 


398147
23952
23965
CTACAGGACAATAC
2-10-2 MOE
957


 


147713
24434
24445
CTCCCACACCAT
1-10-1 MOE
985


 


389965
24543
24554
CTGCAACATGAT
1-10-1 MOE
1018


 


147713
24602
24613
CTCCCACACCAT
1-10-1 MOE
985


 


389965
24711
24722
CTGCAACATGAT
1-10-1 MOE
1018


 


147746
25384
25395
TAAAAACAACAA
1-10-1 MOE
1073


 


398143
25505
25518
GTCAGTCCCAGCTA
2-10-2 MOE
924


 


147691
25610
25621
GAGGTGGGAAAA
1-10-1 MOE
966


 


398130
25672
25685
TTAGTATGACAGCT
2-10-2 MOE
925


 


147746
25810
25821
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
25978
25989
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
26172
26183
TAAAAACAACAA
1-10-1 MOE
1073


 


398151
26718
26731
TCAGTGTAGGAAGA
2-10-2 MOE
926


 


147728
26917
26928
GCCAGACAGAAG
1-10-1 MOE
1013


 


398152
27708
27721
TGAATATACAGATG
2-10-2 MOE
927


 


147698
28629
28640
CCCGCCACCACC
1-10-1 MOE
928


 


389965
28714
28725
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
28714
28725
CTGCAACATGAT
1-9-2 MOE
1018


 


389764
28861
28872
CTGCAACATGAT
1-9-2 MOE
1018


 


390030
29945
29956
TTTATAAAACTG
1-10-1 MOE
1074


 


147744
30654
30665
AGGAAGGGCTTC
1-10-1 MOE
1043


 


147093
30836
30847
TTGTTCCCTCTA
1-10-1 MOE
929


 


147746
30957
30968
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
31105
31116
TAAAAACAACAA
1-10-1 MOE
1073


 


390030
31477
31488
TTTATAAAACTG
1-10-1 MOE
1074


 


384545
31829
31840
CAAGTAGGATGT
1-10-1 MOE
951


 


384545
31977
31988
CAAGTAGGATGT
1-10-1 MOE
951


 


401382
32094
32107
TCTACCTGAGTCCA
2-10-2 MOE
930


 


147089
32387
32398
TCCCTCTACACC
1-10-1 MOE
956


 


389950
32949
32960
CCCTGAAGGTTC
1-10-1 MOE
1063


 


398165
33002
33013
GTTCTTAGGAAG
1-10-1 MOE
968


 


147081
33073
33084
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
33074
33085
AGCTCCTTCCAC
1-10-1 MOE
1036


 


389950
33097
33108
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147736
33160
33171
AGGTAGGAGAAG
1-10-1 MOE
963


 


147081
33221
33232
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
33221
33234
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
33222
33233
AGCTCCTTCCAC
1-10-1 MOE
1036


 


398138
33244
33257
AACATCAAGCTTGA
2-10-2 MOE
931


 


147746
33250
33261
TAAAAACAACAA
1-10-1 MOE
1073


 


398138
33392
33405
AACATCAAGCTTGA
2-10-2 MOE
931


 


401383
33588
33601
GATCACCTTCAGAG
2-10-2 MOE
932


 


147746
33886
33897
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
34606
34617
TAAAAACAACAA
1-10-1 MOE
1073


 


398165
34704
34715
GTTCTTAGGAAG
1-10-1 MOE
968


 


147717
34745
34756
ATCTTCAGAGAT
1-10-1 MOE
996


 


147746
34754
34765
TAAAAACAACAA
1-10-1 MOE
1073


 


398165
34852
34863
GTTCTTAGGAAG
1-10-1 MOE
968


 


147717
34893
34904
ATCTTCAGAGAT
1-10-1 MOE
996


 


401384
34905
34918
TGAACACATCACTA
2-10-2 MOE
933


 


147738
35391
35402
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147736
35396
35407
AGGTAGGAGAAG
1-10-1 MOE
963


 


147738
35539
35550
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147691
35554
35565
GAGGTGGGAAAA
1-10-1 MOE
966


 


147691
35702
35713
GAGGTGGGAAAA
1-10-1 MOE
966


 


147746
35814
35825
TAAAAACAACAA
1-10-1 MOE
1073


 


401385
36109
36122
CCCAGTGGGTTTGA
2-10-2 MOE
890


 


147691
36360
36371
GAGGTGGGAAAA
1-10-1 MOE
966


 


147746
36416
36427
TAAAAACAACAA
1-10-1 MOE
1073


 


147731
36620
36631
TTTCCTCTTGTC
1-10-1 MOE
934


 


147714
37881
37892
TTCTGCTCCCAC
1-10-1 MOE
986


 


147714
38029
38040
TTCTGCTCCCAC
1-10-1 MOE
986


 


147681
38512
38523
ATGTCATTAAAC
1-10-1 MOE
965


 


401386
38516
38529
TAATTGATGTCAAT
2-10-2 MOE
935


 


401387
38518
38531
AGTAATTGATGTCA
2-10-2 MOE
936


 


401388
38520
38533
ACAGTAATTGATGT
2-10-2 MOE
937


 


401389
38522
38535
TTACAGTAATTGAT
2-10-2 MOE
938


 


401390
38524
38537
ACTTACAGTAATTG
2-10-2 MOE
939


 


401391
38526
38539
AGACTTACAGTAAT
2-10-2 MOE
940


 


401392
38528
38541
TCAGACTTACAGTA
2-10-2 MOE
941


 


401393
38530
38543
AATCAGACTTACAG
2-10-2 MOE
942


 


401394
38532
38545
TGAATCAGACTTAC
2-10-2 MOE
943


 


401395
38534
38547
AATGAATCAGACTT
2-10-2 MOE
944


 


147738
38909
38920
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147738
39057
39068
TGGGTGGCCGGG
1-10-1 MOE
1069


 


390030
39249
39260
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
39397
39408
TTTATAAAACTG
1-10-1 MOE
1074


 


401396
39488
39501
TGCAGGATGTTGAG
2-10-2 MOE
945


 


147717
39545
39556
ATCTTCAGAGAT
1-10-1 MOE
996


 


147746
39641
39652
TAAAAACAACAA
1-10-1 MOE
1073


 


147717
39693
39704
ATCTTCAGAGAT
1-10-1 MOE
996


 


147746
39729
39740
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
39877
39888
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
40185
40196
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
40478
40489
TAAAAACAACAA
1-10-1 MOE
1073


 


398166
40589
40600
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147735
40662
40673
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147746
40706
40717
TAAAAACAACAA
1-10-1 MOE
1073


 


398166
40737
40748
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147746
40854
40865
TAAAAACAACAA
1-10-1 MOE
1073


 


401397
41012
41025
CTGGTCAGCATTGA
2-10-2 MOE
946


 


147718
41070
41081
TAATATGACTTG
1-10-1 MOE
998


 


147718
41218
41229
TAATATGACTTG
1-10-1 MOE
998


 


147717
41221
41232
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
41369
41380
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
41599
41610
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
41747
41758
ATCTTCAGAGAT
1-10-1 MOE
996


 


401398
41768
41781
CAAAGTCCCTTAGC
2-10-2 MOE
947


 


390030
42056
42067
TTTATAAAACTG
1-10-1 MOE
1074


 


398153
42157
42170
ATTTCTCTTACAGG
2-10-2 MOE
948


 


398153
42305
42318
ATTTCTCTTACAGG
2-10-2 MOE
948


 


147710
42691
42702
TATAGCTCCTCT
1-10-1 MOE
994


 


147079
43322
43333
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
43323
43334
CTCCTTCCACTG
1-10-1 MOE
1021


 


147716
43477
43488
TTAACGAGCCTT
1-10-1 MOE
949


 


147746
43992
44003
TAAAAACAACAA
1-10-1 MOE
1073


 


147736
44137
44148
AGGTAGGAGAAG
1-10-1 MOE
963


 


384545
44242
44253
CAAGTAGGATGT
1-10-1 MOE
951


 


147687
44354
44365
CGACACGGGAAC
1-10-1 MOE
950


 


384545
44390
44401
CAAGTAGGATGT
1-10-1 MOE
951


 


398110
44713
44726
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


147705
45092
45103
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147705
45240
45251
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147074
45977
45988
CCACTGATCCTG
1-10-1 MOE
845


 


147075
45978
45989
TCCACTGATCCT
1-10-1 MOE
1026


 


147076
45979
45990
TTCCACTGATCC
1-10-1 MOE
1029


 


147076
46127
46138
TTCCACTGATCC
1-10-1 MOE
1029


 


401399
46247
46260
ATTAGCCATATCTC
2-10-2 MOE
953


 


147705
46555
46566
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147714
46685
46696
TTCTGCTCCCAC
1-10-1 MOE
986


 


147705
46703
46714
CGGTTTTTGTTC
1-10-1 MOE
1002


 


390030
46859
46870
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
46933
46944
TTTATAAAACTG
1-10-1 MOE
1074


 


147681
46984
46995
ATGTCATTAAAC
1-10-1 MOE
965


 


390030
47007
47018
TTTATAAAACTG
1-10-1 MOE
1074


 


147746
47023
47034
TAAAAACAACAA
1-10-1 MOE
1073


 


390030
47081
47092
TTTATAAAACTG
1-10-1 MOE
1074


 


147681
47132
47143
ATGTCATTAAAC
1-10-1 MOE
965


 


147746
47171
47182
TAAAAACAACAA
1-10-1 MOE
1073


 


401400
47411
47424
AGCATTCAGCAGTG
2-10-2 MOE
954


 


147746
47461
47472
TAAAAACAACAA
1-10-1 MOE
1073


 


147086
47608
47619
CTCTACACCAGG
1-10-1 MOE
969


 


147087
47609
47620
CCTCTACACCAG
1-10-1 MOE
982


 


147088
47610
47621
CCCTCTACACCA
1-10-1 MOE
1050


 


147090
47612
47623
TTCCCTCTACAC
1-10-1 MOE
955


 


147691
47729
47740
GAGGTGGGAAAA
1-10-1 MOE
966


 


147086
47756
47767
CTCTACACCAGG
1-10-1 MOE
969


 


147088
47758
47769
CCCTCTACACCA
1-10-1 MOE
1050


 


147089
47759
47770
TCCCTCTACACC
1-10-1 MOE
956


 


390030
47847
47858
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
47995
48006
TTTATAAAACTG
1-10-1 MOE
1074


 


147691
48393
48404
GAGGTGGGAAAA
1-10-1 MOE
966


 


398147
48887
48900
CTACAGGACAATAC
2-10-2 MOE
957


 


147706
49133
49144
GCTGACATCTCG
1-10-1 MOE
1071


 


147706
49281
49292
GCTGACATCTCG
1-10-1 MOE
1071


 


398168
49742
49753
TCGGACTTTGAA
1-10-1 MOE
1008


 


401401
49791
49804
AACTGGGTTAAGTA
2-10-2 MOE
958


 


147689
49936
49947
CAGAGAAGGTCT
1-10-1 MOE
987


 


401402
50192
50205
TGAACACGCTATCC
2-10-2 MOE
959


 


398117
50241
50254
TTTCCACTTGGGTG
2-10-2 MOE
960


 


147736
50582
50593
AGGTAGGAGAAG
1-10-1 MOE
963


 


398168
50703
50714
TCGGACTTTGAA
1-10-1 MOE
1008


 


398168
50849
50860
TCGGACTTTGAA
1-10-1 MOE
1008


 


147746
51019
51030
TAAAAACAACAA
1-10-1 MOE
1073


 


147708
51101
51112
TTGATATAGTCA
1-10-1 MOE
997


 


147746
51178
51189
TAAAAACAACAA
1-10-1 MOE
1073


 


147708
51247
51258
TTGATATAGTCA
1-10-1 MOE
997


 


147083
51281
51292
TACACCAGGTCA
1-10-1 MOE
973


 


147081
51287
51298
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
51288
51299
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147746
51331
51342
TAAAAACAACAA
1-10-1 MOE
1073


 


147085
51416
51427
TCTACACCAGGT
1-10-1 MOE
961


 


147083
51427
51438
TACACCAGGTCA
1-10-1 MOE
973


 


147081
51433
51444
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
51434
51445
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147728
51522
51533
GCCAGACAGAAG
1-10-1 MOE
1013


 


147085
51562
51573
TCTACACCAGGT
1-10-1 MOE
961


 


147081
51633
51644
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
51633
51646
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
51634
51645
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368361
51635
51648
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


368360
51779
51792
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
51780
51791
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147736
51859
51870
AGGTAGGAGAAG
1-10-1 MOE
963


 


147684
51867
51878
ACCCAGTCAGGG
1-10-1 MOE
964


 


147746
51918
51929
TAAAAACAACAA
1-10-1 MOE
1073


 


147077
51988
51999
CTTCCACTGATC
1-10-1 MOE
1047


 


147746
52064
52075
TAAAAACAACAA
1-10-1 MOE
1073


 


147084
52125
52136
CTACACCAGGTC
1-10-1 MOE
993


 


147079
52136
52147
TCCTTCCACTGA
1-10-1 MOE
1001


 


147681
52231
52242
ATGTCATTAAAC
1-10-1 MOE
965


 


147084
52271
52282
CTACACCAGGTC
1-10-1 MOE
993


 


147691
52312
52323
GAGGTGGGAAAA
1-10-1 MOE
966


 


401403
52318
52331
TTTCCTAGGAGGTG
2-10-2 MOE
967


 


398167
52527
52538
CAGGCCATGTGG
1-10-1 MOE
1059


 


147703
52670
52681
TGGCTTCATGTC
1-10-1 MOE
971


 


398167
52673
52684
CAGGCCATGTGG
1-10-1 MOE
1059


 


398165
52708
52719
GTTCTTAGGAAG
1-10-1 MOE
968


 


398090
52708
52721
TTGTTCTTAGGAAG
2-10-2 MOE
972


 


147705
52716
52727
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147682
52717
52728
CGGGTACTATGG
1-10-1 MOE
992


 


398167
52762
52773
CAGGCCATGTGG
1-10-1 MOE
1059


 


147703
52816
52827
TGGCTTCATGTC
1-10-1 MOE
971


 


398090
52854
52867
TTGTTCTTAGGAAG
2-10-2 MOE
972


 


147704
52856
52867
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
52862
52873
CGGTTTTTGTTC
1-10-1 MOE
1002


 


398167
52908
52919
CAGGCCATGTGG
1-10-1 MOE
1059


 


147084
53704
53715
CTACACCAGGTC
1-10-1 MOE
993


 


147088
53708
53719
CCCTCTACACCA
1-10-1 MOE
1050


 


147083
53849
53860
TACACCAGGTCA
1-10-1 MOE
973


 


147084
53850
53861
CTACACCAGGTC
1-10-1 MOE
993


 


147086
53852
53863
CTCTACACCAGG
1-10-1 MOE
969


 


147088
53854
53865
CCCTCTACACCA
1-10-1 MOE
1050


 


398167
53870
53881
CAGGCCATGTGG
1-10-1 MOE
1059


 


147703
54137
54148
TGGCTTCATGTC
1-10-1 MOE
971


 


398155
54172
54185
TGTTTTTACACAGA
2-10-2 MOE
970


 


390030
54263
54274
TTTATAAAACTG
1-10-1 MOE
1074


 


147705
54275
54286
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147703
54283
54294
TGGCTTCATGTC
1-10-1 MOE
971


 


390030
54409
54420
TTTATAAAACTG
1-10-1 MOE
1074


 


147704
54965
54976
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
54971
54982
CGGTTTTTGTTC
1-10-1 MOE
1002


 


398090
55109
55122
TTGTTCTTAGGAAG
2-10-2 MOE
972


 


147705
55117
55128
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147083
55206
55217
TACACCAGGTCA
1-10-1 MOE
973


 


147084
55207
55218
CTACACCAGGTC
1-10-1 MOE
993


 


147084
55353
55364
CTACACCAGGTC
1-10-1 MOE
993


 


147705
55524
55535
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147685
55602
55613
GGCTGACATTCA
1-10-1 MOE
975


 


401404
55638
55651
TGAGCTACAGTAGG
2-10-2 MOE
974


 


147685
55748
55759
GGCTGACATTCA
1-10-1 MOE
975


 


147712
55819
55830
ACACCATCTCCC
1-10-1 MOE
1005


 


147712
55965
55976
ACACCATCTCCC
1-10-1 MOE
1005


 


147707
56300
56311
TAGTCATTATCT
1-10-1 MOE
977


 


147708
56306
56317
TTGATATAGTCA
1-10-1 MOE
997


 


390030
56321
56332
TTTATAAAACTG
1-10-1 MOE
1074


 


147709
56326
56337
CCATTTTTATCA
1-10-1 MOE
978


 


398091
56333
56346
GGGCTTCTTCCATT
2-10-2 MOE
979


 


401405
56408
56421
TGGTCAACTGAAAG
2-10-2 MOE
976


 


147707
56446
56457
TAGTCATTATCT
1-10-1 MOE
977


 


147708
56452
56463
TTGATATAGTCA
1-10-1 MOE
997


 


147709
56472
56483
CCATTTTTATCA
1-10-1 MOE
978


 


398091
56479
56492
GGGCTTCTTCCATT
2-10-2 MOE
979


 


401406
56570
56583
GGTGTGGATAACAG
2-10-2 MOE
980


 


368366
56664
56677
CTGATCCTTAGAAG
2-10-2 MOE
1019


 


398148
57157
57170
TCATAACTATTAAG
2-10-2 MOE
981


 


147082
57220
57231
AGCTCCTTCCAC
1-10-1 MOE
1036


 


398148
57303
57316
TCATAACTATTAAG
2-10-2 MOE
981


 


147082
57366
57377
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147743
57758
57769
AGGGCTTCCAGT
1-10-1 MOE
1042


 


398093
57963
57976
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398093
58109
58122
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


147735
58279
58290
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147087
58821
58832
CCTCTACACCAG
1-10-1 MOE
982


 


147087
58967
58978
CCTCTACACCAG
1-10-1 MOE
982


 


390030
59180
59191
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
59326
59337
TTTATAAAACTG
1-10-1 MOE
1074


 


147711
59357
59368
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147743
59382
59393
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147711
59503
59514
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
59675
59686
AAGGGCCCTGGG
1-10-1 MOE
1040


 


401407
59710
59723
CAGCTTAGGCAGAG
2-10-2 MOE
983


 


147712
59711
59722
ACACCATCTCCC
1-10-1 MOE
1005


 


147713
59716
59727
CTCCCACACCAT
1-10-1 MOE
985


 


147714
59721
59732
TTCTGCTCCCAC
1-10-1 MOE
986


 


147695
59722
59733
TCATTCCCCACT
1-10-1 MOE
984


 


147715
59746
59757
GTTGAGCATGAC
1-10-1 MOE
1077


 


147711
59821
59832
AAGGGCCCTGGG
1-10-1 MOE
1040


 


390030
59847
59858
TTTATAAAACTG
1-10-1 MOE
1074


 


147712
59857
59868
ACACCATCTCCC
1-10-1 MOE
1005


 


147713
59862
59873
CTCCCACACCAT
1-10-1 MOE
985


 


147714
59867
59878
TTCTGCTCCCAC
1-10-1 MOE
986


 


390030
59993
60004
TTTATAAAACTG
1-10-1 MOE
1074


 


389949
60471
60482
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147746
60619
60630
TAAAAACAACAA
1-10-1 MOE
1073


 


147689
61113
61124
CAGAGAAGGTCT
1-10-1 MOE
987


 


398105
61267
61280
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147680
61473
61484
GTATGCACTGCT
1-10-1 MOE
988


 


147080
61757
61768
CTCCTTCCACTG
1-10-1 MOE
1021


 


147078
61901
61912
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
61902
61913
TCCTTCCACTGA
1-10-1 MOE
1001


 


147088
62215
62226
CCCTCTACACCA
1-10-1 MOE
1050


 


401408
62600
62613
CAATGAAGCACAGG
2-10-2 MOE
989


 


147688
62843
62854
TCCCAAACAAAT
1-10-1 MOE
990


 


147746
63102
63113
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
63248
63259
TAAAAACAACAA
1-10-1 MOE
1073


 


401409
63430
63443
ATTCTTAACACAGA
2-10-2 MOE
991


 


147682
63483
63494
CGGGTACTATGG
1-10-1 MOE
992


 


147084
63677
63688
CTACACCAGGTC
1-10-1 MOE
993


 


147710
64847
64858
TATAGCTCCTCT
1-10-1 MOE
994


 


147710
64993
65004
TATAGCTCCTCT
1-10-1 MOE
994


 


147746
65151
65162
TAAAAACAACAA
1-10-1 MOE
1073


 


401410
65263
65276
CATTTAGGGTCTAA
2-10-2 MOE
995


 


147717
65862
65873
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
65895
65906
ATCTTCAGAGAT
1-10-1 MOE
996


 


147708
65900
65911
TTGATATAGTCA
1-10-1 MOE
997


 


147718
65909
65920
TAATATGACTTG
1-10-1 MOE
998


 


147717
66008
66019
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
66041
66052
ATCTTCAGAGAT
1-10-1 MOE
996


 


147708
66046
66057
TTGATATAGTCA
1-10-1 MOE
997


 


147718
66055
66066
TAATATGACTTG
1-10-1 MOE
998


 


401411
66123
66136
AGCCGCCTGAAGTG
2-10-2 MOE
999


 


147697
66497
66508
CCCCAGCAGCGG
1-10-1 MOE
1000


 


368377
66562
66577
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
66563
66574
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
66563
66576
TCCTTCCACTGATC
2-10-2 MOE
1031


 


147078
66564
66575
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
66565
66576
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
66566
66577
CTCCTTCCACTG
1-10-1 MOE
1021


 


147697
66643
66654
CCCCAGCAGCGG
1-10-1 MOE
1000


 


368358
66709
66722
TCCTTCCACTGATC
2-10-2 MOE
1031


 


147078
66710
66721
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
66711
66722
TCCTTCCACTGA
1-10-1 MOE
1001


 


147075
66999
67010
TCCACTGATCCT
1-10-1 MOE
1026


 


147705
67067
67078
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147088
67409
67420
CCCTCTACACCA
1-10-1 MOE
1050


 


147080
67430
67441
CTCCTTCCACTG
1-10-1 MOE
1021


 


147082
67432
67443
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147737
67455
67466
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147088
67555
67566
CCCTCTACACCA
1-10-1 MOE
1050


 


147082
67578
67589
AGCTCCTTCCAC
1-10-1 MOE
1036


 


401412
67637
67650
TAAATCCTCTAGCA
2-10-2 MOE
1003


 


147091
67729
67740
GTTCCCTCTACA
1-10-1 MOE
1004


 


147742
67737
67748
AACTTCAGTGTC
1-10-1 MOE
1041


 


147712
68527
68538
ACACCATCTCCC
1-10-1 MOE
1005


 


147712
68673
68684
ACACCATCTCCC
1-10-1 MOE
1005


 


147711
68760
68771
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
68906
68917
AAGGGCCCTGGG
1-10-1 MOE
1040


 


389965
69271
69282
CTGCAACATGAT
1-10-1 MOE
1018


 


389965
69417
69428
CTGCAACATGAT
1-10-1 MOE
1018


 


368353
69519
69532
CACTGATCCTGCAC
2-10-2 MOE
1007


 


147080
69630
69641
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
69631
69642
GCTCCTTCCACT
1-10-1 MOE
1006


 


368353
69665
69678
CACTGATCCTGCAC
2-10-2 MOE
1007


 


398167
69757
69768
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
69758
69771
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


398093
69811
69824
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
69813
69824
TCGGACTTTGAA
1-10-1 MOE
1008


 


398167
69903
69914
CAGGCCATGTGG
1-10-1 MOE
1059


 


398093
69957
69970
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398094
70047
70060
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398095
70065
70078
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


147704
70137
70148
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147728
70450
70461
GCCAGACAGAAG
1-10-1 MOE
1013


 


398164
70464
70475
TTGTCGATCTGC
1-10-1 MOE
1014


 


398096
70562
70575
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
70564
70575
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147737
70575
70586
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147735
70710
70721
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147737
70721
70732
ACAGCCAGGTAG
1-10-1 MOE
1067


 


404131
70729
70742
ACCTTCGATCACAG
2-10-2 MOE
831


 


368349
70762
70775
CTGCACTGACGAGT
2-10-2 MOE
1017


 


389965
70930
70941
CTGCAACATGAT
1-10-1 MOE
1018


 


368366
70995
71008
CTGATCCTTAGAAG
2-10-2 MOE
1019


 


368354
70999
71012
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368375
71000
71015
CCTTCCACTGATCCTG
3-10-3 MOE
1020


 


368356
71001
71014
CTTCCACTGATCCT
2-10-2 MOE
1027


 


368376
71001
71016
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


368357
71002
71015
CCTTCCACTGATCC
2-10-2 MOE
1046


 


368377
71002
71017
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
71003
71014
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
71003
71016
TCCTTCCACTGATC
2-10-2 MOE
1031


 


368378
71003
71018
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


147078
71004
71015
CCTTCCACTGAT
1-10-1 MOE
1044


 


368359
71005
71018
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


368379
71005
71020
AAGCTCCTTCCACTGA
3-10-3 MOE
1034


 


147080
71006
71017
CTCCTTCCACTG
1-10-1 MOE
1021


 


147082
71008
71019
AGCTCCTTCCAC
1-10-1 MOE
1036


 


401413
71019
71032
TGCAGCCATGTACT
2-10-2 MOE
1022


 


147738
71067
71078
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147739
71071
71082
CGTTTGGGTGGC
1-10-1 MOE
1023


 


147741
71129
71140
CACCCACTGGTG
1-10-1 MOE
1055


 


368354
71145
71158
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368355
71146
71159
TTCCACTGATCCTG
2-10-2 MOE
1025


 


147075
71147
71158
TCCACTGATCCT
1-10-1 MOE
1026


 


368356
71147
71160
CTTCCACTGATCCT
2-10-2 MOE
1027


 


368376
71147
71162
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


147076
71148
71159
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
71148
71161
CCTTCCACTGATCC
2-10-2 MOE
1046


 


368377
71148
71163
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
71149
71160
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
71149
71162
TCCTTCCACTGATC
2-10-2 MOE
1031


 


368378
71149
71164
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


147078
71150
71161
CCTTCCACTGAT
1-10-1 MOE
1044


 


368359
71151
71164
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


368379
71151
71166
AAGCTCCTTCCACTGA
3-10-3 MOE
1034


 


368360
71153
71166
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
71154
71165
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368381
71155
71170
GGGAAAGCTCCTTCCA
3-10-3 MOE
1037


 


390030
71986
71997
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
72132
72143
TTTATAAAACTG
1-10-1 MOE
1074


 


147711
72300
72311
AAGGGCCCTGGG
1-10-1 MOE
1040


 


401414
72347
72360
TTGCAATGTCTGGC
2-10-2 MOE
1038


 


147741
72400
72411
CACCCACTGGTG
1-10-1 MOE
1055


 


401415
72415
72428
GATTTATCTGGCTG
2-10-2 MOE
1039


 


147711
72446
72457
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147742
72575
72586
AACTTCAGTGTC
1-10-1 MOE
1041


 


147743
72690
72701
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147744
72694
72705
AGGAAGGGCTTC
1-10-1 MOE
1043


 


147745
72700
72711
TTGACCAGGAAG
1-10-1 MOE
1058


 


147742
72721
72732
AACTTCAGTGTC
1-10-1 MOE
1041


 


147743
72836
72847
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147744
72840
72851
AGGAAGGGCTTC
1-10-1 MOE
1043


 


368357
72898
72911
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147078
72900
72911
CCTTCCACTGAT
1-10-1 MOE
1044


 


398157
72903
72916
GGAAACATACCCTG
2-10-2 MOE
1045


 


368357
73044
73057
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
73045
73056
CTTCCACTGATC
1-10-1 MOE
1047


 


147746
73052
73063
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
73101
73112
TAAAAACAACAA
1-10-1 MOE
1073


 


398160
73139
73152
GAATAGGTTAAGGC
2-10-2 MOE
1048


 


147746
73198
73209
TAAAAACAACAA
1-10-1 MOE
1073


 


398161
73238
73251
AACAATGTGTTGTA
2-10-2 MOE
1049


 


147088
73419
73430
CCCTCTACACCA
1-10-1 MOE
1050


 


404140
73457
73470
GCACACAGCTGAGG
2-10-2 MOE
1051


 


404139
73459
73472
GTGCACACAGCTGA
2-10-2 MOE
1052


 


399301
73461
73474
GTGTGCACACAGCT
2-10-2 MOE
1542


 


404137
73463
73476
CAGTGTGCACACAG
2-10-2 MOE
1053


 


404138
73465
73478
CTCAGTGTGCACAC
2-10-2 MOE
1054


 


147741
73705
73716
CACCCACTGGTG
1-10-1 MOE
1055


 


404135
73858
73871
CATTTCCATGGCCA
2-10-2 MOE
1056


 


398167
74008
74019
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
74009
74022
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


398162
74114
74127
ACCAAACAGTTCAG
2-10-2 MOE
1057


 


147745
74137
74148
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
74154
74165
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
74155
74168
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


389949
74310
74321
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147740
74485
74496
TGTGAGGCTCCA
1-10-1 MOE
1062


 


389950
74527
74538
CCCTGAAGGTTC
1-10-1 MOE
1063


 


398101
74656
74669
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


398104
74805
74818
CAAGAAGACCTTAC
2-10-2 MOE
1065


 


147737
74893
74904
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398105
74894
74907
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147737
74919
74930
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398106
74974
74987
TGGAAAACTGCACC
2-10-2 MOE
1068


 


404199
75045
75058
GGTCATGCACAGGC
2-10-2 MOE
867


 


404134
75048
75061
TCAGGTCATGCACA
2-10-2 MOE
873


 


398106
75120
75133
TGGAAAACTGCACC
2-10-2 MOE
1068


 


147738
75155
75166
TGGGTGGCCGGG
1-10-1 MOE
1069


 


404132
75227
75240
CCTTGGAATGTCTG
2-10-2 MOE
852


 


147738
75301
75312
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398166
75499
75510
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147746
75617
75628
TAAAAACAACAA
1-10-1 MOE
1073


 


147706
75686
75697
GCTGACATCTCG
1-10-1 MOE
1071


 


398112
75730
75743
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


147746
75763
75774
TAAAAACAACAA
1-10-1 MOE
1073


 


398115
75786
75799
AGTAAATATTGGCT
2-10-2 MOE
1076


 


390030
75839
75850
TTTATAAAACTG
1-10-1 MOE
1074


 


398114
75916
75929
AGGCATATAGCAGA
2-10-2 MOE
1075


 


398115
75932
75945
AGTAAATATTGGCT
2-10-2 MOE
1076


 


404133
75968
75981
TATTCCATGGCCAT
2-10-2 MOE
872


 


147715
77045
77056
GTTGAGCATGAC
1-10-1 MOE
1077


 


147715
77190
77201
GTTGAGCATGAC
1-10-1 MOE
1077


 


147693
77385
77396
GTGCGCTCCCAT
1-10-1 MOE
1078


 


398173
40201
40212
CAGCCTGGGCAC
1-10-1 MOE
1543


 


398173
72764
72775
CAGCCTGGGCAC
1-10-1 MOE
1543


 


399096
1986
1999
TGCTCGAACTCCTT
2-10-2 MOE
1544


 


399102
52822
52835
GAAGTCACTGGCTT
2-10-2 MOE
1545


 


399103
52824
52837
GGGAAGTCACTGGC
2-10-2 MOE
1546


 


399113
59827
59840
GTTAGGCAAAGGGC
2-10-2 MOE
1547


 


399132
69977
69990
GGGCTGAGTGACCC
2-10-2 MOE
1548


 


399173
74592
74605
ATGCTAGTGCACTA
2-10-2 MOE
1549


 


399208
75900
75913
AGCTCGCTACCTCT
2-10-2 MOE
1550


 


399276
27559
27572
GAGGTATCCCATCT
2-10-2 MOE
1551


 


399315
74039
74052
GGCAACTTCAACCT
2-10-2 MOE
1552
















TABLE 19







Short antisense compounds targeted to SEQ ID NO:


12 and having 1 or 2 mismatches













5′ Target
3′ Target

Gapmer
Seq ID


ISIS NO.
Site
Site
Sequence (5′-3′)
Motif
NO















398163
20
31
ATGTCAACCGGC
1-10-1 MOE
908


 


384545
23
34
CAAGTAGGATGT
1-10-1 MOE
951


 


147733
26
37
TTCTTGATGTCC
1-10-1 MOE
891


 


147721
59
70
AATGCAGGATCT
1-10-1 MOE
1118


 


147700
110
121
GCGCTAGGCCGC
1-10-1 MOE
1110


 


384545
130
141
CAAGTAGGATGT
1-10-1 MOE
951


 


147705
159
170
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147701
167
178
CCATGGCGGGAC
1-10-1 MOE
921


 


398164
198
209
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
199
210
CTTGTCCATCAG
1-10-1 MOE
1121


 


147702
226
237
CTGGTAAATAGC
1-10-1 MOE
898


 


147703
245
256
TGGCTTCATGTC
1-10-1 MOE
971


 


147705
266
277
CGGTTTTTGTTC
1-10-1 MOE
1002


 


398165
283
294
GTTCTTAGGAAG
1-10-1 MOE
968


 


147704
285
296
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
291
302
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147709
311
322
CCATTTTTATCA
1-10-1 MOE
978


 


147733
349
360
TTCTTGATGTCC
1-10-1 MOE
891


 


147707
360
371
TAGTCATTATCT
1-10-1 MOE
977


 


147708
366
377
TTGATATAGTCA
1-10-1 MOE
997


 


390030
381
392
TTTATAAAACTG
1-10-1 MOE
1074


 


147709
386
397
CCATTTTTATCA
1-10-1 MOE
978


 


147081
393
404
GCTCCTTCCACT
1-10-1 MOE
1006


 


398091
393
406
GGGCTTCTTCCATT
2-10-2 MOE
979


 


398166
395
406
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147712
461
472
ACACCATCTCCC
1-10-1 MOE
1005


 


147713
466
477
CTCCCACACCAT
1-10-1 MOE
985


 


147714
471
482
TTCTGCTCCCAC
1-10-1 MOE
986


 


147710
502
513
TATAGCTCCTCT
1-10-1 MOE
994


 


147736
551
562
AGGTAGGAGAAG
1-10-1 MOE
963


 


147717
574
585
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
607
618
ATCTTCAGAGAT
1-10-1 MOE
996


 


147710
609
620
TATAGCTCCTCT
1-10-1 MOE
994


 


147708
612
623
TTGATATAGTCA
1-10-1 MOE
997


 


147718
621
632
TAATATGACTTG
1-10-1 MOE
998


 


147746
625
636
TAAAAACAACAA
1-10-1 MOE
1073


 


147736
658
669
AGGTAGGAGAAG
1-10-1 MOE
963


 


147720
676
687
GATCTCTCGAGT
1-10-1 MOE
1117


 


147721
683
694
AATGCAGGATCT
1-10-1 MOE
1118


 


398167
704
715
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
705
718
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


147722
709
720
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147723
715
726
GACTCCAAAGTC
1-10-1 MOE
892


 


147746
733
744
TAAAAACAACAA
1-10-1 MOE
1073


 


398093
758
771
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
760
771
TCGGACTTTGAA
1-10-1 MOE
1008


 


147725
761
772
CTCGGACTTTGA
1-10-1 MOE
1119


 


147726
766
777
TGACTCTCGGAC
1-10-1 MOE
1120


 


147738
780
791
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147727
807
818
CAGTGGACCACA
1-10-1 MOE
1128


 


147728
846
857
GCCAGACAGAAG
1-10-1 MOE
1013


 


398094
848
861
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398169
849
860
TCAGCCAGACAG
1-10-1 MOE
909


 


147729
863
874
GTAAGAGGCAGG
1-10-1 MOE
920


 


398095
866
879
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398164
873
884
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
874
885
CTTGTCCATCAG
1-10-1 MOE
1121


 


147731
880
891
TTTCCTCTTGTC
1-10-1 MOE
934


 


147732
885
896
GGGTCTTTCCTC
1-10-1 MOE
1122


 


147738
888
899
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147733
906
917
TTCTTGATGTCC
1-10-1 MOE
891


 


398096
971
984
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
973
984
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147736
978
989
AGGTAGGAGAAG
1-10-1 MOE
963


 


147729
979
990
GTAAGAGGCAGG
1-10-1 MOE
920


 


147737
984
995
ACAGCCAGGTAG
1-10-1 MOE
1067


 


368349
1025
1038
CTGCACTGACGAGT
2-10-2 MOE
1017


 


368369
1025
1040
TCCTGCACTGACGAGT
3-10-3 MOE
893


 


368350
1027
1040
TCCTGCACTGACGA
2-10-2 MOE
1079


 


368370
1027
1042
GATCCTGCACTGACGA
3-10-3 MOE
1080


 


368351
1029
1042
GATCCTGCACTGAC
2-10-2 MOE
1081


 


368371
1029
1044
CTGATCCTGCACTGAC
3-10-3 MOE
1082


 


368352
1031
1044
CTGATCCTGCACTG
2-10-2 MOE
1105


 


368372
1031
1046
CACTGATCCTGCACTG
3-10-3 MOE
894


 


368353
1033
1046
CACTGATCCTGCAC
2-10-2 MOE
1007


 


368373
1033
1048
TCCACTGATCCTGCAC
3-10-3 MOE
1083


 


368354
1035
1048
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368368
1035
1048
TCCACTGATCCTTA
2-10-2 MOE
1127


 


368374
1035
1050
CTTCCACTGATCCTGC
3-10-3 MOE
1126


 


368388
1035
1050
CTTCCACTGATCCTTA
3-10-3 MOE
895


 


147074
1036
1047
CCACTGATCCTG
1-10-1 MOE
845


 


368355
1036
1049
TTCCACTGATCCTG
2-10-2 MOE
1025


 


368375
1036
1051
CCTTCCACTGATCCTG
3-10-3 MOE
1020


 


147075
1037
1048
TCCACTGATCCT
1-10-1 MOE
1026


 


368356
1037
1050
CTTCCACTGATCCT
2-10-2 MOE
1027


 


368376
1037
1052
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


147076
1038
1049
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
1038
1051
CCTTCCACTGATCC
2-10-2 MOE
1046


 


368377
1038
1053
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
1039
1050
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
1039
1052
TCCTTCCACTGATC
2-10-2 MOE
1031


 


368378
1039
1054
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


147078
1040
1051
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
1041
1052
TCCTTCCACTGA
1-10-1 MOE
1001


 


368359
1041
1054
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


368379
1041
1056
AAGCTCCTTCCACTGA
3-10-3 MOE
1034


 


147080
1042
1053
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
1043
1054
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
1043
1056
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


368380
1043
1058
GAAAGCTCCTTCCACT
3-10-3 MOE
896


 


147082
1044
1055
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368361
1045
1058
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


368381
1045
1060
GGGAAAGCTCCTTCCA
3-10-3 MOE
1037


 


147729
1087
1098
GTAAGAGGCAGG
1-10-1 MOE
920


 


147738
1103
1114
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147739
1107
1118
CGTTTGGGTGGC
1-10-1 MOE
1023


 


147740
1124
1135
TGTGAGGCTCCA
1-10-1 MOE
1062


 


398117
1164
1177
TTTCCACTTGGGTG
2-10-2 MOE
960


 


147741
1165
1176
CACCCACTGGTG
1-10-1 MOE
1055


 


398097
1194
1207
GGCAGTCTTTATCC
2-10-2 MOE
897


 


398098
1272
1285
TAACTTCAGTGTCT
2-10-2 MOE
1131


 


398117
1272
1285
TTTCCACTTGGGTG
2-10-2 MOE
960


 


147742
1273
1284
AACTTCAGTGTC
1-10-1 MOE
1041


 


147698
1293
1304
CCCGCCACCACC
1-10-1 MOE
928


 


147743
1388
1399
AGGGCTTCCAGT
1-10-1 MOE
1042


 


398099
1388
1401
GAAGGGCTTCCAGT
2-10-2 MOE
1132


 


147744
1392
1403
AGGAAGGGCTTC
1-10-1 MOE
1043


 


398100
1395
1408
TGACCAGGAAGGGC
2-10-2 MOE
1133


 


147745
1398
1409
TTGACCAGGAAG
1-10-1 MOE
1058


 


398157
1455
1468
GGAAACATACCCTG
2-10-2 MOE
1045


 


147745
1458
1469
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
1475
1486
CAGGCCATGTGG
1-10-1 MOE
1059


 


398118
1564
1577
CGCGAGATATCTAA
2-10-2 MOE
1084


 


147697
1575
1586
CCCCAGCAGCGG
1-10-1 MOE
1000


 


147076
1596
1607
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
1596
1609
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
1597
1608
CTTCCACTGATC
1-10-1 MOE
1047


 


147078
1598
1609
CCTTCCACTGAT
1-10-1 MOE
1044


 


398118
1672
1685
CGCGAGATATCTAA
2-10-2 MOE
1084


 


398158
1681
1694
AGGCCCTGAGATTA
2-10-2 MOE
1134


 


147697
1683
1694
CCCCAGCAGCGG
1-10-1 MOE
1000


 


398159
1686
1699
GGTTAAGGCCCTGA
2-10-2 MOE
1135


 


398160
1691
1704
GAATAGGTTAAGGC
2-10-2 MOE
1048


 


398163
1711
1722
ATGTCAACCGGC
1-10-1 MOE
908


 


147733
1717
1728
TTCTTGATGTCC
1-10-1 MOE
891


 


147089
1747
1758
TCCCTCTACACC
1-10-1 MOE
956


 


147090
1748
1759
TTCCCTCTACAC
1-10-1 MOE
955


 


147746
1750
1761
TAAAAACAACAA
1-10-1 MOE
1073


 


389949
1777
1788
GCGCGAGCCCGA
1-10-1 MOE
1061


 


398161
1790
1803
AACAATGTGTTGTA
2-10-2 MOE
1049


 


147746
1799
1810
TAAAAACAACAA
1-10-1 MOE
1073


 


147700
1801
1812
GCGCTAGGCCGC
1-10-1 MOE
1110


 


147740
1806
1817
TGTGAGGCTCCA
1-10-1 MOE
1062


 


398163
1819
1830
ATGTCAACCGGC
1-10-1 MOE
908


 


147733
1825
1836
TTCTTGATGTCC
1-10-1 MOE
891


 


389950
1848
1859
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147701
1858
1869
CCATGGCGGGAC
1-10-1 MOE
921


 


398164
1889
1900
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
1890
1901
CTTGTCCATCAG
1-10-1 MOE
1121


 


147700
1909
1920
GCGCTAGGCCGC
1-10-1 MOE
1110


 


398119
1920
1933
CGCACCTGGTAAAT
2-10-2 MOE
1085


 


147685
1957
1968
GGCTGACATTCA
1-10-1 MOE
975


 


147701
1966
1977
CCATGGCGGGAC
1-10-1 MOE
921


 


398120
1966
1979
GTTCAAGCGGCCTA
2-10-2 MOE
1086


 


398101
1977
1990
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


398164
1997
2008
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
1998
2009
CTTGTCCATCAG
1-10-1 MOE
1121


 


147702
2025
2036
CTGGTAAATAGC
1-10-1 MOE
898


 


398119
2028
2041
CGCACCTGGTAAAT
2-10-2 MOE
1085


 


398120
2074
2087
GTTCAAGCGGCCTA
2-10-2 MOE
1086


 


398105
2099
2112
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147736
2204
2215
AGGTAGGAGAAG
1-10-1 MOE
963


 


147741
2257
2268
CACCCACTGGTG
1-10-1 MOE
1055


 


398104
2272
2285
CAAGAAGACCTTAC
2-10-2 MOE
1065


 


147737
2360
2371
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398105
2361
2374
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147737
2386
2397
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398095
2407
2420
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398106
2441
2454
TGGAAAACTGCACC
2-10-2 MOE
1068


 


398107
2447
2460
TATTCCTGGAAAAC
2-10-2 MOE
902


 


398121
2474
2487
GTGCCTAGCACAGA
2-10-2 MOE
1097


 


147745
2497
2508
TTGACCAGGAAG
1-10-1 MOE
1058


 


147712
2499
2510
ACACCATCTCCC
1-10-1 MOE
1005


 


398108
2544
2557
GGAATGTCTGAGTT
2-10-2 MOE
1136


 


147691
2575
2586
GAGGTGGGAAAA
1-10-1 MOE
966


 


398121
2582
2595
GTGCCTAGCACAGA
2-10-2 MOE
1097


 


147738
2622
2633
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398162
2666
2679
ACCAAACAGTTCAG
2-10-2 MOE
1057


 


147745
2689
2700
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
2706
2717
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
2707
2720
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


398109
2714
2727
CAAGAAGTGTGGTT
2-10-2 MOE
903


 


398110
2852
2865
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


147091
2854
2865
GTTCCCTCTACA
1-10-1 MOE
1004


 


147723
2924
2935
GACTCCAAAGTC
1-10-1 MOE
892


 


398111
2937
2950
GTGAAAATGCTGGC
2-10-2 MOE
904


 


398166
2966
2977
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147089
2978
2989
TCCCTCTACACC
1-10-1 MOE
956


 


147090
2979
2990
TTCCCTCTACAC
1-10-1 MOE
955


 


147706
3007
3018
GCTGACATCTCG
1-10-1 MOE
1071


 


389949
3008
3019
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147723
3032
3043
GACTCCAAAGTC
1-10-1 MOE
892


 


147740
3037
3048
TGTGAGGCTCCA
1-10-1 MOE
1062


 


398112
3051
3064
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


389950
3079
3090
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147746
3084
3095
TAAAAACAACAA
1-10-1 MOE
1073


 


398122
3148
3161
CCCTTTACACAAGT
2-10-2 MOE
1087


 


147089
3151
3162
TCCCTCTACACC
1-10-1 MOE
956


 


147090
3152
3163
TTCCCTCTACAC
1-10-1 MOE
955


 


398113
3160
3173
AGGAGGTTAAACCA
2-10-2 MOE
905


 


147685
3188
3199
GGCTGACATTCA
1-10-1 MOE
975


 


398101
3208
3221
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


398102
3234
3247
CTACCTGAGGATTT
2-10-2 MOE
899


 


398123
3235
3248
CTCAAAATAGATTT
2-10-2 MOE
1088


 


398114
3237
3250
AGGCATATAGCAGA
2-10-2 MOE
1075


 


398103
3241
3254
CCCAGTACTACCTG
2-10-2 MOE
900


 


398115
3253
3266
AGTAAATATTGGCT
2-10-2 MOE
1076


 


398122
3256
3269
CCCTTTACACAAGT
2-10-2 MOE
1087


 


147089
3259
3270
TCCCTCTACACC
1-10-1 MOE
956


 


147090
3260
3271
TTCCCTCTACAC
1-10-1 MOE
955


 


398116
3266
3279
TAATGACCTGATGA
2-10-2 MOE
1137


 


390030
3306
3317
TTTATAAAACTG
1-10-1 MOE
1074


 


398123
3343
3356
CTCAAAATAGATTT
2-10-2 MOE
1088


 


147736
3435
3446
AGGTAGGAGAAG
1-10-1 MOE
963


 


398104
3503
3516
CAAGAAGACCTTAC
2-10-2 MOE
1065


 


147737
3591
3602
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398105
3592
3605
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147719
3608
3619
CCAACTCCAACT
1-10-1 MOE
1116


 


147737
3617
3628
ACAGCCAGGTAG
1-10-1 MOE
1067


 


401398
3621
3634
CAAAGTCCCTTAGC
2-10-2 MOE
947


 


147079
3637
3648
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
3638
3649
CTCCTTCCACTG
1-10-1 MOE
1021


 


398095
3638
3651
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398106
3672
3685
TGGAAAACTGCACC
2-10-2 MOE
1068


 


147733
3687
3698
TTCTTGATGTCC
1-10-1 MOE
891


 


147731
3688
3699
TTTCCTCTTGTC
1-10-1 MOE
934


 


147719
3716
3727
CCAACTCCAACT
1-10-1 MOE
1116


 


147745
3728
3739
TTGACCAGGAAG
1-10-1 MOE
1058


 


147683
3740
3751
GCTTACGATTGT
1-10-1 MOE
922


 


147079
3745
3756
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
3746
3757
CTCCTTCCACTG
1-10-1 MOE
1021


 


398108
3775
3788
GGAATGTCTGAGTT
2-10-2 MOE
1136


 


147733
3795
3806
TTCTTGATGTCC
1-10-1 MOE
891


 


147731
3796
3807
TTTCCTCTTGTC
1-10-1 MOE
934


 


147691
3806
3817
GAGGTGGGAAAA
1-10-1 MOE
966


 


147738
3853
3864
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398167
3926
3937
CAGGCCATGTGG
1-10-1 MOE
1059


 


147691
3978
3989
GAGGTGGGAAAA
1-10-1 MOE
966


 


398167
4034
4045
CAGGCCATGTGG
1-10-1 MOE
1059


 


147091
4085
4096
GTTCCCTCTACA
1-10-1 MOE
1004


 


147691
4086
4097
GAGGTGGGAAAA
1-10-1 MOE
966


 


398111
4168
4181
GTGAAAATGCTGGC
2-10-2 MOE
904


 


398166
4197
4208
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147091
4223
4234
GTTCCCTCTACA
1-10-1 MOE
1004


 


147092
4224
4235
TGTTCCCTCTAC
1-10-1 MOE
901


 


398112
4282
4295
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


147746
4315
4326
TAAAAACAACAA
1-10-1 MOE
1073


 


398113
4391
4404
AGGAGGTTAAACCA
2-10-2 MOE
905


 


147723
4422
4433
GACTCCAAAGTC
1-10-1 MOE
892


 


398114
4468
4481
AGGCATATAGCAGA
2-10-2 MOE
1075


 


398115
4484
4497
AGTAAATATTGGCT
2-10-2 MOE
1076


 


390030
4491
4502
TTTATAAAACTG
1-10-1 MOE
1074


 


398116
4497
4510
TAATGACCTGATGA
2-10-2 MOE
1137


 


147723
4530
4541
GACTCCAAAGTC
1-10-1 MOE
892


 


390030
4599
4610
TTTATAAAACTG
1-10-1 MOE
1074


 


398124
4761
4774
CACATGAGCTATTC
2-10-2 MOE
1089


 


398124
4869
4882
CACATGAGCTATTC
2-10-2 MOE
1089


 


147703
4926
4937
TGGCTTCATGTC
1-10-1 MOE
971


 


147692
4928
4939
CTCACCTTCATG
1-10-1 MOE
1113


 


147696
4975
4986
TGGATGATTGGC
1-10-1 MOE
906


 


147703
5034
5045
TGGCTTCATGTC
1-10-1 MOE
971


 


147692
5036
5047
CTCACCTTCATG
1-10-1 MOE
1113


 


147098
5173
5184
AGTTGTTGTTCC
1-10-1 MOE
1112


 


398125
5183
5196
CAGTAAGGAATTTT
2-10-2 MOE
913


 


398126
5216
5229
GTGAAGTGAGTCAT
2-10-2 MOE
1090


 


147098
5281
5292
AGTTGTTGTTCC
1-10-1 MOE
1112


 


398127
5283
5296
GGTCACTCAAGATG
2-10-2 MOE
1091


 


398126
5324
5337
GTGAAGTGAGTCAT
2-10-2 MOE
1090


 


398128
5335
5348
CTAAATTTAGTTCA
2-10-2 MOE
911


 


398127
5391
5404
GGTCACTCAAGATG
2-10-2 MOE
1091


 


398128
5443
5456
CTAAATTTAGTTCA
2-10-2 MOE
911


 


147712
5474
5485
ACACCATCTCCC
1-10-1 MOE
1005


 


147736
5600
5611
AGGTAGGAGAAG
1-10-1 MOE
963


 


147746
5606
5617
TAAAAACAACAA
1-10-1 MOE
1073


 


398129
5628
5641
TTTGAGGAGCTATT
2-10-2 MOE
1106


 


147085
5654
5665
TCTACACCAGGT
1-10-1 MOE
961


 


147736
5708
5719
AGGTAGGAGAAG
1-10-1 MOE
963


 


398129
5736
5749
TTTGAGGAGCTATT
2-10-2 MOE
1106


 


147679
5934
5945
CAAAAGGATCCC
1-10-1 MOE
907


 


147723
6229
6240
GACTCCAAAGTC
1-10-1 MOE
892


 


147723
6338
6349
GACTCCAAAGTC
1-10-1 MOE
892


 


390030
6803
6814
TTTATAAAACTG
1-10-1 MOE
1074


 


398142
6885
6898
CCAGCACACTGGAA
2-10-2 MOE
923


 


390030
6912
6923
TTTATAAAACTG
1-10-1 MOE
1074


 


398142
6994
7007
CCAGCACACTGGAA
2-10-2 MOE
923


 


147695
7054
7065
TCATTCCCCACT
1-10-1 MOE
984


 


147695
7163
7174
TCATTCCCCACT
1-10-1 MOE
984


 


398166
7197
7208
GGGCTTCTTCCA
1-10-1 MOE
1070


 


398166
7306
7317
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147684
7442
7453
ACCCAGTCAGGG
1-10-1 MOE
964


 


398130
7694
7707
TTAGTATGACAGCT
2-10-2 MOE
925


 


398131
7711
7724
GGACTCACTCAGCA
2-10-2 MOE
1092


 


398130
7802
7815
TTAGTATGACAGCT
2-10-2 MOE
925


 


398125
7804
7817
CAGTAAGGAATTTT
2-10-2 MOE
913


 


398131
7819
7832
GGACTCACTCAGCA
2-10-2 MOE
1092


 


390030
7877
7888
TTTATAAAACTG
1-10-1 MOE
1074


 


398125
7912
7925
CAGTAAGGAATTTT
2-10-2 MOE
913


 


390030
7985
7996
TTTATAAAACTG
1-10-1 MOE
1074


 


398132
8031
8044
TCAGGGCTACTCAT
2-10-2 MOE
1093


 


398132
8139
8152
TCAGGGCTACTCAT
2-10-2 MOE
1093


 


147684
8148
8159
ACCCAGTCAGGG
1-10-1 MOE
964


 


147684
8256
8267
ACCCAGTCAGGG
1-10-1 MOE
964


 


398163
8365
8376
ATGTCAACCGGC
1-10-1 MOE
908


 


398166
8447
8458
GGGCTTCTTCCA
1-10-1 MOE
1070


 


398163
8473
8484
ATGTCAACCGGC
1-10-1 MOE
908


 


398166
8555
8566
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147718
8631
8642
TAATATGACTTG
1-10-1 MOE
998


 


147691
8698
8709
GAGGTGGGAAAA
1-10-1 MOE
966


 


147691
8806
8817
GAGGTGGGAAAA
1-10-1 MOE
966


 


147728
8835
8846
GCCAGACAGAAG
1-10-1 MOE
1013


 


147727
8876
8887
CAGTGGACCACA
1-10-1 MOE
1128


 


147728
8943
8954
GCCAGACAGAAG
1-10-1 MOE
1013


 


398169
8946
8957
TCAGCCAGACAG
1-10-1 MOE
909


 


147727
8984
8995
CAGTGGACCACA
1-10-1 MOE
1128


 


147742
9060
9071
AACTTCAGTGTC
1-10-1 MOE
1041


 


398133
9112
9125
CAGCACTAGATTCA
2-10-2 MOE
1094


 


384545
9135
9146
CAAGTAGGATGT
1-10-1 MOE
951


 


147742
9168
9179
AACTTCAGTGTC
1-10-1 MOE
1041


 


398133
9220
9233
CAGCACTAGATTCA
2-10-2 MOE
1094


 


384545
9243
9254
CAAGTAGGATGT
1-10-1 MOE
951


 


398125
9368
9381
CAGTAAGGAATTTT
2-10-2 MOE
913


 


398125
9476
9489
CAGTAAGGAATTTT
2-10-2 MOE
913


 


401409
9516
9529
ATTCTTAACACAGA
2-10-2 MOE
991


 


147096
9594
9605
TTGTTGTTCCCT
1-10-1 MOE
1107


 


147733
9597
9608
TTCTTGATGTCC
1-10-1 MOE
891


 


147720
9689
9700
GATCTCTCGAGT
1-10-1 MOE
1117


 


147096
9702
9713
TTGTTGTTCCCT
1-10-1 MOE
1107


 


147733
9705
9716
TTCTTGATGTCC
1-10-1 MOE
891


 


147720
9797
9808
GATCTCTCGAGT
1-10-1 MOE
1117


 


147746
9963
9974
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9966
9977
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9969
9980
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
9991
10002
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10071
10082
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10074
10085
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10077
10088
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
10099
10110
TAAAAACAACAA
1-10-1 MOE
1073


 


398134
10153
10166
TAGCTTAATGTAAC
2-10-2 MOE
1095


 


147085
10221
10232
TCTACACCAGGT
1-10-1 MOE
961


 


398134
10261
10274
TAGCTTAATGTAAC
2-10-2 MOE
1095


 


390030
10278
10289
TTTATAAAACTG
1-10-1 MOE
1074


 


147084
10328
10339
CTACACCAGGTC
1-10-1 MOE
993


 


147711
10684
10695
AAGGGCCCTGGG
1-10-1 MOE
1040


 


398128
11333
11346
CTAAATTTAGTTCA
2-10-2 MOE
911


 


398128
11340
11353
CTAAATTTAGTTCA
2-10-2 MOE
911


 


147730
11783
11794
CTTGTCCATCAG
1-10-1 MOE
1121


 


147731
11789
11800
TTTCCTCTTGTC
1-10-1 MOE
934


 


147730
11790
11801
CTTGTCCATCAG
1-10-1 MOE
1121


 


147731
11796
11807
TTTCCTCTTGTC
1-10-1 MOE
934


 


147707
11960
11971
TAGTCATTATCT
1-10-1 MOE
977


 


147090
12008
12019
TTCCCTCTACAC
1-10-1 MOE
955


 


147091
12009
12020
GTTCCCTCTACA
1-10-1 MOE
1004


 


147091
12014
12025
GTTCCCTCTACA
1-10-1 MOE
1004


 


398096
12141
12154
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
12143
12154
GGAGAAGCGCAG
1-10-1 MOE
1016


 


398096
12146
12159
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
12148
12159
GGAGAAGCGCAG
1-10-1 MOE
1016


 


398166
12209
12220
GGGCTTCTTCCA
1-10-1 MOE
1070


 


398166
12214
12225
GGGCTTCTTCCA
1-10-1 MOE
1070


 


398135
12303
12316
GACTACATTTTACA
2-10-2 MOE
912


 


147741
12389
12400
CACCCACTGGTG
1-10-1 MOE
1055


 


147741
12394
12405
CACCCACTGGTG
1-10-1 MOE
1055


 


398125
12431
12444
CAGTAAGGAATTTT
2-10-2 MOE
913


 


147714
12585
12596
TTCTGCTCCCAC
1-10-1 MOE
986


 


147718
12594
12605
TAATATGACTTG
1-10-1 MOE
998


 


398125
12612
12625
CAGTAAGGAATTTT
2-10-2 MOE
913


 


147737
12803
12814
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147746
12876
12887
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
12900
12911
GAGGTGGGAAAA
1-10-1 MOE
966


 


398136
12915
12928
TTGTGACATCTAGG
2-10-2 MOE
1096


 


147737
12984
12995
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147746
13057
13068
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
13081
13092
GAGGTGGGAAAA
1-10-1 MOE
966


 


398136
13096
13109
TTGTGACATCTAGG
2-10-2 MOE
1096


 


398138
13254
13267
AACATCAAGCTTGA
2-10-2 MOE
931


 


398138
13435
13448
AACATCAAGCTTGA
2-10-2 MOE
931


 


147691
13488
13499
GAGGTGGGAAAA
1-10-1 MOE
966


 


147681
13659
13670
ATGTCATTAAAC
1-10-1 MOE
965


 


147691
13669
13680
GAGGTGGGAAAA
1-10-1 MOE
966


 


389965
13839
13850
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
13839
13850
CTGCAACATGAT
1-9-2 MOE
1018


 


147681
13840
13851
ATGTCATTAAAC
1-10-1 MOE
965


 


389965
14020
14031
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
14020
14031
CTGCAACATGAT
1-9-2 MOE
1018


 


389948
14067
14078
CCGTTGGACCCC
1-10-1 MOE
915


 


147736
14123
14134
AGGTAGGAGAAG
1-10-1 MOE
963


 


389948
14248
14259
CCGTTGGACCCC
1-10-1 MOE
915


 


147738
14279
14290
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147736
14304
14315
AGGTAGGAGAAG
1-10-1 MOE
963


 


147731
14411
14422
TTTCCTCTTGTC
1-10-1 MOE
934


 


147738
14461
14472
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147692
14475
14486
CTCACCTTCATG
1-10-1 MOE
1113


 


147731
14593
14604
TTTCCTCTTGTC
1-10-1 MOE
934


 


389950
14614
14625
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147692
14657
14668
CTCACCTTCATG
1-10-1 MOE
1113


 


147717
14750
14761
ATCTTCAGAGAT
1-10-1 MOE
996


 


147698
14754
14765
CCCGCCACCACC
1-10-1 MOE
928


 


389950
14796
14807
CCCTGAAGGTTC
1-10-1 MOE
1063


 


398112
14863
14876
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


398121
14875
14888
GTGCCTAGCACAGA
2-10-2 MOE
1097


 


147717
14932
14943
ATCTTCAGAGAT
1-10-1 MOE
996


 


398112
15045
15058
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


398121
15057
15070
GTGCCTAGCACAGA
2-10-2 MOE
1097


 


147730
15117
15128
CTTGTCCATCAG
1-10-1 MOE
1121


 


147730
15299
15310
CTTGTCCATCAG
1-10-1 MOE
1121


 


401407
15339
15352
CAGCTTAGGCAGAG
2-10-2 MOE
983


 


398167
15556
15567
CAGGCCATGTGG
1-10-1 MOE
1059


 


147736
16444
16455
AGGTAGGAGAAG
1-10-1 MOE
963


 


147746
16510
16521
TAAAAACAACAA
1-10-1 MOE
1073


 


147738
16590
16601
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147736
16610
16621
AGGTAGGAGAAG
1-10-1 MOE
963


 


398167
16631
16642
CAGGCCATGTGG
1-10-1 MOE
1059


 


401411
16657
16670
AGCCGCCTGAAGTG
2-10-2 MOE
999


 


147746
16676
16687
TAAAAACAACAA
1-10-1 MOE
1073


 


398144
16745
16758
GACAGCTTCTATAA
2-10-2 MOE
916


 


147738
16756
16767
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398167
16797
16808
CAGGCCATGTGG
1-10-1 MOE
1059


 


398144
16911
16924
GACAGCTTCTATAA
2-10-2 MOE
916


 


389965
17096
17107
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
17096
17107
CTGCAACATGAT
1-9-2 MOE
1018


 


389965
17264
17275
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
17264
17275
CTGCAACATGAT
1-9-2 MOE
1018


 


147709
17406
17417
CCATTTTTATCA
1-10-1 MOE
978


 


147745
17443
17454
TTGACCAGGAAG
1-10-1 MOE
1058


 


147746
17497
17508
TAAAAACAACAA
1-10-1 MOE
1073


 


147720
17589
17600
GATCTCTCGAGT
1-10-1 MOE
1117


 


147745
17611
17622
TTGACCAGGAAG
1-10-1 MOE
1058


 


147695
17634
17645
TCATTCCCCACT
1-10-1 MOE
984


 


147746
17665
17676
TAAAAACAACAA
1-10-1 MOE
1073


 


147088
17707
17718
CCCTCTACACCA
1-10-1 MOE
1050


 


147720
17757
17768
GATCTCTCGAGT
1-10-1 MOE
1117


 


147711
17808
17819
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
17976
17987
AAGGGCCCTGGG
1-10-1 MOE
1040


 


398139
18049
18062
AGTGACTGACCACA
2-10-2 MOE
917


 


398139
18217
18230
AGTGACTGACCACA
2-10-2 MOE
917


 


398140
18596
18609
GTAGCATAGAGCCT
2-10-2 MOE
918


 


398140
18764
18777
GTAGCATAGAGCCT
2-10-2 MOE
918


 


398167
18927
18938
CAGGCCATGTGG
1-10-1 MOE
1059


 


398167
19095
19106
CAGGCCATGTGG
1-10-1 MOE
1059


 


147724
19147
19158
GAAATTGAGGAA
1-10-1 MOE
1139


 


147746
19207
19218
TAAAAACAACAA
1-10-1 MOE
1073


 


147724
19315
19326
GAAATTGAGGAA
1-10-1 MOE
1139


 


147740
19348
19359
TGTGAGGCTCCA
1-10-1 MOE
1062


 


147746
19375
19386
TAAAAACAACAA
1-10-1 MOE
1073


 


147729
19386
19397
GTAAGAGGCAGG
1-10-1 MOE
920


 


147701
19503
19514
CCATGGCGGGAC
1-10-1 MOE
921


 


147711
19508
19519
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147740
19516
19527
TGTGAGGCTCCA
1-10-1 MOE
1062


 


147718
19617
19628
TAATATGACTTG
1-10-1 MOE
998


 


390030
19618
19629
TTTATAAAACTG
1-10-1 MOE
1074


 


147679
19635
19646
CAAAAGGATCCC
1-10-1 MOE
907


 


147711
19676
19687
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147694
19747
19758
CAGCCTACCAGT
1-10-1 MOE
1098


 


147718
19785
19796
TAATATGACTTG
1-10-1 MOE
998


 


390030
19786
19797
TTTATAAAACTG
1-10-1 MOE
1074


 


147679
19803
19814
CAAAAGGATCCC
1-10-1 MOE
907


 


147698
19852
19863
CCCGCCACCACC
1-10-1 MOE
928


 


147694
19915
19926
CAGCCTACCAGT
1-10-1 MOE
1098


 


147704
20011
20022
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147698
20020
20031
CCCGCCACCACC
1-10-1 MOE
928


 


398142
20485
20498
CCAGCACACTGGAA
2-10-2 MOE
923


 


147078
20514
20525
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
20515
20526
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
20516
20527
CTCCTTCCACTG
1-10-1 MOE
1021


 


398143
20561
20574
GTCAGTCCCAGCTA
2-10-2 MOE
924


 


389965
20620
20631
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
20620
20631
CTGCAACATGAT
1-9-2 MOE
1018


 


398142
20653
20666
CCAGCACACTGGAA
2-10-2 MOE
923


 


147078
20682
20693
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
20683
20694
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
20684
20695
CTCCTTCCACTG
1-10-1 MOE
1021


 


147080
20704
20715
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
20705
20716
GCTCCTTCCACT
1-10-1 MOE
1006


 


398143
20729
20742
GTCAGTCCCAGCTA
2-10-2 MOE
924


 


389965
20788
20799
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
20788
20799
CTGCAACATGAT
1-9-2 MOE
1018


 


147746
20870
20881
TAAAAACAACAA
1-10-1 MOE
1073


 


147080
20872
20883
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
20873
20884
GCTCCTTCCACT
1-10-1 MOE
1006


 


147746
21038
21049
TAAAAACAACAA
1-10-1 MOE
1073


 


147717
21080
21091
ATCTTCAGAGAT
1-10-1 MOE
996


 


147076
21222
21233
TTCCACTGATCC
1-10-1 MOE
1029


 


147076
21390
21401
TTCCACTGATCC
1-10-1 MOE
1029


 


398094
21441
21454
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


147746
21465
21476
TAAAAACAACAA
1-10-1 MOE
1073


 


398094
21609
21622
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398169
21610
21621
TCAGCCAGACAG
1-10-1 MOE
909


 


147746
21633
21644
TAAAAACAACAA
1-10-1 MOE
1073


 


147738
21884
21895
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147743
22045
22056
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147738
22052
22063
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147683
22107
22118
GCTTACGATTGT
1-10-1 MOE
922


 


147743
22213
22224
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147681
22566
22577
ATGTCATTAAAC
1-10-1 MOE
965


 


389950
22619
22630
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147681
22734
22745
ATGTCATTAAAC
1-10-1 MOE
965


 


147736
22759
22770
AGGTAGGAGAAG
1-10-1 MOE
963


 


389950
22787
22798
CCCTGAAGGTTC
1-10-1 MOE
1063


 


389949
22794
22805
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147736
22927
22938
AGGTAGGAGAAG
1-10-1 MOE
963


 


389949
22962
22973
GCGCGAGCCCGA
1-10-1 MOE
1061


 


398144
22962
22975
GACAGCTTCTATAA
2-10-2 MOE
916


 


398142
23008
23021
CCAGCACACTGGAA
2-10-2 MOE
923


 


147727
23019
23030
CAGTGGACCACA
1-10-1 MOE
1128


 


398169
23064
23075
TCAGCCAGACAG
1-10-1 MOE
909


 


398144
23130
23143
GACAGCTTCTATAA
2-10-2 MOE
916


 


398145
23154
23167
ACATGTCAGTAATT
2-10-2 MOE
1099


 


398142
23176
23189
CCAGCACACTGGAA
2-10-2 MOE
923


 


147727
23187
23198
CAGTGGACCACA
1-10-1 MOE
1128


 


147735
23243
23254
GGAGAAGCGCAG
1-10-1 MOE
1016


 


398145
23322
23335
ACATGTCAGTAATT
2-10-2 MOE
1099


 


147735
23411
23422
GGAGAAGCGCAG
1-10-1 MOE
1016


 


398146
23478
23491
CTCATGGACACAAA
2-10-2 MOE
1100


 


398146
23646
23659
CTCATGGACACAAA
2-10-2 MOE
1100


 


398147
23784
23797
CTACAGGACAATAC
2-10-2 MOE
957


 


398114
23853
23866
AGGCATATAGCAGA
2-10-2 MOE
1075


 


398147
23952
23965
CTACAGGACAATAC
2-10-2 MOE
957


 


398114
24021
24034
AGGCATATAGCAGA
2-10-2 MOE
1075


 


147702
24319
24330
CTGGTAAATAGC
1-10-1 MOE
898


 


147702
24487
24498
CTGGTAAATAGC
1-10-1 MOE
898


 


389965
24543
24554
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
24543
24554
CTGCAACATGAT
1-9-2 MOE
1018


 


147713
24602
24613
CTCCCACACCAT
1-10-1 MOE
985


 


389965
24711
24722
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
24711
24722
CTGCAACATGAT
1-9-2 MOE
1018


 


147684
24918
24929
ACCCAGTCAGGG
1-10-1 MOE
964


 


147684
25086
25097
ACCCAGTCAGGG
1-10-1 MOE
964


 


398148
25152
25165
TCATAACTATTAAG
2-10-2 MOE
981


 


398144
25192
25205
GACAGCTTCTATAA
2-10-2 MOE
916


 


147746
25216
25227
TAAAAACAACAA
1-10-1 MOE
1073


 


147736
25313
25324
AGGTAGGAGAAG
1-10-1 MOE
963


 


398148
25320
25333
TCATAACTATTAAG
2-10-2 MOE
981


 


398143
25337
25350
GTCAGTCCCAGCTA
2-10-2 MOE
924


 


398144
25360
25373
GACAGCTTCTATAA
2-10-2 MOE
916


 


147746
25384
25395
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
25442
25453
GAGGTGGGAAAA
1-10-1 MOE
966


 


147736
25481
25492
AGGTAGGAGAAG
1-10-1 MOE
963


 


398130
25504
25517
TTAGTATGACAGCT
2-10-2 MOE
925


 


147691
25610
25621
GAGGTGGGAAAA
1-10-1 MOE
966


 


147721
25662
25673
AATGCAGGATCT
1-10-1 MOE
1118


 


398130
25672
25685
TTAGTATGACAGCT
2-10-2 MOE
925


 


147688
25750
25761
TCCCAAACAAAT
1-10-1 MOE
990


 


147746
25810
25821
TAAAAACAACAA
1-10-1 MOE
1073


 


147721
25830
25841
AATGCAGGATCT
1-10-1 MOE
1118


 


147688
25918
25929
TCCCAAACAAAT
1-10-1 MOE
990


 


147746
25978
25989
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
26172
26183
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
26340
26351
TAAAAACAACAA
1-10-1 MOE
1073


 


398149
26492
26505
GGAAGTTTTCAAGT
2-10-2 MOE
1101


 


398150
26526
26539
GAATCTGGAGGTAA
2-10-2 MOE
1102


 


398149
26641
26654
GGAAGTTTTCAAGT
2-10-2 MOE
1101


 


398150
26675
26688
GAATCTGGAGGTAA
2-10-2 MOE
1102


 


147729
26712
26723
GTAAGAGGCAGG
1-10-1 MOE
920


 


398151
26718
26731
TCAGTGTAGGAAGA
2-10-2 MOE
926


 


147729
26861
26872
GTAAGAGGCAGG
1-10-1 MOE
920


 


398151
26867
26880
TCAGTGTAGGAAGA
2-10-2 MOE
926


 


147728
26917
26928
GCCAGACAGAAG
1-10-1 MOE
1013


 


147728
27066
27077
GCCAGACAGAAG
1-10-1 MOE
1013


 


147076
27258
27269
TTCCACTGATCC
1-10-1 MOE
1029


 


147731
27267
27278
TTTCCTCTTGTC
1-10-1 MOE
934


 


147076
27407
27418
TTCCACTGATCC
1-10-1 MOE
1029


 


147731
27416
27427
TTTCCTCTTGTC
1-10-1 MOE
934


 


398152
27559
27572
TGAATATACAGATG
2-10-2 MOE
927


 


398152
27708
27721
TGAATATACAGATG
2-10-2 MOE
927


 


147696
28265
28276
TGGATGATTGGC
1-10-1 MOE
906


 


147696
28414
28425
TGGATGATTGGC
1-10-1 MOE
906


 


147698
28481
28492
CCCGCCACCACC
1-10-1 MOE
928


 


147720
28662
28673
GATCTCTCGAGT
1-10-1 MOE
1117


 


389965
28714
28725
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
28714
28725
CTGCAACATGAT
1-9-2 MOE
1018


 


389965
28861
28872
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
28861
28872
CTGCAACATGAT
1-9-2 MOE
1018


 


398153
28980
28993
ATTTCTCTTACAGG
2-10-2 MOE
948


 


398153
29126
29139
ATTTCTCTTACAGG
2-10-2 MOE
948


 


147719
29570
29581
CCAACTCCAACT
1-10-1 MOE
1116


 


398154
29692
29705
AGCCCCTTGGCCGT
2-10-2 MOE
1103


 


147719
29715
29726
CCAACTCCAACT
1-10-1 MOE
1116


 


398155
29785
29798
TGTTTTTACACAGA
2-10-2 MOE
970


 


398154
29837
29850
AGCCCCTTGGCCGT
2-10-2 MOE
1103


 


401384
29905
29918
TGAACACATCACTA
2-10-2 MOE
933


 


398155
29930
29943
TGTTTTTACACAGA
2-10-2 MOE
970


 


390030
29945
29956
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
30090
30101
TTTATAAAACTG
1-10-1 MOE
1074


 


398156
30141
30154
GAATACTTCAAATC
2-10-2 MOE
1104


 


398156
30286
30299
GAATACTTCAAATC
2-10-2 MOE
1104


 


389948
30384
30395
CCGTTGGACCCC
1-10-1 MOE
915


 


389948
30530
30541
CCGTTGGACCCC
1-10-1 MOE
915


 


398142
30591
30604
CCAGCACACTGGAA
2-10-2 MOE
923


 


147744
30654
30665
AGGAAGGGCTTC
1-10-1 MOE
1043


 


147093
30689
30700
TTGTTCCCTCTA
1-10-1 MOE
929


 


398142
30738
30751
CCAGCACACTGGAA
2-10-2 MOE
923


 


147744
30801
30812
AGGAAGGGCTTC
1-10-1 MOE
1043


 


398168
31082
31093
TCGGACTTTGAA
1-10-1 MOE
1008


 


147746
31105
31116
TAAAAACAACAA
1-10-1 MOE
1073


 


398168
31230
31241
TCGGACTTTGAA
1-10-1 MOE
1008


 


390030
31329
31340
TTTATAAAACTG
1-10-1 MOE
1074


 


147736
31458
31469
AGGTAGGAGAAG
1-10-1 MOE
963


 


390030
31477
31488
TTTATAAAACTG
1-10-1 MOE
1074


 


147736
31606
31617
AGGTAGGAGAAG
1-10-1 MOE
963


 


147698
31713
31724
CCCGCCACCACC
1-10-1 MOE
928


 


384545
31829
31840
CAAGTAGGATGT
1-10-1 MOE
951


 


147698
31861
31872
CCCGCCACCACC
1-10-1 MOE
928


 


147723
31941
31952
GACTCCAAAGTC
1-10-1 MOE
892


 


384545
31977
31988
CAAGTAGGATGT
1-10-1 MOE
951


 


147692
32061
32072
CTCACCTTCATG
1-10-1 MOE
1113


 


147723
32089
32100
GACTCCAAAGTC
1-10-1 MOE
892


 


147692
32209
32220
CTCACCTTCATG
1-10-1 MOE
1113


 


147089
32535
32546
TCCCTCTACACC
1-10-1 MOE
956


 


401396
32569
32582
TGCAGGATGTTGAG
2-10-2 MOE
945


 


147730
32714
32725
CTTGTCCATCAG
1-10-1 MOE
1121


 


398165
32854
32865
GTTCTTAGGAAG
1-10-1 MOE
968


 


147730
32862
32873
CTTGTCCATCAG
1-10-1 MOE
1121


 


389950
32949
32960
CCCTGAAGGTTC
1-10-1 MOE
1063


 


398165
33002
33013
GTTCTTAGGAAG
1-10-1 MOE
968


 


147736
33012
33023
AGGTAGGAGAAG
1-10-1 MOE
963


 


368352
33056
33069
CTGATCCTGCACTG
2-10-2 MOE
1105


 


147081
33073
33084
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
33073
33086
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
33074
33085
AGCTCCTTCCAC
1-10-1 MOE
1036


 


389950
33097
33108
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147736
33160
33171
AGGTAGGAGAAG
1-10-1 MOE
963


 


368352
33204
33217
CTGATCCTGCACTG
2-10-2 MOE
1105


 


147081
33221
33232
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
33222
33233
AGCTCCTTCCAC
1-10-1 MOE
1036


 


398138
33244
33257
AACATCAAGCTTGA
2-10-2 MOE
931


 


147746
33250
33261
TAAAAACAACAA
1-10-1 MOE
1073


 


398138
33392
33405
AACATCAAGCTTGA
2-10-2 MOE
931


 


147746
33398
33409
TAAAAACAACAA
1-10-1 MOE
1073


 


147732
33652
33663
GGGTCTTTCCTC
1-10-1 MOE
1122


 


147724
33733
33744
GAAATTGAGGAA
1-10-1 MOE
1139


 


147732
33800
33811
GGGTCTTTCCTC
1-10-1 MOE
1122


 


147724
33881
33892
GAAATTGAGGAA
1-10-1 MOE
1139


 


147719
33976
33987
CCAACTCCAACT
1-10-1 MOE
1116


 


147746
34034
34045
TAAAAACAACAA
1-10-1 MOE
1073


 


398129
34045
34058
TTTGAGGAGCTATT
2-10-2 MOE
1106


 


147719
34124
34135
CCAACTCCAACT
1-10-1 MOE
1116


 


147721
34156
34167
AATGCAGGATCT
1-10-1 MOE
1118


 


398129
34193
34206
TTTGAGGAGCTATT
2-10-2 MOE
1106


 


147721
34304
34315
AATGCAGGATCT
1-10-1 MOE
1118


 


147746
34606
34617
TAAAAACAACAA
1-10-1 MOE
1073


 


398165
34704
34715
GTTCTTAGGAAG
1-10-1 MOE
968


 


147746
34754
34765
TAAAAACAACAA
1-10-1 MOE
1073


 


398165
34852
34863
GTTCTTAGGAAG
1-10-1 MOE
968


 


147717
34893
34904
ATCTTCAGAGAT
1-10-1 MOE
996


 


147719
34976
34987
CCAACTCCAACT
1-10-1 MOE
1116


 


147092
34987
34998
TGTTCCCTCTAC
1-10-1 MOE
901


 


147719
35124
35135
CCAACTCCAACT
1-10-1 MOE
1116


 


147092
35135
35146
TGTTCCCTCTAC
1-10-1 MOE
901


 


147736
35248
35259
AGGTAGGAGAAG
1-10-1 MOE
963


 


147738
35391
35402
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147736
35396
35407
AGGTAGGAGAAG
1-10-1 MOE
963


 


147738
35539
35550
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147691
35554
35565
GAGGTGGGAAAA
1-10-1 MOE
966


 


147691
35702
35713
GAGGTGGGAAAA
1-10-1 MOE
966


 


147746
35814
35825
TAAAAACAACAA
1-10-1 MOE
1073


 


147733
35889
35900
TTCTTGATGTCC
1-10-1 MOE
891


 


147733
35923
35934
TTCTTGATGTCC
1-10-1 MOE
891


 


147746
35962
35973
TAAAAACAACAA
1-10-1 MOE
1073


 


147726
35978
35989
TGACTCTCGGAC
1-10-1 MOE
1120


 


147733
36037
36048
TTCTTGATGTCC
1-10-1 MOE
891


 


147733
36071
36082
TTCTTGATGTCC
1-10-1 MOE
891


 


147726
36126
36137
TGACTCTCGGAC
1-10-1 MOE
1120


 


147736
36359
36370
AGGTAGGAGAAG
1-10-1 MOE
963


 


147691
36360
36371
GAGGTGGGAAAA
1-10-1 MOE
966


 


147736
36507
36518
AGGTAGGAGAAG
1-10-1 MOE
963


 


147691
36508
36519
GAGGTGGGAAAA
1-10-1 MOE
966


 


147746
36564
36575
TAAAAACAACAA
1-10-1 MOE
1073


 


147723
36575
36586
GACTCCAAAGTC
1-10-1 MOE
892


 


147731
36620
36631
TTTCCTCTTGTC
1-10-1 MOE
934


 


147723
36723
36734
GACTCCAAAGTC
1-10-1 MOE
892


 


147731
36768
36779
TTTCCTCTTGTC
1-10-1 MOE
934


 


398169
37174
37185
TCAGCCAGACAG
1-10-1 MOE
909


 


147688
37380
37391
TCCCAAACAAAT
1-10-1 MOE
990


 


147688
37528
37539
TCCCAAACAAAT
1-10-1 MOE
990


 


147714
37881
37892
TTCTGCTCCCAC
1-10-1 MOE
986


 


147714
38029
38040
TTCTGCTCCCAC
1-10-1 MOE
986


 


147681
38364
38375
ATGTCATTAAAC
1-10-1 MOE
965


 


147736
38766
38777
AGGTAGGAGAAG
1-10-1 MOE
963


 


147738
38909
38920
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147736
38914
38925
AGGTAGGAGAAG
1-10-1 MOE
963


 


147738
39057
39068
TGGGTGGCCGGG
1-10-1 MOE
1069


 


390030
39249
39260
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
39397
39408
TTTATAAAACTG
1-10-1 MOE
1074


 


147717
39545
39556
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
39693
39704
ATCTTCAGAGAT
1-10-1 MOE
996


 


147746
39729
39740
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
39789
39800
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
39829
39840
GAGGTGGGAAAA
1-10-1 MOE
966


 


147746
39877
39888
TAAAAACAACAA
1-10-1 MOE
1073


 


147691
39977
39988
GAGGTGGGAAAA
1-10-1 MOE
966


 


147727
39983
39994
CAGTGGACCACA
1-10-1 MOE
1128


 


147727
40131
40142
CAGTGGACCACA
1-10-1 MOE
1128


 


147746
40333
40344
TAAAAACAACAA
1-10-1 MOE
1073


 


147719
40457
40468
CCAACTCCAACT
1-10-1 MOE
1116


 


147679
40467
40478
CAAAAGGATCCC
1-10-1 MOE
907


 


147746
40478
40489
TAAAAACAACAA
1-10-1 MOE
1073


 


147741
40565
40576
CACCCACTGGTG
1-10-1 MOE
1055


 


398166
40589
40600
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147719
40605
40616
CCAACTCCAACT
1-10-1 MOE
1116


 


147679
40615
40626
CAAAAGGATCCC
1-10-1 MOE
907


 


147746
40626
40637
TAAAAACAACAA
1-10-1 MOE
1073


 


147735
40662
40673
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147746
40706
40717
TAAAAACAACAA
1-10-1 MOE
1073


 


147741
40713
40724
CACCCACTGGTG
1-10-1 MOE
1055


 


398166
40737
40748
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147735
40810
40821
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147746
40854
40865
TAAAAACAACAA
1-10-1 MOE
1073


 


147718
41218
41229
TAATATGACTTG
1-10-1 MOE
998


 


147717
41221
41232
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
41369
41380
ATCTTCAGAGAT
1-10-1 MOE
996


 


147723
41627
41638
GACTCCAAAGTC
1-10-1 MOE
892


 


147717
41747
41758
ATCTTCAGAGAT
1-10-1 MOE
996


 


147723
41775
41786
GACTCCAAAGTC
1-10-1 MOE
892


 


390030
41908
41919
TTTATAAAACTG
1-10-1 MOE
1074


 


390030
42056
42067
TTTATAAAACTG
1-10-1 MOE
1074


 


398153
42157
42170
ATTTCTCTTACAGG
2-10-2 MOE
948


 


398153
42305
42318
ATTTCTCTTACAGG
2-10-2 MOE
948


 


147690
42423
42434
TGAAGTTAATTC
1-10-1 MOE
1138


 


147695
42521
42532
TCATTCCCCACT
1-10-1 MOE
984


 


147710
42543
42554
TATAGCTCCTCT
1-10-1 MOE
994


 


147690
42571
42582
TGAAGTTAATTC
1-10-1 MOE
1138


 


147695
42669
42680
TCATTCCCCACT
1-10-1 MOE
984


 


147078
43321
43332
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
43322
43333
TCCTTCCACTGA
1-10-1 MOE
1001


 


147716
43329
43340
TTAACGAGCCTT
1-10-1 MOE
949


 


147078
43469
43480
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
43470
43481
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
43471
43482
CTCCTTCCACTG
1-10-1 MOE
1021


 


398102
43837
43850
CTACCTGAGGATTT
2-10-2 MOE
899


 


147074
43848
43859
CCACTGATCCTG
1-10-1 MOE
845


 


401408
43871
43884
CAATGAAGCACAGG
2-10-2 MOE
989


 


398102
43985
43998
CTACCTGAGGATTT
2-10-2 MOE
899


 


147736
44137
44148
AGGTAGGAGAAG
1-10-1 MOE
963


 


147746
44140
44151
TAAAAACAACAA
1-10-1 MOE
1073


 


147687
44206
44217
CGACACGGGAAC
1-10-1 MOE
950


 


147743
44223
44234
AGGGCTTCCAGT
1-10-1 MOE
1042


 


384545
44242
44253
CAAGTAGGATGT
1-10-1 MOE
951


 


147736
44285
44296
AGGTAGGAGAAG
1-10-1 MOE
963


 


147743
44371
44382
AGGGCTTCCAGT
1-10-1 MOE
1042


 


384545
44390
44401
CAAGTAGGATGT
1-10-1 MOE
951


 


147728
44589
44600
GCCAGACAGAAG
1-10-1 MOE
1013


 


389948
44628
44639
CCGTTGGACCCC
1-10-1 MOE
915


 


147720
44703
44714
GATCTCTCGAGT
1-10-1 MOE
1117


 


147728
44729
44740
GCCAGACAGAAG
1-10-1 MOE
1013


 


147728
44737
44748
GCCAGACAGAAG
1-10-1 MOE
1013


 


389948
44776
44787
CCGTTGGACCCC
1-10-1 MOE
915


 


147720
44851
44862
GATCTCTCGAGT
1-10-1 MOE
1117


 


398110
44861
44874
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


147728
44877
44888
GCCAGACAGAAG
1-10-1 MOE
1013


 


147705
45092
45103
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147705
45240
45251
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147681
45337
45348
ATGTCATTAAAC
1-10-1 MOE
965


 


147681
45485
45496
ATGTCATTAAAC
1-10-1 MOE
965


 


147096
45660
45671
TTGTTGTTCCCT
1-10-1 MOE
1107


 


147096
45808
45819
TTGTTGTTCCCT
1-10-1 MOE
1107


 


368368
45976
45989
TCCACTGATCCTTA
2-10-2 MOE
1127


 


147074
45977
45988
CCACTGATCCTG
1-10-1 MOE
845


 


147075
45978
45989
TCCACTGATCCT
1-10-1 MOE
1026


 


147076
45979
45990
TTCCACTGATCC
1-10-1 MOE
1029


 


368368
46124
46137
TCCACTGATCCTTA
2-10-2 MOE
1127


 


147075
46126
46137
TCCACTGATCCT
1-10-1 MOE
1026


 


147076
46127
46138
TTCCACTGATCC
1-10-1 MOE
1029


 


147705
46555
46566
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147714
46685
46696
TTCTGCTCCCAC
1-10-1 MOE
986


 


147705
46703
46714
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147714
46833
46844
TTCTGCTCCCAC
1-10-1 MOE
986


 


390030
47007
47018
TTTATAAAACTG
1-10-1 MOE
1074


 


147746
47023
47034
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
47171
47182
TAAAAACAACAA
1-10-1 MOE
1073


 


147085
47607
47618
TCTACACCAGGT
1-10-1 MOE
961


 


147746
47609
47620
TAAAAACAACAA
1-10-1 MOE
1073


 


147089
47611
47622
TCCCTCTACACC
1-10-1 MOE
956


 


147091
47613
47624
GTTCCCTCTACA
1-10-1 MOE
1004


 


401384
47689
47702
TGAACACATCACTA
2-10-2 MOE
933


 


147691
47729
47740
GAGGTGGGAAAA
1-10-1 MOE
966


 


147085
47755
47766
TCTACACCAGGT
1-10-1 MOE
961


 


147087
47757
47768
CCTCTACACCAG
1-10-1 MOE
982


 


147090
47760
47771
TTCCCTCTACAC
1-10-1 MOE
955


 


147091
47761
47772
GTTCCCTCTACA
1-10-1 MOE
1004


 


147099
47770
47781
GAGTTGTTGTTC
1-10-1 MOE
1108


 


147100
47771
47782
CGAGTTGTTGTT
1-10-1 MOE
1109


 


390030
47847
47858
TTTATAAAACTG
1-10-1 MOE
1074


 


147691
47877
47888
GAGGTGGGAAAA
1-10-1 MOE
966


 


147099
47918
47929
GAGTTGTTGTTC
1-10-1 MOE
1108


 


147100
47919
47930
CGAGTTGTTGTT
1-10-1 MOE
1109


 


390030
47995
48006
TTTATAAAACTG
1-10-1 MOE
1074


 


147074
48222
48233
CCACTGATCCTG
1-10-1 MOE
845


 


147731
48340
48351
TTTCCTCTTGTC
1-10-1 MOE
934


 


147691
48393
48404
GAGGTGGGAAAA
1-10-1 MOE
966


 


147731
48488
48499
TTTCCTCTTGTC
1-10-1 MOE
934


 


147691
48541
48552
GAGGTGGGAAAA
1-10-1 MOE
966


 


398147
48887
48900
CTACAGGACAATAC
2-10-2 MOE
957


 


398147
49035
49048
CTACAGGACAATAC
2-10-2 MOE
957


 


147074
49525
49536
CCACTGATCCTG
1-10-1 MOE
845


 


398168
49742
49753
TCGGACTTTGAA
1-10-1 MOE
1008


 


384545
49858
49869
CAAGTAGGATGT
1-10-1 MOE
951


 


398168
49890
49901
TCGGACTTTGAA
1-10-1 MOE
1008


 


147724
49974
49985
GAAATTGAGGAA
1-10-1 MOE
1139


 


384545
50006
50017
CAAGTAGGATGT
1-10-1 MOE
951


 


147689
50084
50095
CAGAGAAGGTCT
1-10-1 MOE
987


 


147687
50102
50113
CGACACGGGAAC
1-10-1 MOE
950


 


147724
50122
50133
GAAATTGAGGAA
1-10-1 MOE
1139


 


147687
50250
50261
CGACACGGGAAC
1-10-1 MOE
950


 


398117
50389
50402
TTTCCACTTGGGTG
2-10-2 MOE
960


 


147736
50436
50447
AGGTAGGAGAAG
1-10-1 MOE
963


 


147736
50582
50593
AGGTAGGAGAAG
1-10-1 MOE
963


 


398168
50703
50714
TCGGACTTTGAA
1-10-1 MOE
1008


 


401397
50822
50835
CTGGTCAGCATTGA
2-10-2 MOE
946


 


147746
51019
51030
TAAAAACAACAA
1-10-1 MOE
1073


 


147708
51101
51112
TTGATATAGTCA
1-10-1 MOE
997


 


147746
51165
51176
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
51185
51196
TAAAAACAACAA
1-10-1 MOE
1073


 


147708
51247
51258
TTGATATAGTCA
1-10-1 MOE
997


 


147081
51287
51298
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
51288
51299
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147746
51324
51335
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
51331
51342
TAAAAACAACAA
1-10-1 MOE
1073


 


147728
51376
51387
GCCAGACAGAAG
1-10-1 MOE
1013


 


147729
51406
51417
GTAAGAGGCAGG
1-10-1 MOE
920


 


147081
51433
51444
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
51434
51445
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147728
51492
51503
GCCAGACAGAAG
1-10-1 MOE
1013


 


147728
51522
51533
GCCAGACAGAAG
1-10-1 MOE
1013


 


147729
51552
51563
GTAAGAGGCAGG
1-10-1 MOE
920


 


368360
51633
51646
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
51634
51645
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368361
51635
51648
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


147728
51638
51649
GCCAGACAGAAG
1-10-1 MOE
1013


 


147695
51644
51655
TCATTCCCCACT
1-10-1 MOE
984


 


147736
51713
51724
AGGTAGGAGAAG
1-10-1 MOE
963


 


147684
51721
51732
ACCCAGTCAGGG
1-10-1 MOE
964


 


147081
51779
51790
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
51779
51792
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
51780
51791
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368361
51781
51794
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


147695
51790
51801
TCATTCCCCACT
1-10-1 MOE
984


 


147736
51859
51870
AGGTAGGAGAAG
1-10-1 MOE
963


 


147077
51988
51999
CTTCCACTGATC
1-10-1 MOE
1047


 


147079
51990
52001
TCCTTCCACTGA
1-10-1 MOE
1001


 


147746
52064
52075
TAAAAACAACAA
1-10-1 MOE
1073


 


147681
52085
52096
ATGTCATTAAAC
1-10-1 MOE
965


 


147077
52134
52145
CTTCCACTGATC
1-10-1 MOE
1047


 


147079
52136
52147
TCCTTCCACTGA
1-10-1 MOE
1001


 


147691
52166
52177
GAGGTGGGAAAA
1-10-1 MOE
966


 


147719
52252
52263
CCAACTCCAACT
1-10-1 MOE
1116


 


147691
52312
52323
GAGGTGGGAAAA
1-10-1 MOE
966


 


147719
52398
52409
CCAACTCCAACT
1-10-1 MOE
1116


 


147728
52428
52439
GCCAGACAGAAG
1-10-1 MOE
1013


 


147729
52483
52494
GTAAGAGGCAGG
1-10-1 MOE
920


 


398167
52527
52538
CAGGCCATGTGG
1-10-1 MOE
1059


 


147682
52571
52582
CGGGTACTATGG
1-10-1 MOE
992


 


147728
52574
52585
GCCAGACAGAAG
1-10-1 MOE
1013


 


147724
52615
52626
GAAATTGAGGAA
1-10-1 MOE
1139


 


147729
52629
52640
GTAAGAGGCAGG
1-10-1 MOE
920


 


147703
52670
52681
TGGCTTCATGTC
1-10-1 MOE
971


 


398167
52673
52684
CAGGCCATGTGG
1-10-1 MOE
1059


 


398165
52708
52719
GTTCTTAGGAAG
1-10-1 MOE
968


 


147704
52710
52721
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
52716
52727
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147724
52761
52772
GAAATTGAGGAA
1-10-1 MOE
1139


 


398167
52762
52773
CAGGCCATGTGG
1-10-1 MOE
1059


 


147703
52816
52827
TGGCTTCATGTC
1-10-1 MOE
971


 


398165
52854
52865
GTTCTTAGGAAG
1-10-1 MOE
968


 


147704
52856
52867
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
52862
52873
CGGTTTTTGTTC
1-10-1 MOE
1002


 


398167
52908
52919
CAGGCCATGTGG
1-10-1 MOE
1059


 


147689
53063
53074
CAGAGAAGGTCT
1-10-1 MOE
987


 


147727
53111
53122
CAGTGGACCACA
1-10-1 MOE
1128


 


147727
53158
53169
CAGTGGACCACA
1-10-1 MOE
1128


 


147689
53209
53220
CAGAGAAGGTCT
1-10-1 MOE
987


 


147727
53257
53268
CAGTGGACCACA
1-10-1 MOE
1128


 


147727
53304
53315
CAGTGGACCACA
1-10-1 MOE
1128


 


147680
53638
53649
GTATGCACTGCT
1-10-1 MOE
988


 


147722
53650
53661
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147083
53703
53714
TACACCAGGTCA
1-10-1 MOE
973


 


147085
53705
53716
TCTACACCAGGT
1-10-1 MOE
961


 


147086
53706
53717
CTCTACACCAGG
1-10-1 MOE
969


 


398167
53724
53735
CAGGCCATGTGG
1-10-1 MOE
1059


 


147684
53747
53758
ACCCAGTCAGGG
1-10-1 MOE
964


 


147680
53784
53795
GTATGCACTGCT
1-10-1 MOE
988


 


147722
53796
53807
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147085
53851
53862
TCTACACCAGGT
1-10-1 MOE
961


 


398167
53870
53881
CAGGCCATGTGG
1-10-1 MOE
1059


 


147684
53893
53904
ACCCAGTCAGGG
1-10-1 MOE
964


 


398155
54026
54039
TGTTTTTACACAGA
2-10-2 MOE
970


 


147703
54137
54148
TGGCTTCATGTC
1-10-1 MOE
971


 


398155
54172
54185
TGTTTTTACACAGA
2-10-2 MOE
970


 


147705
54275
54286
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147703
54283
54294
TGGCTTCATGTC
1-10-1 MOE
971


 


147705
54421
54432
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147727
54853
54864
CAGTGGACCACA
1-10-1 MOE
1128


 


398165
54963
54974
GTTCTTAGGAAG
1-10-1 MOE
968


 


398090
54963
54976
TTGTTCTTAGGAAG
2-10-2 MOE
972


 


147704
54965
54976
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
54971
54982
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147727
54999
55010
CAGTGGACCACA
1-10-1 MOE
1128


 


398165
55109
55120
GTTCTTAGGAAG
1-10-1 MOE
968


 


147704
55111
55122
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147705
55117
55128
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147083
55352
55363
TACACCAGGTCA
1-10-1 MOE
973


 


147705
55378
55389
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147705
55524
55535
CGGTTTTTGTTC
1-10-1 MOE
1002


 


147712
55819
55830
ACACCATCTCCC
1-10-1 MOE
1005


 


147712
55965
55976
ACACCATCTCCC
1-10-1 MOE
1005


 


147733
56289
56300
TTCTTGATGTCC
1-10-1 MOE
891


 


147707
56300
56311
TAGTCATTATCT
1-10-1 MOE
977


 


147708
56306
56317
TTGATATAGTCA
1-10-1 MOE
997


 


390030
56321
56332
TTTATAAAACTG
1-10-1 MOE
1074


 


147081
56333
56344
GCTCCTTCCACT
1-10-1 MOE
1006


 


398166
56335
56346
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147733
56435
56446
TTCTTGATGTCC
1-10-1 MOE
891


 


147707
56446
56457
TAGTCATTATCT
1-10-1 MOE
977


 


147708
56452
56463
TTGATATAGTCA
1-10-1 MOE
997


 


390030
56467
56478
TTTATAAAACTG
1-10-1 MOE
1074


 


147081
56479
56490
GCTCCTTCCACT
1-10-1 MOE
1006


 


398091
56479
56492
GGGCTTCTTCCATT
2-10-2 MOE
979


 


398166
56481
56492
GGGCTTCTTCCA
1-10-1 MOE
1070


 


368366
56518
56531
CTGATCCTTAGAAG
2-10-2 MOE
1019


 


147743
57612
57623
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147700
57709
57720
GCGCTAGGCCGC
1-10-1 MOE
1110


 


147743
57758
57769
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147700
57855
57866
GCGCTAGGCCGC
1-10-1 MOE
1110


 


398093
57963
57976
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
57965
57976
TCGGACTTTGAA
1-10-1 MOE
1008


 


147698
58105
58116
CCCGCCACCACC
1-10-1 MOE
928


 


398093
58109
58122
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
58111
58122
TCGGACTTTGAA
1-10-1 MOE
1008


 


147698
58251
58262
CCCGCCACCACC
1-10-1 MOE
928


 


147735
58279
58290
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147735
58425
58436
GGAGAAGCGCAG
1-10-1 MOE
1016


 


404135
58946
58959
CATTTCCATGGCCA
2-10-2 MOE
1056


 


390030
59326
59337
TTTATAAAACTG
1-10-1 MOE
1074


 


147711
59357
59368
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147743
59382
59393
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147711
59503
59514
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147743
59528
59539
AGGGCTTCCAGT
1-10-1 MOE
1042


 


147695
59576
59587
TCATTCCCCACT
1-10-1 MOE
984


 


147713
59716
59727
CTCCCACACCAT
1-10-1 MOE
985


 


147714
59721
59732
TTCTGCTCCCAC
1-10-1 MOE
986


 


147715
59746
59757
GTTGAGCATGAC
1-10-1 MOE
1077


 


147716
59771
59782
TTAACGAGCCTT
1-10-1 MOE
949


 


147712
59857
59868
ACACCATCTCCC
1-10-1 MOE
1005


 


147714
59867
59878
TTCTGCTCCCAC
1-10-1 MOE
986


 


147715
59892
59903
GTTGAGCATGAC
1-10-1 MOE
1077


 


147716
59917
59928
TTAACGAGCCTT
1-10-1 MOE
949


 


390030
59993
60004
TTTATAAAACTG
1-10-1 MOE
1074


 


147690
60270
60281
TGAAGTTAATTC
1-10-1 MOE
1138


 


389949
60325
60336
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147690
60416
60427
TGAAGTTAATTC
1-10-1 MOE
1138


 


389949
60471
60482
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147746
60619
60630
TAAAAACAACAA
1-10-1 MOE
1073


 


384545
60676
60687
CAAGTAGGATGT
1-10-1 MOE
951


 


147746
60765
60776
TAAAAACAACAA
1-10-1 MOE
1073


 


384545
60822
60833
CAAGTAGGATGT
1-10-1 MOE
951


 


147689
60967
60978
CAGAGAAGGTCT
1-10-1 MOE
987


 


147689
61008
61019
CAGAGAAGGTCT
1-10-1 MOE
987


 


147689
61049
61060
CAGAGAAGGTCT
1-10-1 MOE
987


 


398105
61121
61134
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147689
61154
61165
CAGAGAAGGTCT
1-10-1 MOE
987


 


147689
61195
61206
CAGAGAAGGTCT
1-10-1 MOE
987


 


398105
61267
61280
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147692
61365
61376
CTCACCTTCATG
1-10-1 MOE
1113


 


147692
61511
61522
CTCACCTTCATG
1-10-1 MOE
1113


 


147680
61619
61630
GTATGCACTGCT
1-10-1 MOE
988


 


147078
61755
61766
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
61756
61767
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
61757
61768
CTCCTTCCACTG
1-10-1 MOE
1021


 


147078
61901
61912
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
61902
61913
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
61903
61914
CTCCTTCCACTG
1-10-1 MOE
1021


 


147088
62361
62372
CCCTCTACACCA
1-10-1 MOE
1050


 


401384
62573
62586
TGAACACATCACTA
2-10-2 MOE
933


 


147688
62697
62708
TCCCAAACAAAT
1-10-1 MOE
990


 


147746
63102
63113
TAAAAACAACAA
1-10-1 MOE
1073


 


147721
63225
63236
AATGCAGGATCT
1-10-1 MOE
1118


 


147742
63226
63237
AACTTCAGTGTC
1-10-1 MOE
1041


 


147746
63248
63259
TAAAAACAACAA
1-10-1 MOE
1073


 


147682
63337
63348
CGGGTACTATGG
1-10-1 MOE
992


 


147721
63371
63382
AATGCAGGATCT
1-10-1 MOE
1118


 


147742
63372
63383
AACTTCAGTGTC
1-10-1 MOE
1041


 


147688
63401
63412
TCCCAAACAAAT
1-10-1 MOE
990


 


147097
63449
63460
GTTGTTGTTCCC
1-10-1 MOE
1111


 


147098
63450
63461
AGTTGTTGTTCC
1-10-1 MOE
1112


 


401409
63458
63471
ATTCTTAACACAGA
2-10-2 MOE
991


 


147084
63531
63542
CTACACCAGGTC
1-10-1 MOE
993


 


147688
63547
63558
TCCCAAACAAAT
1-10-1 MOE
990


 


147097
63595
63606
GTTGTTGTTCCC
1-10-1 MOE
1111


 


147098
63596
63607
AGTTGTTGTTCC
1-10-1 MOE
1112


 


147721
64086
64097
AATGCAGGATCT
1-10-1 MOE
1118


 


147721
64232
64243
AATGCAGGATCT
1-10-1 MOE
1118


 


147692
64233
64244
CTCACCTTCATG
1-10-1 MOE
1113


 


147692
64379
64390
CTCACCTTCATG
1-10-1 MOE
1113


 


147729
64633
64644
GTAAGAGGCAGG
1-10-1 MOE
920


 


401403
64746
64759
TTTCCTAGGAGGTG
2-10-2 MOE
967


 


147729
64779
64790
GTAAGAGGCAGG
1-10-1 MOE
920


 


147746
65151
65162
TAAAAACAACAA
1-10-1 MOE
1073


 


147746
65297
65308
TAAAAACAACAA
1-10-1 MOE
1073


 


147689
65302
65313
CAGAGAAGGTCT
1-10-1 MOE
987


 


147689
65448
65459
CAGAGAAGGTCT
1-10-1 MOE
987


 


147717
65862
65873
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
65895
65906
ATCTTCAGAGAT
1-10-1 MOE
996


 


147729
66000
66011
GTAAGAGGCAGG
1-10-1 MOE
920


 


147717
66008
66019
ATCTTCAGAGAT
1-10-1 MOE
996


 


147717
66041
66052
ATCTTCAGAGAT
1-10-1 MOE
996


 


147708
66046
66057
TTGATATAGTCA
1-10-1 MOE
997


 


147718
66055
66066
TAATATGACTTG
1-10-1 MOE
998


 


147729
66146
66157
GTAAGAGGCAGG
1-10-1 MOE
920


 


147089
66236
66247
TCCCTCTACACC
1-10-1 MOE
956


 


368363
66281
66294
CTTAGAAGGCAGCA
2-10-2 MOE
1114


 


147727
66293
66304
CAGTGGACCACA
1-10-1 MOE
1128


 


147093
66319
66330
TTGTTCCCTCTA
1-10-1 MOE
929


 


147094
66320
66331
GTTGTTCCCTCT
1-10-1 MOE
1115


 


147089
66382
66393
TCCCTCTACACC
1-10-1 MOE
956


 


368363
66427
66440
CTTAGAAGGCAGCA
2-10-2 MOE
1114


 


147727
66439
66450
CAGTGGACCACA
1-10-1 MOE
1128


 


147719
66441
66452
CCAACTCCAACT
1-10-1 MOE
1116


 


147093
66465
66476
TTGTTCCCTCTA
1-10-1 MOE
929


 


147094
66466
66477
GTTGTTCCCTCT
1-10-1 MOE
1115


 


147075
66561
66572
TCCACTGATCCT
1-10-1 MOE
1026


 


368357
66562
66575
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147076
66562
66573
TTCCACTGATCC
1-10-1 MOE
1029


 


368377
66562
66577
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
66563
66574
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
66563
66576
TCCTTCCACTGATC
2-10-2 MOE
1031


 


147078
66564
66575
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
66565
66576
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
66566
66577
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
66567
66578
GCTCCTTCCACT
1-10-1 MOE
1006


 


147719
66587
66598
CCAACTCCAACT
1-10-1 MOE
1116


 


147075
66707
66718
TCCACTGATCCT
1-10-1 MOE
1026


 


368377
66708
66723
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147076
66708
66719
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
66708
66721
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
66709
66720
CTTCCACTGATC
1-10-1 MOE
1047


 


147078
66710
66721
CCTTCCACTGAT
1-10-1 MOE
1044


 


147079
66711
66722
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
66712
66723
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
66713
66724
GCTCCTTCCACT
1-10-1 MOE
1006


 


147089
66842
66853
TCCCTCTACACC
1-10-1 MOE
956


 


147089
66988
66999
TCCCTCTACACC
1-10-1 MOE
956


 


147075
66999
67010
TCCACTGATCCT
1-10-1 MOE
1026


 


147075
67145
67156
TCCACTGATCCT
1-10-1 MOE
1026


 


147705
67213
67224
CGGTTTTTGTTC
1-10-1 MOE
1002


 


401413
67301
67314
TGCAGCCATGTACT
2-10-2 MOE
1022


 


147737
67309
67320
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147080
67430
67441
CTCCTTCCACTG
1-10-1 MOE
1021


 


147737
67455
67466
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147080
67576
67587
CTCCTTCCACTG
1-10-1 MOE
1021


 


147082
67578
67589
AGCTCCTTCCAC
1-10-1 MOE
1036


 


147090
67582
67593
TTCCCTCTACAC
1-10-1 MOE
955


 


147091
67583
67594
GTTCCCTCTACA
1-10-1 MOE
1004


 


147742
67591
67602
AACTTCAGTGTC
1-10-1 MOE
1041


 


147090
67728
67739
TTCCCTCTACAC
1-10-1 MOE
955


 


147698
68036
68047
CCCGCCACCACC
1-10-1 MOE
928


 


147698
68182
68193
CCCGCCACCACC
1-10-1 MOE
928


 


147681
68267
68278
ATGTCATTAAAC
1-10-1 MOE
965


 


147721
68386
68397
AATGCAGGATCT
1-10-1 MOE
1118


 


147681
68413
68424
ATGTCATTAAAC
1-10-1 MOE
965


 


147712
68527
68538
ACACCATCTCCC
1-10-1 MOE
1005


 


147721
68532
68543
AATGCAGGATCT
1-10-1 MOE
1118


 


147711
68760
68771
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147711
68906
68917
AAGGGCCCTGGG
1-10-1 MOE
1040


 


147696
69045
69056
TGGATGATTGGC
1-10-1 MOE
906


 


147696
69191
69202
TGGATGATTGGC
1-10-1 MOE
906


 


147723
69194
69205
GACTCCAAAGTC
1-10-1 MOE
892


 


147723
69210
69221
GACTCCAAAGTC
1-10-1 MOE
892


 


389965
69271
69282
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
69271
69282
CTGCAACATGAT
1-9-2 MOE
1018


 


147723
69340
69351
GACTCCAAAGTC
1-10-1 MOE
892


 


147723
69356
69367
GACTCCAAAGTC
1-10-1 MOE
892


 


398101
69357
69370
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


389965
69417
69428
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
69417
69428
CTGCAACATGAT
1-9-2 MOE
1018


 


398101
69503
69516
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


368353
69519
69532
CACTGATCCTGCAC
2-10-2 MOE
1007


 


147074
69522
69533
CCACTGATCCTG
1-10-1 MOE
845


 


147081
69631
69642
GCTCCTTCCACT
1-10-1 MOE
1006


 


368353
69665
69678
CACTGATCCTGCAC
2-10-2 MOE
1007


 


147720
69729
69740
GATCTCTCGAGT
1-10-1 MOE
1117


 


147721
69736
69747
AATGCAGGATCT
1-10-1 MOE
1118


 


398167
69757
69768
CAGGCCATGTGG
1-10-1 MOE
1059


 


147722
69762
69773
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147723
69768
69779
GACTCCAAAGTC
1-10-1 MOE
892


 


147080
69776
69787
CTCCTTCCACTG
1-10-1 MOE
1021


 


147081
69777
69788
GCTCCTTCCACT
1-10-1 MOE
1006


 


398093
69811
69824
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
69813
69824
TCGGACTTTGAA
1-10-1 MOE
1008


 


147725
69814
69825
CTCGGACTTTGA
1-10-1 MOE
1119


 


147726
69819
69830
TGACTCTCGGAC
1-10-1 MOE
1120


 


147727
69860
69871
CAGTGGACCACA
1-10-1 MOE
1128


 


147720
69875
69886
GATCTCTCGAGT
1-10-1 MOE
1117


 


147721
69882
69893
AATGCAGGATCT
1-10-1 MOE
1118


 


147728
69899
69910
GCCAGACAGAAG
1-10-1 MOE
1013


 


398094
69901
69914
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398167
69903
69914
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
69904
69917
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


147722
69908
69919
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147723
69914
69925
GACTCCAAAGTC
1-10-1 MOE
892


 


147729
69916
69927
GTAAGAGGCAGG
1-10-1 MOE
920


 


398095
69919
69932
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398093
69957
69970
TCGGACTTTGAAAA
2-10-2 MOE
1009


 


398168
69959
69970
TCGGACTTTGAA
1-10-1 MOE
1008


 


147725
69960
69971
CTCGGACTTTGA
1-10-1 MOE
1119


 


147726
69965
69976
TGACTCTCGGAC
1-10-1 MOE
1120


 


147704
69991
70002
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147727
70006
70017
CAGTGGACCACA
1-10-1 MOE
1128


 


147728
70045
70056
GCCAGACAGAAG
1-10-1 MOE
1013


 


398094
70047
70060
ATCAGCCAGACAGA
2-10-2 MOE
1010


 


398169
70048
70059
TCAGCCAGACAG
1-10-1 MOE
909


 


147729
70062
70073
GTAAGAGGCAGG
1-10-1 MOE
920


 


398095
70065
70078
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


147704
70137
70148
TTGTTCTTAGGA
1-10-1 MOE
1012


 


147697
70161
70172
CCCCAGCAGCGG
1-10-1 MOE
1000


 


147697
70307
70318
CCCCAGCAGCGG
1-10-1 MOE
1000


 


147728
70450
70461
GCCAGACAGAAG
1-10-1 MOE
1013


 


398164
70464
70475
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
70465
70476
CTTGTCCATCAG
1-10-1 MOE
1121


 


147731
70471
70482
TTTCCTCTTGTC
1-10-1 MOE
934


 


147732
70476
70487
GGGTCTTTCCTC
1-10-1 MOE
1122


 


147733
70497
70508
TTCTTGATGTCC
1-10-1 MOE
891


 


398096
70562
70575
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
70564
70575
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147736
70569
70580
AGGTAGGAGAAG
1-10-1 MOE
963


 


147737
70575
70586
ACAGCCAGGTAG
1-10-1 MOE
1067


 


147728
70596
70607
GCCAGACAGAAG
1-10-1 MOE
1013


 


398164
70610
70621
TTGTCGATCTGC
1-10-1 MOE
1014


 


147730
70611
70622
CTTGTCCATCAG
1-10-1 MOE
1121


 


368349
70616
70629
CTGCACTGACGAGT
2-10-2 MOE
1017


 


147731
70617
70628
TTTCCTCTTGTC
1-10-1 MOE
934


 


147732
70622
70633
GGGTCTTTCCTC
1-10-1 MOE
1122


 


147733
70643
70654
TTCTTGATGTCC
1-10-1 MOE
891


 


398096
70708
70721
GGAGAAGCGCAGCT
2-10-2 MOE
1015


 


147735
70710
70721
GGAGAAGCGCAG
1-10-1 MOE
1016


 


147736
70715
70726
AGGTAGGAGAAG
1-10-1 MOE
963


 


147737
70721
70732
ACAGCCAGGTAG
1-10-1 MOE
1067


 


389764
70784
70795
CTGCAACATGAT
1-9-2 MOE
1018


 


389965
70784
70795
CTGCAACATGAT
1-10-1 MOE
1018


 


389965
70930
70941
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
70930
70941
CTGCAACATGAT
1-9-2 MOE
1018


 


368386
70995
71010
CACTGATCCTTAGAAG
3-10-3 MOE
1123


 


368367
70997
71010
CACTGATCCTTAGA
2-10-2 MOE
1124


 


368387
70997
71012
TCCACTGATCCTTAGA
3-10-3 MOE
1125


 


368354
70999
71012
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368374
70999
71014
CTTCCACTGATCCTGC
3-10-3 MOE
1126


 


368368
70999
71012
TCCACTGATCCTTA
2-10-2 MOE
1127


 


368388
70999
71014
CTTCCACTGATCCTTA
3-10-3 MOE
895


 


368355
71000
71013
TTCCACTGATCCTG
2-10-2 MOE
1025


 


147074
71000
71011
CCACTGATCCTG
1-10-1 MOE
845


 


368375
71000
71015
CCTTCCACTGATCCTG
3-10-3 MOE
1020


 


147075
71001
71012
TCCACTGATCCT
1-10-1 MOE
1026


 


368376
71001
71016
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


147076
71002
71013
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
71002
71015
CCTTCCACTGATCC
2-10-2 MOE
1046


 


368377
71002
71017
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
71003
71014
CTTCCACTGATC
1-10-1 MOE
1047


 


368378
71003
71018
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


147078
71004
71015
CCTTCCACTGAT
1-10-1 MOE
1044


 


368359
71005
71018
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


368379
71005
71020
AAGCTCCTTCCACTGA
3-10-3 MOE
1034


 


147079
71005
71016
TCCTTCCACTGA
1-10-1 MOE
1001


 


147080
71006
71017
CTCCTTCCACTG
1-10-1 MOE
1021


 


368360
71007
71020
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


368380
71007
71022
GAAAGCTCCTTCCACT
3-10-3 MOE
896


 


147081
71007
71018
GCTCCTTCCACT
1-10-1 MOE
1006


 


147082
71008
71019
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368361
71009
71022
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


368381
71009
71024
GGGAAAGCTCCTTCCA
3-10-3 MOE
1037


 


147738
71067
71078
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147739
71071
71082
CGTTTGGGTGGC
1-10-1 MOE
1023


 


147740
71088
71099
TGTGAGGCTCCA
1-10-1 MOE
1062


 


147741
71129
71140
CACCCACTGGTG
1-10-1 MOE
1055


 


368366
71141
71154
CTGATCCTTAGAAG
2-10-2 MOE
1019


 


368386
71141
71156
CACTGATCCTTAGAAG
3-10-3 MOE
1123


 


368367
71143
71156
CACTGATCCTTAGA
2-10-2 MOE
1124


 


368387
71143
71158
TCCACTGATCCTTAGA
3-10-3 MOE
1125


 


368374
71145
71160
CTTCCACTGATCCTGC
3-10-3 MOE
1126


 


368354
71145
71158
TCCACTGATCCTGC
2-10-2 MOE
1024


 


368368
71145
71158
TCCACTGATCCTTA
2-10-2 MOE
1127


 


368388
71145
71160
CTTCCACTGATCCTTA
3-10-3 MOE
895


 


368355
71146
71159
TTCCACTGATCCTG
2-10-2 MOE
1025


 


368375
71146
71161
CCTTCCACTGATCCTG
3-10-3 MOE
1020


 


147075
71147
71158
TCCACTGATCCT
1-10-1 MOE
1026


 


368356
71147
71160
CTTCCACTGATCCT
2-10-2 MOE
1027


 


368376
71147
71162
TCCTTCCACTGATCCT
3-10-3 MOE
1028


 


147076
71148
71159
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
71148
71161
CCTTCCACTGATCC
2-10-2 MOE
1046


 


368377
71148
71163
CTCCTTCCACTGATCC
3-10-3 MOE
1030


 


147077
71149
71160
CTTCCACTGATC
1-10-1 MOE
1047


 


368358
71149
71162
TCCTTCCACTGATC
2-10-2 MOE
1031


 


368378
71149
71164
GCTCCTTCCACTGATC
3-10-3 MOE
1032


 


147078
71150
71161
CCTTCCACTGAT
1-10-1 MOE
1044


 


368359
71151
71164
GCTCCTTCCACTGA
2-10-2 MOE
1033


 


147079
71151
71162
TCCTTCCACTGA
1-10-1 MOE
1001


 


368379
71151
71166
AAGCTCCTTCCACTGA
3-10-3 MOE
1034


 


147080
71152
71163
CTCCTTCCACTG
1-10-1 MOE
1021


 


368380
71153
71168
GAAAGCTCCTTCCACT
3-10-3 MOE
896


 


147081
71153
71164
GCTCCTTCCACT
1-10-1 MOE
1006


 


368360
71153
71166
AAGCTCCTTCCACT
2-10-2 MOE
1035


 


147082
71154
71165
AGCTCCTTCCAC
1-10-1 MOE
1036


 


368381
71155
71170
GGGAAAGCTCCTTCCA
3-10-3 MOE
1037


 


368361
71155
71168
GAAAGCTCCTTCCA
2-10-2 MOE
962


 


398097
71158
71171
GGCAGTCTTTATCC
2-10-2 MOE
897


 


147738
71213
71224
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147739
71217
71228
CGTTTGGGTGGC
1-10-1 MOE
1023


 


147740
71234
71245
TGTGAGGCTCCA
1-10-1 MOE
1062


 


147741
71275
71286
CACCCACTGGTG
1-10-1 MOE
1055


 


398097
71304
71317
GGCAGTCTTTATCC
2-10-2 MOE
897


 


147727
71702
71713
CAGTGGACCACA
1-10-1 MOE
1128


 


147727
71848
71859
CAGTGGACCACA
1-10-1 MOE
1128


 


390030
71986
71997
TTTATAAAACTG
1-10-1 MOE
1074


 


147102
72015
72026
TGCGAGTTGTTG
1-10-1 MOE
1129


 


390030
72132
72143
TTTATAAAACTG
1-10-1 MOE
1074


 


147102
72161
72172
TGCGAGTTGTTG
1-10-1 MOE
1129


 


147722
72199
72210
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147696
72232
72243
TGGATGATTGGC
1-10-1 MOE
906


 


147741
72254
72265
CACCCACTGGTG
1-10-1 MOE
1055


 


147722
72345
72356
AAAGTCAGGCCA
1-10-1 MOE
1130


 


147696
72378
72389
TGGATGATTGGC
1-10-1 MOE
906


 


147741
72400
72411
CACCCACTGGTG
1-10-1 MOE
1055


 


147711
72446
72457
AAGGGCCCTGGG
1-10-1 MOE
1040


 


398098
72574
72587
TAACTTCAGTGTCT
2-10-2 MOE
1131


 


147742
72575
72586
AACTTCAGTGTC
1-10-1 MOE
1041


 


147698
72595
72606
CCCGCCACCACC
1-10-1 MOE
928


 


147743
72690
72701
AGGGCTTCCAGT
1-10-1 MOE
1042


 


398099
72690
72703
GAAGGGCTTCCAGT
2-10-2 MOE
1132


 


147744
72694
72705
AGGAAGGGCTTC
1-10-1 MOE
1043


 


398100
72697
72710
TGACCAGGAAGGGC
2-10-2 MOE
1133


 


147745
72700
72711
TTGACCAGGAAG
1-10-1 MOE
1058


 


398098
72720
72733
TAACTTCAGTGTCT
2-10-2 MOE
1131


 


147742
72721
72732
AACTTCAGTGTC
1-10-1 MOE
1041


 


147698
72741
72752
CCCGCCACCACC
1-10-1 MOE
928


 


398157
72757
72770
GGAAACATACCCTG
2-10-2 MOE
1045


 


147743
72836
72847
AGGGCTTCCAGT
1-10-1 MOE
1042


 


398099
72836
72849
GAAGGGCTTCCAGT
2-10-2 MOE
1132


 


147744
72840
72851
AGGAAGGGCTTC
1-10-1 MOE
1043


 


398100
72843
72856
TGACCAGGAAGGGC
2-10-2 MOE
1133


 


147745
72846
72857
TTGACCAGGAAG
1-10-1 MOE
1058


 


147076
72898
72909
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
72898
72911
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
72899
72910
CTTCCACTGATC
1-10-1 MOE
1047


 


147078
72900
72911
CCTTCCACTGAT
1-10-1 MOE
1044


 


398157
72903
72916
GGAAACATACCCTG
2-10-2 MOE
1045


 


398158
72983
72996
AGGCCCTGAGATTA
2-10-2 MOE
1134


 


398159
72988
73001
GGTTAAGGCCCTGA
2-10-2 MOE
1135


 


398160
72993
73006
GAATAGGTTAAGGC
2-10-2 MOE
1048


 


147076
73044
73055
TTCCACTGATCC
1-10-1 MOE
1029


 


368357
73044
73057
CCTTCCACTGATCC
2-10-2 MOE
1046


 


147077
73045
73056
CTTCCACTGATC
1-10-1 MOE
1047


 


147078
73046
73057
CCTTCCACTGAT
1-10-1 MOE
1044


 


147746
73052
73063
TAAAAACAACAA
1-10-1 MOE
1073


 


398161
73092
73105
AACAATGTGTTGTA
2-10-2 MOE
1049


 


147746
73101
73112
TAAAAACAACAA
1-10-1 MOE
1073


 


398158
73129
73142
AGGCCCTGAGATTA
2-10-2 MOE
1134


 


398159
73134
73147
GGTTAAGGCCCTGA
2-10-2 MOE
1135


 


398160
73139
73152
GAATAGGTTAAGGC
2-10-2 MOE
1048


 


147746
73198
73209
TAAAAACAACAA
1-10-1 MOE
1073


 


398161
73238
73251
AACAATGTGTTGTA
2-10-2 MOE
1049


 


147746
73247
73258
TAAAAACAACAA
1-10-1 MOE
1073


 


147088
73273
73284
CCCTCTACACCA
1-10-1 MOE
1050


 


398105
73401
73414
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


398105
73547
73560
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147741
73559
73570
CACCCACTGGTG
1-10-1 MOE
1055


 


147741
73705
73716
CACCCACTGGTG
1-10-1 MOE
1055


 


398162
73968
73981
ACCAAACAGTTCAG
2-10-2 MOE
1057


 


147745
73991
74002
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
74008
74019
CAGGCCATGTGG
1-10-1 MOE
1059


 


398092
74009
74022
AGTCAGGCCATGTG
2-10-2 MOE
1060


 


398162
74114
74127
ACCAAACAGTTCAG
2-10-2 MOE
1057


 


147745
74137
74148
TTGACCAGGAAG
1-10-1 MOE
1058


 


398167
74154
74165
CAGGCCATGTGG
1-10-1 MOE
1059


 


147089
74280
74291
TCCCTCTACACC
1-10-1 MOE
956


 


147090
74281
74292
TTCCCTCTACAC
1-10-1 MOE
955


 


389949
74310
74321
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147740
74339
74350
TGTGAGGCTCCA
1-10-1 MOE
1062


 


389950
74381
74392
CCCTGAAGGTTC
1-10-1 MOE
1063


 


147089
74426
74437
TCCCTCTACACC
1-10-1 MOE
956


 


147090
74427
74438
TTCCCTCTACAC
1-10-1 MOE
955


 


389949
74456
74467
GCGCGAGCCCGA
1-10-1 MOE
1061


 


147685
74490
74501
GGCTGACATTCA
1-10-1 MOE
975


 


398101
74510
74523
TTTGATAAAGCCCT
2-10-2 MOE
1064


 


398102
74536
74549
CTACCTGAGGATTT
2-10-2 MOE
899


 


398103
74543
74556
CCCAGTACTACCTG
2-10-2 MOE
900


 


147685
74636
74647
GGCTGACATTCA
1-10-1 MOE
975


 


398102
74682
74695
CTACCTGAGGATTT
2-10-2 MOE
899


 


398103
74689
74702
CCCAGTACTACCTG
2-10-2 MOE
900


 


147736
74737
74748
AGGTAGGAGAAG
1-10-1 MOE
963


 


398104
74805
74818
CAAGAAGACCTTAC
2-10-2 MOE
1065


 


147736
74883
74894
AGGTAGGAGAAG
1-10-1 MOE
963


 


147737
74893
74904
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398105
74894
74907
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147737
74919
74930
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398095
74940
74953
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


398104
74951
74964
CAAGAAGACCTTAC
2-10-2 MOE
1065


 


398106
74974
74987
TGGAAAACTGCACC
2-10-2 MOE
1068


 


398107
74980
74993
TATTCCTGGAAAAC
2-10-2 MOE
902


 


147745
75030
75041
TTGACCAGGAAG
1-10-1 MOE
1058


 


147737
75039
75050
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398105
75040
75053
TGCACAGGCAGGTT
2-10-2 MOE
1066


 


147737
75065
75076
ACAGCCAGGTAG
1-10-1 MOE
1067


 


398108
75077
75090
GGAATGTCTGAGTT
2-10-2 MOE
1136


 


398095
75086
75099
CATCAGCAAGAGGC
2-10-2 MOE
1011


 


147691
75108
75119
GAGGTGGGAAAA
1-10-1 MOE
966


 


398106
75120
75133
TGGAAAACTGCACC
2-10-2 MOE
1068


 


398107
75126
75139
TATTCCTGGAAAAC
2-10-2 MOE
902


 


147738
75155
75166
TGGGTGGCCGGG
1-10-1 MOE
1069


 


147745
75176
75187
TTGACCAGGAAG
1-10-1 MOE
1058


 


398108
75223
75236
GGAATGTCTGAGTT
2-10-2 MOE
1136


 


398109
75247
75260
CAAGAAGTGTGGTT
2-10-2 MOE
903


 


147691
75254
75265
GAGGTGGGAAAA
1-10-1 MOE
966


 


147738
75301
75312
TGGGTGGCCGGG
1-10-1 MOE
1069


 


398110
75385
75398
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


147091
75387
75398
GTTCCCTCTACA
1-10-1 MOE
1004


 


398109
75393
75406
CAAGAAGTGTGGTT
2-10-2 MOE
903


 


398111
75470
75483
GTGAAAATGCTGGC
2-10-2 MOE
904


 


401385
75494
75507
CCCAGTGGGTTTGA
2-10-2 MOE
890


 


398166
75499
75510
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147091
75525
75536
GTTCCCTCTACA
1-10-1 MOE
1004


 


147092
75526
75537
TGTTCCCTCTAC
1-10-1 MOE
901


 


398110
75531
75544
GTTCCCTTTGCAGG
2-10-2 MOE
952


 


147091
75533
75544
GTTCCCTCTACA
1-10-1 MOE
1004


 


147706
75540
75551
GCTGACATCTCG
1-10-1 MOE
1071


 


398112
75584
75597
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


398111
75616
75629
GTGAAAATGCTGGC
2-10-2 MOE
904


 


147746
75617
75628
TAAAAACAACAA
1-10-1 MOE
1073


 


398166
75645
75656
GGGCTTCTTCCA
1-10-1 MOE
1070


 


147091
75671
75682
GTTCCCTCTACA
1-10-1 MOE
1004


 


147092
75672
75683
TGTTCCCTCTAC
1-10-1 MOE
901


 


398113
75693
75706
AGGAGGTTAAACCA
2-10-2 MOE
905


 


398112
75730
75743
CAGCCTGGCACCTA
2-10-2 MOE
1072


 


147746
75763
75774
TAAAAACAACAA
1-10-1 MOE
1073


 


398114
75770
75783
AGGCATATAGCAGA
2-10-2 MOE
1075


 


398115
75786
75799
AGTAAATATTGGCT
2-10-2 MOE
1076


 


398116
75799
75812
TAATGACCTGATGA
2-10-2 MOE
1137


 


398113
75839
75852
AGGAGGTTAAACCA
2-10-2 MOE
905


 


390030
75839
75850
TTTATAAAACTG
1-10-1 MOE
1074


 


398115
75932
75945
AGTAAATATTGGCT
2-10-2 MOE
1076


 


398116
75945
75958
TAATGACCTGATGA
2-10-2 MOE
1137


 


398106
75982
75995
TGGAAAACTGCACC
2-10-2 MOE
1068


 


390030
75985
75996
TTTATAAAACTG
1-10-1 MOE
1074


 


398106
76127
76140
TGGAAAACTGCACC
2-10-2 MOE
1068


 


147690
76196
76207
TGAAGTTAATTC
1-10-1 MOE
1138


 


147690
76341
76352
TGAAGTTAATTC
1-10-1 MOE
1138


 


147724
76740
76751
GAAATTGAGGAA
1-10-1 MOE
1139


 


147089
76873
76884
TCCCTCTACACC
1-10-1 MOE
956


 


147679
76881
76892
CAAAAGGATCCC
1-10-1 MOE
907


 


147724
76885
76896
GAAATTGAGGAA
1-10-1 MOE
1139


 


147089
77018
77029
TCCCTCTACACC
1-10-1 MOE
956


 


147679
77026
77037
CAAAAGGATCCC
1-10-1 MOE
907


 


147693
77240
77251
GTGCGCTCCCAT
1-10-1 MOE
1078


 


147697
77759
77770
CCCCAGCAGCGG
1-10-1 MOE
1000









In certain embodiments, a target region is nucleotides 177-190 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 177-190 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 177-190 comprises a nucleotide sequence selected from SEQ ID NO 886, 859, or 853. In certain such embodiments, a short antisense compound targeted to nucleotides 177-190 of SEQ ID NO: 11 is selected from Isis No 147022, 147023, or 147024.
In certain embodiments, a target region is nucleotides 195-228 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 195-228 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 195-228 comprises a nucleotide sequence selected from SEQ ID NO 877, 868, 882, 886, 859, 853, 865, 835, 843, 846, 842, 848, 874, 849, 863, 855, 850, 864, or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 195-228 of SEQ ID NO: 11 is selected from Isis No 147019, 147020, 147021, 147022, 147023, 147024, 147025, 147026, 147027, 147028, 147073, 147029, 147030, 147036, 147037, 147038, 147039, 147040, or 147041.
In certain embodiments, a target region is nucleotides 323-353 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 323-353 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 323-353 comprises a nucleotide sequence selected from SEQ ID NO 866, 881, 869, 883, 858, 833, 875, 837, 829, 871, 884, 887, 839, 830, 840, 861, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 323-353 of SEQ ID NO: 11 is selected from Isis No 147042, 147043, 147044, 147045, 147046, 147047, 147051, 147052, 147053, 147054, 147055, 147056, 147057, 147058, 147059, 147060, or 147061.
In certain embodiments, a target region is nucleotides 322-353 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 322-353 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 322-353 comprises a nucleotide sequence selected from SEQ ID NO 842, 866, 881, 869, 883, 858, 833, 875, 837, 829, 871, 884, 887, 839, 830, 840, 861, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 322-353 of SEQ ID NO: 11 is selected from Isis No 147073, 147042, 147043, 147044, 147045, 147046, 147047, 147051, 147052, 147053, 147054, 147055, 147056, 147057, 147058, 147059, 147060, or 147061.
In certain embodiments, a target region is nucleotides 679-799 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 679-799 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 679-799 comprises a nucleotide sequence selected from SEQ ID NO 883, 858, 883, or 858. In certain such embodiments, a short antisense compound targeted to nucleotides 679-799 of SEQ ID NO: 11 is selected from Isis No 147045, 147046, 147045, or 147046.
In certain embodiments, a target region is nucleotides 679-827 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 679-827 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 679-827 comprises a nucleotide sequence selected from SEQ ID NO 883, 858, 883, 858, or 851. In certain such embodiments, a short antisense compound targeted to nucleotides 679-827 of SEQ ID NO: 11 is selected from Isis No 147045, 147046, 147045, 147046, or 147066.
In certain embodiments, a target region is nucleotides 1024-1046 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1024-1046 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1024-1046 comprises a nucleotide sequence selected from SEQ ID NO 841, 862, 880, 857, 851, 876, 838, 860, 878, 856, 832, or 842. In certain such embodiments, a short antisense compound targeted to nucleotides 1024-1046 of SEQ ID NO: 11 is selected from Isis No 147062, 147063, 147064, 147065, 147066, 147067, 147068, 147069, 147070, 147071, 147072, or 147073.
In certain embodiments, a target region is nucleotides 992-1046 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 992-1046 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 992-1046 comprises a nucleotide sequence selected from SEQ ID NO 831, 841, 862, 880, 857, 851, 876, 838, 860, 878, 856, 832, or 842. In certain such embodiments, a short antisense compound targeted to nucleotides 992-1046 of SEQ ID NO: 11 is selected from Isis No 404131, 147062, 147063, 147064, 147065, 147066, 147067, 147068, 147069, 147070, 147071, 147072, or 147073.
In certain embodiments, a target region is nucleotides 1868-1881 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1868-1881 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1868-1881 comprises a nucleotide sequence selected from SEQ ID NO 886, 859, or 853. In certain such embodiments, a short antisense compound targeted to nucleotides 1868-1881 of SEQ ID NO: 11 is selected from Isis No 147022, 147023, or 147024.
In certain embodiments, a target region is nucleotides 1886-1919 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1886-1919 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1886-1919 comprises a nucleotide sequence selected from SEQ ID NO 877, 868, 882, 886, 859, 865, 843, 846, 874, 863, 855, 864, or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 1886-1919 of SEQ ID NO: 11 is selected from Isis No 147019, 147020, 147021, 147022, 147023, 147025, 147027, 147028, 147030, 147037, 147038, 147040, or 147041.
In certain embodiments, a target region is nucleotides 1869-1919 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1869-1919 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1869-1919 comprises a nucleotide sequence selected from SEQ ID NO 859, 853, 877, 868, 882, 886, 859, 865, 843, 846, 874, 863, 855, 864, or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 1869-1919 of SEQ ID NO: 11 is selected from Isis No 147023, 147024, 147019, 147020, 147021, 147022, 147023, 147025, 147027, 147028, 147030, 147037, 147038, 147040, or 147041.
In certain embodiments, a target region is nucleotides 1976-1989 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1976-1989 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1976-1989 comprises a nucleotide sequence selected from SEQ ID NO 886, 859, or 853. In certain such embodiments, a short antisense compound targeted to nucleotides 1976-1989 of SEQ ID NO: 11 is selected from Isis No 147022, 147023, or 147024.
In certain embodiments, a target region is nucleotides 1995-2027 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 1995-2027 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 1995-2027 comprises a nucleotide sequence selected from SEQ ID NO 868, 882, 886, 859, 853, 865, 835, 843, 846, 848, 874, 849, 863, 855, 850, 864, or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 1995-2027 of SEQ ID NO: 11 is selected from Isis No 147020, 147021, 147022, 147023, 147024, 147025, 147026, 147027, 147028, 147029, 147030, 147036, 147037, 147038, 147039, 147040, or 147041.
In certain embodiments, a target region is nucleotides 2366-2382 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 2366-2382 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 2366-2382 comprises a nucleotide sequence selected from SEQ ID NO 867 or 873. In certain such embodiments, a short antisense compound targeted to nucleotides 2366-2382 of SEQ ID NO: 11 is selected from Isis No 404199 or 404134.
In certain embodiments, a target region is nucleotides 6220-6233 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 6220-6233 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 6220-6233 comprises a nucleotide sequence selected from SEQ ID NO 870, 836, or 844. In certain such embodiments, a short antisense compound targeted to nucleotides 6220-6233 of SEQ ID NO: 11 is selected from Isis No 147032, 147033, or 147034.
In certain embodiments, a target region is nucleotides 6288-6300 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 6288-6300 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 6288-6300 comprises a nucleotide sequence selected from SEQ ID NO 869 or 883. In certain such embodiments, a short antisense compound targeted to nucleotides 6288-6300 of SEQ ID NO: 11 is selected from Isis No 147044 or 147045.
In certain embodiments, a target region is nucleotides 6329-6342 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 6329-6342 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 6329-6342 comprises a nucleotide sequence selected from SEQ ID NO 870, 836, or 844. In certain such embodiments, a short antisense compound targeted to nucleotides 6329-6342 of SEQ ID NO: 11 is selected from Isis No 147032, 147033, or 147034.
In certain embodiments, a target region is nucleotides 6397-6409 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 6397-6409 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to nucleotides 6397-6409 comprises a nucleotide sequence selected from SEQ ID NO 869 or 883. In certain such embodiments, a short antisense compound targeted to nucleotides 6397-6409 of SEQ ID NO: 11 is selected from Isis No 147044 or 147045.
In certain embodiments, a target region is nucleotides 7057-7178 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 7057-7178 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 7057-7178 comprises a nucleotide sequence selected from SEQ ID NO 830, 840, 861, 830, or 840. In certain such embodiments, a short antisense compound targeted to nucleotides 7057-7178 of SEQ ID NO: 11 is selected from Isis No 147058, 147059, 147060, 147058, or 147059.
In certain embodiments, a target region is nucleotides 8630-8750 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 8630-8750 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 8630-8750 comprises a nucleotide sequence selected from SEQ ID NO 843, 846, 843, or 846. In certain such embodiments, a short antisense compound targeted to nucleotides 8630-8750 of SEQ ID NO: 11 is selected from Isis No 147027, 147028, 147027, or 147028.
In certain embodiments, a target region is nucleotides 10957-11077 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 10957-11077 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 10957-11077 comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 881, or 869. In certain such embodiments, a short antisense compound targeted to nucleotides 10957-11077 of SEQ ID NO: 11 is selected from Isis No 147043, 147044, 147043, or 147044.
In certain embodiments, a target region is nucleotides 11605-11623 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 11605-11623 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 11605-11623 comprises a nucleotide sequence selected from SEQ ID NO 856, 878, or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 11605-11623 of SEQ ID NO: 11 is selected from Isis No 147071, 147070, or 147071.
In certain embodiments, a target region is nucleotides 12805-12817 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 12805-12817 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 12805-12817 comprises a nucleotide sequence selected from SEQ ID NO 874 or 885. In certain such embodiments, a short antisense compound targeted to nucleotides 12805-12817 of SEQ ID NO: 11 is selected from Isis No 147030 or 147031.
In certain embodiments, a target region is nucleotides 12986-12998 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 12986-12998 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 12986-12998 comprises a nucleotide sequence selected from SEQ ID NO 874 or 885. In certain such embodiments, a short antisense compound targeted to nucleotides 12986-12998 of SEQ ID NO: 11 is selected from Isis No 147030 or 147031.
In certain embodiments, a target region is nucleotides 15560-15572 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 15560-15572 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 15560-15572 comprises a nucleotide sequence selected from SEQ ID NO 876 or 838. In certain such embodiments, a short antisense compound targeted to nucleotides 15560-15572 of SEQ ID NO: 11 is selected from Isis No 147067 or 147068.
In certain embodiments, a target region is nucleotides 17787-17941 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 17787-17941 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 17787-17941 comprises a nucleotide sequence selected from SEQ ID NO 874 or 880. In certain such embodiments, a short antisense compound targeted to nucleotides 17787-17941 of SEQ ID NO: 11 is selected from Isis No 147030 or 147064.
In certain embodiments, a target region is nucleotides 21190-21202 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 21190-21202 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 21190-21202 comprises a nucleotide sequence selected from SEQ ID NO 843 or 846. In certain such embodiments, a short antisense compound targeted to nucleotides 21190-21202 of SEQ ID NO: 11 is selected from Isis No 147027 or 147028.
In certain embodiments, a target region is nucleotides 21358-21370 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 21358-21370 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 21358-21370 comprises a nucleotide sequence selected from SEQ ID NO 843 or 846. In certain such embodiments, a short antisense compound targeted to nucleotides 21358-21370 of SEQ ID NO: 11 is selected from Isis No 017027 or 147028.
In certain embodiments, a target region is nucleotides 24318-24332 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 24318-24332 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 24318-24332 comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 883, or 858. In certain such embodiments, a short antisense compound targeted to nucleotides 24318-24332 of SEQ ID NO: 11 is selected from Isis No 147043, 147044, 147045, or 147046.
In certain embodiments, a target region is nucleotides 24486-24501 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 24486-24501 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 24486-24501 comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 858, or 833. In certain such embodiments, a short antisense compound targeted to nucleotides 24486-24501 of SEQ ID NO: 11 is selected from Isis No 147043, 147044, 147046, or 147047.
In certain embodiments, a target region is nucleotides 25065-25077 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 25065-25077 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 25065-25077 comprises a nucleotide sequence selected from SEQ ID NO 864 or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 25065-25077 of SEQ ID NO: 11 is selected from Isis No 147040 or 147041.
In certain embodiments, a target region is nucleotides 25232-25245 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 25232-25245 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 25232-25245 comprises a nucleotide sequence selected from SEQ ID NO 850, 864, or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 25232-25245 of SEQ ID NO: 11 is selected from Isis No 147039, 147040, or 147041.
In certain embodiments, a target region is nucleotides 25508-25523 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 25508-25523 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 25508-25523 comprises a nucleotide sequence selected from SEQ ID NO 839 or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 25508-25523 of SEQ ID NO: 11 is selected from Isis No 147057 or 147061.
In certain embodiments, a target region is nucleotides 25676-28890 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 25676-28890 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 25676-28890 comprises a nucleotide sequence selected from SEQ ID NO 839, 860, or 878. In certain such embodiments, a short antisense compound targeted to nucleotides 25676-28890 of SEQ ID NO: 11 is selected from Isis No 147057, 147069, or 147070.
In certain embodiments, a target region is nucleotides 33056-33069 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33056-33069 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 33056-33069 comprises a nucleotide sequence selected from SEQ ID NO 860, 878, or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 33056-33069 of SEQ ID NO: 11 is selected from Isis No 147069, 147070, or 147071.
In certain embodiments, a target region is nucleotides 33205-33217 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33205-33217 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 33205-33217 comprises a nucleotide sequence selected from SEQ ID NO 878 or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 33205-33217 of SEQ ID NO: 11 is selected from Isis No 14707 or 147071.
In certain embodiments, a target region is nucleotides 33318-33334 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33318-33334 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted to 33318-33334 comprises a nucleotide sequence selected from SEQ ID NO 858, 854, or 875. In certain such embodiments, a short antisense compound targeted to nucleotides 33318-33334 of SEQ ID NO: 11 is selected from Isis No 147046, 147049, or 147051.
In certain embodiments, a target region is nucleotides 33466-33482 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33466-33482 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 33466-33482 comprises a nucleotide sequence selected from SEQ ID NO 858, 833, or 875. In certain such embodiments, a short antisense compound targeted to nucleotides 33466-33482 of SEQ ID NO: 11 is selected from Isis No 147046, 147047, or 147051.
In certain embodiments, a target region is nucleotides 33640-33656 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33640-33656 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 33640-33656 comprises a nucleotide sequence selected from SEQ ID NO 858 or 875. In certain such embodiments, a short antisense compound targeted to nucleotides 33640-33656 of SEQ ID NO: 11 is selected from Isis No 147046 or 147051.
In certain embodiments, a target region is nucleotides 33788-33804 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 33788-33804 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 33788-33804 comprises a nucleotide sequence selected from SEQ ID NO 858 or 875. In certain such embodiments, a short antisense compound targeted to nucleotides 33788-33804 of SEQ ID NO: 11 is selected from Isis No 147046 or 147051.
In certain embodiments, a target region is nucleotides 35437-35449 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 35437-35449 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 35437-35449 comprises a nucleotide sequence selected from SEQ ID NO 840 or 861. In certain such embodiments, a short antisense compound targeted to nucleotides 35437-35449 of SEQ ID NO: 11 is selected from Isis No 147059 or 147060.
In certain embodiments, a target region is nucleotides 40353-40373 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 40353-40373 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 40353-40373 comprises a nucleotide sequence selected from SEQ ID NO 879 or 881. In certain such embodiments, a short antisense compound targeted to nucleotides 40353-40373 of SEQ ID NO: 11 is selected from Isis No 147061 or 147043.
In certain embodiments, a target region is nucleotides 42527-42541 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 42527-42541 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 42527-42541 comprises a nucleotide sequence selected from SEQ ID NO 885, 870, or 844. In certain such embodiments, a short antisense compound targeted to nucleotides 42527-42541 of SEQ ID NO: 11 is selected from Isis No 147031, 147032, or 147034.
In certain embodiments, a target region is nucleotides 42675-42689 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 42675-42689 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 42675-42689 comprises a nucleotide sequence selected from SEQ ID NO 885, 870, 836, or 844. In certain such embodiments, a short antisense compound targeted to nucleotides 42675-42689 of SEQ ID NO: 11 is selected from Isis No 147031, 147032, 147033, or 147034.
In certain embodiments, a target region is nucleotides 46313-46328 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 46313-46328 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 46313-46328 comprises a nucleotide sequence selected from SEQ ID NO 839, 830, 840, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 46313-46328 of SEQ ID NO: 11 is selected from Isis No 147057, 147058, 147059, or 147061.
In certain embodiments, a target region is nucleotides 46461-46476 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 46461-46476 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 46461-46476 comprises a nucleotide sequence selected from SEQ ID NO 839, 840, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 46461-46476 of SEQ ID NO: 11 is selected from Isis No 147057, 147059, or 147061.
In certain embodiments, a target region is nucleotides 48369-48381 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 48369-48381 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 48369-48381 comprises a nucleotide sequence selected from SEQ ID NO 842 or 845. In certain such embodiments, a short antisense compound targeted to nucleotides 48369-48381 of SEQ ID NO: 11 is selected from Isis No 147073 or 147074.
In certain embodiments, a target region is nucleotides 48714-48726 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 48714-48726 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 48714-48726 comprises a nucleotide sequence selected from SEQ ID NO 843 or 846. In certain such embodiments, a short antisense compound targeted to nucleotides 48714-48726 of SEQ ID NO: 11 is selected from Isis No 147027 or 147028.
In certain embodiments, a target region is nucleotides 49050-49062 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 49050-49062 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 49050-49062 of comprises a nucleotide sequence selected from SEQ ID NO 876 or 838. In certain such embodiments, a short antisense compound targeted to nucleotides 49050-49062 of SEQ ID NO: 11 is selected from Isis No 147067 or 147068.
In certain embodiments, a target region is nucleotides 49672-49684 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 49672-49684 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 49672-49684 of comprises a nucleotide sequence selected from SEQ ID NO 842 or 845. In certain such embodiments, a short antisense compound targeted to nucleotides 49672-49684 of SEQ ID NO: 11 is selected from Isis No 147073 or 147074.
In certain embodiments, a target region is nucleotides 52292-52304 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 52292-52304 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 52292-52304 of comprises a nucleotide sequence selected from SEQ ID NO 849 or 863. In certain such embodiments, a short antisense compound targeted to nucleotides 52292-52304 of SEQ ID NO: 11 is selected from Isis No 147036 or 147037.
In certain embodiments, a target region is nucleotides 52438-52450 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 52438-52450 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 52438-52450 of comprises a nucleotide sequence selected from SEQ ID NO 849 or 863. In certain such embodiments, a short antisense compound targeted to nucleotides 52438-52450 of SEQ ID NO: 11 is selected from Isis No 147036 or 147037.
In certain embodiments, a target region is nucleotides 53445-53458 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 53445-53458 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 53445-53458 of comprises a nucleotide sequence selected from SEQ ID NO 866, 881, or 869. In certain such embodiments, a short antisense compound targeted to nucleotides 53445-53458 of SEQ ID NO: 11 is selected from Isis No 147042, 147043, or 147044.
In certain embodiments, a target region is nucleotides 53591-53604 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 53591-53604 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 53591-53604 of comprises a nucleotide sequence selected from SEQ ID NO 866, 874, 881, 885, or 869. In certain such embodiments, a short antisense compound targeted to nucleotides 53591-53604 of SEQ ID NO: 11 is selected from Isis No 147042, 147030, 147043, 147031, or 147044.
In certain embodiments, a target region is nucleotides 53738-53750 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 53738-53750 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 53738-53750 of comprises a nucleotide sequence selected from SEQ ID NO 874 or 885. In certain such embodiments, a short antisense compound targeted to nucleotides 53738-53750 of SEQ ID NO: 11 is selected from Isis No 147030 or 147031.
In certain embodiments, a target region is nucleotides 53783-53795 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 53783-53795 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 53783-53795 of comprises a nucleotide sequence selected from SEQ ID NO 864 or 834. In certain such embodiments, a short antisense compound targeted to nucleotides 53783-53795 of SEQ ID NO: 11 is selected from Isis No 147040 or 147041.
In certain embodiments, a target region is nucleotides 55008-55020 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 55008-55020 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 55008-55020 of comprises a nucleotide sequence selected from SEQ ID NO 866 or 881. In certain such embodiments, a short antisense compound targeted to nucleotides 55008-55020 of SEQ ID NO: 11 is selected from Isis No 147042 or 147043.
In certain embodiments, a target region is nucleotides 55154-55166 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 55154-55166 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 55154-55166 of comprises a nucleotide sequence selected from SEQ ID NO 866 or 881. In certain such embodiments, a short antisense compound targeted to nucleotides 55154-55166 of SEQ ID NO: 11 is selected from Isis No 147042 or 147043.
In certain embodiments, a target region is nucleotides 55682-55695 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 55682-55695 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 55682-55695 of comprises a nucleotide sequence selected from SEQ ID NO 877 or 882. In certain such embodiments, a short antisense compound targeted to nucleotides 55682-55695 of SEQ ID NO: 11 is selected from Isis No 147019 or 147021.
In certain embodiments, a target region is nucleotides 56275-56293 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 56275-56293 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 56275-56293 of comprises a nucleotide sequence selected from SEQ ID NO 871, 884, 887, 830, 840, 861, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 56275-56293 of SEQ ID NO: 11 is selected from Isis No 147054, 147055, 147056, 147058, 147059, 147060, or 147061.
In certain embodiments, a target region is nucleotides 56418-56439 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 56418-56439 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 56418-56439 of comprises a nucleotide sequence selected from SEQ ID NO 875, 829, 871, 884, 887, 839, 830, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 56418-56439 of SEQ ID NO: 11 is selected from Isis No 147051, 147053, 147054, 147055, 147056, 147057, 147058, or 147061.
In certain embodiments, a target region is nucleotides 57264-57276 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 57264-57276 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 57264-57276 of comprises a nucleotide sequence selected from SEQ ID NO 883 or 858. In certain such embodiments, a short antisense compound targeted to nucleotides 57264-57276 of SEQ ID NO: 11 is selected from Isis No 147045 or 147046.
In certain embodiments, a target region is nucleotides 61276-61293 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 61276-61293 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 61276-61293 of comprises a nucleotide sequence selected from SEQ ID NO 856, 847, 849, 863, 855, 850, or 864. In certain such embodiments, a short antisense compound targeted to nucleotides 61276-61293 of SEQ ID NO: 11 is selected from Isis No 147071, 147035, 147036, 147037, 147038, 147039, or 147040.
In certain embodiments, a target region is nucleotides 61257-61320 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 61257-61320 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 61257-61320 of comprises a nucleotide sequence selected from SEQ ID NO 881, 856, 847, 849, 863, 855, 850, 864, or 886. In certain such embodiments, a short antisense compound targeted to nucleotides 61257-61320 of SEQ ID NO: 11 is selected from Isis No 147043, 147071, 147035, 147036, 147037, 147038, 147039, 147040, or 147071.
In certain embodiments, a target region is nucleotides 61422-61439 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 61422-61439 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 61422-61439 of comprises a nucleotide sequence selected from SEQ ID NO 844, 847, 849, 863, 855, or 864. In certain such embodiments, a short antisense compound targeted to nucleotides 61422-61439 of SEQ ID NO: 11 is selected from Isis No 147034, 147035, 147036, 147037, 147038, or 147040.
In certain embodiments, a target region is nucleotides 61422-61466 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 61422-61466 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 61422-61466 of comprises a nucleotide sequence selected from SEQ ID NO 844, 847, 849, 863, 855, 864, or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 61422-61466 of SEQ ID NO: 11 is selected from Isis No 147034, 147035, 147036, 147037, 147038, 147040, or 147071.
In certain embodiments, a target region is nucleotides 63065-63078 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 63065-63078 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 63065-63078 of comprises a nucleotide sequence selected from SEQ ID NO 851 or 838. In certain such embodiments, a short antisense compound targeted to nucleotides 63065-63078 of SEQ ID NO: 11 is selected from Isis No 147066 or 147068.
In certain embodiments, a target region is nucleotides 63207-63222 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 63207-63222 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 63207-63222 of comprises a nucleotide sequence selected from SEQ ID NO 841 or 851. In certain such embodiments, a short antisense compound targeted to nucleotides 63207-63222 of SEQ ID NO: 11 is selected from Isis No 147062 or 147066.
In certain embodiments, a target region is nucleotides 64538-64550 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 64538-64550 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 64538-64550 of comprises a nucleotide sequence selected from SEQ ID NO 849 or 863. In certain such embodiments, a short antisense compound targeted to nucleotides 64538-64550 of SEQ ID NO: 11 is selected from Isis No 147036 or 147037.
In certain embodiments, a target region is nucleotides 64864-64876 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 64864-64876 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 64864-64876 of comprises a nucleotide sequence selected from SEQ ID NO 851 or 876. In certain such embodiments, a short antisense compound targeted to nucleotides 64864-64876 of SEQ ID NO: 11 is selected from Isis No 147066 or 147067.
In certain embodiments, a target region is nucleotides 65010-65028 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 65010-65028 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 65010-65028 of comprises a nucleotide sequence selected from SEQ ID NO 851, 876, or 883. In certain such embodiments, a short antisense compound targeted to nucleotides 65010-65028 of SEQ ID NO: 11 is selected from Isis No 147066, 147067, or 147045.
In certain embodiments, a target region is nucleotides 65163-65175 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 65163-65175 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 65163-65175 of comprises a nucleotide sequence selected from SEQ ID NO 883 or 858. In certain such embodiments, a short antisense compound targeted to nucleotides 65163-65175 of SEQ ID NO: 11 is selected from Isis No 147045 or 147046.
In certain embodiments, a target region is nucleotides 65408-65422 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 65408-65422 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 65408-65422 of comprises a nucleotide sequence selected from SEQ ID NO 883 or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 65408-65422 of SEQ ID NO: 11 is selected from Isis No 147068 or 147071.
In certain embodiments, a target region is nucleotides 65549-65568 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 65549-65568 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 65549-65568 of comprises a nucleotide sequence selected from SEQ ID NO 860, 838, or 856. In certain such embodiments, a short antisense compound targeted to nucleotides 65549-65568 of SEQ ID NO: 11 is selected from Isis No 147069, 147068, or 147071.
In certain embodiments, a target region is nucleotides 67741-67754 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 67741-67754 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 67741-67754 of comprises a nucleotide sequence selected from SEQ ID NO 848, 874, or 885. In certain such embodiments, a short antisense compound targeted to nucleotides 67741-67754 of SEQ ID NO: 11 is selected from Isis No 147029, 147030, or 147031.
In certain embodiments, a target region is nucleotides 67886-67900 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 67886-67900 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 67886-67900 of comprises a nucleotide sequence selected from SEQ ID NO 846, 848, 874, or 885. In certain such embodiments, a short antisense compound targeted to nucleotides 67886-67900 of SEQ ID NO: 11 is selected from Isis No 147028, 147029, 147030, or 147031.
In certain embodiments, a target region is nucleotides 68867-68880 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 68867-68880 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 68867-68880 of comprises a nucleotide sequence selected from SEQ ID NO 881, 869, or 883. In certain such embodiments, a short antisense compound targeted to nucleotides 68867-68880 of SEQ ID NO: 11 is selected from Isis No 147043, 147044, or 147045.
In certain embodiments, a target region is nucleotides 69013-69532 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 69013-69532 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 69013-69532 of comprises a nucleotide sequence selected from SEQ ID NO 881, 869, 883, 858, 856, 832, or 842. In certain such embodiments, a short antisense compound targeted to nucleotides 69013-69532 of SEQ ID NO: 11 is selected from Isis No 147043, 147044, 147045, 147046, 147071, 147072, or 147073.
In certain embodiments, a target region is nucleotides 69665-69880 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 69665-69880 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 69665-69880 of comprises a nucleotide sequence selected from SEQ ID NO 856, 832, 842, 845, or 851. In certain such embodiments, a short antisense compound targeted to nucleotides 69665-69880 of SEQ ID NO: 11 is selected from Isis No 147071, 147072, 147073, 147074, or 147066.
In certain embodiments, a target region is nucleotides 70611-70630 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 70611-70630 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 70611-70630 of comprises a nucleotide sequence selected from SEQ ID NO 859, 841, 862, 880, 857, or 851. In certain such embodiments, a short antisense compound targeted to nucleotides 70611-70630 of SEQ ID NO: 11 is selected from Isis No 147023, 147062, 147063, 147064, 147065, or 147066.
In certain embodiments, a target region is nucleotides 70762-70776 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 70762-70776 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 70762-70776 of comprises a nucleotide sequence selected from SEQ ID NO 862, 880, 857, or 851. In certain such embodiments, a short antisense compound targeted to nucleotides 70762-70776 of SEQ ID NO: 11 is selected from Isis No 147063, 147064, 147065, or 147066.
In certain embodiments, a target region is nucleotides 70998-71010 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 70998-71010 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 70998-71010 of comprises a nucleotide sequence selected from SEQ ID NO 832 or 842. In certain such embodiments, a short antisense compound targeted to nucleotides 70998-71010 of SEQ ID NO: 11 is selected from Isis No 147072 or 147073.
In certain embodiments, a target region is nucleotides 71144-714364 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 71144-714364 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 71144-714364 of comprises a nucleotide sequence selected from SEQ ID NO 832, 842, 845, 863, 855, or 850. In certain such embodiments, a short antisense compound targeted to nucleotides 71144-714364 of SEQ ID NO: 11 is selected from Isis No 147072, 147073, 147074, 147037, 147038, or 147039.
In certain embodiments, a target region is nucleotides 71497-71652 of SEQ ID NO: 11. In certain embodiments, a short antisense compound is targeted to nucleotides 71497-71652 of SEQ ID NO: 11. In certain such embodiments, a short antisense compound targeted 71497-71652 of comprises a nucleotide sequence selected from SEQ ID NO 863, 855, 850, or 879. In certain such embodiments, a short antisense compound targeted to nucleotides 71497-71652 of SEQ ID NO: 11 is selected from Isis No 147037, 147038, 147039, or 147061.
In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 8 to 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 9 to 14 nucleotides in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 10 to 14 nucleotides in length. In certain embodiments, such short antisense compounds are short antisense oligonucleotides.
In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are short gapmers. In certain such embodiments, short gapmers targeted to a PTP1B nucleic acid comprise at least one high affinity modification in one or more wings of the compound. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise 1 to 3 high-affinity modifications in each wing. In certain such embodiments, the nucleosides or nucleotides of the wing comprise a 2′ modification. In certain such embodiments, the monomers of the wing are BNA's. In certain such embodiments, the monomers of the wing are selected from α-L-Methyleneoxy (4′-CH2—O-2′) BNA, β-D-Methyleneoxy (4′-CH2—O-2′) BNA, Ethyleneoxy (4′-(CH2)2—O-2′) BNA, Aminooxy (4′-CH2—O—N(R)-2′) BNA and Oxyamino (4′-CH2—N(R)—O-2′) BNA. In certain embodiments, the monomers of a wing comprise a substituent at the 2′ position selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rm), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. In certain embodiments, the monomers of a wing are 2′MOE nucleotides.
In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise a gap between the 5′ wing and the 3′ wing. In certain embodiments the gap comprises five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen monomers. In certain embodiments, the monomers of the gap are unmodified deoxyribonucleotides. In certain embodiments, the monomers of the gap are unmodified ribonucleotides. In certain embodiments, gap modifications (if any) gap result in an antisense compound that, when bound to its target nucleic acid, supports cleavage by an RNase, including, but not limited to, RNase H.
In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid have uniform monomeric linkages. In certain such embodiments, those linkages are all phosphorothioate linkages. In certain embodiments, the linkages are all phosphodiester linkages. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid have mixed backbones.
In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 8 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 9 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 10 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 11 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 13 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 14 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 15 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid are 16 monomers in length. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise 9 to 15 monomers. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise 10 to 15 monomers. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise 12 to 14 monomers. In certain embodiments, short antisense compounds targeted to a PTP1B nucleic acid comprise 12 to 14 nucleotides or nucleosides.
In certain embodiments, the invention provides methods of modulating expression of PTP1B. In certain embodiments, such methods comprise use of one or more short antisense compound targeted to a PTP1B nucleic acid, wherein the short antisense compound targeted to a PTP1B nucleic acid is from about 8 to about 16, preferably 9 to 15, more preferably 9 to 14, more preferably 10 to 14 monomers (i.e. from about 8 to about 16 linked monomers). One of ordinary skill in the art will appreciate that this comprehends methods of modulating expression of PTP1B using one or more short antisense compounds targeted to a PTP1B nucleic acid of 8, 9, 10, 11, 12, 13, 14, 15 or 16 monomers.
In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 8 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 9 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 10 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 11 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 12 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 13 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 14 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 15 monomers in length. In certain embodiments, methods of modulating PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid that is 16 monomers in length.
In certain embodiments, methods of modulating expression of PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid comprising 9 to 15 monomers. In certain embodiments, methods of modulating expression of PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid comprising 10 to 15 monomers. In certain embodiments, methods of modulating expression of PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid comprising 12 to 14 monomers. In certain embodiments, methods of modulating expression of PTP1B comprise use of a short antisense compound targeted to a PTP1B nucleic acid comprising 12 or 14 nucleotides or nucleosides.
10. PTEN
In certain embodiments, the invention provides short antisense compounds targeted to a nucleic acid encoding PTEN. In certain embodiments, such compounds are used to modulate PTEN expression if cells. In certain such embodiments, short antisense compounds targeted to a PTEN nucleic acid are administered to an animal. In certain embodiments, short antisense compounds targeted to a PTEN nucleic acid are useful for studying PTEN, for studying certain nucleases and/or for assessing antisense activity. In certain such embodiments, short antisense compounds targeted to PTEN nucleic acids are useful for assessing certain motifs and/or chemical modifications. In certain embodiments, administration of a short antisense compound targeted to PTEN nucleic acid to an animal results in a measurable phenotypic change.
The short antisense compounds targeting PTEN may have any one or more properties or characteristics of the short antisense compounds generally described herein. In certain embodiments, short antisense compounds targeting a PTP1B nucleic acid have a motif (wing-deoxy gap-wing) selected from 1-12-1, 1-1-10-2, 2-10-1-1, 3-10-3, 2-10-3, 2-10-2, 1-10-1, 1-10-2, 3-8-3, 2-8-2, 1-8-1, 3-6-3 or 1-6-1, more preferably 1-10-1, 2-10-2, 3-10-3, and 1-9-2.
Certain Short Antisense Compounds Targeted to a PTEN Nucleic Acid
In certain embodiments, short antisense compounds are targeted to a PTEN nucleic acid having the sequence of GENBANK® Accession No. NM_000314.4, incorporated herein as SEQ ID NO: 14. In certain embodiments, short antisense compounds are targeted to a PTEN nucleic acid having the sequence of nucleotides 8063255 to 8167140 of the sequence of GENBANK® Accession No. NT 033890.3, incorporated herein as SEQ ID NO: 15. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 14 is at least 90% complementary to SEQ ID NO: 14. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 14 is at least 95% complementary to SEQ ID NO: 14. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 15 is 100% complementary to SEQ ID NO: 15. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 15 is at least 90% complementary to SEQ ID NO: 15. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 15 is at least 95% complementary to SEQ ID NO: 15. In certain such embodiments, a short antisense compound targeted to SEQ ID NO: 15 is 100% complementary to SEQ ID NO: 15.
In certain embodiments, a short antisense compound targeted to SEQ ID NO: 14 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Tables 20 and 21. In certain embodiments, a short antisense compound targeted to SEQ ID NO: 15 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Tables 22 and 23.
Each nucleotide sequence set forth in Tables 20, 21, 22, and 23 is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, short antisense compounds comprising a nucleotide sequence as set forth in Tables 20, 21, 22, and 23 may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis NO.) indicate a combination of nucleobase sequence and one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
Table 20 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 14. Table 22 illustrates short antisense compounds that are 100% complementary to SEQ ID NO: 15. The column labeled ‘gapmer motif’ indicates the wing-gap-wing motif of each short antisense compounds. The gap segment comprises 2′-deoxynucleotides and each nucleotide of each wing segment comprises a 2′-modified sugar. The particular 2′-modified sugar is also indicated in the ‘gapmer motif’ column. For example, ‘2-10-2 MOE’ means a 2-10-2 gapmer motif, where a gap segment of ten 2′-deoxynucleotides is flanked by wing segments of two nucleotides, where the nucleotides of the wing segments are 2′-MOE nucleotides. Internucleoside linkages are phosphorothioate. The short antisense compounds comprise 5-methylcytidine in place of unmodified cytosine, unless “unmodified cytosine” is listed in the gapmer motif column, in which case the indicated cytosines are unmodified cytosines. For example, “5-mC in gap only” indicates that the gap segment has 5-methylcytosines, while the wing segments have unmodified cytosines.
The 2′-modified nucleotides and abbreviations include: 2′-O-methoxyethyl (MOE); 2′-O-methyl (OMe); 2′-O-(2,2,3,3,3-pentafluoropropyl) (PentaF); 2′-O-[(2-methoxy)ethyl]-4′-thio (2′-MOE-4′-thio); (R)-CMOE-BNA. As illustrated in Tables 20 and 22, a wing may comprise monomers comprising more than type of 2′ substituent. For example, 1-2-10-2 MOE/PentaF/MOE indicates one MOE-modified nucleotide, followed by two PentaF-modified nucleotides, followed by a gap of ten deoxynucleotides, followed by two PentaF-modified nucleotides. For example, 1-1-10-2 2′-(butylacetomido)-palmitamide Methyleneoxy BNA/Methyleneoxy BNA indicates that the 5′-most nucleotide is 2′-(butylacetomide)-palmitamide, the second nucleotide is a methyleneoxy BNA nucleotide, and the 3′ wing is methyleneoxy BNA. Unless otherwise indicated, cytosines are 5-methylcytosines and internucleoside linkages are phosphorothioate.







TABLE 20







Short Antisense Compounds Targeted to SEQ ID NO: 14













5′
3′


SEQ


ISIS
Target
Target


ID


No
Site
Site
Sequence (5′-3′)
Gapmer Motif
NO















390092
5530
5541
AGAATGAGACTT
1-10-1 MOE
1514


 


390091
5435
5446
TGAGGCATTATC
1-10-1 MOE
1522


 


390090
5346
5357
AGAGTATCTGAA
1-10-1 MOE
1227


 


390088
5162
5173
CACATTAACAGT
1-10-1 MOE
1511


 


390087
5126
5137
GTGGCAACCACA
1-10-1 MOE
1501


 


390085
5031
5042
ATTTGATGCTGC
1-10-1 MOE
1505


 


390084
4982
4993
CAAAGAATGGTG
1-10-1 MOE
1215


 


390082
4910
4921
AGGACTTGGGAT
1-10-1 MOE
1503


 


390080
4833
4844
TGCTGCACATCC
1-10-1 MOE
1150


 


392067
4832
4845
CTGCTGCACATCCA
2-10-2 Methyleneoxy BNA
1510






Unmodified cytosines in gap



 


390078
4714
4725
CTTTCAGTCATA
1-10-1 MOE
1520


 


390077
4693
4704
GTCAAATTCTAT
1-10-1 MOE
1252


 


390076
4599
4610
TTCCAATGACTA
1-10-1 MOE
1506


 


390075
4576
4587
GTAAGCAAGGCT
1-10-1 MOE
#N/A


 


390074
4533
4544
ACCCTCATTCAG
1-10-1 MOE
1513


 


390068
4191
4202
GTAAATCCTAAG
1-10-1 MOE
1515


 


390064
4001
4012
ACCACAGCTAGT
1-10-1 MOE
1498


 


390063
3977
3988
CACCAATAAGTT
1-10-1 MOE
1219


 


390058
3828
3839
AGTAGTTGTACT
1-10-1 MOE
1192


 


390056
3793
3804
GGGCATATCAAA
1-10-1 MOE
1521


 


390054
3705
3716
AACACTGCACAT
1-10-1 MOE
1493


 


390052
3623
3634
GACAATTTCTAC
1-10-1 MOE
1492


 


390050
3503
3514
GTATTCAAGTAA
1-10-1 MOE
1140


 


390049
3479
3490
GTTAATGACATT
1-10-1 MOE
1491


 


390047
3428
3439
TGTGTAAGGTCA
1-10-1 MOE
1490


 


390041
3175
3186
TTAGCACTGGCC
1-10-1 MOE
1489


 


398076
3171
3182
CACTGGCCTTGA
1-10-1 MOE
1488


 


398009
3170
3183
GCACTGGCCTTGAT
2-10-2 MOE
1487


 


398075
3111
3122
AAATCATTGTCA
1-10-1 MOE
1233


 


398008
3110
3123
TAAATCATTGTCAA
2-10-2 MOE
1486


 


398074
2913
2924
GCACCAATATGC
1-10-1 MOE
1248


 


398007
2912
2925
AGCACCAATATGCT
2-10-2 MOE
1247


 


398073
2681
2692
TTAGCCAACTGC
1-10-1 MOE
1485


 


398006
2680
2693
CTTAGCCAACTGCA
2-10-2 MOE
1484


 


390033
2679
2690
AGCCAACTGCAA
1-10-1 MOE
1483


 


398072
2671
2682
GCAAACTTATCT
1-10-1 MOE
1482


 


398005
2670
2683
TGCAAACTTATCTG
2-10-2 MOE
1481


 


390030
2534
2545
TTTATAAAACTG
1-10-1 MOE
1074


 


398071
2533
2544
TTATAAAACTGG
1-10-1 MOE
1480


 


398004
2532
2545
TTTATAAAACTGGA
2-10-2 MOE
1479


 


390029
2510
2521
AAAGTGCCATCT
1-10-1 MOE
1478


 


390028
2491
2502
TCCTAATTGAAT
1-10-1 MOE
1477


 


398070
2481
2492
ATTTTAAATGTC
1-10-1 MOE
1476


 


398003
2480
2493
AATTTTAAATGTCC
2-10-2 MOE
1475


 


390027
2455
2466
AGGTATATACAT
1-10-1 MOE
1206


 


398069
2451
2462
ATATACATGACA
1-10-1 MOE
1474


 


398002
2450
2463
TATATACATGACAC
2-10-2 MOE
1473


 


398068
2440
2451
ACAGCTACACAA
1-10-1 MOE
1472


 


398001
2439
2452
CACAGCTACACAAC
2-10-2 MOE
1471


 


390026
2438
2449
AGCTACACAACC
1-10-1 MOE
1470


 


390025
2406
2417
GTGTCAAAACCC
1-10-1 MOE
1211


 


398067
2405
2416
TGTCAAAACCCT
1-10-1 MOE
1210


 


398000
2404
2417
GTGTCAAAACCCTG
2-10-2 MOE
1469


 


398066
2372
2383
AGATTGGTCAGG
1-10-1 MOE
1468


 


397999
2371
2384
AAGATTGGTCAGGA
2-10-2 MOE
1467


 


398065
2349
2360
GTTCCTATAACT
1-10-1 MOE
1466


 


397998
2348
2361
TGTTCCTATAACTG
2-10-2 MOE
1465


 


398064
2331
2342
CTGACACAATGT
1-10-1 MOE
1464


 


397997
2330
2343
TCTGACACAATGTC
2-10-2 MOE
1463


 


398063
2321
2332
GTCCTATTGCCA
1-10-1 MOE
1205


 


397996
2320
2333
TGTCCTATTGCCAT
2-10-2 MOE
1462


 


390022
2286
2297
CAGTTTATTCAA
1-10-1 MOE
1142


 


336221
2230
2243
TCAGACTTTTGTAA
3-8-3 MOE
1461


 


336220
2224
2237
TTTTGTAATTTGTG
3-8-3 MOE
1460


 


336219
2209
2222
ATGCTGATCTTCAT
3-8-3 MOE
1459


 


390021
2203
2214
CTTCATCAAAAG
1-10-1 MOE
1458


 


336218
2201
2214
CTTCATCAAAAGGT
3-8-3 MOE
1457


 


389779
2201
2212
TCATCAAAAGGT
1-9-2 MOE
1176


 


389979
2201
2212
TCATCAAAAGGT
1-10-1 MOE
1176


 


397995
2200
2213
TTCATCAAAAGGTT
2-10-2 MOE
1456


 


336217
2192
2205
AAGGTTCATTCTCT
3-8-3 MOE
1455


 


390020
2183
2194
TCTGGATCAGAG
1-10-1 MOE
1149


 


336216
2182
2195
CTCTGGATCAGAGT
3-8-3 MOE
1454


 


336215
2169
2182
TCAGTGGTGTCAGA
3-8-3 MOE
1453


 


398062
2166
2177
GGTGTCAGAATA
1-10-1 MOE
1255


 


397994
2165
2178
TGGTGTCAGAATAT
2-10-2 MOE
1452


 


390019
2163
2174
GTCAGAATATCT
1-10-1 MOE
1173


 


336214
2157
2170
GAATATCTATAATG
3-8-3 MOE
1573


 


398061
2151
2162
ATAATGATCAGG
1-10-1 MOE
1451


 


397993
2150
2163
TATAATGATCAGGT
2-10-2 MOE
1450


 


336213
2146
2159
ATGATCAGGTTCAT
3-8-3 MOE
1449


 


389778
2144
2155
TCAGGTTCATTG
1-9-2 MOE
1448


 


389978
2144
2155
TCAGGTTCATTG
1-10-1 MOE
1448


 


398060
2137
2148
CATTGTCACTAA
1-10-1 MOE
1447


 


336212
2136
2149
TCATTGTCACTAAC
3-8-3 MOE
1446


 


397992
2136
2149
TCATTGTCACTAAC
2-10-2 MOE
1446


 


336211
2112
2125
ACAGAAGTTGAACT
3-8-3 MOE
1445


 


390017
2111
2122
GAAGTTGAACTG
1-10-1 MOE
1444


 


398059
2108
2119
GTTGAACTGCTA
1-10-1 MOE
1443


 


397991
2107
2120
AGTTGAACTGCTAG
2-10-2 MOE
1442


 


336210
2104
2117
TGAACTGCTAGCCT
3-8-3 MOE
1441


 


335340
2104
2118
TTGAACTGCTAGCCT
1-10-4 MOE
1440


 


335339
2103
2117
TGAACTGCTAGCCTC
1-10-4 MOE
1439


 


335338
2102
2116
GAACTGCTAGCCTCT
1-10-4 MOE
1438


 


335337
2101
2115
AACTGCTAGCCTCTG
1-10-4 MOE
1437


 


335336
2100
2114
ACTGCTAGCCTCTGG
1-10-4 MOE
1436


 


390430
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines



 


390431
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







C in wing 9-







(aminoethoxy)phenoxazine



 


390432
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163


 


390433
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 6 is 9-(aminoethoxy)phenoxazine



 


390434
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 7 is 9-(aminoethoxy)phenoxazine



 


390435
2099
2111
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 9 is 9-(aminoethoxy)phenoxazine



 


335335
2099
2113
CTGCTAGCCTCTGGA
1-10-4 MOE
1435


 


389777
2098
2109
TAGCCTCTGGAT
1-9-2 MOE
1434


 


389954
2098
2109
TAGCCTCTGGAT
1-10-1 MOE
1434


 


335334
2098
2112
TGCTAGCCTCTGGAT
1-10-4 MOE
1433


 


331429
2097
2110
CTAGCCTCTGGATT
2-10-2 MOE
1431


 


335349
2097
2110
CTAGCCTCTGGATT
2-10-2 MOE
1431


 


335367
2097
2110
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431


 


335378
2097
2110
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431


 


392061
2097
2110
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431






Unmodified cytosines in gap



 


383991
2097
2109
TAGCCTCTGGATT
1-10-2
1432






2′-(acetylamino-butyl-acetamido)-







cholesterol/MOE



 


383992
2097
2109
TAGCCTCTGGATT
1-10-2
1432






2′-(acetylamino-butyl-acetamido)-







cholic acid/MOE



 


386970
2097
2109
TAGCCTCTGGATT
1-10-2 MOE
1432


 


390578
2097
2109
TAGCCTCTGGATT
1-10-2 MOE
1432






Unmodified cytosines







Ts in wings are 2-thiothymines



 


390614
2097
2109
TAGCCTCTGGATT
1-10-2 PentaF
1432


 


335333
2097
2111
GCTAGCCTCTGGATT
1-10-4 MOE
1430


 


386683
2097
2109
TAGCCTCTGGATT
1-10-2 2′-(butylacetamido)-
1432






palmitamide/MOE



 


371975
2096
2110
CTAGCCTCTGGATTT
3-10-2 MOE
1429


 


335341
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


335350
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


335368
2096
2111
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Phosphodiester linkages in wings



 


335379
2096
2111
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428


 


383739
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






5-methylcytosine in gap



 


384071
2096
2111
GCTAGCCTCTGGATTT
3-10-3 OMe
1428






5-methylcytosine in gap



 


384073
2096
2111
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






5-methylcytosine in gap



 


390576
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






5-methylcytosine in gap







T's in wings are 2-thiothymines



 


390580
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Pyrimidines in wings are 5-thiazole







Unmodified cytosines in gap



 


390581
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Unmodified cytosines in gap



 


391863
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Unmodified cytosines



 


391864
2096
2111
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Unmodified cytosines in gap



 


391865
2096
2111
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Unmodified cytosines



 


375560
2096
2110
CTAGCCTCTGGATTT
2-10-3 MOE
1429


 


391172
2096
2110
CTAGCCTCTGGATTT
2-10-2 Methyleneoxy BNA
1429






Unmodified cytosines



 


391175
2096
2110
CTAGCCTCTGGATTT
2-10-3 Methyleneoxy BNA
1429


 


391449
2096
2110
CTAGCCTCTGGATTT
2-10-3 MOE
1429






Unmodified cytosines



 


392054
2096
2110
CTAGCCTCTGGATTT
2-10-3 Methyleneoxy BNA
1429






Unmodified cytosines in gap



 


392055
2096
2110
CTAGCCTCTGGATTT
2-10-3 MOE
1429






Unmodified cytosines in gap



 


362977
2096
2111
GCTAGCCTCTGGATTT
2-12-2 MOE
1428


 


386770
2096
2109
TAGCCTCTGGATTT
1-11-2 MOE
1427


 


390577
2096
2109
TAGCCTCTGGATTT
1-10-3 MOE
1427






Unmodified cytosines







T's in wings are 2-thiothymines



 


335332
2096
2110
CTAGCCTCTGGATTT
1-10-4 MOE
1429


 


390579
2096
2111
GCTAGCCTCTGGATTT
1-1-1-10-3 MOE/4′-thio/2′-O-[(2-
1428






methoxy)ethyl]-4′-thio/2′-O-[(2-







methoxy)ethyl]-4′-thio







Unmodified cytosines in wings







Phosphorodiester linkage in wings



 


391173
2096
2110
CTAGCCTCTGGATTT
2-10-3 (5′R)-5′-methyl-
1429






Methyleneoxy BNA







Unmodified cytosines



 


391174
2096
2110
CTAGCCTCTGGATTT
2-10-3 (5′S)-5′-methyl-
1429






Methyleneoxy BNA







Unmodified cytosines



 


390607
2096
2111
GCTAGCCTCTGGATTT
3-10-3 MOE/pentaF
1428






Unmodified cytosines in wing



 


390609
2096
2111
GCTAGCCTCTGGATTT
3-10-2-1 MOE/MOE/pentaF
1428






Unmodified cytosines in wing



 


384072
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 MOE/pentaF/pentaF
1428






Unmodified cytosines in wings



 


390606
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 MOE/pentaF/pentaF
1428






Unmodified cytosines in wing



 


390608
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 MOE/pentaF/pentaF
1428






Unmodified cytosines in wing



 


391869
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 Methyleneoxy BNA/(5′S)-
1428






5′-methyl-Methyleneoxy BNA/







(5′S)-5′-methyl-Methyleneoxy







BNA







Unmodified cytosines



 


385036
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 OMe/2′-O-methyl-4′-
1428






thio/2′-O-methyl-4′-thio







Unmodified cytosines in wing



 


385871
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 OMe/2′-O-[(2-
1428






methoxy)ethyl]-4′-thio/2′-O-[(2-







methoxy)ethyl]-4′-thio







Unmodified cytosines in wing



 


386682
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 2′-(butylacetamido)-
1428






palmitamide/MOE/MOE



 


390582
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 MOE/2′-O-[(2-
1428






methoxy)ethyl]-4′-thio/2′-O-[(2-







methoxy)ethyl]-4′-thio







Unmodified cytosines in wings







Phosphodiester linkage in wings



 


391868
2096
2111
GCTAGCCTCTGGATTT
1-2-10-3 (5′R)-5′-methyl-
1428






Methyleneoxy BNA/Methyleneoxy







BNA/(5′R)-5′-methyl-







Methyleneoxy BNA







Unmodified cytosines



 


336209
2095
2108
AGCCTCTGGATTTG
3-8-3 MOE
1425


 


335331
2095
2109
TAGCCTCTGGATTTG
1-10-4 MOE
1426


 


335376
2095
2109
TAGCCTCTGGATTTG
1-10-4 Methyleneoxy BNA
1426


 


335377
2095
2109
TAGCCTCTGGATTTG
1-10-4 Methyleneoxy BNA
1426






Phosphodiester in 3′ wing



 


335330
2094
2108
AGCCTCTGGATTTGA
1-10-4 MOE
1424


 


336208
2079
2092
GGCTCCTCTACTGT
3-8-3 MOE
1423


 


336207
2073
2086
TCTACTGTTTTTGT
3-8-3 MOE
1422


 


336206
2047
2060
CACCTTAAAATTTG
3-8-3 MOE
1518


 


389776
2046
2057
CTTAAAATTTGG
1-9-2 MOE
1421


 


389977
2046
2057
CTTAAAATTTGG
1-10-1 MOE
1421


 


397990
2045
2058
CCTTAAAATTTGGA
2-10-2 MOE
1420


 


336205
2043
2056
TTAAAATTTGGAGA
3-8-3 MOE
1419


 


398058
2029
2040
AGTATCGGTTGG
1-10-1 MOE
1418


 


336204
2028
2041
AAGTATCGGTTGGC
3-8-3 MOE
1417


 


397989
2028
2041
AAGTATCGGTTGGC
2-10-2 MOE
1417


 


336203
2002
2015
TGCTTTGTCAAGAT
3-8-3 MOE
1416


 


389775
2002
2013
CTTTGTCAAGAT
1-9-2 MOE
1177


 


389976
2002
2013
CTTTGTCAAGAT
1-10-1 MOE
1177


 


397988
2001
2014
GCTTTGTCAAGATC
2-10-2 MOE
1415


 


336202
1959
1972
TCCTTGTCATTATC
3-8-3 MOE
1414


 


389774
1945
1956
CACGCTCTATAC
1-9-2 MOE
1413


 


389975
1945
1956
CACGCTCTATAC
1-10-1 MOE
1413


 


336201
1944
1957
GCACGCTCTATACT
3-8-3 MOE
1412


 


336200
1929
1942
CAAATGCTATCGAT
3-8-3 MOE
1411


 


389773
1904
1915
AGACTTCCATTT
1-9-2 MOE
1410


 


389974
1904
1915
AGACTTCCATTT
1-10-1 MOE
1410


 


336199
1902
1915
AGACTTCCATTTTC
3-8-3 MOE
1409


 


336198
1884
1897
TTTTCTGAGGTTTC
3-8-3 MOE
1408


 


398057
1878
1889
GGTTTCCTCTGG
1-10-1 MOE
1407


 


397987
1877
1890
AGGTTTCCTCTGGT
2-10-2 MOE
1406


 


336197
1873
1886
TTCCTCTGGTCCTG
3-8-3 MOE
1405


 


390015
1868
1879
GGTCCTGGTATG
1-10-1 MOE
1404


 


398056
1865
1876
CCTGGTATGAAG
1-10-1 MOE
1403


 


336196
1864
1877
TCCTGGTATGAAGA
3-8-3 MOE
1402


 


397986
1864
1877
TCCTGGTATGAAGA
2-10-2 MOE
1402


 


398055
1849
1860
TATTTACCCAAA
1-10-1 MOE
1401


 


397985
1848
1861
GTATTTACCCAAAA
2-10-2 MOE
1400


 


336195
1847
1860
TATTTACCCAAAAG
3-8-3 MOE
1399


 


389772
1846
1857
TTACCCAAAAGT
1-9-2 MOE
1398


 


389973
1846
1857
TTACCCAAAAGT
1-10-1 MOE
1398


 


336194
1838
1851
AAAAGTGAAACATT
3-8-3 MOE
1145


 


398054
1836
1847
GTGAAACATTTT
1-10-1 MOE
1144


 


397984
1835
1848
AGTGAAACATTTTG
2-10-2 MOE
1397


 


336193
1828
1841
CATTTTGTCCTTTT
3-8-3 MOE
1182


 


336192
1810
1823
CATCTTGTTCTGTT
3-8-3 MOE
1396


 


336191
1800
1813
TGTTTGTGGAAGAA
3-8-3 MOE
1395


 


398053
1796
1807
TGGAAGAACTCT
1-10-1 MOE
1394


 


397983
1795
1808
GTGGAAGAACTCTA
2-10-2 MOE
1393


 


389771
1794
1805
GAAGAACTCTAC
1-9-2 MOE
1392


 


389972
1794
1805
GAAGAACTCTAC
1-10-1 MOE
1392


 


336190
1789
1802
GAACTCTACTTTGA
3-8-3 MOE
1391


 


336189
1773
1786
TCACCACACACAGG
3-8-3 MOE
1390


 


336188
1754
1767
GCTGAGGGAACTCA
3-8-3 MOE
1389


 


398052
1751
1762
GGGAACTCAAAG
1-10-1 MOE
1388


 


389770
1750
1761
GGAACTCAAAGT
1-9-2 MOE
1386


 


389971
1750
1761
GGAACTCAAAGT
1-10-1 MOE
1386


 


397982
1750
1763
AGGGAACTCAAAGT
2-10-2 MOE
1387


 


336187
1747
1760
GAACTCAAAGTACA
3-8-3 MOE
1385


 


390012
1745
1756
TCAAAGTACATG
1-10-1 MOE
1384


 


336186
1688
1701
TCTTCACCTTTAGC
3-8-3 MOE
1383


 


398051
1684
1695
CCTTTAGCTGGC
1-10-1 MOE
1220


 


397981
1683
1696
ACCTTTAGCTGGCA
2-10-2 MOE
1382


 


336185
1677
1690
AGCTGGCAGACCAC
3-8-3 MOE
1381


 


389769
1676
1687
TGGCAGACCACA
1-9-2 MOE
1249


 


389970
1676
1687
TGGCAGACCACA
1-10-1 MOE
1249


 


392060
1675
1688
CTGGCAGACCACAA
2-10-2 Methyleneoxy BNA
1380






Unmodified cytosines in gap



 


398050
1672
1683
AGACCACAAACT
1-10-1 MOE
1379


 


397980
1671
1684
CAGACCACAAACTG
2-10-2 MOE
1378


 


390011
1658
1669
GGATTGCAAGTT
1-10-1 MOE
1238


 


336184
1655
1668
GATTGCAAGTTCCG
3-8-3 MOE
1508


 


336183
1644
1657
CCGCCACTGAACAT
3-8-3 MOE
1377


 


390010
1643
1654
CCACTGAACATT
1-10-1 MOE
1240


 


398049
1641
1652
ACTGAACATTGG
1-10-1 MOE
1376


 


397979
1640
1653
CACTGAACATTGGA
2-10-2 MOE
1375


 


336182
1633
1646
CATTGGAATAGTTT
3-8-3 MOE
1374


 


389768
1630
1641
GAATAGTTTCAA
1-9-2 MOE
1373


 


389969
1630
1641
GAATAGTTTCAA
1-10-1 MOE
1373


 


398048
1626
1637
AGTTTCAAACAT
1-10-1 MOE
1372


 


397978
1625
1638
TAGTTTCAAACATC
2-10-2 MOE
1371


 


336181
1623
1636
GTTTCAAACATCAT
3-8-3 MOE
1370


 


398047
1614
1625
CATCTTGTGAAA
1-10-1 MOE
1369


 


336180
1613
1626
TCATCTTGTGAAAC
3-8-3 MOE
1368


 


390009
1613
1624
ATCTTGTGAAAC
1-10-1 MOE
1175


 


397977
1613
1626
TCATCTTGTGAAAC
2-10-2 MOE
1368


 


390007
1563
1574
CAGGTAGCTATA
1-10-1 MOE
1367


 


336179
1561
1574
CAGGTAGCTATAAT
3-8-3 MOE
1366


 


336178
1541
1554
CATAGCGCCTCTGA
3-8-3 MOE
1365


 


336177
1534
1547
CCTCTGACTGGGAA
3-8-3 MOE
1364


 


389767
1534
1545
TCTGACTGGGAA
1-9-2 MOE
1151


 


389968
1534
1545
TCTGACTGGGAA
1-10-1 MOE
1151


 


335344
1503
1516
TCTCTGGTCCTTAC
2-10-2 MOE
1363


 


335355
1503
1516
TCTCTGGTCCTTAC
2-10-2 MOE
1363






Phosphodiester linkage in wings



 


335370
1503
1516
TCTCTGGTCCTTAC
2-10-2 Methyleneoxy BNA
1363






Phosphodiester linkage in wings



 


335381
1503
1516
TCTCTGGTCCTTAC
2-10-2 Methyleneoxy BNA
1363


 


335411
1503
1516
TCTCTGGTCCTTAC
2-10-2 MOE
1363






3′ C is 9-(aminoethoxy)phenoxazine



 


335412
1503
1516
TCTCTGGTCCTTAC
2-10-2 MOE
1363






C in 5′ wing is 9-







(aminoethoxy)phenoxazine



 


335413
1503
1516
TCTCTGGTCCTTAC
2-10-2 MOE
1363






C in wings are







9-(aminoethoxy)phenoxazine



 


336176
1502
1515
CTCTGGTCCTTACT
3-8-3 MOE
1361


 


335345
1502
1517
GTCTCTGGTCCTTACT
3-10-3 MOE
1362


 


335356
1502
1517
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






Phosphodiester linkage in wings



 


335371
1502
1517
GTCTCTGGTCCTTACT
3-10-3 Methyleneoxy BNA
1362






Phosphodiester linkage in wings



 


335382
1502
1517
GTCTCTGGTCCTTACT
3-10-3 Methyleneoxy BNA
1362


 


335414
1502
1517
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C in 3′ wing is 9-







(aminoethoxy)phenoxazine



 


335415
1502
1517
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C in 5′ wing is 9-







(aminoethoxy)phenoxazine



 


335416
1502
1517
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C's in wings are







9-(aminoethoxy)phenoxazine



 


336175
1495
1508
CCTTACTTCCCCAT
3-8-3 MOE
1360


 


336174
1472
1485
GGGCCTCTTGTGCC
3-8-3 MOE
1359


 


336173
1465
1478
TTGTGCCTTTAAAA
3-8-3 MOE
1358


 


398046
1465
1476
GTGCCTTTAAAA
1-10-1 MOE
1199


 


389766
1464
1475
TGCCTTTAAAAA
1-9-2 MOE
1217


 


389967
1464
1475
TGCCTTTAAAAA
1-10-1 MOE
1217


 


397976
1464
1477
TGTGCCTTTAAAAA
2-10-2 MOE
1357


 


336172
1437
1450
AATAAATATGCACA
3-8-3 MOE
1356


 


398045
1423
1434
TCATTACACCAG
1-10-1 MOE
1355


 


336171
1422
1435
ATCATTACACCAGT
3-8-3 MOE
1354


 


389765
1422
1433
CATTACACCAGT
1-9-2 MOE
1353


 


389966
1422
1433
CATTACACCAGT
1-10-1 MOE
1353


 


397975
1422
1435
ATCATTACACCAGT
2-10-2 MOE
1354


 


390005
1400
1411
CCAGCTTTACAG
1-10-1 MOE
1352


 


336170
1392
1405
TTACAGTGAATTGC
3-8-3 MOE
1351


 


398044
1382
1393
GCTGCAACATGA
1-10-1 MOE
1350


 


336169
1381
1394
TGCTGCAACATGAT
3-8-3 MOE
1349


 


389764
1381
1392
CTGCAACATGAT
1-9-2 MOE
1018


 


389965
1381
1392
CTGCAACATGAT
1-10-1 MOE
1018


 


397974
1381
1394
TGCTGCAACATGAT
2-10-2 MOE
1349


 


336168
1362
1375
TCTTCACTTAGCCA
3-8-3 MOE
1348


 


390004
1362
1373
TTCACTTAGCCA
1-10-1 MOE
1208


 


336167
1353
1366
AGCCATTGGTCAAG
3-8-3 MOE
1347


 


398043
1345
1356
CAAGATCTTCAC
1-10-1 MOE
1244


 


336166
1344
1357
TCAAGATCTTCACA
3-8-3 MOE
1346


 


390003
1344
1355
AAGATCTTCACA
1-10-1 MOE
1243


 


397973
1344
1357
TCAAGATCTTCACA
2-10-2 MOE
1346


 


336165
1329
1342
AAGGGTTTGATAAG
3-8-3 MOE
1345


 


390002
1322
1333
ATAAGTTCTAGC
1-10-1 MOE
1344


 


336164
1318
1331
AAGTTCTAGCTGTG
3-8-3 MOE
1343


 


398042
1305
1316
TGGGTTATGGTC
1-10-1 MOE
1214


 


336163
1304
1317
GTGGGTTATGGTCT
3-8-3 MOE
1342


 


397972
1304
1317
GTGGGTTATGGTCT
2-10-2 MOE
1342


 


398089
1298
1309
TGGTCTTCAAAA
1-10-1 MOE
1341


 


389763
1296
1307
GTCTTCAAAAGG
1-9-2 MOE
1197


 


389964
1296
1307
GTCTTCAAAAGG
1-10-1 MOE
1197


 


398041
1294
1305
CTTCAAAAGGAT
1-10-1 MOE
1196


 


336162
1293
1306
TCTTCAAAAGGATA
3-8-3 MOE
1340


 


397971
1293
1306
TCTTCAAAAGGATA
2-10-2 MOE
1340


 


398040
1279
1290
GTGCAACTCTGC
1-10-1 MOE
1236


 


336161
1278
1291
TGTGCAACTCTGCA
3-8-3 MOE
1235


 


397970
1278
1291
TGTGCAACTCTGCA
2-10-2 MOE
1235


 


398039
1264
1275
TAAATTTGGCGG
1-10-1 MOE
1339


 


397969
1263
1276
TTAAATTTGGCGGT
2-10-2 MOE
1338


 


336160
1261
1274
AAATTTGGCGGTGT
3-8-3 MOE
1337


 


336159
1253
1266
CGGTGTCATAATGT
3-8-3 MOE
1336


 


398038
1252
1263
TGTCATAATGTC
1-10-1 MOE
1200


 


390000
1251
1262
GTCATAATGTCT
1-10-1 MOE
1194


 


397968
1251
1264
GTGTCATAATGTCT
2-10-2 MOE
1195


 


336158
1227
1240
AGATTGTATATCTT
3-8-3 MOE
1335


 


389762
1220
1231
ATCTTGTAATGG
1-9-2 MOE
1334


 


389963
1220
1231
ATCTTGTAATGG
1-10-1 MOE
1334


 


336157
1215
1228
TTGTAATGGTTTTT
3-8-3 MOE
1333


 


336156
1202
1215
TATGCTTTGAATCC
3-8-3 MOE
1332


 


389998
1199
1210
TTTGAATCCAAA
1-10-1 MOE
1331


 


397967
1198
1211
CTTTGAATCCAAAA
2-10-2 MOE
1330


 


336155
1190
1203
CCAAAAACCTTACT
3-8-3 MOE
1500


 


336154
1176
1189
ACATCATCAATATT
3-8-3 MOE
1329


 


389761
1171
1182
CAATATTGTTCC
1-9-2 MOE
1328


 


389962
1171
1182
CAATATTGTTCC
1-10-1 MOE
1328


 


398037
1170
1181
AATATTGTTCCT
1-10-1 MOE
1202


 


397966
1169
1182
CAATATTGTTCCTG
2-10-2 MOE
1327


 


336153
1164
1177
TTGTTCCTGTATAC
3-8-3 MOE
1326


 


336152
1149
1162
CCTTCAAGTCTTTC
3-8-3 MOE
1325


 


389996
1141
1152
TTTCTGCAGGAA
1-10-1 MOE
1165


 


336151
1138
1151
TTCTGCAGGAAATC
3-8-3 MOE
1324


 


398036
1138
1149
CTGCAGGAAATC
1-10-1 MOE
1323


 


397965
1137
1150
TCTGCAGGAAATCC
2-10-2 MOE
1322


 


389760
1129
1140
ATCCCATAGCAA
1-9-2 MOE
1321


 


389961
1129
1140
ATCCCATAGCAA
1-10-1 MOE
1321


 


398035
1126
1137
CCATAGCAATAA
1-10-1 MOE
1320


 


336150
1125
1138
CCCATAGCAATAAT
3-8-3 MOE
1319


 


397964
1125
1138
CCCATAGCAATAAT
2-10-2 MOE
1319


 


336149
1110
1123
TTTGGATAAATATA
3-8-3 MOE
1496


 


389995
1106
1117
TAAATATAGGTC
1-10-1 MOE
1516


 


336148
1100
1113
TATAGGTCAAGTCT
3-8-3 MOE
1495


 


398034
1099
1110
AGGTCAAGTCTA
1-10-1 MOE
1300


 


397963
1098
1111
TAGGTCAAGTCTAA
2-10-2 MOE
1494


 


389994
1095
1106
CAAGTCTAAGTC
1-10-1 MOE
1299


 


336147
1090
1103
GTCTAAGTCGAATC
3-8-3 MOE
1298


 


389993
1083
1094
GAATCCATCCTC
1-10-1 MOE
1297


 


336146
1080
1093
AATCCATCCTCTTG
3-8-3 MOE
1296


 


398033
1077
1088
ATCCTCTTGATA
1-10-1 MOE
1198


 


397962
1076
1089
CATCCTCTTGATAT
2-10-2 MOE
1295


 


336145
1070
1083
CTTGATATCTCCTT
3-8-3 MOE
1294


 


336144
1057
1070
TTTGTTTCTGCTAA
3-8-3 MOE
1293


 


389759
1056
1067
GTTTCTGCTAAC
1-9-2 MOE
1292


 


389960
1056
1067
GTTTCTGCTAAC
1-10-1 MOE
1292


 


392059
1055
1068
TGTTTCTGCTAACG
2-10-2 Methyleneoxy BNA
1291






Unmodified cytosines in gap



 


336143
1044
1057
ACGATCTCTTTGAT
3-8-3 MOE
1290


 


398032
1038
1049
TTTGATGATGGC
1-10-1 MOE
1222


 


397961
1037
1050
CTTTGATGATGGCT
2-10-2 MOE
1289


 


389992
1036
1047
TGATGATGGCTG
1-10-1 MOE
1288


 


336142
1032
1045
ATGATGGCTGTCAT
3-8-3 MOE
1287


 


389991
1021
1032
TGTCTGGGAGCC
1-10-1 MOE
1286


 


392058
1020
1033
ATGTCTGGGAGCCT
2-10-2 Methyleneoxy BNA
1285






Unmodified cytosines in gap



 


397960
1020
1033
ATGTCTGGGAGCCT
2-10-2 MOE
1285


 


389990
1007
1018
TGGCTGAAGAAA
1-10-1 MOE
1284


 


397959
1006
1019
GTGGCTGAAGAAAA
2-10-2 MOE
1283


 


398031
987
998
GAGAGATGGCAG
1-10-1 MOE
1282


 


397958
986
999
AGAGAGATGGCAGA
2-10-2 MOE
1281


 


389758
983
994
GATGGCAGAAGC
1-9-2 MOE
1280


 


389959
983
994
GATGGCAGAAGC
1-10-1 MOE
1280


 


398030
976
987
GAAGCTGCTGGT
1-10-1 MOE
1143


 


397957
975
988
AGAAGCTGCTGGTG
2-10-2 MOE
1279


 


389989
953
964
TTCTGCAGGATG
1-10-1 MOE
1170


 


389757
941
952
GAAATGGCTCTG
1-9-2 MOE
1278


 


389958
941
952
GAAATGGCTCTG
1-10-1 MOE
1278


 


397956
940
953
GGAAATGGCTCTGG
2-10-2 MOE
1277


 


398029
931
942
TGGACTTGGCGG
1-10-1 MOE
1186


 


397955
930
943
CTGGACTTGGCGGT
2-10-2 MOE
1276


 


398028
914
925
GATGCCCCTCGC
1-10-1 MOE
1275


 


397954
913
926
TGATGCCCCTCGCT
2-10-2 MOE
1274


 


398027
883
894
GGACCGCAGCCG
1-10-1 MOE
1155


 


397953
882
895
TGGACCGCAGCCGG
2-10-2 MOE
1273


 


389756
874
885
CCGGGTAATGGC
1-9-2 MOE
1272


 


389957
874
885
CCGGGTAATGGC
1-10-1 MOE
1272


 


398026
867
878
ATGGCTGCTGCG
1-10-1 MOE
1160


 


397952
866
879
AATGGCTGCTGCGG
2-10-2 MOE
1271


 


389987
848
859
CTGGATGGTTGC
1-10-1 MOE
1270


 


389755
806
817
AGAGGCCTGGCA
1-9-2 MOE
1269


 


389956
806
817
AGAGGCCTGGCA
1-10-1 MOE
1269


 


389985
584
595
ATGGTGACAGGC
1-10-1 MOE
1268


 


398025
581
592
GTGACAGGCGAC
1-10-1 MOE
1267


 


397951
580
593
GGTGACAGGCGACT
2-10-2 MOE
1266


 


389754
312
323
TGCTCACAGGCG
1-9-2 MOE
1158


 


389955
312
323
TGCTCACAGGCG
1-10-1 MOE
1158


 


398024
231
242
CAGCGGCTCAAC
1-10-1 MOE
1265


 


397950
230
243
ACAGCGGCTCAACT
2-10-2 MOE
1264


 


389982
205
216
CATGGCTGCAGC
1-10-1 MOE
1161


 


392056
204
217
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263


 


394424
204
217
TCATGGCTGCAGCT
2-10-2 MOE
1263


 


396007
204
217
TCATGGCTGCAGCT
2-10-2 (R)-CMOE BNA
1263






Unmodified cytosines



 


396008
204
217
TCATGGCTGCAGCT
2-10-2 (S)-CMOE BNA
1263






Unmodified cytosines



 


396009
204
217
TCATGGCTGCAGCT
2-10-2 α-L-methyleneoxy BNA
1263






Unmodified cytosines



 


396566
204
217
TCATGGCTGCAGCT
2-10-2 Oxyamino BNA
1263






Unmodified cytosines



 


396567
204
217
TCATGGCTGCAGCT
2-10-2 N-Methyl-Oxyamino BNA
1263






Unmodified cytosines



 


396568
204
217
TCATGGCTGCAGCT
2-10-2 (6R)-6-Methyl
1263






Methyleneoxy BNA







Unmodified cytosines



 


397913
204
217
TCATGGCTGCAGCT
2-10-2 OMe
1263






Unmodified cytosines in gap



 


401974
204
217
TCATGGCTGCAGCT
2-10-2 OMe
1263






Unmodified cytosines



 


403737
204
217
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-thiazole nucleobases in wings



 


403747
204
217
TCATGGCTGCAGCT
2-10-2 N-methoxyamino BNA
1263






Unmodified cytosines



 


404121
204
217
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-methylcytosine in gaps







3′ Terminal THF phosphorothioate



 


404228
204
217
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-methylcytosinse in gaps







5′-terminal reverse abasic



 


396024
204
217
TCATGGCTGCAGCT
2-10-2 (6′S)-6′-methyl-
1263






Methyleneoxy BNA







Unmodified cytosines



 


396569
204
217
TCATGGCTGCAGCT
2-10-2 (5′S)-5′-methyl-
1263






Methyleneoxy BNA







Unmodified cytosines



 


396577
204
217
TCATGGCTGCAGCT
2-10-1-1 Methyleneoxy BNA/
1263






Methyleneoxy BNA/2′-







(butylacetamido)-palmitamide/







Unmodified cytosines in gap



 


396576
204
217
TCATGGCTGCAGCT
1-1-10-2 2′-(butylacetamido)-
1263






palmitamide/Methyleneoxy BNA/







Methyleneoxy BNA







Unmodified cytosines in gap



 


398023
191
202
CCGAGAGGAGAG
1-10-1 MOE
1262


 


397949
190
203
TCCGAGAGGAGAGA
2-10-2 MOE
1261


 


398022
126
137
AAGAGTCCCGCC
1-10-1 MOE
1260


 


397948
125
138
AAAGAGTCCCGCCA
2-10-2 MOE
1259
















TABLE 22







Short Antisense Compounds targeted to SEQ ID NO: 15













5′
3′


SEQ


ISIS
Target
Target


ID


No.
Site
Site
Sequence (5′-3′)
Gapmer Motif
NO















397948
525
538
AAAGAGTCCCGCCA
2-10-2 MOE
1259


 


398022
526
537
AAGAGTCCCGCC
1-10-1 MOE
1260


 


397949
590
603
TCCGAGAGGAGAGA
2-10-2 MOE
1261


 


398023
591
602
CCGAGAGGAGAG
1-10-1 MOE
1262


 


394424
604
617
TCATGGCTGCAGCT
2-10-2 MOE
1263


 


397913
604
617
TCATGGCTGCAGCT
2-10-2 OMe
1263






Unmodified cytosines in gap



 


401974
604
617
TCATGGCTGCAGCT
2-10-2 Ome
1263






Unmodified cytosines



 


403737
604
617
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-thiazole nucleobases in







wings



 


392056
604
617
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






Unmodified cytosines in gap



 


396576
604
617
TCATGGCTGCAGCT
1-1-10-2 2′-
1263






(butylacetamido)-







palmitamide/Methyleneoxy







BNA/Methyleneoxy BNA







Unmodified cytosines in gap



 


396577
604
617
TCATGGCTGCAGCT
2-10-1-2 Methyleneoxy
1263






BNA/Methyleneoxy BNA/







2′-(butylacetamido)-







palmitamide/







Unmodified cytosines in gap



 


404121
604
617
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-methylcytosine in gaps







3′ Terminal THF







phosphorothioate



 


404228
604
617
TCATGGCTGCAGCT
2-10-2 Methyleneoxy BNA
1263






5-methylcytosinse in gaps







5′-terminal reverse abasic



 


396007
604
617
TCATGGCTGCAGCT
2-10-2 (R)-CMOE BNA
1263






Unmodified cytosines



 


396008
604
617
TCATGGCTGCAGCT
2-10-2 (S)-CMOE BNA
1263






Unmodified cytosines



 


396009
604
617
TCATGGCTGCAGCT
2-10-2 α-L-methyleneoxy
1263






BNA







Unmodified cytosines



 


396024
604
617
TCATGGCTGCAGCT
2-10-2 (6′S)-6′-methyl-
1263






Methyleneoxy BNA







Unmodified cytosines



 


396566
604
617
TCATGGCTGCAGCT
2-10-2 Oxyamino BNA
1263






Unmodified cytosines



 


396567
604
617
TCATGGCTGCAGCT
2-10-2 N-Methyl-Oxyamino
1263






BNA







Unmodified cytosines



 


396568
604
617
TCATGGCTGCAGCT
2-10-2 (6R)-6-Methyl
1263






Methyleneoxy BNA







Unmodified cytosines



 


396569
604
617
TCATGGCTGCAGCT
2-10-2 (5′S)-5′-methyl-
1263






Methyleneoxy BNA







Unmodified cytosines



 


403747
604
617
TCATGGCTGCAGCT
2-10-2 N-methoxyamino
1263






BNA







Unmodified nucleobases



 


389982
605
616
CATGGCTGCAGC
1-10-1 MOE
1161


 


397950
630
643
ACAGCGGCTCAACT
2-10-2 MOE
1264


 


398024
631
642
CAGCGGCTCAAC
1-10-1 MOE
1265


 


389955
712
723
TGCTCACAGGCG
1-10-1 MOE
1158


 


389754
712
723
TGCTCACAGGCG
1-9-2 MOE
1158


 


397951
980
993
GGTGACAGGCGACT
2-10-2 MOE
1266


 


398025
981
992
GTGACAGGCGAC
1-10-1 MOE
1267


 


389985
984
995
ATGGTGACAGGC
1-10-1 MOE
1268


 


389956
1206
1217
AGAGGCCTGGCA
1-10-1 MOE
1269


 


389755
1206
1217
AGAGGCCTGGCA
1-9-2 MOE
1269


 


389987
1248
1259
CTGGATGGTTGC
1-10-1 MOE
1270


 


397952
1266
1279
AATGGCTGCTGCGG
2-10-2 MOE
1271


 


398026
1267
1278
ATGGCTGCTGCG
1-10-1 MOE
1160


 


389957
1274
1285
CCGGGTAATGGC
1-10-1 MOE
1272


 


389756
1274
1285
CCGGGTAATGGC
1-9-2 MOE
1272


 


397953
1282
1295
TGGACCGCAGCCGG
2-10-2 MOE
1273


 


398027
1283
1294
GGACCGCAGCCG
1-10-1 MOE
1155


 


397954
1313
1326
TGATGCCCCTCGCT
2-10-2 MOE
1274


 


398028
1314
1325
GATGCCCCTCGC
1-10-1 MOE
1275


 


397955
1330
1343
CTGGACTTGGCGGT
2-10-2 MOE
1276


 


398029
1331
1342
TGGACTTGGCGG
1-10-1 MOE
1186


 


397956
1340
1353
GGAAATGGCTCTGG
2-10-2 MOE
1277


 


389958
1341
1352
GAAATGGCTCTG
1-10-1 MOE
1278


 


389757
1341
1352
GAAATGGCTCTG
1-9-2 MOE
1278


 


389989
1353
1364
TTCTGCAGGATG
1-10-1 MOE
1170


 


397957
1375
1388
AGAAGCTGCTGGTG
2-10-2 MOE
1279


 


398030
1376
1387
GAAGCTGCTGGT
1-10-1 MOE
1143


 


389959
1383
1394
GATGGCAGAAGC
1-10-1 MOE
1280


 


389758
1383
1394
GATGGCAGAAGC
1-9-2 MOE
1280


 


397958
1386
1399
AGAGAGATGGCAGA
2-10-2 MOE
1281


 


398031
1387
1398
GAGAGATGGCAG
1-10-1 MOE
1282


 


397959
1406
1419
GTGGCTGAAGAAAA
2-10-2 MOE
1283


 


389990
1407
1418
TGGCTGAAGAAA
1-10-1 MOE
1284


 


397960
1420
1433
ATGTCTGGGAGCCT
2-10-2 MOE
1285


 


392058
1420
1433
ATGTCTGGGAGCCT
2-10-2 Methyleneoxy BNA
1285






5-methylcytosine in wing



 


389991
1421
1432
TGTCTGGGAGCC
1-10-1 MOE
1286


 


336142
1432
1445
ATGATGGCTGTCAT
3-8-3 MOE
1287


 


389992
1436
1447
TGATGATGGCTG
1-10-1 MOE
1288


 


397961
1437
1450
CTTTGATGATGGCT
2-10-2 MOE
1289


 


398032
1438
1449
TTTGATGATGGC
1-10-1 MOE
1222


 


336143
1444
1457
ACGATCTCTTTGAT
3-8-3 MOE
1290


 


392059
1455
1468
TGTTTCTGCTAACG
2-10-2 Methyleneoxy BNA
1291






5-methylcytosine in wing



 


389960
1456
1467
GTTTCTGCTAAC
1-10-1 MOE
1292


 


389759
1456
1467
GTTTCTGCTAAC
1-9-2 MOE
1292


 


336144
1457
1470
TTTGTTTCTGCTAA
3-8-3 MOE
1293


 


336145
1470
1483
CTTGATATCTCCTT
3-8-3 MOE
1294


 


397962
1476
1489
CATCCTCTTGATAT
2-10-2 MOE
1295


 


398033
1477
1488
ATCCTCTTGATA
1-10-1 MOE
1198


 


336146
1480
1493
AATCCATCCTCTTG
3-8-3 MOE
1296


 


389993
1483
1494
GAATCCATCCTC
1-10-1 MOE
1297


 


336147
1490
1503
GTCTAAGTCGAATC
3-8-3 MOE
1298


 


389994
1495
1506
CAAGTCTAAGTC
1-10-1 MOE
1299


 


398034
1499
1510
AGGTCAAGTCTA
1-10-1 MOE
1300


 


398010
1500
1513
TACAGGTCAAGTCT
2-10-2 MOE
1166


 


398077
1501
1512
ACAGGTCAAGTC
1-10-1 MOE
1167


 


398011
1512
1525
CGCAGAAATGGATA
2-10-2 MOE
1301


 


398078
1513
1524
GCAGAAATGGAT
1-10-1 MOE
1302


 


398012
1570
1583
TTCGCATCCGTCTA
2-10-2 MOE
1303


 


398079
1571
1582
TCGCATCCGTCT
1-10-1 MOE
1304


 


398013
1663
1676
CCCTAGGTTGAATA
2-10-2 MOE
1305


 


398080
1664
1675
CCTAGGTTGAAT
1-10-1 MOE
1306


 


398014
2025
2038
GTTATGCAAATCAG
2-10-2 MOE
1307


 


398081
2026
2037
TTATGCAAATCA
1-10-1 MOE
1308


 


398015
2620
2633
TGACTCAGTAAATT
2-10-2 MOE
1309


 


398082
2621
2632
GACTCAGTAAAT
1-10-1 MOE
1310


 


398016
2655
2668
TTAAAATTCTTGGG
2-10-2 MOE
1311


 


398083
2656
2667
TAAAATTCTTGG
1-10-1 MOE
1312


 


398017
2687
2700
CCTAACTTTTAGAC
2-10-2 MOE
1313


 


398084
2688
2699
CTAACTTTTAGA
1-10-1 MOE
1314


 


398018
2745
2758
ACCTGAAACTGCAA
2-10-2 MOE
1315


 


398085
2746
2757
CCTGAAACTGCA
1-10-1 MOE
1157


 


398019
13166
13179
GTGTCAAAACCACT
2-10-2 MOE
1316


 


398086
13167
13178
TGTCAAAACCAC
1-10-1 MOE
1204


 


398020
14675
14688
CCTATTCCCACTGA
2-10-2 MOE
1317


 


398087
14676
14687
CTATTCCCACTG
1-10-1 MOE
1318


 


390033
15351
15362
AGCCAACTGCAA
1-10-1 MOE
1483


 


398021
30985
30998
TTGGATAAATATCT
2-10-2 MOE
1168


 


398088
30986
30997
TGGATAAATATC
1-10-1 MOE
1169


 


397964
31001
31014
CCCATAGCAATAAT
2-10-2 MOE
1319


 


336150
31001
31014
CCCATAGCAATAAT
3-8-3 MOE
1319


 


398035
31002
31013
CCATAGCAATAA
1-10-1 MOE
1320


 


389961
31005
31016
ATCCCATAGCAA
1-10-1 MOE
1321


 


389760
31005
31016
ATCCCATAGCAA
1-9-2 MOE
1321


 


397965
31013
31026
TCTGCAGGAAATCC
2-10-2 MOE
1322


 


398036
31014
31025
CTGCAGGAAATC
1-10-1 MOE
1323


 


336151
31014
31027
TTCTGCAGGAAATC
3-8-3 MOE
1324


 


389996
31017
31028
TTTCTGCAGGAA
1-10-1 MOE
1165


 


336152
31025
31038
CCTTCAAGTCTTTC
3-8-3 MOE
1325


 


336153
31040
31053
TTGTTCCTGTATAC
3-8-3 MOE
1326


 


397966
31045
31058
CAATATTGTTCCTG
2-10-2 MOE
1327


 


398037
31046
31057
AATATTGTTCCT
1-10-1 MOE
1202


 


389962
31047
31058
CAATATTGTTCC
1-10-1 MOE
1328


 


389761
31047
31058
CAATATTGTTCC
1-9-2 MOE
1328


 


336154
31052
31065
ACATCATCAATATT
3-8-3 MOE
1329


 


389977
31480
31491
CTTAAAATTTGG
1-10-1 MOE
1421


 


389776
31480
31491
CTTAAAATTTGG
1-9-2 MOE
1421


 


397967
62446
62459
CTTTGAATCCAAAA
2-10-2 MOE
1330


 


389998
62447
62458
TTTGAATCCAAA
1-10-1 MOE
1331


 


336156
62450
62463
TATGCTTTGAATCC
3-8-3 MOE
1332


 


336157
62463
62476
TTGTAATGGTTTTT
3-8-3 MOE
1333


 


389963
62468
62479
ATCTTGTAATGG
1-10-1 MOE
1334


 


389762
62468
62479
ATCTTGTAATGG
1-9-2 MOE
1334


 


336158
62475
62488
AGATTGTATATCTT
3-8-3 MOE
1335


 


390000
67987
67998
GTCATAATGTCT
1-10-1 MOE
1194


 


397968
67987
68000
GTGTCATAATGTCT
2-10-2 MOE
1195


 


398038
67988
67999
TGTCATAATGTC
1-10-1 MOE
1200


 


336159
67989
68002
CGGTGTCATAATGT
3-8-3 MOE
1336


 


336160
67997
68010
AAATTTGGCGGTGT
3-8-3 MOE
1337


 


397969
67999
68012
TTAAATTTGGCGGT
2-10-2 MOE
1338


 


398039
68000
68011
TAAATTTGGCGG
1-10-1 MOE
1339


 


397971
69952
69965
TCTTCAAAAGGATA
2-10-2 MOE
1340


 


336162
69952
69965
TCTTCAAAAGGATA
3-8-3 MOE
1340


 


398041
69953
69964
CTTCAAAAGGAT
1-10-1 MOE
1196


 


389964
69955
69966
GTCTTCAAAAGG
1-10-1 MOE
1197


 


389763
69955
69966
GTCTTCAAAAGG
1-9-2 MOE
1197


 


398089
69957
69968
TGGTCTTCAAAA
1-10-1 MOE
1341


 


397972
69963
69976
GTGGGTTATGGTCT
2-10-2 MOE
1342


 


336163
69963
69976
GTGGGTTATGGTCT
3-8-3 MOE
1342


 


398042
69964
69975
TGGGTTATGGTC
1-10-1 MOE
1214


 


336164
69977
69990
AAGTTCTAGCTGTG
3-8-3 MOE
1343


 


390002
69981
69992
ATAAGTTCTAGC
1-10-1 MOE
1344


 


336165
69988
70001
AAGGGTTTGATAAG
3-8-3 MOE
1345


 


390003
70003
70014
AAGATCTTCACA
1-10-1 MOE
1243


 


397973
70003
70016
TCAAGATCTTCACA
2-10-2 MOE
1346


 


336166
70003
70016
TCAAGATCTTCACA
3-8-3 MOE
1346


 


398043
70004
70015
CAAGATCTTCAC
1-10-1 MOE
1244


 


336167
70012
70025
AGCCATTGGTCAAG
3-8-3 MOE
1347


 


390004
70021
70032
TTCACTTAGCCA
1-10-1 MOE
1208


 


336168
70021
70034
TCTTCACTTAGCCA
3-8-3 MOE
1348


 


389965
70040
70051
CTGCAACATGAT
1-10-1 MOE
1018


 


389764
70040
70051
CTGCAACATGAT
1-9-2 MOE
1018


 


397974
70040
70053
TGCTGCAACATGAT
2-10-2 MOE
1349


 


336169
70040
70053
TGCTGCAACATGAT
3-8-3 MOE
1349


 


398044
70041
70052
GCTGCAACATGA
1-10-1 MOE
1350


 


336170
70051
70064
TTACAGTGAATTGC
3-8-3 MOE
1351


 


390005
70059
70070
CCAGCTTTACAG
1-10-1 MOE
1352


 


389966
70081
70092
CATTACACCAGT
1-10-1 MOE
1353


 


389765
70081
70092
CATTACACCAGT
1-9-2 MOE
1353


 


397975
70081
70094
ATCATTACACCAGT
2-10-2 MOE
1354


 


336171
70081
70094
ATCATTACACCAGT
3-8-3 MOE
1354


 


398045
70082
70093
TCATTACACCAG
1-10-1 MOE
1355


 


336172
70096
70109
AATAAATATGCACA
3-8-3 MOE
1356


 


389967
70123
70134
TGCCTTTAAAAA
1-10-1 MOE
1217


 


389766
70123
70134
TGCCTTTAAAAA
1-9-2 MOE
1217


 


397976
70123
70136
TGTGCCTTTAAAAA
2-10-2 MOE
1357


 


398046
70124
70135
GTGCCTTTAAAA
1-10-1 MOE
1199


 


336173
70124
70137
TTGTGCCTTTAAAA
3-8-3 MOE
1358


 


336174
70131
70144
GGGCCTCTTGTGCC
3-8-3 MOE
1359


 


336175
70154
70167
CCTTACTTCCCCAT
3-8-3 MOE
1360


 


335345
70161
70176
GTCTCTGGTCCTTACT
3-10-3 MOE
1362


 


335356
70161
70176
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






Phosphodiester linkage in







wings



 


335414
70161
70176
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C in 3′ wing is 9-







(aminoethoxy)phenoxazine



 


335415
70161
70176
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C in 5′ wing is 9-







(aminoethoxy)phenoxazine



 


335416
70161
70176
GTCTCTGGTCCTTACT
3-10-3 MOE
1362






C's in wings are







9-







(aminoethoxy)phenoxazine



 


336176
70161
70174
CTCTGGTCCTTACT
3-8-3 MOE
1361


 


335371
70161
70176
GTCTCTGGTCCTTACT
3-10-3 Methyleneoxy BNA
1362






Phosphodiester linkage in







wings



 


335382
70161
70176
GTCTCTGGTCCTTACT
3-10-3 Methyleneoxy BNA
1362


 


335344
70162
70175
TCTCTGGTCCTTAC
2-10-2 MOE
1363


 


335355
70162
70175
TCTCTGGTCCTTAC
2-10-2 MOE
1363






Phosphodiester linkage in







wings



 


335411
70162
70175
TCTCTGGTCCTTAC
2-10-2 MOE
1363






3′ C is 9-







(aminoethoxy)phenoxazine



 


335412
70162
70175
TCTCTGGTCCTTAC
2-10-2 MOE
1363






2nd C is 9-







(aminoethoxy)phenoxazine



 


335413
70162
70175
TCTCTGGTCCTTAC
2-10-2 MOE
1363






2nd and 3′ terminal C's are







9-







(aminoethoxy)phenoxazine



 


335370
70162
70175
TCTCTGGTCCTTAC
2-10-2 Methyleneoxy BNA
1363






Phosphodiester linkage in







wings



 


335381
70162
70175
TCTCTGGTCCTTAC
2-10-2 Methyleneoxy BNA
1363


 


398068
79799
79810
ACAGCTACACAA
1-10-1 MOE
1472


 


389968
89056
89067
TCTGACTGGGAA
1-10-1 MOE
1151


 


389767
89056
89067
TCTGACTGGGAA
1-9-2 MOE
1151


 


336177
89056
89069
CCTCTGACTGGGAA
3-8-3 MOE
1364


 


336178
89063
89076
CATAGCGCCTCTGA
3-8-3 MOE
1365


 


336179
89083
89096
CAGGTAGCTATAAT
3-8-3 MOE
1366


 


390007
89085
89096
CAGGTAGCTATA
1-10-1 MOE
1367


 


390009
89135
89146
ATCTTGTGAAAC
1-10-1 MOE
1175


 


397977
89135
89148
TCATCTTGTGAAAC
2-10-2 MOE
1368


 


336180
89135
89148
TCATCTTGTGAAAC
3-8-3 MOE
1368


 


398047
89136
89147
CATCTTGTGAAA
1-10-1 MOE
1369


 


336181
89145
89158
GTTTCAAACATCAT
3-8-3 MOE
1370


 


397978
89147
89160
TAGTTTCAAACATC
2-10-2 MOE
1371


 


398048
89148
89159
AGTTTCAAACAT
1-10-1 MOE
1372


 


389969
89152
89163
GAATAGTTTCAA
1-10-1 MOE
1373


 


389768
89152
89163
GAATAGTTTCAA
1-9-2 MOE
1373


 


336182
89155
89168
CATTGGAATAGTTT
3-8-3 MOE
1374


 


397979
89162
89175
CACTGAACATTGGA
2-10-2 MOE
1375


 


398049
89163
89174
ACTGAACATTGG
1-10-1 MOE
1376


 


390010
89165
89176
CCACTGAACATT
1-10-1 MOE
1240


 


336183
89166
89179
CCGCCACTGAACAT
3-8-3 MOE
1377


 


397980
94786
94799
CAGACCACAAACTG
2-10-2 MOE
1378


 


398050
94787
94798
AGACCACAAACT
1-10-1 MOE
1379


 


392060
94790
94803
CTGGCAGACCACAA
2-10-2 Methyleneoxy BNA
1380






Unmodified cytosines in gap



 


389970
94791
94802
TGGCAGACCACA
1-10-1 MOE
1249


 


389769
94791
94802
TGGCAGACCACA
1-9-2 MOE
1249


 


336185
94792
94805
AGCTGGCAGACCAC
3-8-3 MOE
1381


 


397981
94798
94811
ACCTTTAGCTGGCA
2-10-2 MOE
1382


 


398051
94799
94810
CCTTTAGCTGGC
1-10-1 MOE
1220


 


336186
94803
94816
TCTTCACCTTTAGC
3-8-3 MOE
1383


 


390012
94860
94871
TCAAAGTACATG
1-10-1 MOE
1384


 


336187
94862
94875
GAACTCAAAGTACA
3-8-3 MOE
1385


 


389971
94865
94876
GGAACTCAAAGT
1-10-1 MOE
1386


 


389770
94865
94876
GGAACTCAAAGT
1-9-2 MOE
1386


 


397982
94865
94878
AGGGAACTCAAAGT
2-10-2 MOE
1387


 


398052
94866
94877
GGGAACTCAAAG
1-10-1 MOE
1388


 


336188
94869
94882
GCTGAGGGAACTCA
3-8-3 MOE
1389


 


336189
94888
94901
TCACCACACACAGG
3-8-3 MOE
1390


 


336190
94904
94917
GAACTCTACTTTGA
3-8-3 MOE
1391


 


389972
94909
94920
GAAGAACTCTAC
1-10-1 MOE
1392


 


389771
94909
94920
GAAGAACTCTAC
1-9-2 MOE
1392


 


397983
94910
94923
GTGGAAGAACTCTA
2-10-2 MOE
1393


 


398053
94911
94922
TGGAAGAACTCT
1-10-1 MOE
1394


 


336191
94915
94928
TGTTTGTGGAAGAA
3-8-3 MOE
1395


 


336192
94925
94938
CATCTTGTTCTGTT
3-8-3 MOE
1396


 


397984
97824
97837
AGTGAAACATTTTG
2-10-2 MOE
1397


 


398054
97825
97836
GTGAAACATTTT
1-10-1 MOE
1144


 


336194
97827
97840
AAAAGTGAAACATT
3-8-3 MOE
1145


 


389973
97835
97846
TTACCCAAAAGT
1-10-1 MOE
1398


 


389772
97835
97846
TTACCCAAAAGT
1-9-2 MOE
1398


 


336195
97836
97849
TATTTACCCAAAAG
3-8-3 MOE
1399


 


397985
97837
97850
GTATTTACCCAAAA
2-10-2 MOE
1400


 


398055
97838
97849
TATTTACCCAAA
1-10-1 MOE
1401


 


397986
97853
97866
TCCTGGTATGAAGA
2-10-2 MOE
1402


 


336196
97853
97866
TCCTGGTATGAAGA
3-8-3 MOE
1402


 


398056
97854
97865
CCTGGTATGAAG
1-10-1 MOE
1403


 


390015
97857
97868
GGTCCTGGTATG
1-10-1 MOE
1404


 


336197
97862
97875
TTCCTCTGGTCCTG
3-8-3 MOE
1405


 


397987
97866
97879
AGGTTTCCTCTGGT
2-10-2 MOE
1406


 


398057
97867
97878
GGTTTCCTCTGG
1-10-1 MOE
1407


 


336198
97873
97886
TTTTCTGAGGTTTC
3-8-3 MOE
1408


 


336199
97891
97904
AGACTTCCATTTTC
3-8-3 MOE
1409


 


389974
97893
97904
AGACTTCCATTT
1-10-1 MOE
1410


 


389773
97893
97904
AGACTTCCATTT
1-9-2 MOE
1410


 


336200
97918
97931
CAAATGCTATCGAT
3-8-3 MOE
1411


 


336201
97933
97946
GCACGCTCTATACT
3-8-3 MOE
1412


 


389975
97934
97945
CACGCTCTATAC
1-10-1 MOE
1413


 


389774
97934
97945
CACGCTCTATAC
1-9-2 MOE
1413


 


336202
97948
97961
TCCTTGTCATTATC
3-8-3 MOE
1414


 


397988
97990
98003
GCTTTGTCAAGATC
2-10-2 MOE
1415


 


389976
97991
98002
CTTTGTCAAGAT
1-10-1 MOE
1177


 


389775
97991
98002
CTTTGTCAAGAT
1-9-2 MOE
1177


 


336203
97991
98004
TGCTTTGTCAAGAT
3-8-3 MOE
1416


 


397989
98017
98030
AAGTATCGGTTGGC
2-10-2 MOE
1417


 


336204
98017
98030
AAGTATCGGTTGGC
3-8-3 MOE
1417


 


398058
98018
98029
AGTATCGGTTGG
1-10-1 MOE
1418


 


336205
98032
98045
TTAAAATTTGGAGA
3-8-3 MOE
1419


 


397990
98034
98047
CCTTAAAATTTGGA
2-10-2 MOE
1420


 


389977
98035
98046
CTTAAAATTTGG
1-10-1 MOE
1421


 


389776
98035
98046
CTTAAAATTTGG
1-9-2 MOE
1421


 


336207
102230
102243
TCTACTGTTTTTGT
3-8-3 MOE
1422


 


336208
102236
102249
GGCTCCTCTACTGT
3-8-3 MOE
1423


 


335330
102251
102265
AGCCTCTGGATTTGA
1-10-4 MOE
1424


 


335331
102252
102266
TAGCCTCTGGATTTG
1-10-4 MOE
1426


 


336209
102252
102265
AGCCTCTGGATTTG
3-8-3 MOE
1425


 


335377
102252
102266
TAGCCTCTGGATTTG
1-10-4 Methyleneoxy BNA
1426






Phosphodiester in 3′ wing



 


335376
102252
102266
TAGCCTCTGGATTTG
1-10-4 Methyleneoxy BNA
1426


 


390577
102253
102266
TAGCCTCTGGATTT
1-10-3 MOE
1427






Unmodified cytosines







T's in wings are 2-







thiothymines



 


335332
102253
102267
CTAGCCTCTGGATTT
1-10-4 MOE
1429


 


386770
102253
102266
TAGCCTCTGGATTT
1-11-2 MOE
1427


 


375560
102253
102267
CTAGCCTCTGGATTT
2-10-3 MOE
1429


 


391449
102253
102267
CTAGCCTCTGGATTT
2-10-3 MOE
1429






Unmodified cytosines



 


392055
102253
102267
CTAGCCTCTGGATTT
2-10-3 MOE
1429






Unmodified cytosines in gap



 


362977
102253
102268
GCTAGCCTCTGGATTT
2-12-2 MOE
1428


 


371975
102253
102267
CTAGCCTCTGGATTT
3-10-2 MOE
1429


 


386556
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


335341
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


335350
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


383739
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






5-methylcytosine in gap



 


390576
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






5-methylcytosine in gap







T's in wings are 2-







thiothymines



 


390580
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Pyrimidines in wings are 5-







thiazole







Unmodified cytosines in gap



 


390581
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Unmodified cytosines in gap



 


391096
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


391098
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428


 


391863
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE
1428






Unmodified cytosines



 


384071
102253
102268
GCTAGCCTCTGGATTT
3-10-3 OMe
1428






5-methylcytosine in gap



 


385036
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 OMe/2′-O-methyl-
1428






4′-thio/2′-O-methyl-4′-thio







Unmodified cytosines in







wing



 


335368
102253
102268
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Phosphodiester linkages in







wings



 


391864
102253
102268
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Unmodified cytosines in gap



 


392054
102253
102267
CTAGCCTCTGGATTT
2-10-3 Methyleneoxy BNA
1429






Unmodified cytosines in gap



 


391172
102253
102267
CTAGCCTCTGGATTT
2-10-3 Methyleneoxy BNA
1429






Unmodified cytosines



 


391865
102253
102268
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






Unmodified cytosines



 


391868
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 (5′R)-5′-methyl-
1428






Methyleneoxy BNA/







Methyleneoxy BNA/(5′R)-







5′-methyl-Methyleneoxy







BNA







Unmodified cytosines



 


391869
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 Methyleneoxy
1428






BNA/(5′S)-5′-methyl-







Methyleneoxy BNA/(5′S)-







5′-methyl-Methyleneoxy







BNA







Unmodified cytosines



 


384073
102253
102268
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428






5-methylcytosine in gap



 


335379
102253
102268
GCTAGCCTCTGGATTT
3-10-3 Methyleneoxy BNA
1428


 


390579
102253
102268
GCTAGCCTCTGGATTT
1-1-1-10-3 MOE/4′thio/2′-
1428






O-[(2-methoxy)ethyl]-4′-







thio/2′-O-[(2-







methoxy)ethyl]-4′-thio







Unmodified cytosines in







wings







Phosphorodiester linkage in







wings



 


390582
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 MOE/4′thio/2′-O-
1428






[(2-methoxy)ethyl]-4′-thio







Unmodified cytosines in







wings







Phosphorodiester linkage in







wings



 


390606
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3
1428






MOE/pentaF/pentaF







Unmodified cytosines in







wings







Phosphodiester linkage in







wings



 


384072
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3
1428






MOE/pentaF/pentaF







Unmodified cytosines in







wings



 


385871
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 OMe/2′-O-[(2-
1428






methoxy)ethyl]-4′-thio/2′-O-







[(2-methoxy)ethyl]-4′-thio







Unmodified cytosines in







wing



 


390607
102253
102268
GCTAGCCTCTGGATTT
3-10-3 MOE/pentaF
1428






Unmodified cytosines in







wing



 


390608
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3
1428






MOE/pentaF/pentaF







Unmodified cytosines in







wing



 


390609
102253
102268
GCTAGCCTCTGGATTT
3-10-2-1 MOE/MOE/pentaF
1428






Unmodified cytosines in







wing



 


386682
102253
102268
GCTAGCCTCTGGATTT
1-2-10-3 2′-
1428






(butylacetamido)-







palmitamide/MOE/MOE



 


391173
102253
102267
CTAGCCTCTGGATTT
2-10-3 (5′R)-5′-methyl-
1429






Methyleneoxy BNA







Unmodified cytosines



 


391174
102253
102267
CTAGCCTCTGGATTT
2-10-3 (5′S)-5′-methyl-
1429






Methyleneoxy BNA







Unmodified cytosines



 


386970
102254
102266
TAGCCTCTGGATT
1-10-2 MOE
1432


 


390578
102254
102266
TAGCCTCTGGATT
1-10-2 MOE
1432






Unmodified cytosines







Ts in wings are 2-







thiothymines



 


335333
102254
102268
GCTAGCCTCTGGATT
1-10-4 MOE
1430


 


331429
102254
102267
CTAGCCTCTGGATT
2-10-2 MOE
1431


 


335349
102254
102267
CTAGCCTCTGGATT
2-10-2 MOE
1431


 


335367
102254
102267
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431






Phosphodiester linkages in







wings



 


392061
102254
102267
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431






Unmodified cytosines in gap



 


335378
102254
102267
CTAGCCTCTGGATT
2-10-2 Methyleneoxy BNA
1431


 


383991
102254
102266
TAGCCTCTGGATT
1-10-2
1432






2′-(acetylamino-butyl-







acetamido)-cholesterol/







MOE



 


383992
102254
102266
TAGCCTCTGGATT
1-10-2
1432






2′-(acetylamino-butyl-







acetamido)-cholic acid/MOE



 


386683
102254
102266
TAGCCTCTGGATT
1-10-2
1432






5′ terminal 2′-







(butylacetamido)-







palmitamide/MOE



 


390614
102254
102266
TAGCCTCTGGATT
1-10-2 PentaF
1432


 


389954
102255
102266
TAGCCTCTGGAT
1-10-1 MOE
1434


 


335334
102255
102269
TGCTAGCCTCTGGAT
1-10-4 MOE
1433


 


389777
102255
102266
TAGCCTCTGGAT
1-9-2 MOE
1434


 


390430
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines



 


390431
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







C in wing 9-







(aminoethoxy)phenoxazine



 


390432
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163


 


390433
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 6 is 9-







(aminoethoxy)phenoxazine



 


390434
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 7 is 9-







(aminoethoxy)phenoxazine



 


390435
102256
102268
GCTAGCCTCTGGA
1-10-2 MOE
1163






Unmodified cytosines







Nt 9 is 9-







(aminoethoxy)phenoxazine



 


335335
102256
102270
CTGCTAGCCTCTGGA
1-10-4 MOE
1435


 


335336
102257
102271
ACTGCTAGCCTCTGG
1-10-4 MOE
1436


 


335337
102258
102272
AACTGCTAGCCTCTG
1-10-4 MOE
1437


 


335338
102259
102273
GAACTGCTAGCCTCT
1-10-4 MOE
1438


 


335339
102260
102274
TGAACTGCTAGCCTC
1-10-4 MOE
1439


 


335340
102261
102275
TTGAACTGCTAGCCT
1-10-4 MOE
1440


 


336210
102261
102274
TGAACTGCTAGCCT
3-8-3 MOE
1441


 


397991
102264
102277
AGTTGAACTGCTAG
2-10-2 MOE
1442


 


398059
102265
102276
GTTGAACTGCTA
1-10-1 MOE
1443


 


390017
102268
102279
GAAGTTGAACTG
1-10-1 MOE
1444


 


336211
102269
102282
ACAGAAGTTGAACT
3-8-3 MOE
1445


 


397992
102293
102306
TCATTGTCACTAAC
2-10-2 MOE
1446


 


336212
102293
102306
TCATTGTCACTAAC
3-8-3 MOE
1446


 


398060
102294
102305
CATTGTCACTAA
1-10-1 MOE
1447


 


389978
102301
102312
TCAGGTTCATTG
1-10-1 MOE
1448


 


389778
102301
102312
TCAGGTTCATTG
1-9-2 MOE
1448


 


336213
102303
102316
ATGATCAGGTTCAT
3-8-3 MOE
1449


 


397993
102307
102320
TATAATGATCAGGT
2-10-2 MOE
1450


 


398061
102308
102319
ATAATGATCAGG
1-10-1 MOE
1451


 


336214
102314
102327
GAATATCTATAATG
3-8-3 MOE
1139


 


390019
102320
102331
GTCAGAATATCT
1-10-1 MOE
1173


 


397994
102322
102335
TGGTGTCAGAATAT
2-10-2 MOE
1452


 


398062
102323
102334
GGTGTCAGAATA
1-10-1 MOE
1255


 


336215
102326
102339
TCAGTGGTGTCAGA
3-8-3 MOE
1453


 


336216
102339
102352
CTCTGGATCAGAGT
3-8-3 MOE
1454


 


390020
102340
102351
TCTGGATCAGAG
1-10-1 MOE
1149


 


336217
102349
102362
AAGGTTCATTCTCT
3-8-3 MOE
1455


 


397995
102357
102370
TTCATCAAAAGGTT
2-10-2 MOE
1456


 


389979
102358
102369
TCATCAAAAGGT
1-10-1 MOE
1176


 


389779
102358
102369
TCATCAAAAGGT
1-9-2 MOE
1176


 


336218
102358
102371
CTTCATCAAAAGGT
3-8-3 MOE
1457


 


390021
102360
102371
CTTCATCAAAAG
1-10-1 MOE
1458


 


336219
102366
102379
ATGCTGATCTTCAT
3-8-3 MOE
1459


 


336220
102381
102394
TTTTGTAATTTGTG
3-8-3 MOE
1460


 


336221
102387
102400
TCAGACTTTTGTAA
3-8-3 MOE
1461


 


390022
102443
102454
CAGTTTATTCAA
1-10-1 MOE
1142


 


397996
102477
102490
TGTCCTATTGCCAT
2-10-2 MOE
1462


 


398063
102478
102489
GTCCTATTGCCA
1-10-1 MOE
1205


 


397997
102487
102500
TCTGACACAATGTC
2-10-2 MOE
1463


 


398064
102488
102499
CTGACACAATGT
1-10-1 MOE
1464


 


397998
102505
102518
TGTTCCTATAACTG
2-10-2 MOE
1465


 


398065
102506
102517
GTTCCTATAACT
1-10-1 MOE
1466


 


397999
102528
102541
AAGATTGGTCAGGA
2-10-2 MOE
1467


 


398066
102529
102540
AGATTGGTCAGG
1-10-1 MOE
1468


 


398000
102561
102574
GTGTCAAAACCCTG
2-10-2 MOE
1469


 


398067
102562
102573
TGTCAAAACCCT
1-10-1 MOE
1210


 


390025
102563
102574
GTGTCAAAACCC
1-10-1 MOE
1211


 


390026
102595
102606
AGCTACACAACC
1-10-1 MOE
1470


 


398001
102596
102609
CACAGCTACACAAC
2-10-2 MOE
1471


 


398068
102597
102608
ACAGCTACACAA
1-10-1 MOE
1472


 


398002
102607
102620
TATATACATGACAC
2-10-2 MOE
1473


 


398069
102608
102619
ATATACATGACA
1-10-1 MOE
1474


 


390027
102612
102623
AGGTATATACAT
1-10-1 MOE
1206


 


398003
102637
102650
AATTTTAAATGTCC
2-10-2 MOE
1475


 


398070
102638
102649
ATTTTAAATGTC
1-10-1 MOE
1476


 


390028
102648
102659
TCCTAATTGAAT
1-10-1 MOE
1477


 


390029
102667
102678
AAAGTGCCATCT
1-10-1 MOE
1478


 


398004
102689
102702
TTTATAAAACTGGA
2-10-2 MOE
1479


 


398071
102690
102701
TTATAAAACTGG
1-10-1 MOE
1480


 


390030
102691
102702
TTTATAAAACTG
1-10-1 MOE
1074


 


398005
102827
102840
TGCAAACTTATCTG
2-10-2 MOE
1481


 


398072
102828
102839
GCAAACTTATCT
1-10-1 MOE
1482


 


390033
102836
102847
AGCCAACTGCAA
1-10-1 MOE
1483


 


398006
102837
102850
CTTAGCCAACTGCA
2-10-2 MOE
1484


 


398073
102838
102849
TTAGCCAACTGC
1-10-1 MOE
1485


 


398007
103069
103082
AGCACCAATATGCT
2-10-2 MOE
1247


 


398074
103070
103081
GCACCAATATGC
1-10-1 MOE
1248


 


398008
103267
103280
TAAATCATTGTCAA
2-10-2 MOE
1486


 


398075
103268
103279
AAATCATTGTCA
1-10-1 MOE
1233


 


398009
103327
103340
GCACTGGCCTTGAT
2-10-2 MOE
1487


 


398076
103328
103339
CACTGGCCTTGA
1-10-1 MOE
1488


 


390041
103332
103343
TTAGCACTGGCC
1-10-1 MOE
1489


 


390047
103585
103596
TGTGTAAGGTCA
1-10-1 MOE
1490


 


390049
103636
103647
GTTAATGACATT
1-10-1 MOE
1491


 


390050
103660
103671
GTATTCAAGTAA
1-10-1 MOE
1140


 


390052
103780
103791
GACAATTTCTAC
1-10-1 MOE
1492


 


390054
103862
103873
AACACTGCACAT
1-10-1 MOE
1493









Salts, Prodrugs and Bioequivalents

The antisense compounds provided herein comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the antisense compounds, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes, chemicals, and/or conditions. In particular, prodrug versions of the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764. Prodrugs can also include antisense compounds wherein one or both ends comprise nucleobases that are cleaved (e.g., by incorporating phosphodiester backbone linkages at the ends) to produce the active compound. In certain embodiments, one or more non-drug moieties is cleaved from a prodrug to yield the active form. In certain such embodiments, such non-drug moieties is not a nucleotide or oligonucleotide.
The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds described herein: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans.
In certain embodiments, salts, including, but not limited to sodium salts, of double stranded nucleic acids (including but not limited to dsRNA compounds) are also provided.
G. CERTAIN PHARMACEUTICAL COMPOSITIONS
In certain embodiments, pharmaceutical compositions of the present invention comprise one or more short antisense compound and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
In certain embodiments, a pharmaceutical composition of the present invention is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tabletting processes.
In certain embodiments, a pharmaceutical composition of the present invention is a liquid (e.g., a suspension, elixir and/or solution). In certain of such embodiments, a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
In certain embodiments, a pharmaceutical composition of the present invention is a solid (e.g., a powder, tablet, and/or capsule). In certain of such embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.
In certain embodiments, a pharmaceutical composition of the present invention is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
In certain embodiments, a pharmaceutical composition of the present invention comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
In certain embodiments, a pharmaceutical composition of the present invention comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
In certain embodiments, a pharmaceutical composition of the present invention comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM, and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
In certain embodiments, a pharmaceutical composition of the present invention comprises a sustained-release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers. In certain embodiments, sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.
In certain embodiments, a pharmaceutical composition of the present invention is prepared for oral administration. In certain of such embodiments, a pharmaceutical composition is formulated by combining one or more oligonucleotides with one or more pharmaceutically acceptable carriers. Certain of such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject. In certain embodiments, pharmaceutical compositions for oral use are obtained by mixing oligonucleotide and one or more solid excipient. Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). In certain embodiments, such a mixture is optionally ground and auxiliaries are optionally added. In certain embodiments, pharmaceutical compositions are formed to obtain tablets or dragee cores. In certain embodiments, disintegrating agents (e.g., cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate) are added.
In certain embodiments, dragee cores are provided with coatings. In certain such embodiments, concentrated sugar solutions may be used, which may optionally comprise gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to tablets or dragee coatings.
In certain embodiments, pharmaceutical compositions for oral administration are push-fit capsules made of gelatin. Certain of such push-fit capsules comprise one or more pharmaceutical agents of the present invention in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In certain embodiments, pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In certain soft capsules, one or more pharmaceutical agents of the present invention are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
In certain embodiments, pharmaceutical compositions are prepared for buccal administration. Certain of such pharmaceutical compositions are tablets or lozenges formulated in conventional manner.
In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may comprise formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may comprise substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also comprise suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In certain embodiments, a pharmaceutical composition is prepared for administration by inhalation. Certain of such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer. Certain of such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In certain embodiments using a pressurized aerosol, the dosage unit may be determined with a valve that delivers a metered amount. In certain embodiments, capsules and cartridges for use in an inhaler or insufflator may be formulated. Certain of such formulations comprise a powder mixture of a pharmaceutical agent of the invention and a suitable powder base such as lactose or starch.
In certain embodiments, a pharmaceutical composition is prepared for rectal administration, such as a suppositories or retention enema. Certain of such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides.
In certain embodiments, a pharmaceutical composition is prepared for topical administration. Certain of such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams. Exemplary suitable ointment bases include, but are not limited to, petrolatum, petrolatum plus volatile silicones, lanolin and water in oil emulsions such as EucerinTM, available from Beiersdorf (Cincinnati, Ohio). Exemplary suitable cream bases include, but are not limited to, NiveaTM Cream, available from Beiersdorf (Cincinnati, Ohio), cold cream (USP), Purpose CreamTM, available from Johnson & Johnson (New Brunswick, N.J.), hydrophilic ointment (USP) and LubridermTM, available from Pfizer (Morris Plains, N.J.).
In certain embodiments, a pharmaceutical composition of the present invention comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more short antisense compound of the present invention is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of the short antisense compound. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).
In certain embodiments, a pharmaceutical composition comprising one or more pharmaceutical agents of the present invention is useful for treating a conditions or disorders in a mammalian, and particularly in a human, subject. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., in the renal or cardiac area).
In certain embodiments, short antisense compounds, compared to their parent oligonucleotides, make them particularly suited to oral administration. In certain embodiments, short antisense compounds are better suited for oral administration than their parent oligonucleotides because they have increased potency compared to those parent oligonucleotides. In certain embodiments, short antisense compounds are better suited for oral administration than their parent oligonucleotides because they have better stability, availability or solubility properties compared to those parent oligonucleotides.
In a further aspect, a pharmaceutical agent is sterile lyophilized oligonucleotide that is reconstituted with a suitable diluent, e.g., sterile water for injection. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline. The lyophilized drug product consists of the oligonucleotide which has been prepared in water for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized. The lyophilized oligonucleotide may be 25-800 mg of the oligonucleotide. It is understood that this encompasses 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of lyophilized oligonucleotide. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum FLIP-OFF® overseal.
The compositions of the present invention may additionally comprise other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may comprise additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may comprise additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
The antisense compounds provided herein may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds.
Also described herein are pharmaceutical compositions and formulations which include the antisense compounds provided herein. The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. In a preferred embodiment, administration is topical to the surface of the respiratory tract, particularly pulmonary, e.g., by nebulization, inhalation, or insufflation of powders or aerosols, by mouth and/or nose.
The pharmaceutical formulations described herein, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product (e.g., into a specific particle size for delivery). In a preferred embodiment, the pharmaceutical formulations are prepared for pulmonary administration in an appropriate solvent, e.g., water or normal saline, possibly in a sterile formulation, with carriers or other agents to allow for the formation of droplets of the desired diameter for delivery using inhalers, nasal delivery devices, nebulizers, and other devices for pulmonary delivery. Alternatively, the pharmaceutical formulations may be formulated as dry powders for use in dry powder inhalers.
A “pharmaceutical carrier” or “excipient” can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an individual and are known in the art. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
H. CERTAIN THERAPEUTIC USES
In certain embodiments, antisense compounds are used to modulate the expression of a target gene in an animal, such as a human. In certain embodiments, such compounds can be used to treat metabolic disorders or modulate one or more disease indications. For example, the methods comprise the step of administering to said animal in need of therapy for a disease or condition associated with a target gene an effective amount of an antisense compound that modulates expression of the target gene. Antisense compounds provided herein which effectively modulate expression of a target RNA or protein products of expression are considered active antisense compounds. Active antisense compounds also include compounds which effectively modulate one or more of a number of disease indications, including metabolic and cardiovascular disease indications, examples of which are described below.
Modulation of expression of a target gene can be measured in a bodily fluid, which may or may not contain cells; tissue; or organ of the animal. Methods of obtaining samples for analysis, such as body fluids (e.g., sputum, serum, urine), tissues (e.g., biopsy), or organs, and methods of preparation of the samples to allow for analysis are well known to those skilled in the art. Methods for analysis of RNA and protein levels are discussed above and are well known to those skilled in the art. The effects of treatment can be assessed by measuring biomarkers, or disease indications, associated with the target gene expression in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds described herein, by routine clinical methods known in the art. These biomarkers include but are not limited to: liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein, chemokines, cytokines, and other markers of inflammation.
The antisense compounds provided herein can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Acceptable carriers and diluents are well known to those skilled in the art. Selection of a diluent or carrier is based on a number of factors, including, but not limited to, the solubility of the compound and the route of administration. Such considerations are well understood by those skilled in the art. In one aspect, the antisense compounds described herein inhibit expression of a target gene. The compounds can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to a target gene.
Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the antisense compounds or compositions provided herein are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds resulting in modulation of target gene expression in the cells of bodily fluids, organs or tissues. An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan.
Co-Administration
In certain embodiments, two or more antisense compounds are co-administered. In certain embodiments, pharmaceutical compositions include one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more antisense compounds targeted to a second nucleic acid target. One or more of those antisense compounds may be a short antisense compound. In certain embodiments, pharmaceutical compositions include two or more antisense compounds targeted to different regions of the same nucleic acid target. One or more of such antisense compounds may be a short antisense compound. Two or more combined compounds may be used together or sequentially.
In certain embodiments, one or more pharmaceutical compositions are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include lipid-lowering agents. In certain such embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain such embodiments, the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.
In certain embodiments, a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoA reductase inhibitor is a statin. In certain such embodiments the statin is selected from atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin. In certain embodiments, a co-administered lipid-lowering agent is a cholesterol absorption inhibitor. In certain such embodiments, cholesterol absorption inhibitor is ezetimibe. In certain embodiments, a co-administered lipid-lowering agent is a co-formulated HMG-CoA reductase inhibitor and cholesterol absorption inhibitor. In certain such embodiments the co-formulated lipid-lowering agent is ezetimibe/simvastatin. In certain embodiments, a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor.
In certain embodiments, a co-administered pharmaceutical agent is a bile acid sequestrant. In certain such embodiments, the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.
In certain embodiments, a co-administered pharmaceutical agent is a nicotinic acid. In certain such embodiments, the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
In certain embodiments, a co-administered pharmaceutical agent is a fibric acid. In certain such embodiments, a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
Further examples of pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sedatives; poison oak or poison sumac products; antibodies; and vaccines.
In certain embodiments, the pharmaceutical compositions of the present invention may be administered in conjunction with a lipid-lowering therapy. In certain such embodiments, a lipid-lowering therapy is therapeutic lifestyle change. In certain such embodiments, a lipid-lowering therapy is LDL apheresis.
I. KITS, RESEARCH REAGENTS AND DIAGNOSTICS
The antisense compounds provided herein can be utilized for diagnostics, and as research reagents and kits. Furthermore, antisense compounds, which are able to inhibit gene expression or modulate gene expression with specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the antisense compounds described herein, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues. Methods of gene expression analysis are well known to those skilled in the art.
J. CERTAIN ADVANTAGES OF SHORT ANTISENSE COMPOUNDS
In certain embodiments, short antisense compounds have advantages when compared to their parent oligonucleotides. For example, in certain embodiments, short antisense compounds have greater affinity for a target nucleic acid than their parent oligonucleotide. In certain embodiments, short antisense compounds have greater potency in vitro than their parent oligonucleotide. In certain such embodiments, that increased in vitro potency is not entirely explained by increased affinity. In certain embodiments, such increased in vitro potency may be attributable to increased ability of short antisense compounds to penetrate cells and/or increased ability to access target nucleic acids in a cell. In certain embodiments, short antisense compounds have greater potency in vivo than their parent oligonucleotides. In certain embodiments, such greater in vivo potency is not attributable to increased in vitro potency or increased affinity. In certain embodiments, short antisense compounds have even greater in vivo potency compared to their parent oligonucleotides than would be predicted based on in vitro potencies or on affinities. In certain embodiments, such increased in vivo potency may be attributable to increased bioavailability, better penetration into the cell, better access to target nucleic acid once in the cell, or other factors.
In certain embodiments, one would expect short antisense compounds to be less specific for their target nucleic acid compared to their parent oligonucleotides. In certain such embodiments, one would expect increased side-effects, including potential for toxic effects, from short antisense compounds. In certain embodiments, such additional side-effects are not observed. In certain embodiments, non-target nucleic acids to which a particular short antisense compound may bind are not available to the short antisense compound. In such embodiments, side-effects, including toxicity, are less problematic than would be predicted.
In certain embodiments, because they are smaller, short antisense compounds are less likely to bind proteins. In certain such embodiments, such less binding of proteins results in lower toxicity, since protein binding may have undesired consequences. In certain embodiments, such less binding of proteins results in greater potency, since it leaves more antisense compound available for therapeutic effect. In certain embodiments, less binding of proteins results in decreased drug-drug interaction toxicity.
NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
Example 1
Cell Culture and Treatment with Short Antisense Compounds
The effect of short antisense compounds on target nucleic acid expression can be tested in any one of a number of cultured or primary cell lines. Cells lines can be obtained from publicly available sources, such as the American Type Culture Collection (Manassas, Va.). Cells are cultured according to methods well known to those of ordinary skill in the art.
When cells reached appropriate confluency, they were treated with oligonucleotide using LIPOFECTIN® as described. When cells reached 65-75% confluency, they were treated with oligonucleotide. Oligonucleotide was mixed with LIPOFECTIN® Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEMO®-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN® concentration of 2.5 or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture was incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM®-1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates were treated similarly, using appropriate volumes of medium and oligonucleotide. Cells were treated and data were obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture was replaced with fresh culture medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
Control oligonucleotides are used to determine the optimal oligomeric compound concentration for a particular cell line. Furthermore, when oligomeric compounds are tested in oligomeric compound screening experiments or phenotypic assays, control oligonucleotides are tested in parallel.
The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. The concentration of positive control oligonucleotide that results in 80% inhibition of the target mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of the target mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM when the antisense oligonucleotide is transfected using a liposome reagent and 1 nM to 40 nM when the antisense oligonucleotide is transfected by electroporation.
Example 2
Real-Time Quantitative PCR Analysis of Target mRNA Levels
Quantitation of target mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured were evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. After isolation the RNA is subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR was carried out in the same by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen® (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT, real-time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA was quantified using RiboGreen® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).
170 μL of RiboGreen® working reagent (RiboGreen® reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well plate containing 30 μL purified cellular RNA. The plate was read in a CytoFluor® 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
The GAPDH PCR probes have JOE covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where JOE is the fluorescent reporter dye and TAMRA or MGB is the quencher dye. In some cell types, primers and probe designed to a GAPDH sequence from a different species are used to measure GAPDH expression. For example, a human GAPDH primer and probe set is used to measure GAPDH expression in monkey-derived cells and cell lines.
Probes and primers for use in real-time PCR were designed to hybridize to target nucleic acids using routine methods. For example, PrimerExpress® (Applied Biosystems, Foster City, Calif.) software is routinely used to design probes and primers for use in real-time PCR. Examples of primer and probe sequences and the target nucleic acids to which they hybridize are presented in Table 24. The target-specific PCR probes have FAM covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.







TABLE 24







Target-specific primers and probes for use in real-time PCR











Target

Sequence

SEQ ID


Name
Species
Description
Sequence (5′ to 3′)
NO





ApoB
Mouse
Forward
CGTGGGCTCCAGCATTCTA
1524




Primer




ApoB
Mouse
Reverse
AGTCATTTCTGCCTTTGCGTC
1525




Primer




ApoB
Mouse
Probe
CCAATGGTCGGGCACTGCTCAA
1526


 


ApoB
Mouse
Forward
GAAAATAGACTTCCTGAATAACTATGCATT
1527




Primer




ApoB
Mouse
Reverse
ACTCGCTTGCCAGCTTGC
1528




Primer




ApoB
Mouse
Probe
TTTCTGAGTCCCCGTGCCCAACA
1529


 


GCGR
Mouse
Forward
TGAGCCTTGCCACCTTCTCT
1530




Primer




GCGR
Mouse
Reverse
GCGCACCCCAGCCAA
1531




Primer




GCGR
Mouse
Probe
AGAGGAGCTTCTTTTCCCTCTACCTGGGC
1532


 


GCGR
Mouse
Forward
ATTTCCTGCCCCTGGTACCT
1533




Primer




GCGR
Mouse
Reverse
CGGGCCCACACCTCTTG
1534




Primer




GCGR
Mouse
Probe
CCACAAAGTGCAGCACCGCCTAGTGT
1535


 


PTEN
Mouse
Forward
GCCACAGGCTCCCAGACAT
1536




Primer




PTEN
Mouse
Reverse
TCCATCCTCTTGATATCTCCTTTTG
1537




Primer




PTEN
Mouse
Probe
ACAGCCATCATCAAAGAGATCGTTAGCAGAA
1538


 


PTEN
Mouse
Forward
ATGACAATCATGTTGCAGCAATTC
1539




Primer




PTEN
Mouse
Reverse
CGATGCAATAAATATGCACAAATCA
1540




Primer




PTEN
Mouse
Probe
CTGTAAAGCTGGAAAGGGACGGACTGGT
1541









Example 3
Short Antisense Compounds Targeted to an ApoB Nucleic Acid and Having 2′-MOE or methyleneoxy (4′-CH2—O-2′) BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected intraperitoneally (i.p.) with antisense compounds targeted to ApoB, at a frequency of twice per week for three weeks. Antisense compound doses included 2.4, 1.2, 0.6, 0.3 and 0.15 μmol/kg. For antisense compounds 14 nucleotides in length, these doses equate to approximately 12, 6, 3, 1.5 or 0.75 mg/kg, respectively. Shown in Table 25 are the sequences and motifs of the antisense compounds used in this study. The antisense compounds are either 20 or 14 nucleotides in length and have a central “gap” region consisting of ten 2′-deoxynucleotides flanked by wings having 2′-O-methoxyethyl (2′-MOE) or BNA modified “wings.” For example, the 2-10-2 MOE gapmer motif indicates an antisense compound with a gap of ten nucleotides flanked by 2 nucleotide wings with 2′-MOE modifications. Bolded residues indicate 2′-O-methoxyethyl moieties and italicized residues indicate methyleneoxy (4′-CH2—O-2′) BNAs. The internucleoside linkages of each compound are phosphorothioate throughout. All cytosine residues of ISIS 147764 and ISIS 372938 are replaced by 5-methyl cytosines. For ISIS 387462, only the cytosine residue in the wing of the compound is replaced by 5-methyl cytosine. ApoB antisense compounds are targeted to publicly available ApoB-100 sequences, including Genbank Accession No. XM_137955.5 (SEQ ID NO: 2).







TABLE 25







Antisense Compounds Targeted to an ApoB nucleic acid













Target
5′





ISIS
SEQ
Target


SEQ


NO
ID NO
Site
Sequence (5′-3′)
Gapmer Motif
ID NO















147764
2
8865
GTCCCTGAAGATGTCAATGC
5-10-5 MOE
1561


 


372938
2
8235
GGTACATGGAAGTC
2-10-2 MOE
190


 


387462
2
8235
GGTACATGGAAGTC
2-10-2
190






methyleneoxy (4′-







CH2—O-2′) BNA









Forty-eight hours following the final injection, mice were sacrificed to evaluate transaminases (Table 26); liver and kidney weight (Table 27); triglyceride, LDL, HDL and free fatty acid levels (Table 28); target mRNA level in liver (Table 29); target protein level in plasma; and oligonucleotide tissue concentration (Table 30). These endpoints were determined using methods described herein and well known to those of ordinary skill in the art.







TABLE 26







ALT and AST Levels (IU/L)













Dose





ISIS NO
μmol/kg
ALT
AST







Saline
N/A
27.8
46.3



147764
2.4
29.5
64.0



372938
2.4
26.0
49.0



372938
1.2
24.8
49.5



372938
0.6
28.0
79.3



372938
0.3
28.3
60.0



372938
0.15
28.3
50.3



387462
2.4
41.3
84.0



387462
1.2
35.3
63.5



387462
0.6
32.0
77.3



387462
0.3
27.8
55.0



387462
0.15
29.3
68.3

















TABLE 27







Liver and Kidney Weight (% of saline control)













Dose





ISIS NO
μmol/kg
Liver
Kidney
















Saline
N/A
100
100



147764
2.4
102
105



372938
2.4
100
100



372938
1.2
90
101



372938
0.6
96
112



372938
0.3
91
107



372938
0.15
96
98



387462
2.4
116
90



387462
1.2
113
90



387462
0.6
106
97



387462
0.3
101
126



387462
0.15
95
100










Total body weight and food consumption did not differ significantly between saline-treated or oligonucleotide-treated animals. Glucose levels also were similar among all treatment groups.







TABLE 28







Triglyceride (TRIG), Total Cholesterol (CHOL), 


HDL, LDL and Free Fatty Acid (FFA) Levels














Dose
TRIG
CHOL
HDL
LDL
FFA


ISIS NO
μmol/kg
(mg/dL)
(mg/dL)
(mg/dL) 
(mg/dL)
(mg/dL)
















Saline
N/A
167
107
81.8
11.0
1.76


147764
2.4
167
107
81.3
10.3
1.29


372938
2.4
153
104
79.0
10.3
1.28


372938
1.2
136
101
77.8
9.5
1.70


372938
0.6
184
110
83.3
10.8
1.66


372938
0.3
138
109
84.3
11.0
1.53


372938
0.15
151
106
82.8
10.8
1.57


387462
2.4
49
14
9.0
1.5
0.74


387462
1.2
71
23
16.5
2.0
0.76


387462
0.6
150
55
39.3
3.7
1.43


387462
0.3
136
92
72.8 
7.5
1.14


387462
0.15
163
104
81.5
9.3
1.47
















TABLE 29







% ApoB mRNA Level (relative to saline control)












ISIS
2.4 μmol/
1.2 μmol/
0.6 μmol/




NO
kg
kg
kg
0.3 μmol/kg
0.15 μmol/kg















147764
57.7
ND
ND
ND
ND


372938
77.0
90.0
87.3
92.6
93.1


387462
1.5
8.5
27.4
58.9
75.8









Treatment with ISIS 387462 resulted in a significant and dose-dependent decrease in triglycerides, total cholesterol, HDL, LDL and free fatty acids. In accordance with these phenotypic findings, treatment with ISIS 387462 also led to a dose-dependent reduction in ApoB mRNA (Table 29) and protein (not shown) levels in mouse plasma. To determine whether the observed increase in efficiency with the methyleneoxy (4′-CH2—O-2′) BNA gapmer is due to an increase in oligonucleotide accumulation, full-length and total oligonucleotide concentration in the liver and kidney were determined







TABLE 30







Full-length and Total Antisense Compound Tissue Concentration 


(μM) Relative to ApoB mRNA level (% of saline control)















Kidney







Dose
Full-
Liver
Kidney
Liver
ApoB


ISIS NO
μmol/kg
Length
Full-Length
Total
Total
mRNA
















147764
2.4
28.6
22.9
33.5
31.3
58


372938
2.4
32.0
5.49
34.0
7.76
77


387462
2.4
37.2
5.69
38.9
7.31
1.5


387462
1.2
29.8
3.71
31.3
4.91
8.5


387462
0.6
18.9
1.97
20.0
2.57
27


387462
0.3
9.11
0.73
9.49
0.78
59


387462
0.15
6.97
0.19
7.43
0.24
76









Levels of the 2-10-2 methyleneoxy (4′-CH2—O-2′) BNA gapmer were similar to the 5-10-5 and 2-10-2 MOE gapmers in the kidney, but significantly reduced in the liver. The EC50 for ISIS 387462 in the liver was determined by comparing oligonucleotide concentration in the liver to inhibition of ApoB mRNA. The approximate EC50 for ISIS 387462 is 1 μM. In contrast, an effective 5-10-5 MOE gapmer compound typically has an EC50 of approximately 15 μM in the liver.
Taken together, these results demonstrate that the ApoB short gapmer having methyleneoxy (4′-CH2—O-2′) in the wings is a potent inhibitor of target mRNA expression and can effectively lower triglycerides, cholesterol and free fatty acids. The potency of the short antisense compound does not appear to be a result of increased tissue accumulation since similar levels of the compound were observed in kidney and reduced levels were found in the liver, relative to the 5-10-5 MOE gapmer. In addition, the methyleneoxy (4′-CH2—O-2′) BNA gapmer exhibited little to no adverse side effects.
Example 4
Short Antisense Compounds Targeted to a GCGR Nucleic Acid and Having 2′-MOE Modifications
Eight-week old male C57/BL6 mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single dose of GCGR oligonucleotide by intraperitoneal injection at a concentration of 6.25, 12.5, 25 or 50 mg. Each dose group consisted of four animals. Shown in Table 31 are the sequences, motifs and conjugates of the GCGR antisense compounds used in this study. Bolded residues indicate 2′-O-methoxyethyl (2′-MOE) moieties. All compounds comprise phosphorothioate internucleoside linkages throughout and each cytosine is replaced with 5-methylcytosine. ISIS 386626, ISIS 386627 and ISIS 386628 further comprise a C16 conjugate group attached to the 2′-O position of the sugar via a diamide linkage (2′-OCH2C(═O)N(H)(CH2)4N(H)C(═O)—(CH2)15CH3). GCGR antisense compounds target published GCGR sequences, including Genbank® Accession No. BC031885.1 (SEQ ID NO: 7).







TABLE 31







Short antisense compounds targeted to a GCGR nucleic acid














Target
5′



SEQ


ISIS
SEQ
Target

Gapmer

ID


NO
ID NO
Site
Sequence (5′-3′)
Motif
Conjugate
NO
















148364
7
393
TGCACTTTGTGGTACCAAGG
5-10-5 MOE
None
1562


 


386626
7
1768
GC16CTTCTCCATCATA
2-10-2 MOE
C16
1563


 


386627
7
1244
GC16GGCATGCTCGTCA
2-10-2 MOE
C16
653


 


386593
7
1244
GGGCATGCTCGTCA
2-10-2 MOE
None
649


 


386628
7
1680
TC16GTCTTGCTGCTTT
2-10-2 MOE
C16
1564


 


386594
7
1680
TGTCTTGCTGCTTT
2-10-2 MOE
None
1565









Mice were sacrificed 48 hours following injection to determine serum transaminase levels (Table 32); liver, white adipose tissue (WAT), spleen and kidney weight (Table 33); cholesterol, triglyceride and glucose levels (Table 34); GCGR mRNA levels (Tables 35-41); and full-length and total oligonucleotide concentration in liver and kidney (Table 42). Endpoints were assessed using methods described herein and well known to those of ordinary skill in the art. Data is included from a pre-treatment bleed (Pre-Bleed) and post-treatment bleed (Post-Bleed).







TABLE 32







ALT & AST Levels (IU/L)













Dose
ALT
ALT
AST
AST


ISIS NO
(mg/kg)
Pre-Bleed
Post-Bleed
Pre-Bleed
Post-Bleed















Saline
N/A
36
51
55
85


148364
50
24
40
40
115


148364
25
26
35
42
87


148364
12.5
23
32
44
69


148364
6.25
28
34
47
76


386626
50
28
40
48
120


386626
25
30
36
44
92


386626
12.5
28
34
44
90


386626
6.25
26
42
46
69


386627
50
27
457
42
451


386627
25
29
97
45
142


386627
12.5
29
62
46
81


386627
6.25
23
87
38
96


386593
50
23
33
46
58


386593
25
25
32
41
95


386593
12.5
26
33
43
74


386593
6.25
28
31
43
53


386628
50
28
68
44
76


386628
25
24
32
40
57


386628
12.5
28
35
42
75


386628
6.25
22
29
40
59


386594
50
29
34
46
92


386594
25
27
31
47
82


386594
12.5
28
33
45
74


386594
6.25
23
48
42
67
















TABLE 33







Organ Weights (% saline control)












ISIS NO
Dose (mg/kg)
Liver
WAT
Kidney
Spleen















Saline
N/A
100
100
100
100


148364
50
103
80
108
123


148364
25
103
75
112
115


148364
12.5
100
84
108
96


148364
6.25
101
89
104
113


386626
50
112
77
104
130


386626
25
109
97
103
120


386626
12.5
96
73
97
114


386626
6.25
100
90
100
95


386627
50
90
113
102
165


386627
25
99
87
99
143


386627
12.5
109
93
102
136


386627
6.25
103
96
102
131


386593
50
96
98
102
118


386593
25
83
94
100
104


386593
12.5
99
82
101
129


386593
6.25
96
77
98
144


386628
50
104
100
99
126


386628
25
102
97
109
113


386628
12.5
101
111
99
114


386628
6.25
98
106
102
151


386594
50
90
80
99
131


386594
25
93
76
99
128


386594
12.5
94
98
100
113


386594
6.25
102
85
101
119









Overall, the GCGR antisense compounds exhibited little to no adverse side effects.







TABLE 34







Triglyceride (TRIG), Cholesterol (CHOL) and Glucose Levels (IU/L)
















TRIG
TRIG
CHOL
CHOL
Glucose
Glucose


ISIS NO
Dose (mg/kg)
Pre-Bleed
Post-Bleed
Pre-Bleed
Post-Bleed
Pre-Bleed
Post-Bleed

















Saline
N/A
132
181
91
96
208
285


148364
50
110
177
81
94
207
228


148364
25
115
200
83
96
219
239


148364
12.5
106
179
85
89
198
256


148364
6.25
86
162
86
89
226
215


386626
50
87
163
79
57
239
179


386626
25
100
187
87
72
235
186


386626
12.5
100
148
82
76
232
185


386626
6.25
86
162
85
90
222
221


386627
50
106
120
83
126
227
150


386627
25
101
148
90
115
218
203


386627
12.5
99
203
86
98
237
219


386627
6.25
111
165
88
104
238
228


386593
50
130
128
100
95
244
213


386593
25
119
135
83
77
206
208


386593
12.5
122
128
83
79
222
233


386593
6.25
120
138
84
78
214
219


386628
50
102
98
88
95
209
232


386628
25
102
129
84
85
210
223


386628
12.5
90
123
90
94
231
240


386628
6.25
117
121
83
85
228
229


386594
50
93
99
84
85
203
274


386594
25
106
94
90
86
219
272


386594
12.5
118
133
85
95
200
292


386594
6.25
112
146
78
94
222
275









GCGR 2-10-2 MOE gapmers exhibited a trend toward lower post-bleed triglyceride levels, relative to the 5-10-5 MOE gapmer, with ISIS 386628 and ISIS 386594 having the greatest dose-dependent effect. Glucose levels also were decreased in a dose-dependent manner following treatment with ISIS 386626 and ISIS 386627. Treatment with ISIS 386628, ISIS 386593 and ISIS 386594 also generally led to a decrease in post-bleed glucose levels. Cholesterol levels did not appear to significantly differ among treatment groups.
To determine whether the phenotypic changes shown above correlated with a decrease in GCGR mRNA, treated animals were evaluated for levels of target mRNA in liver by real time PCR according to methods described herein. Tables 35 to 41 show results from direct comparisons of the antisense compounds targeting GCGR nucleic acid for their effect on target expression. Results are expressed as percent of saline control.







TABLE 35







GCGR mRNA levels following treatment with


ISIS 148364 & ISIS 386626











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














148364
36
79
87
62


386626
0
8
3
7
















TABLE 36







GCGR mRNA levels following treatment with


ISIS 148364 & ISIS 386627











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














148364
63
87
105
86


386627
3
30
57
74
















TABLE 37







GCGR mRNA levels following treatment with


ISIS 148364 & ISIS 386593











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














148364
56
74
105
86


386593
9
38
74
90
















TABLE 38







GCGR mRNA levels following treatment with


ISIS 148364 & ISIS 386628











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














148364
42
77
98
101


386628
2
18
53
77
















TABLE 39







GCGR mRNA levels following treatment with


ISIS 148364 & ISIS 386594











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














148364
59
98
102
96


386594
25
47
50
96
















TABLE 40







GCGR mRNA levels following treatment with


ISIS 386627 & ISIS 386593











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














386627
5
40
58
42


386593
10
29
34
71
















TABLE 41







GCGR mRNA levels following treatment with


ISIS 386628 & ISIS 386594











ISIS NO
50 mg/kg
25 mg/kg
12.5 mg/kg
6.25 mg/kg














386628
4
13
38
97


386594
19
50
56
99









Treatment with the 2-10-2 MOE gapmers led to a significant dose-dependent decrease in GCGR mRNA expression. ISIS 386626 exhibited the greatest decrease in target mRNA. To determine whether the observed increase in efficiency with the short antisense compounds is due to an increase in antisense compound accumulation, full-length and total antisense compound concentration in the liver and kidney were determined







TABLE 42







Total and Full-length Antisense Compound Concentrations


in Liver and Kidney (μg/g)















Full-



Total
Total
Full-length
length


ISIS NO
Kidney
Liver
Kidney
Liver














148364
90
54
58
46


386626
757
274
355
125


386593
91
12
77
12


386628
496
286
305
202









The results shown in Table 42 demonstrate that short antisense compounds comprising a C16 conjugate exhibit a significant increase in antisense compound accumulation in both liver and kidney. However, ISIS 386593, which was effective at reducing target mRNA, triglycerides and glucose levels, accumulates to a level similar to the 5-10-5 MOE gapmer in liver and to a lower level in kidney. These results suggest that while conjugation with C16 can increase liver and kidney antisense compound concentration, it does not entirely account for the effectiveness of the short antisense compounds.
Taken together, these results demonstrate that GCGR short antisense compounds are capable of significantly inhibiting target mRNA expression while also lowering triglyceride and glucose levels. In addition, with the exception of ISIS 386627, the short MOE gapmers exhibited little to no toxic effects.
Example 5
Short Antisense Compounds Targeting to a GCGR Nucleic Acid and Having 2′-MOE and Methyleneoxy (4′-CH2—O-2′) BNA Modifications
Eight-week old male C57/BL6 mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single dose of GCGR antisense compound by intraperitonel (i.p.) injection at a concentration of 10, 3.2, 1, and 0.32 μmol·kg. Each dose group consisted of four animals. Shown in Table 43 are the sequences, motifs and conjugates of the GCGR antisense compounds used in this study. Bolded residues indicate 2′-O-methoxyethyl (2′-MOE) modifications and the italicized residues indicate methyleneoxy (4′-CH2—O-2′) BNA modifications. All antisense compounds comprise phosphorothioate internucleoside linkages throughout and each cytosine is replaced with 5-methylcytosine. GCGR antisense compounds target published GCGR nucleic acids, including Genbank Accession No. BC031885.1 (SEQ ID NO: 7).







TABLE 43







Antisense Compounds targeted to a GCGR nucleic acid













Target
5′





ISIS
SEQ ID
Target





NO
NO
Site
Sequence (5′-3′)
Gapmer Motif
SEQ ID NO















148364
7
393
TGCACTTTGTGGTACCAAGG
5-10-5 MOE
1562


 


396144
7
1768
GCTTCTCCATCATA
2-10-2 MOE
1566


 


396148
7
1768
GCTTCTCCATCATA
2-10-2
1567






Methyleneoxy







(4′-CH2—O-2′)







BNA



 


396145
7
1765
ATGGCTTCTCCATCATATCC
5-10-5 MOE
1568


 


396146
7
1244
GGGCATGCTCGTCA
2-10-2 MOE
650


 


396149
7
1244
GGGCATGCTCGTCA
2-10-2
652






Methyleneoxy







(4′-CH2—O-2′)







BNA



 


396147
7
1241
CTTGGGCATGCTCGTCAGTC
5-10-5 MOE
1569









To determine whether the phenotypic changes shown above correlated with a decrease in GCGR mRNA, treated animals were evaluated for levels of target mRNA in liver by RT, real time PCR according to methods described herein. Table 44 show results from direct comparisons of the antisense compounds targeting GCGR nucleic acid for their effect on target expression. Results are expressed as percent of saline control.







TABLE 44







GCGR mRNA levels











ISIS NO.
0.32 μmol/kg
1 μmol/kg
3.2 μmol/kg
10 μmol/kg














148364
105 
106
73
38


396144
122 
117
40
35


396148
20
 6
2
1


396145
nd
Nd
33
8


396146
98
135
95
35


396149
91
 41
30
7


396147
nd
Nd
68
28









As shown in Table 44, each short antisense compound having methyleneoxy (4′-CH2—O-2′) BNA modifications demonstrated a dose-dependent reduction in GCGR mRNA levels. Furthermore, the short antisense compounds were more effective at target reduction than the 5-10-5 MOE gapmer. Each short antisense compound comprising methyleneoxy (4′-CH2—O-2′) BNA in the wings resulted in a significant reduction in GCGR protein relative to both saline control and ISIS 148364 treatment. Next, estimated ED50 concentrations for each antisense were calculated using Graphpad Prism; ED50 is the dose at which 50% mRNA reduction is observed. The results are shown below in Table 45.







TABLE 45







Estimated ED50 Concentration











ISIS




Gapmer Motif
NO
ED50 (μmole/kg)
ED50 (mg/kg)













5-10-5 MOE
148364
7
50.6


2-10-2 MOE
396144
4
18.1


2-10-2 methyleneoxy BNA
396148
0.1
0.4


5-10-5 MOE
396145
2.1
9.3


2-10-2 MOE
396146
8.3
40


2-10-2 methylenexy BNA
396149
1.1
5


5-10-5 MOE
396147
5.2
37.5









Example 6
Short Antisense Compounds Targeting a PTEN Nucleic Acid and Having Methyleneoxy (4′-CH2—O-2′) BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single i.p. injection of PTEN antisense compound at a dose of 8 μmol/kg. Each dose group consisted of four animals. Shown in Table 46 are the sequences and motifs of the PTEN antisense compounds used in this study. Bolded residues indicate 2′-O-methoxyethyl moieties (2′-MOE) and italicized residues indicate Methyleneoxy BNA nucleotides. Each antisense compound comprises phosphorothioate linkages throughout. In addition, the cytosine residues in the gap of ISIS 384073 and in the wings of ISIS 392056, ISIS 392057, ISIS 392061 and ISIS 392063 are replaced with 5-methylcytosines. Antisense compounds target published PTEN nucleic acids, including Genbank Accession No. U92437.1 (SEQ ID NO: 13).







TABLE 46







Antisense Compounds targeted to a PTEN nucleic acid













Target
5′





ISIS
SEQ ID
Target


SEQ


NO
NO
Site
Sequence (5′-3′)
Gapmer Motif
ID NO





141923
Control
N/A
CCTTCCCTGAAGGTTCCTCC
5-10-5 MOE
1570


 


116847
29
2011
TCAAATCCAGAGGCTAGCAG
5-10-5 MOE
1571


 


384073
29
2013
AAATCCAGAGGCTAGC
3-10-3 methyleneoxy
1428






(4′-CH2—O-2′) BNA



 


391172
29
2013
AAATCCAGAGGCTAG
2-10-3 methyleneoxy
1429






(4′-CH2—O-2′) BNA



 


392056
29
140
AGCTGCAGCCATGA
2-10-2 methyleneoxy
1263






(4′-CH2—O-2′) BNA



 


392057
29
807
GGTCCAGGGCCAAG
2-10-2 methyleneoxy
1162






(4′-CH2—O-2′) BNA



 


392061
29
2014
AATCCAGAGGCTAG
2-10-2 methyleneoxy
1431






(4′-CH2—O-2′) BNA



 


392063
29
3099
AGGCCAGTGCTAAG
2-10-2 methyleneoxy
1226






(4′-CH2—O-2′) BNA









Mice were sacrificed 72 hours following injection to determine serum transaminase levels (Table 47); liver and spleen weights (Table 47); and PTEN mRNA levels in liver, kidney and fat (Table 48), according to procedures described herein and well know to one of ordinary skill in the art.







TABLE 47







Transaminase Levels and Organ Weights














Liver



ISIS
AST
ALT
Weight
Spleen Weight


NO
(IU/L)
(IU/L)
% Saline
% Saline














Saline
98.5
37.5
100
100


141923
89.5
34.8
101
108


116847
59.8
29.5
109
108


384073
57.8
29.3
115
111


391172
48.5
32.8
120
112


392056
516
892
125
167


392057
63.8
34.5
125
101


392061
189
42.0
123
111


392063
67.3
21.8
127
134









Overall, the short antisense compounds with methyleneoxy (4′-CH2—O-2′) BNA modifications exhibited little to no adverse effects. In addition, total body weight did not significantly differ between treatment groups.







TABLE 48







% PTEN mRNA levels in Liver, Kidney and Fat












ISIS NO
Liver
Kidney
Fat
















Saline
100
100
100



141923
102
133
118



116847
37
96
85



384073
24
74
77



391172
18
63
101



392056
27
88
74



392057
33
79
96



392061
24
61
85



392063
6.5
52
72










As shown in Table 48, each antisense compound targeted to a PTEN nucleic acid led to a significant reduction in target mRNA levels in liver as compared with saline treated and control treated animals. The antisense compounds had various effects on target mRNA levels in kidney and fat.
Example 7
Short Antisense Compounds Targeting a PTEN Nucleic Acid and Having BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal (i.p.) injection of antisense compound targeted a PTEN nucleic acid at a dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted of four animals. Shown in Table 49 are the sequence, wing chemistry and motif of each antisense compound used in this study. Bold residues indicate 2′-MOE modified nucleotides, italicized letters indicate methyleneoxy (4′-CH2—O-2′) BNA modifications. All antisense compounds comprise phosphorothioate linkages at each position. Each cytosine of ISIS 116847 and the cytosine residues in the methyleneoxy (4′-CH2—O-2′) BNA wings of ISIS 392063 are replaced with 5-methylcytosines, while the thymidine residues in the methyleneoxy (4′-CH2—O-2′) BNA wings of ISIS 392745 are replaced with 5-methyl thymidines. Antisense compounds target published PTEN nucleic acids, including Genbank Accession No. U92437.1 (SEQ ID NO: 13).







TABLE 49







Antisense Compounds Targeted to a PTEN Nucleic Acid













Target



SEQ


ISIS
SEQ
Target


ID


NO
ID NO
Site
Sequence (5′-3′)
Gapmer Motif
NO





116847
13
2011
TCAAATCCAGAGGCTAGCAG
5-10-5
1571






MOE



 


392063
13
3099
CTTAGCACTGGCCT
2-10-2
1226






Methyleneoxy BNA



 


392745
13
3099
CTTAGCACTGGCCT
2-10-2 methyleneoxy
1226






BNA









Mice were sacrificed 72 hours following injection to determine serum transaminase levels (Table 50); liver, kidney and spleen weights (Table 50); PTEN mRNA levels in liver (Table 51); and estimated ED50 oligonucleotide concentration (Table 52). These endpoints were measured using methods described herein and well known to those of ordinary skill in the art.







TABLE 50







AST, ALT and Bilirubin Levels and Organ Weights



















Liver









Weight
Kidney
Spleen


ISIS
Dose
AST
ALT
Bilirubin
%
Weight
Weight


NO
μmol/kg
(IU/L)
(IU/L)
(mg/dL)
Saline
% Saline
% Saline

















Saline
N/A
64.0
31.8
0.15
100
100
100


116847
8
73.0
32.0
0.1
114
92
106


392063
8
50.3
17.3
0.1
115
98
115


392063
4
100.8
31.3
0.15
122
94
116


392063
2
60.5
32.8
0.1
112
99
106


392063
1
57.5
29.3
0.1
104
95
107


392745
8
75.5
23.5
0.13
125
99
100


392745
4
77.0
29.3
0.13
121
100
96


392745
2
69.0
32.0
0.13
110
98
103


392745
1
52.0
27.3
0.1
109
97
104









Overall, the PTEN antisense compounds did not show significant signs of toxicity. Kidney, liver and spleen weights were all within normal ranges. Total body weight did not significantly differ between treatment groups.







TABLE 51







% PTEN mRNA levels in Liver (relative to saline control)











ISIS NO
8 μmol/kg
4 μmol/kg
2 μmol/kg
1 μmol/kg














116847
36
ND
ND
ND


392063
7.4
16
32
60


392745
5.2
11
31
60









As shown in Table 51, each short antisense compound having methyleneoxy (4′-CH2—O-2′) BNA modifications demonstrated a dose-dependent reduction in PTEN mRNA levels. Furthermore, the short antisense compounds were more effective at target reduction than the 5-10-5 MOE gapmer. Levels of PTEN protein in liver were also determined following administration of each antisense compound at a dose of 8 μmol/kg. Each short antisense compound comprising methyleneoxy (4′-CH2—O-2′) BNA in the wings resulted in a significant reduction in PTEN protein relative to both saline control and ISIS 116847 treatment. Next, estimated ED50 concentrations for each oligonucleotide were calculated using Graphpad Prism. The results are shown below in Table 52.







TABLE 52







Estimated ED50 Concentration










Wing Chemistry
ISIS NO
ED50 (μmole/kg)
ED50 (mg/kg)













MOE (with 5-MeC)
116847
6.3
45.2


methyleneoxy BNA
392063
1.3
5.8


(with 5-MeC)


methyleneoxy BNA
392745
1.2
5.6









To further investigate different types of bicyclic nucleic acid compounds, an additional set of short antisense compounds targeting a PTEN nucleic acid was designed and tested. Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal (i.p.) injection of antisense compound at a dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted of four animals. Shown in Table 53 are the sequence, wing chemistry and motif of each antisense compound used in this study. All antisense compounds comprise phosphorothioate linkages at each position. The cytosine residues in the methyleneoxy (4′-CH2—O-2′) BNA wings of ISIS 392063 are replaced with 5-methylcytosines. The antisense compound target published PTEN nucleic acids, including Genbank Accession No. U92437.1 (SEQ ID NO: 13).







TABLE 53







Antisense Compounds Targeting a PTEN Nucleic Acid













Target
5′





ISIS
SEQ
Target


SEQ ID


NO
ID NO
Site
Sequence (5′-3′)
Gapmer Motif
NO





392063
29
3099
CTTAGCACTGGCCT
2-10-2
1226






Methyleneoxy BNA



 


396564
29
3099
CTTAGCACTGGCCT
2-10-2
1226






Oxyamino







(4′-CH2—N(R)—O-2′)







BNA



 


396006
29
3099
CTTAGCACTGGCCT
2-10-2α-L-
1226






Methyleneoxy







BNA









Mice were sacrificed 72 hours following injection to determine serum transaminase levels (Table 54); liver and spleen weights (Table 54); and PTEN mRNA levels in liver (Table 55), according to methods described herein and well known to those of ordinary skill in the art.







TABLE 54







AST and ALT Levels and Organ Weights












ISIS
Dose
AST
ALT
Liver
Spleen


NO
μmol/kg
(IU/L)
(IU/L)
Weight
Weight















Saline
N/A
71
33
100
100


392063
8
97
38
118
103


392063
4
179
36
115
107


392063
2
67
32
109
116


392063
1
68
27
102
105


396564
8
67
25
100
104


396564
4
96
30
102
106


396564
2
68
27
100
119


396564
1
79
39
97
109


396006
8
56
28
110
104


396006
2
139
36
97
105
















TABLE 55







% PTEN mRNA levels in Liver (relative to saline control)











ISIS NO
8 μmol/kg
4 μmol/kg
2 μmol/kg
1 μmol/kg














392063
6.9
18
39
71


396564
86
97
100
96


396006
6.5
ND
ND
70









As shown above, short antisense compounds having α-L-methyleneoxy (4′-CH2—O-2′) BNA modifications led to a dose-dependent reduction in target mRNA levels. Treatment with the short antisense compound having oxyamino BNA modifications led to a modest reduction in target expression.
Example 8
Single Dose Administration Dose Response Study with Short Antisense Compounds Targeting ApoB and PTEN Nucleic Acids
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal (i.p.) injection of antisense compound at a dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted of four animals. Shown in Table 56 are the sequence, wing chemistry and motif of each antisense compound used in this study. Italicized residues indicate methyleneoxy (4′-CH2—O-2′) BNA modifications, underlined residues indicate N-methyl-oxyamino (4′-CH2—N(CH3)—O-2′) BNA modifications, and boxed residues indicate α-L-methyleneoxy (4′-CH2—O-2′) BNA modifications. All antisense compounds comprise phosphorothioate linkages at each position. Each cytosine of ISIS 116847 and the cytosine residues in the methyleneoxy (4′-CH2—O-2′) BNA wings of ISIS 392063 are replaced with 5-methylcytosines, while the thymidine residues in the methyleneoxy (4′-CH2—O-2′) BNA wings of ISIS 392745 are replaced with 5-methyl thymidines. PTEN antisense compounds target published PTEN nucleic acid, including Genbank Accession No. U92437.1 (SEQ ID NO: 13). ApoB antisense compounds target published ApoB nucleic acid, including Genbank Accession No. XM_137955.5 (SEQ ID NO: 2).







TABLE 56







Short Antisense Compounds Targeted to ApoB and PTEN Nucleic Acids
















5′ 


SEQ


ISIS

Target
Target


ID


NO
Target
Seq ID
Site
SEQUENCE
Gapmer
NO





387462
ApoB
19
8235
GGTACATGGAAGTC
2-10-2
 193







Methyleneoxy BNA



 


392063
PTEN
29
3099
CTTAGCACTGGCCT
2-10-2
1226







Methyleneoxy BNA



 


396565
PTEN
29
3099
CUTAGCACTGGCCU
2-10-2
1226







N-Me-oxyamino BNA



 


396006
PTEN
29
3099




2-10-2 α-L-methyleneoxy BNA
1226
















TABLE 57







% ApoB and PTEN mRNA Reduction (relative to saline control)













% PTEN


ISIS
Dose
% ApoB mRNA Reduction
mRNA Reduction


NO
(μmol/kg)
(relative to saline)
(relative to saline)













387462
8
0.62
92.8



4
6.55
103



2
18.6
105



1
42.0
98.0


392063
8
126
6.79



4
111
18.1



2
112
42.4



1
114
62.3


396565
8
116
23.8



4
1.04
46.6



2
94.4
76.1



1
115
89.5


396006
8
94.3
62.9



4
101
18.2



2
79.7
52.4



1
111
82.4









As shown in Table 57, each short antisense compound having Methyleneoxy BNA modifications demonstrated a dose-dependent reduction in target mRNA levels. Notably, the short antisense compound with N-methyl-oxyamino BNA wings (ISIS 396565) also demonstrated dose-dependent reduction in PTEN expression similar to both the β-D-methyleneoxy BNA and α-L-methyleneoxy BNA short antisense compounds. Next, estimated ED50 concentrations for each antisense were calculated using Graphpad Prism. The results are shown below in Table 58.







TABLE 58







Estimated ED50 Concentrations












ED50
ED50


Wing Chemistry
ISIS NO
(μmole/kg)
(mg/kg)













Methyleneoxy BNA
387462
0.8
3.9


Methyleneoxy BNA
392063
1.5
7


N-Me-oxyamino BNA
396565
3.8
17.4


α-L-methyleneoxy BNA
396006
2.1
9.3









Example 9
Administration of a Parent and Parent Mixed Backbone Antisense Compound Targeting SGLT-2 mRNA
ISIS 257016 was administered to db/db mice (Charles River Laboratories, Wilmington, Mass.) intraperitoneally at a dose of 1, 7.5, 14 or 17 mg/kg twice a week. Control groups included a group receiving saline on the same dosing schedule and a group receiving ISIS 145733. ISIS 257016 and ISIS 145733 both comprise the sequence GAAGTAGCCACCAACTGTGC (SEQ ID NO: 1572) further comprising a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The internucleoside (backbone) linkages are phosphorothioate (P=5) throughout the oligonucleotide for ISIS 145733; however ISIS 257016 has a mixed backbone. The internucleoside linkages for ISIS 257016 are phosphodiester (P═O) in the wings and phosphorothioate in the gap. Forty-eight hours following administration of the last dose the mice were sacrificed and kidney tissue was analyzed for SGLT-2 mRNA levels. The results are shown below in Table 59.







TABLE 59







Antisense inhibition of SGLT2 mRNA expression


in vivo by 5-10-5 MOE gapmers










% change in SGLT2 expression



Dose of
relative to saline









oligonucleotide
ISIS
ISIS


nmol/kg
145733
257016












17
−37.5
−76


14
−31.25
−74


7.5
−12.5
−62.5


1
+3
−44









Both ISIS 257016 and ISIS 145733 markedly reduced SGLT-2 levels compared to saline control. (mRNA levels determined using RT, real-time PCR as described above) However, ISIS 257016 has been shown to be about 20-50 times more potent for reducing SGLT-2 mRNA compared to ISIS 145733. An associated reduction in plasma glucose levels was seen for the treatment groups (661±14 for the saline group compared to 470±23 for the group receiving ISIS 257016). Accumulation of ISIS 257016 and ISIS 145733 in the kidney was similar over the dose range, however little of the full length 257016 antisense was detected in the kidney which supports the theory that a degradation product is responsible for the increased activity. Also the onset of action following a single dose of 25 mg/kg correlated to a time pint were little intact 257016 antisense compound was left.
Similar studies were performed in lean mice, ob/ob mice and in ZDF rats (Charles Rivers Laboratories) using ISIS 257016, ISIS 145733 or saline in a similar same dosing schedule as described above. The sequence of the binding site for ISIS 145733 and ISIS 257016 is conserved between mouse and rat (see Table 60). Reduction of SGLT-2 mRNA in the kidney was similar to that seen above. In a study utilizing rats, at a dose of 10 mg/kg given two times a week for two weeks, ISIS 145733 was shown to reduce SGLT-2 mRNA levels by about 40% whereas the reduction achieved with ISIS 257016 was greater than 80%. ISIS 257016 reduces SGLT2 expression maximally at a low dose of 12.5 mg/kg. Additional studies at lower dosing ranges show significant reduction of SGLT2 mRNA levels with the mixed backbone antisense compound at doses less than 1 mg/kg/wk.
Example 10
Administration of a Parent and Short Antisense Compound Targeting SGLT-2 mRNA
Pharmacokinetic studies indicated that ISIS 257016 was acting as a prodrug that was metabolized to a 12 nucleobase pharmacophore. In a next study, ZDF rats were dosed intraperitoneally twice per week with 1.5 mg/kg of either ISIS 257016 or ISIS 370717, or with saline at a similar dosing schedule. ISIS 370717 is a 12 nucleobase antisense compound targeted to SGLT-2 nucleic acid comprising the sequence TAGCCACCAACT (SEQ ID NO: 154) and further comprising central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by one-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. All cytidine residues are 5-methylcytidines. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide.
Following five weeks of dosing the animals were sacrificed and kidney tissue was analyzed for SGLT-2 mRNA levels. The pharmacological activity of ISIS 257016 and ISIS 370717 were similar, however, the 12 nucleotide antisense compound displayed a faster onset of action. ISIS 370717 displayed nearly 80% inhibition of SGLT2 expression in kidney on day two after a single dose of 2.8 umoles/kg whereas ISIS 257016 displayed only about 25% inhibition on day 2 after the same single dose administration. The date support that ISIS 257016 is a prodrug having a 12 nucleotide pharmacophore.
Example 11
Potency and Bioavailability of a Short Antisense Compound
The improved potency displayed by ISIS 370717 and the improved oral bioavailability for these short antisense compounds makes these compounds useful for oral administration. Normal rats received ISIS 370717, ISIS 145733 or saline at 100 mg/kg twice per week via intrajejunal administration. About 48 hours following the last dose, the animals were sacrificed and kidney tissue was analyzed for antisense compound concentration and SGLT-2 mRNA levels. There was a significantly higher accumulation of ISIS 370717 in the kidney tissue (approximately 500 micro grams per gram of tissue) compared to the controls. Moreover, SGLT-2 mRNA was reduced by more than 80% over the controls.
Example 12
Wing, Gap and Total Length Variations Around a 12 Nucleotide Short Antisense Compound
ISIS 370717 1-10-1 MOE gapmer was used as a template to make sequence related oligos with varying motifs. These variations are provided in Table 60. The antisense compounds were designed to target different regions of the mouse or rat SGLT2 nucleic acid, using published sequences (GenBank accession number U29881.1, incorporated herein as SEQ ID NO: 1575, and GenBank accession number AJ292928.1, incorporated herein as SEQ ID NO: 1576, respectively).







TABLE 60







Short Antisense compounds targeting SGLT2 nucleic acids













5′ Target Site
5′ Target Site






on mouse
on rat


SEQ


ISIS
SEQ ID NO:
SEQ ID NO:
Gapmer

ID


NO
1575
1576
Motif
Sequence (5′-3′)
NO





257016
2680
148
5-10-5
GAAGTAGCCACCAACTGTGC
1553





MOE


 


370717
2684
152
1-10-1
TAGCCACCAACT
1554





MOE


 


386169
2684
152
2-8-2
TAGCCACCAACT
1555





MOE


 


386176
2685
153
1-8-1
AGCCACCAAC
1556





MOE


 


386196
2684
152
3-6-3
TAGCCACCAACT
1557





MOE









The antisense compounds were analyzed for their effect on mouse SGLT2 mRNA levels. Data are ranges taken from three experiments in which mice were dosed twice per week for three weeks with 2.5, 0.5 or 0.1 umol/kg of the above MOE gapmers given by intraperitoneal injection. Mice were sacrificed 48 hours following last administration and evaluated for SGLT2 levels in kidney. SGLT2 mRNA levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 61.







TABLE 61







Antisense inhibition of SGLT2 in vivo


by 1-10-1 and 1-10-2 MOE gapmers









% change in SGLT2 expression


Dose of
relative to saline












oligonucleotide
ISIS
ISIS
ISIS
ISIS
ISIS


umol/kg
370717
386169
386176
386196
386197















2.5
−82
−85
−80
−50
−20


0.5
−70
−80
−68
−30
−15


0.1
−55
−70
−65
−35
−20









These results illustrate that all the various motifs tested inhibit the expression of SGLT2 in vivo in a dose-dependent manner. The 1-10-1, 2-8-2 and 1-8-1 gapmers were found to be particularly potent.
Example 13
Antisense Inhibition of Rat SGLT-2 by 1-10-1 and 1-10-2 MOE Gapmers
1-10-1 and 1-10-2 MOE gapmer antisense compounds, provided in Table 62, were designed to target different regions of the mouse or rat SGLT2 RNA. All short antisense compounds in Table 62 are chimeric oligonucleotides (“gapmers”) either 12 or 13 nucleotides in length, composed of a central “gap” segment consisting of ten 2′-deoxynucleotides, which are flanked on the 5′ side by a one-nucleoside “wing” and on the 3′ side by a two or one-nucleotide “wing”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.







TABLE 62







Antisense compounds targeting SGLT2 nucleic acid













5′ Target Site
5′ Target Site






on SEQ ID
on SEQ ID



NO: 1576
NO: 1575
Gapmer

SEQ


ISIS NO
(mouse)
(rat)
Motif
Sequence (5′-3′)
ID NO















370717
2684
152
1-10-1 MOE
TAGCCACCAACT
1554


 


382675
2683
151
1-10-1 MOE
TAGCCACCAACTG
1559


 


379692

508
1-10-1 MOE
TGTTCCAGCCCA
246


 


382676

507
1-10-2 MOE
TGTTCCAGCCCAG
246


 


379699

1112
1-10-2 MOE
GGCATGAGCTTC
281


 


382677

1111
1-10-2 MOE
GGCATGAGCTTCA
281


 


382677

958
1-10-2 MOE
GGCATGAGCTTCA
281









The short antisense compounds were analyzed for their effect on rat SGLT2 mRNA levels. Data are ranges taken from three experiments in which Male Sprague-Dawley rats (170-200 g) were dosed twice per week for three weeks with 450, 150 or 50 nmol/kg of either a 1-10-1 or 1-10-2 MOE gapmer given by intraperitoneal injection. Rats were sacrificed 48 hours following last administration and evaluated for SGLT2 mRNA levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 63.







TABLE 63







Antisense inhibition of SGLT2 mRNA in 


vivo by 1-10-1 and 1-10-2 MOE gapmers









% change in SGLT2 expression



relative to saline













Dose of 
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


oligonucleotide
370717
382675
379692
382676
379699
382677


nmol/kg
1-10-1
1-10-2
1-10-1
1-10-2
1-10-1
1-10-2





450
−70
−80
−90
−85
−83
−75


150
−70
−65
−85
−80
−75
−60


 50
−55
−50
−80
−65
−60
−40









These results illustrate that both the 1-10-1 and 1-10-2 MOE gapmers reduce SGLT2 mRNA in vivo in a dose-dependent manner.
Rats were further evaluated for total body weight, liver, spleen and kidney weight. Significant changes in spleen, liver or body weight can indicate that a particular compound causes toxic effects. All changes were within the margin of error of the experiment. No significant changes in body weight were observed during the treatment or at study termination. No significant changes in liver or spleen weights were observed.
Toxic effects of short antisense compounds administered in vivo can also be assessed by measuring the levels of enzymes and proteins associated with disease or injury of the liver or kidney. Elevations in the levels of the serum transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are often indicators of liver disease or injury. Serum total bilirubin is an indicator of liver and biliary function, and albumin and blood urea nitrogen (BUN) are indicators of renal function. Glucose and triglyceride levels are sometimes altered due to toxicity of a treatment. Serum glucose also depends in part upon the activity of SGLT2. The levels of ALT, AST, total bilirubin, albumin, BUN, glucose and triglyceride were measured in rats treated with the short antisense compounds. The levels of routine clinical indicators of liver and kidney injury and disease were within normal ranges and are not significantly changed relative to saline-treated animals, demonstrating that the short antisense compounds do not significantly affect renal or hepatic function. Triglyceride and glucose levels were not significantly elevated relative to saline-treated animals.
Example 14
Antisense Inhibition of Mouse and Rat SGLT2 by 1-10-1 MOE Gapmers
1-10-1 MOE gapmer antisense compounds designed to target different regions of mouse SGLT2 mRNA are shown in Table 64.







TABLE 64







Composition of Antisense Compounds Targeting SGLT2 mRNA













5′ Target Site
5′ Target Site






on SEQ ID
on SEQ ID


ISIS
NO: 1576
NO: 1575


SEQ


NO
(mouse)
(rat)
Motif
Sequence (5′-3′)
ID NO















370717
2684  
152
1-10-1 MOE
TAGCCACCAACT
1554


 


379692

508
1-10-1 MOE
TGTTCCAGCCCA
246


 


379699

1112
1-10-1 MOE
GGCATGAGCTTC
281


 


379702

1525
1-10-1 MOE
GCACACAGCTGC
293


 


381408
3034**

1-10-1 MOE
TACCGAACACCT
1560





**indicates 3 mismatches to a target sequence






The short antisense compounds were analyzed for their effect on mouse SGLT2 mRNA levels. Data was taken from three experiments in which Male 6-week old Balb/c mice were dosed twice per week for two weeks with 450, 150, or 50 nmol/kg of one of the above 1-10-1 MOE gapmers given by intraperitoneal injection. Mice were sacrificed 48 hours following last administration and evaluated for SGLT2 mRNA levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 65.







TABLE 65







Antisense inhibition of SGLT2 mRNA


in vivo by 1-10-1 MOE gapmers









% change in SGLT2 expression


Dose of
relative to saline












oligonucleotide
ISIS
ISIS
ISIS
ISIS
ISIS


nmol/kg
370717
379692
379699
379702
381408















450
−65
−80
−80
−75
—


150
−55
−70
−62.5
−72.5
—


50
−47.5
−52.5
−42.5
−52.5
—









These results illustrate that all the 1-10-1 MOE gapmers except, ISIS 381408, inhibit the expression of SGLT2 in vivo in a dose-dependent manner in mouse. Activity of ISIS 381408 has been shown in Rat studies (See Table 65).
Evaluation of 1-10-1 Gapmers in Rat
The effect of the above 1-10-1 gapmers (see Table 64 above) on rat SGLT2 mRNA levels. Data are taken from four experiments in which male Sprague-Dawley rats (170-200 g) were dosed twice per week for three weeks with 250 nmol/kg given by intraperitoneal injection. Rats were sacrificed 48 hours following last administration and evaluated for SGLT2 mRNA levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 66.







TABLE 66







Antisense inhibition of SGLT2 mRNA


in vivo by 1-10-1 MOE gapmers









% change in SGLT2 expression


Dose of
relative to saline












oligonucleotide
ISIS
ISIS
ISIS
ISIS
ISIS


nmol/kg
370717
379692
379699
379702
381408















250
−70
−85
−75
−25
−5









These results illustrate that all the 1-10-1 MOE gapmers inhibit the expression of SGLT2 in in vivo rat studies.
Example 15
Antisense Inhibition of Mouse and Rat SGLT2 Expression by Additional 1-10-1 and 2-8-2 MOE Gapmers
1-10-1 and 2-8-2 MOE gapmer short antisense compounds were designed to target different regions of the mouse SGLT2 RNA but have complementarity across species. The short antisense compounds are shown in Table 67. All short antisense compounds in Table 67 are gapmers 12 nucleotides in length, composed of a central “gap” segment consisting of 2′-deoxynucleotides, which are flanked on both sides (5′ and 3′ directions) by wing segments having 2′-modifications. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.







TABLE 67







Short Antisense Compounds Targeting SGLT2 nucleic acid













5′
Target






Target
SEQ ID
Gapmer

SEQ ID


ISIS NO
Site (rat)
(rat)
Motif
Sequence (5′-3′)
NO















379692
508

1-10-1 MOE
TGTTCCAGCCCA
246


 


388625
508

1-10-1 MOE
TGTTCCAGCCCA
246


 


379699
1112

1-10-1 MOE
GGCATGAGCTTC
281


 


388626
1112

2-8-2 MOE
GGCATGAGCTTC
281


 


379702
1525

2-8-2 MOE
GCACACAGCTGC
293


 


388627
1525

2-8-2 MOE
GCACACAGCTGC
293









The short antisense compounds were analyzed for their effect on mouse SGLT2 mRNA levels in vivo. Data was taken from three experiments in which male 6-week old Balb/c mice were dosed twice per week for three weeks with 0.5, 0.1, or 0.02 umol/kg of either a 1-10-1 or 2-8-2 MOE gapmer given by intraperitoneal injection. Mice were sacrificed 48 hours following last administration and evaluated for SGLT2 levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 68.







TABLE 68







Antisense inhibition of SGLT2 mRNA in 


vivo by 1-10-1 and 2-8-2 MOE gapmers









% change in SGLT2 expression


Dose of
relative to saline













oligo-
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


nucleotide
379692
388625
379699
388626
379702
388627


umol/kg
1-10-1
2-8-2
1-10-1
2-8-2
1-10-1
2-8-2





0.5
−85
−90
−75
−80
−70
−65


0.1
−75
−88
−60
−60
−65
−50


0.02
−55
−65
−30
−45
−40
−38









These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmers inhibit the expression of SGLT2 in vivo in a dose-dependent manner.
Mice were further evaluated for total body weight, liver, spleen and kidney weight. All changes were within the margin of error of the experiment. No significant changes in body weight were observed during the treatment or at study termination. No significant changes in liver or spleen weights were observed.
The levels of ALT, AST, BUN, transaminases, plasma creatinine, glucose and triglyceride were measured in mice treated with the short antisense compounds. The levels of routine clinical indicators of liver and kidney injury and disease were within normal ranges and are not significantly changed relative to saline-treated animals, demonstrating that the short antisense compounds do not significantly affect renal or hepatic function. Triglyceride and glucose levels were not significantly elevated relative to saline-treated animals.
Evaluation of ISIS 379692 1-10-1 MOE Gapmer, ISIS 3921701-10-1 Methyleneoxy BNA Gapmer, ISIS 388625 2-8-2 MOE Gapmer and ISIS 392173 2-8-2 Methyleneoxy BNA Gapmer in Mice
The effect of ISIS 379692 1-10-1 MOE gapmer and ISIS 388625 2-8-2 MOE gapmer are compared with the effect of ISIS 392170 1-10-1 Methyleneoxy BNA Gapmer and ISIS 392173 2-8-2 Methyleneoxy BNA Gapmer (see Table 69) on mouse SGLT2 mRNA levels in vivo. Data are taken from three experiments in which male 6-week old Balb/c mice were dosed twice per week for three weeks with 5, 25 and 125 nmol/kg of either the ISIS 379692 1-10-1 MOE gapmer or the ISIS 388625 2-8-2 MOE gapmer given by intraperitoneal injection. Mice were sacrificed 48 hours following last administration and evaluated for SGLT2 mRNA levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The data are expressed as percent change (“+” indicates an increase, “−” indicates a decrease) relative to saline treated animals and are illustrated in Table 69.







TABLE 69







Antisense inhibition of SGLT2 mRNA in


vivo by a 1-10-1 and a 2-8-2 MOE gapmer













ISIS

ISIS



ISIS
392170
ISIS
392173


Dose of
379692
1-10-1
388625
2-8-2


oligonucleotide
1-10-1
Methyleneoxy
2-8-2
Methyleneoxy


nmol/kg
MOE
BNA
MOE
BNA














125
−58
−69
−70
−75


25
−46
−54
−47
−57


5
−7
−23
−18
−44









These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmer inhibit the expression of SGLT2 in vivo at the highest three dosing ranges in a dose-dependent manner. The results also illustrate that the Methyleneoxy BNA constructs are more potent then the MOE constructs. No significant changes in body weight were observed during the treatment or at study termination. No significant changes in liver or spleen weights were observed. The toxicity parameters including levels of ALT, AST, BUN, and creatinine were within normal ranges and are not significantly changed relative to saline-treated animals, demonstrating that the compounds do not significantly affect renal or hepatic function.
Evaluation of ISIS 379692 1-10-1 MOE Gapmer and ISIS 388625 2-8-2 MOE Gapmer in Rat
The effect of ISIS 379692 1-10-1 MOE gapmer and ISIS 388625 MOE 2-8-2 gapmer (see Table 70) on rat SGLT2 mRNA levels in vivo. Data are taken from four experiments in which male Sprague-Dawley rats (170-200 g) were dosed twice per week for three weeks with 200, 50, 12.5, or 3.125 nmol/kg of either the ISIS 379692 1-10-1 MOE gapmer or the ISIS 388625 2-8-2 MOE gapmer given by intraperitoneal injection. Rats were sacrificed 48 hours following last administration and evaluated for SGLT2 levels in kidney. Target levels were determined by RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 70.







TABLE 70







Antisense inhibition of SGLT2 mRNA in


vivo by a 1-10-1 and a 2-8-2 MOE gapmer










% change in SGLT2 expression




relative to saline









Dose of
ISIS
ISIS


oligonucleotide
379692
388625


umol/kg
1-10-1
2-8-2












200
−80
−80


50
−65
−65


12.5
−15
−15


3.125
+30
+25









These results illustrate that both the 1-10-1 and 2-8-2 MOE gapmer inhibit the expression of SGLT2 in vivo at the highest three dosing ranges in a dose-dependent manner.
Rats were further evaluated for total body weight, liver, spleen and kidney weight. All changes were within the margin of error of the experiment. No significant changes in body weight were observed during the treatment or at study termination. No significant changes in liver or spleen weights were observed.
The levels of ALT, AST, BUN, cholesterol, plasma creatinine and triglycerides were measured in rats treated with the short antisense compounds. The levels of routine clinical indicators of liver and kidney injury and disease were within normal ranges and are not significantly changed relative to saline-treated animals, demonstrating that the short antisense compounds do not significantly affect renal or hepatic function.
Example 16
Antisense Inhibition of SGLT2 Expression in ZDF Rat
ISIS 388625, 388626 and control oligo ISIS 388628 were analyzed for their effect on ZDF rat plasma glucose levels and HbA1c. The leptin receptor deficient Zucker diabetic fatty (ZDF) rat is a useful model for the investigation of type 2 diabetes. Diabetes develops spontaneously in these male rats at ages 8-10 weeks, and is associated with hyperphagia, polyuria, polydipsia, and impaired weight gain, symptoms which parallel the clinical symptoms of diabetes (Phillips M S, et al., 1996, Nat Genet 13, 18-19). Six week old ZDF rats were injected intraperitoneally with short antisense compound at a dose of 40 0 nM/kg once a week for twelve weeks. Data are illustrated in Tables 71 and 72.







TABLE 71







Plasma glucose















Plasma glucose recorded



Seq


on specific dates


ISIS
ID


(mg/dl)














NO.
NO
Sequence (5′-3′)
Motif
Day 10
Day 40
Day 55
Day 66





PBS

n/a
n/a
450.7
478.5
392.8
526.2


 


388625
246
TGTTCCAGCCCA
2-8-2 MOE
435.5
278.7
213.8
325.5


 


388626
281
GGCATGAGCTTC
2-8-2 MOE
434.7
300.5
219.8
379.8


 


388628
226
TAGCCGCCCACA
2-8-2 MOE
436.0
502.0
411.2
668.8
















TABLE 72







HbA1c Status















Percentage HbA1c on



Seq


specific dates (%)



ID


p < 0.001













ISIS NO.
NO
Sequence (5′-3′)
Motif
Day 40
Day 55
Day 68
















PBS

n/a
n/a
8.0
8.9
10.0


 


388625
246
TGTTCCAGCCCA
2-8-2 MOE
6.5
5.8
4.3


 


388626
281
GGCATGAGCTTC
2-8-2 MOE
6.6
5.9
4.0


 


388628
226
TAGCCGCCCACA
2-8-2 MOE
8.0
9.1
7.8









ISIS 388625 and 388626 significantly reduced plasma glucose levels and HbA1C compared to PBS and control treated animals.
Example 17
Antisense Inhibition of SGLT2 Expression in Dog Kidney (ISIS 388625)
ISIS 388625 is a 2-8-2 MOE Gapmer with sequence TGTTCCAGCCCA (SEQ ID NO: 246) (e.g. see Table 71). The effect of ISIS 388625 on dog SGLT2 mRNA levels. Data are taken from two dosing groups in which a total of nine male beagle dogs were dosed with either one or ten mg/kg/week of ISIS 388625 or saline given by subcutaneous injection twice weekly. On day 46 of the study all dogs were sacrificed and evaluated for SGLT2 levels in kidney. Target levels were determined by quantitative RT, real-time PCR as described by other examples herein. PCR results were normalized to an internal ISIS control. The results are shown below in Table 73.







TABLE 73







Antisense inhibition of SGLT2 mRNA in vivo by ISIS 388625










Dose of
% change in SGLT2 expression



oligonucleotide
Relative to saline



mg/kg/wk
ISIS 388625














1
−85



10
−95










These results illustrate that greater than 80% reduction of SGLT2 mRNA can be achieved at a 1 mg/kg/wk dose of ISIS 388625. Even greater reduction can be achieved at slightly higher doses. Administration of ISIS 388625 in dog was also shown to improve glucose tolerance. Peak plasma glucose levels were decreased by over 50% on average and the subsequent drop in glucose was lessened compared to saline controls in a standard glucose tolerance test. Urinary glucose excretion was also increased.
Example 18
In Vivo Testing of Short Antisense Compounds Targeted to SGLT2 Nucleic Acid
Twenty 1-10-1 MOE gapmers that are complementary to human/monkey/mouse/rat SGLT2 were designed, synthesized and tested in vivo for suppression of SGLT2 mRNA levels in kidney. Target sites for mouse and rat are indicated in Table 74. Target sites for human are indicated in Tables 4 and 5. Data are averages from two experiments in which male 6-week old Balb/c mice were administered intraperitoneal injections of 350 nmol/kg of oligonucleotide, twice per week, over a period of two weeks (a total of four injections). Mice were sacrificed 48 hours following the last administration and evaluated for SGLT2 mRNA levels in kidney. SGLT2 mRNA levels were determined by quantitative real-time PCR analysis according to standard procedures, using two different PCR primer probe sets, primer probe set (PPS) 534 and PPS 553. SGLT2 mRNA levels were normalized to cyclophilin mRNA levels, which were also measured by quantitative real-time PCR. The results are shown below in Table 74.







TABLE 74







Antisense inhibition of SGLT2 in vivo















5′ Target
5′ Target








Site on
Site on



SEQ ID
SEQ ID


PPS
PPS
SEQ


ISIS
NO: 1576
NO: 1575


534 %
553 %
ID


NO
(mouse)
(rat)
Sequence (5′-3′)
Motif
Saline
Saline
NO

















PBS


N/A

—
—



 


370717
2684
152
TAGCCACCAACT
1-10-1
−84.4
−84.3
1554






MOE


 


379684
2070
64
TGTCAGCAGGAT
1-10-1
−45.0
−43.2
214






MOE


 


379685
2103
97
TGACCAGCAGGA
1-10-1
−10.3
−20.5
219






MOE


 


379686
 2121*
115
ACCACAAGCCAA
1-10-1
−71.9
−75.1
225






MOE


 


379687
2824
216
GATGTTGCTGGC
1-10-1
−47.1
−52.1
230






MOE


 


379688
2876
268
CCAAGCCACTTG
1-10-1
−62.6
−70.4
240






MOE


 


379689

298
AGAGCGCATTCC
1-10-1
−17.5
−30.4
241






MOE


 


379690

415
ACAGGTAGAGGC
1-10-1
−18.9
−22.5
242






MOE


 


379691

454
AGATCTTGGTGA
1-10-1
−35.0
−48.6
243






MOE


 


379692

508
TGTTCCAGCCCA
1-10-1
−88.1
−88.5
246






MOE


 


379693

546
CATGGTGATGCC
1-10-1
−51.6
−59.9
254






MOE


 


379694

609
GACGAAGGTCTG
1-10-1
−42.1
−54.4
264






MOE


 


379695

717
GGACACCGTCAG
1-10-1
−52.5
−64.1
266






MOE


 


379696

954
CAGCTTCAGGTA
1-10-1
−24.6
−36.2
267






MOE


 


379697

982
CTGGCATGACCA
1-10-1
−32.0
−46.3
272






MOE


 


379698

1071
GCAGCCCACCTC
1-10-1
−11.8
−27.0
275






MOE


 


379699

1112
GGCATGAGCTTC
1-10-1
−83.5
−85.8
281






MOE


 


379700

1138
CCAGCATGAGTC
1-10-1
−2.8
−16.4
285






MOE


 


379701

1210
CCATGGTGAAGA
1-10-1
−0.3
−11.9
288






MOE


 


379702

1525
GCACACAGCTGC
1-10-1
−87.8
−89.5
293






MOE


 


379703

1681
GCCGGAGACTGA
1-10-1
−44.2
−45.9
295






MOE





*indicates 1 or 2 mismatches to a target sequence






Example 19
Antisense Inhibition of Human PCSK9 in Hep3B Cells
Short antisense compounds targeted to a PCSK9 nucleic acid were tested for their effects on PCSK9 mRNA in vitro. The short antisense compounds are presented in Table 6. The Isis No, gapmer motif and SEQ ID NO of each short antisense compound are shown again in Table 75. Cultured Hep3B cells were treated with 100 nM of short antisense compound. 5-10-5 MOE gapmers targeted to a PCSK9 nucleic acid were used as positive controls. After the treatment period, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR, as described herein. PCSK9 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in Table 75 as percent inhibition of PCSK9 (% Inhib), relative to untreated control cells. In the “% Inhib” column, a “0” indicates that no reduction of PCSK9 mRNA was observed with that particular short antisense compound.







TABLE 75







Antisense inhibition of PCSK9 by short antisense compounds















5′
3′







Target
Target







Site on
Site on

%



ISIS
SEQ ID
SEQ ID
SEQ ID
Gapmer
Inhibition
%


No.
NO
NO: 4
NO: 4
Motif
Range
Inhib
















400297
329
695
708
2-10-2 MOE

0


400298
330
696
709
2-10-2 MOE

0


400299
331
697
710
2-10-2 MOE

0


400300
332
742
755
2-10-2 MOE

9


400301
333
757
770
2-10-2 MOE
20-30%
27


400302
334
828
841
2-10-2 MOE

0


400303 
335
829
842
2-10-2 MOE

0


400304 
336
830
843
2-10-2 MOE
10-20%
11


400305 
337
937
950
2-10-2 MOE
30-40%
38


400306 
338
952
965
2-10-2 MOE
40-50%
40


400307 
339
988
1001 
2-10-2 MOE
70-80%
76


400308 
340
989
1002 
2-10-2 MOE
50-60%
55


400309 
341
990
1003 
2-10-2 MOE
40-50%
44


400310 
342
991
1004 
2-10-2 MOE

8


400311 
343
992
1005 
2-10-2 MOE
10-20%
18


400312 
344
993
1006
2-10-2 MOE
20-30%
28


400313 
345
994
1007 
2-10-2 MOE
10-20%
10


400314 
346
1057
1070 
2-10-2 MOE
20-30%
26


400315 
347
1075
1088 
2-10-2 MOE

0


400316 
348
1076
1089 
2-10-2 MOE

8


400317 
349
1077
1090 
2-10-2 MOE

7


400318 
350
1078
1091 
2-10-2 MOE
20-30%
26


400319 
351
1093
1106 
2-10-2 MOE

0


400320 
352
1094
1107 
2-10-2 MOE

0


400321 
353
1095
1108 
2-10-2 MOE

0


400322 
354
1096
1109 
2-10-2 MOE

0


400323 
355
1147
1160 
2-10-2 MOE

0


400324 
356
1255
1268 
2-10-2 MOE

7


400325 
357
1334
1347 
2-10-2 MOE

4


400326 
358
1335
1348 
2-10-2 MOE

0


400327 
359
1336
1349 
2-10-2 MOE
30-40%
36


400328 
360
1453
1466 
2-10-2 MOE
10-20%
13


400329 
361
1454
1467 
2-10-2 MOE
10-20%
14


400330 
362
1455
1468 
2-10-2 MOE
40-50%
43


400331 
363
1456
1469 
2-10-2 MOE
30-40%
35


400332 
364
1569
1582 
2-10-2 MOE

0


400333 
365
1570
1583 
2-10-2 MOE

0


400334 
366
1571
1584 
2-10-2 MOE

0


400335 
367
1572
1585 
2-10-2 MOE

0


400336
368
1573
1586 
2-10-2 MOE

4


400337 
369
1574
1587 
2-10-2 MOE

0


400338 
370
1575
1588 
2-10-2 MOE

9


400339 
371
1576
1589 
2-10-2 MOE

0


400340 
372
1577
1590 
2-10-2 MOE

0


400341 
373
1578
1591 
2-10-2 MOE

0


400342 
374
1621
1634 
2-10-2 MOE

0


400343
375
1622
1635
2-10-2 MOE

0


400344
376
1623
1636
2-10-2 MOE

0


400345
377
1624
1637
2-10-2 MOE

0


400346
378
1738
1751
2-10-2 MOE

5


400347
379
1739
1752
2-10-2 MOE

0


400348
380
1740
1753
2-10-2 MOE

0


400349
381
1741
1754
2-10-2 MOE
10-20%
13


400350
382
1834
1847
2-10-2 MOE
10-20%
15


400351
383
1835
1848
2-10-2 MOE
10-20%
14


400352
384
1836
1849
2-10-2 MOE
20-30%
29


400353
385
1837
1850
2-10-2 MOE
10-20%
19


400354
386
1838
1851
2-10-2 MOE
10-20%
19


400355
387
1839
1852
2-10-2 MOE

0


400356
388
1840
1853
2-10-2 MOE

0


400357
389
2083
2096
2-10-2 MOE

0


400358
390
2084
2097
2-10-2 MOE
10-20%
12


400359
391
2085
2098
2-10-2 MOE

0


400360
392
2086
2099
2-10-2 MOE
30-40%
38


400361
393
2316
2329
2-10-2 MOE

2


400362
394
2317
2330
2-10-2 MOE
10-20%
16


400363
395
2318
2331
2-10-2 MOE

8


400364
396
2319
2332
2-10-2 MOE

0


400365
397
2320
2333
2-10-2 MOE
20-30%
25


400366
398
2321
2334
2-10-2 MOE
10-20%
15


400367
399
2322
2335
2-10-2 MOE
10-20%
12


400368
400
2323
2336
2-10-2 MOE
10-20%
11


400369
401
2324
2337
2-10-2 MOE

0


400370
402
2325
2338
2-10-2 MOE
10-20%
13


400371
403
3543
3556
2-10-2 MOE

0









As illustrated in Table 75, short antisense compounds targeted to a PCSK9 nucleic acid, having a 2-10-2 MOE gapmer motif, reduced PCSK9 mRNA in cultured cells.
Short antisense compounds targeted to a PCSK9 nucleic acid were tested in a dose response experiment Hep3B cells. Cells were treated as described herein with nM concentrations of short antisense compound as indicated in Tables 76. After the treatment period, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR, as described herein. PCSK9 mRNA levels were normalized to cyclophilin mRNA levels, as measured by real-time PCR using a cyclophilin-specific primer probe set. Results are presented as percent inhibition of PCSK9, relative to untreated control cells. Also shown is the EC50 (concentration at which 50% reduction of mRNA is observed) for each short antisense compound tested in the dose response experiment, as calculated using Graphpad Prism. As illustrated in the following table, PCSK9 mRNA levels were reduced in a dose-dependent manner.







TABLE 76







Dose-dependent antisense inhibition of 


PCSK9 by short antisense compounds









% Inhibition














160 nM
80 nM
40 nM
20 nM
10 nM
5 nM
















5-10-5
95
96
85
78
58
38


400307
93
92
56
45
39
35


400308
86
77
40
26
10
31


400309
78
72
12
38
23
49


400327
55
43
49
23
37
5


400330
71
82
69
40
32
8


400331
82
75
63
47
40
29


400352
64
63
44
40
16
7


400353
48
54
43
23
27
15









Example 20
Antisense Inhibition of PCSK9 by Short Antisense Compounds Comprising BNAs
Short antisense compounds targeted to a PCSK9 nucleic acid were tested in dose response experiments, in both mouse and human cultured cells. The compounds tested included ISIS 403739 and ISIS 403740. ISIS 403739 is a short antisense compound consisting of the nucleotide sequence of SEQ ID NO: 404 and having a 2-10-2 gapmer motif, where the nucleotides in the wings comprise (6′S)-6′-methyl BNA. ISIS 403740 is a short antisense compound consisting of the nucleotide sequence of SEQ ID NO: 405 and having a 2-10-2 gapmer motif, where the nucleotides in the wings comprise (6′S)-6′-methyl BNA. Also tested was a 5-10-5 MOE gapmer targeted to a PCSK9 nucleic acid.
Mouse hepatocytes were plated and treated as described herein with nM concentrations of short antisense compound as indicated in Table 77. After the treatment period, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR, as described herein. PCSK9 mRNA levels were normalized to cyclophilin mRNA levels, as measured by real-time PCR using a cyclophilin-specific primer probe set. Results are presented as percent inhibition of PCSK9, relative to untreated control cells. Where present, “0” indicates no observed reduction in PCSK9 mRNA. ISIS 403739 exhibited dose-dependent reduction of mouse PCSK9 mRNA at the doses of 30 nM and higher. ISIS 403740 exhibited reduction of mouse PCSK9 mRNA at the two highest doses of short antisense compound.







TABLE 77







Antisense inhibition of mouse PCSK9 by


short antisense compounds comprising BNAs









% Inhibition




















120 
240



3.75 nM
7.5 nM
15 nM
30 nM
60 nM
nM
nM





5-10-5
10
15
21
18
44
43
77


403739
40
19
29
29
32
49
57


403740
 3
 0
29
13
 0
40
33









Human Hep3B cells were treated with nM concentrations of short antisense compound as described herein. After the treatment period, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR, as described herein. PCSK9 mRNA levels were normalized to cyclophilin mRNA levels, as measured by real-time PCR using a cyclophilin-specific primer probe set. Results are presented as percent inhibition of PCSK9, relative to untreated control cells. The data are shown in Table 78 and demonstrate a dose-dependent reduction in human PCSK9 mRNA following treatment with ISIS 403740. ISIS 403739 exhibited dose-dependent reduction at higher doses.







TABLE 78







Antisense inhibition of mouse PCSK9 by


short antisense compounds comprising BNAs









% Inhibition















2.5 nM
5 nM
10 nM
20 nM
40 nM
80 nM
160 nM





5-10-5
 7
 2
21
33
30
59
71


403739
10
 5
 7
 6
25
52
65


403740
 6
12
16
29
45
48
59









Example 21
Antisense Inhibition of GCGR in HepG2 Cells
Short antisense compounds targeted to a GCGR nucleic acid were tested for their effects on GCGR mRNA in vitro.
HepG2 Cells
Cultured HepG2 cells at a density of 10000 cells per well in a 96-well plate were treated as described herein with 25, 50, 100 or 200 nM of antisense oligonucleotide. After the treatment period, RNA was isolated from the cells and GCGR mRNA levels were measured by quantitative real-time PCR, as described herein. GCGR mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent reduction in GCGR mRNA, relative to untreated control cells.
Table 79 presents data following treatment with the indicated doses of ISIS 327161, a 3-10-3 MOE gapmer. ISIS 327161 reduced GCGR mRNA in a dose-dependent manner.







TABLE 79







Antisense inhibition of GCGR in HepG2 cells


by a short antisense compound














ISIS
Seq ID

Gapmer






NO.
NO
Sequence (5′-3′)
Motif
25 nM
50 nM
100 nM
200 nM





327161
520
AGCTGCTGTACATC
3-8-3
−36
−30
−33
−64





MOE









Monkey Hepatocytes

Additional short antisense compounds targeted to a GCGR nucleic acid were tested for their effects on monkey GCGR mRNA in vitro. Cultured primary monkey hepatocytes were treated as described herein with 25, 50, 100 or 200 nM of short antisense compound. After the treatment period, RNA was isolated from the cells and GCGR mRNA levels were measured by quantitative real-time PCR, as described herein. GCGR mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in Table 80 as percent reduction in GCGR mRNA, relative to untreated control cells.







TABLE 80







Antisense inhibition of GCGR in primary monkey hepatocytes


by short antisense compounds














ISIS
Seq ID

Gapmer






NO.
NO
Sequence (5′-3′)
Motif
25 nM
50 nM
100 nM
200 nM

















327131
489
ATGTTGGCCGTGGT
3-8-3
0
−8
−36
−36





MOE


 


327161
520
AGCTGCTGTACATC
3-8-3
−19
−33
−55
−54





MOE









Example 22
Antisense Inhibition of DGAT2 by Short Antisense Compounds
Short antisense compounds targeted to a DGAT2 nucleic acid were tested for their effects on DGAT2 mRNA in vitro. Cultured A10 cells in a 96-well plate were treated with 75 nM of short antisense compound. After a treatment period of approximately 24 hours, RNA was isolated from the cells and DGAT2 mRNA levels were measured by quantitative real-time PCR, as described herein. DGAT2 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of DGAT2, relative to untreated control cells in Table 81.







TABLE 81







Antisense inhibition of DGAT2 in A10 cells











ISIS
Seq ID


%


NO.
NO
Sequence (5′-3′)
Gapmer Motif
Control














372491
795
ACATGAGGATGACACT
3-10-3 MOE
80


 


372500
702
GTGTGTCTTCACCAGC
3-10-3 MOE
16


 


372501
704
TTGTGTGTCTTCACCA
3-10-3 MOE
28


 


372503
708
GCAGGTTGTGTGTCTT
3-10-3 MOE
35


 


372508
719
AGTTCCTGGTGGTCAG
3-10-3 MOE
35


 


372516
805
TACAGAAGGCACCCAG
3-10-3 MOE
27


 


372524
738
GCCAGGCATGGAGCTC
3-10-3 MOE
21


 


372530
746
TCGGCCCCAGGAGCCC
3-10-3 MOE
35


 


372546
825
TTGGTCTTGTGATTGT
3-10-3 MOE
34


 


372563
691
AGCCAGGTGACAGA
2-10-2 MOE
48


 


372569
796
CATGAGGATGACAC
2-10-2 MOE
104


 


372578
703
TGTGTCTTCACCAG
2-10-2 MOE
59


 


372580
707
GGTTGTGTGTCTTC
2-10-2 MOE
48


 


372586
720
GTTCCTGGTGGTCA
2-10-2 MOE
40


 


372594
806
ACAGAAGGCACCCA
2-10-2 MOE
77


 


372602
739
CCAGGCATGGAGCT
2-10-2 MOE
39


 


372618
765
GTGGTACAGGTCGA
2-10-2 MOE
29


 


372624
826
TGGTCTTGTGATTG
2-10-2 MOE
56









Additional short antisense compounds targeted to DGAT2 mRNA were tested in vitro in a dose-response experiment. A10 cells were prepared as described above and treated with 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 nM short antisense compounds to determine if DGAT2 inhibition occurs in a dose-dependent manner. The data demonstrate that each of the short antisense compounds presented in Table 82 reduces rat DGAT2 mRNA in a dose-dependent manner. Results are presented as percent inhibition, relative to untreated control cells. A “0” indicates that DGAT2 mRNA was not reduced.







TABLE 82







Dose-Dependent Inhibition of DGAT2 in A10 cells

















Seq










ISIS
ID

Gapmer


NO.
NO
Sequence (5′-3′)
Motif
6.25 nM
12.5 nM
25.0 nM
50.0 nM
100.0 nM
200.0 nM



















372562
784
GTCTTGGAGGGCCG
2-10-2
0
0
0
36
48
75





MOE


 


372568
794
GACACTGCAGGCCA
2-10-2
0
0
15
26
72
69





MOE


 


372586
720
GTTCCTGGTGGTCA
2-10-2
19
0
7
22
45
77





MOE


 


372602
739
CCAGGCATGGAGCT
2-10-2
0
0
0
18
47
76





MOE


 


372618
765
GTGGTACAGGTCGA
2-10-2
0
5
0
27
65
80





MOE









Additional short antisense compounds targeted to DGAT2 mRNA were tested in vitro. A10 cells were prepared as described above and treated with 0.62, 1.85, 5.56, 16.67, 50.0, and 150.0 nM short antisense compounds to determine if DGAT2 inhibition occurs in a dose-dependent manner. DGAT2 mRNA was measured using quantitative real-time PCR, as described herein. The data demonstrate that each of the short antisense compounds presented in Table 83 below inhibit rat DGAT2 mRNA in a dose-dependent manner. Results are presented as percent inhibition of rat DGAT2, relative to untreated control cells. Where present, “0” indicates that no reduction in DGAT2 mRNA was observed.







TABLE 83







Dose-Dependent Inhibition of DGAT2 in A10 cells

















Seq










ISIS
ID

Gapmer


NO.
NO
Sequence (5′-3′)
Motif
0.62 nM
1.85 nM
5.56 nM
16.67 nM
50 nM
150 nM



















372500
702
GTGTGTCTTCACCAGC
3-10-3
0
0
0
18
64
88





MOE


 


372501
704
TTGTGTGTCTTCACCA
3-10-3
1
5
10
11
25
68





MOE


 


372503
708
GCAGGTTGTGTGTCTT
3-10-3
7
10
4
25
54
80





MOE


 


372508
719
AGTTCCTGGTGGTCAG
3-10-3
0
0
6
14
39
71





MOE


 


372516
805
TACAGAAGGCACCCAG
3-10-3
1
10
0
4
35
81





MOE


 


372524
738
GCCAGGCATGGAGCTC
3-10-3
7
0
5
30
68
91





MOE


 


372530
746
TCGGCCCCAGGAGCCC
3-10-3
0
2
0
10
38
78





MOE


 


372546
825
TTGGTCTTGTGATTGT
3-10-3
0
2
11
4
48
78





MOE


 


372563
691
AGCCAGGTGACAGA
2-10-2
0
0
0
1
4
46





MOE


 


372578
703
TGTGTCTTCACCAG
2-10-2
0
0
0
2
7
42





MOE


 


372580
707
GGTTGTGTGTCTTC
2-10-2
0
5
5
3
16
42





MOE


 


372586
720
GTTCCTGGTGGTCA
2-10-2
0
0
0
0
7
55





MOE


 


372594
806
ACAGAAGGCACCCA
2-10-2
0
0
0
0
2
15





MOE


 


372602
739
CCAGGCATGGAGCT
2-10-2
0
0
10
0
19
51





MOE


 


372618
765
GTGGTACAGGTCGA
2-10-2
0
0
0
0
30
60





MOE


 


372624
826
TGGTCTTGTGATTG
2-10-2
0
0
0
1
16
38





MOE









Example 23
Antisense Inhibition of Human PTP1B in HuVEC Cells
Short antisense compounds targeted to a PTP1B nucleic acid were tested for their effects on PTP1B mRNA in vitro. Cultured HuVEC cells at a density of 5000 cells per well in a 96-well plate were treated as described herein with 3 nM of short antisense compound. After the treatment period, RNA was isolated from the cells and PTP1B mRNA levels were measured by quantitative real-time PCR, as described herein. PTP1B mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of PTP1B (% Inhib), relative to untreated control cells. The data demonstrated that short antisense compounds targeted to a PTP1B nucleic acid and having a 2-10-2 gapmer motif can inhibit PTP1B in HuVEC cells in Table 84.







TABLE 84







Antisense inhibition of PTP1B in HuVEC


cells by short antisense compounds












ISIS NO.
SEQ ID NO
Gapmer Motif
% Inhib
















399301
1542
2-10-2 OMe
55



404137
1053
2-10-2 MOE
76



404138
1054
2-10-2 MOE
76



404139
1052
2-10-2 MOE
80



404140
1051
2-10-2 MOE
73










Example 24
Antisense Inhibition of Human PTP1B in HepG2 Cells
Short antisense compounds targeted to a PTP1B nucleic acid were tested for their effects on PTP1B mRNA in vitro. Cultured HepG2 cells at a density of 10000 cells per well in a 96-well plate were treated with 25 nM of antisense oligonucleotide. After the treatment period, RNA was isolated from the cells and PTP1B mRNA levels were measured by quantitative real-time PCR, as described herein. PTP1B mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition (% Inhib) of PTP1B, relative to untreated control cells. The data demonstrated that short antisense compounds targeted to a PTP1B nucleic acid and having a 2-10-2 gapmer motif can inhibit PTP1B in HepG2 cells in Table 85.







TABLE 85







Antisense inhibition of PTP1B in HepG2


cells by short antisense compounds












ISIS NO.
SEQ ID NO
Gapmer Motif
% Inhib
















399301
1542
2-10-2 OMe
43



404137
1053
2-10-2 MOE
71



404138
1054
2-10-2 MOE
86



404139
1052
2-10-2 MOE
45



404140
1051
2-10-2 MOE
93










Example 25
Antisense Inhibition of PTP1B in HuVEC Cells: Dose Response Experiment
Human vascular endothelial (HuVEC) cells were plated at a density of 5000 cells per well and treated as described herein with nM concentrations of short antisense compound as indicated in Table 86. After the treatment period, RNA was isolated from the cells and PTP1B mRNA levels were measured by quantitative real-time PCR, as described herein. PTP1B mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Two different human PTP1B primer probe sets were used to measure mRNA levels. Results with Primer Probe Set (PPS) 198 are shown in Table 86, and results with Primer Probe Set (PPS) 3000 are shown in Table 87. Results are presented as percent inhibition of PTP1B mRNA expression relative to untreated control cells. Where present, “0” indicates that no PTP1B mRNA reduction was observed. As illustrated in Tables 86 and 87, PTP1B mRNA levels were reduced in a dose-dependent manner







TABLE 86







Dose Response for Human PTP1B in HuVEC cells, using PPS 198


 










ISIS
Seq ID 
Gapmer
% Inhibition













NO.
NO
Motif
1.11 nM
3.33 nM
10.0 nM 
30.0 nM
















398105 
1066
2-10-2 MOE
0
25
79
90


398112
1072
2-10-2 MOE 
1
10
73
93


398120
1086
2-10-2 MOE 
0
31
80
96


399096
1544
2-10-2 MOE
3
30
78
96


399102
1545
2-10-2 MOE
0
15
62
88


399113
1547
2-10-2 MOE
0
31
72
90


399132
1548
2-10-2 MOE
0
32
75
95


399173
1549
2-10-2 MOE
0
24
63
89


399208
1550
2-10-2 MOE
0
37
86
93


399276
1551
2-10-2 MOE
0
8
61
89


399301
1542
2-10-2 MOE 
8
63
91
97


399315 
1552
2-10-2 MOE 
0
20
68
88


398173
1543
1-10-1 MOE
0
4
80
97
















TABLE 87







Dose Response for Human PTP1B in HuVEC cells, using PPS 3000


 











Seq ID
Gapmer
% Inhibition













ISIS NO. 
NO
Motif
1.11 nM 
3.33 nM
10.0 nM
30.0 nM
















398105
1066 
2-10-2 MOE
0
35
79
93


398112
1072 
2-10-2 MOE
0
26
77
94


398120
1086 
2-10-2 MOE
0
35
79
93


399096
1544 
2-10-2 MOE
0
23
75
94


399102
1545 
2-10-2 MOE
0
9
60
87


399113
1547 
2-10-2 MOE
0
9
65
90


399132
1548 
2-10-2 MOE
0
26
76
91


399173
1549 
2-10-2 MOE
0
11
59
92


399208
1550 
2-10-2 MOE
0
47
85
96


399276
1551 
2-10-2 MOE
0
14
64
86


399301
1542 
2-10-2 MOE
16
65
93
99


399315
1552 
2-10-2 MOE
0
25
71
93


398173
1543 
1-10-1 MOE
0
18
80
90









Example 26
Antisense Inhibition of ApoB by Short Antisense Compounds
The short antisense compounds shown in Table 88 were tested for their effects in vivo. Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered intraperitoneal doses of 3.2, 1, 0.32, or 0.1 umol/kg, twice per week for three weeks. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 48 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. ApoB mRNA levels were measured by real-time PCR as described herein. ApoB mRNA levels were normalized to RNA levels as determined by RIBOGREEN, and are presented in Table 89 as percent inhibition relative to ApoB mRNA levels in saline-treated control animals.







TABLE 88







Short Antisense Compounds


Targeting an ApoB nucleic acid










ISIS


SEQ


NO
Sequence (5′-3′)
Gapmer Motif
ID NO





387462
GGTACATGGAAGTC
2-10-2 Methyleneoxy
190




BNA


 


398296
GGTACATGGAAGTC
2-10-2 6′-(S)-methyl
190




Methyleneoxy BNA
















TABLE 89







Antisense inhibition of ApoB by Short


Antisense Compounds Comprising BNA









Isis No
Dose (umol/kg)
% Inhib












379818
1
56


387462
0.1
33



0.32
57



1
93



3.2
99


398296
0.1
17



0.32
35



1
80



3.2
98









Table 89 shows that ApoB mRNA levels were reduced in a dose-dependent manner following treatment with short antisense compounds having a 2-10-2 gapmer motif and BNA modifications in the wings. At the 1 umol/kg dose, ApoB inhibition by the short antisense compounds was greater than observed with a 5-10-5 MOE gapmer at an equivalent dose. Cholesterol was reduced at the 1 and 3.2 umol/kg doses of short antisense compound.
The short antisense compounds exhibited little to no adverse side effects, as judged by organ and body weights, serum transaminases, bilirubin, blood urea nitrogen, and creatinine.
Example 27
Antisense Inhibition of PTEN by Short Antisense Compounds
The short antisense compounds shown in Table 90 were tested for their effects in vivo. Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered intraperitoneal doses of 3.2, 1, 0.32, or 0.1 umol/kg, twice per week for three weeks. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 48 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. PTEN mRNA levels were measured by real-time PCR as described herein. PTEN mRNA levels were normalized to RNA levels as determined by RIBOGREEN, and are presented in Table 91 as percent inhibition relative to PTEN mRNA levels in saline-treated control animals.







TABLE 90







Short Antisense Compounds targeted to a


PTEN nucleic acid










ISIS


SEQ


NO
Sequence (5′-3′)
Gapmer Motif
ID NO





392063
AGGCCAGTGCTAAG
2-10-2 Methyleneoxy
1226




BNA


 


392749
AGGCCAGTGCTAAG
2-10-2
1226




(6′S)-6′-methyl




Methyleneoxy BNA


 


396006
AGGCCAGTGCTAAG
2-10-2
1226




alpha-L-methyleneoxy




BNA
















TABLE 91







Antisense inhibition of PTEN by short antisense


compounds comprising BNA modifications









Isis No
Dose (umol/kg)
% Inhib












116847
1
47


392063
0.1
26



0.32
43



1
74



3.2
96


392749
0.1
17



0.32
34



1
64



3.2
96


396006
0.1
20



0.32
32



1
67



3.2
88









Table 91 shows that PTEN mRNA levels were reduced in a dose-dependent manner following treatment with short antisense compounds having a 2-10-2 gapmer motif and BNA modifications in the wings. At the 1 umol/kg dose, PTEN inhibition by the short antisense compounds was greater than observed with a 5-10-5 MOE gapmer at an equivalent dose.
With the exception of the highest dose of ISIS 392063, no significant increases in serum transaminases were observed. Overall, the short antisense compounds exhibited little to no adverse side effects.
Example 28
Single Dose Administration of Short Antisense Compounds Comprising BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal injection of short antisense compound at a dose of 8, 4, 2 or 1 μmol/kg. The short antisense compounds tested were ISIS 387462 and ISIS 398296. Each dose group consisted of four animals. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 48 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. ApoB mRNA levels were measured by real-time PCR as described herein. ApoB mRNA levels were normalized to RNA levels as determined by RIBOGREEN, and are presented in Table 92 as percent inhibition relative to ApoB mRNA levels in saline-treated control animals.







TABLE 92







Antisense inhibition of ApoB by Short


Antisense Compounds Comprising BNA









Isis No
Dose (umol/kg)
% Inhib












379818
8
77


387462
8
99



4
93



2
81



1
58


398296
8
97



4
81



2
54



1
19









Table 92 shows that ApoB mRNA levels were reduced in a dose-dependent manner following a single administration of short antisense compounds having a 2-10-2 gapmer motif and BNA modifications in the wings. At the 8 umol/kg dose, ApoB inhibition by the short antisense compounds was greater than observed with a 5-10-5 MOE gapmer at an equivalent dose. The ED50 of ISIS 387462 was 3.9 mg/kg, and the ED50 of ISIS 398296 was 8.7 mg/kg. Cholesterol was also reduced in a dose-dependent manner. Triglycerides were reduced at the highest dose.
The short antisense compounds exhibited little to no adverse side effects, as judged by organ and body weights, serum transaminases, bilirubin, blood urea nitrogen, and creatinine.
In a similar single dose administration study, ISIS 392748, having SEQ ID NO: 1226, a 2-10-2 gapmer motif, where the nucleotides of the wings comprise (6′R)-6′-methyl methyleneoxy BNA modifications, reduced PTEN mRNA in a dose-dependent manner. Additionally, ISIS 392749, having SEQ ID NO: 1226, a 2-10-2 gapmer motif, where the nucleotides of the wings comprise (6′S)-6′-methyl methyleneoxy BNA modifications, reduced PTEN mRNA in a dose-dependent manner. A short antisense compound having 2-10-2 gapmer motifs, the sequence of SEQ ID NO: 1226, and 6-(S)—CH2—O—CH3—BNA modifications also reduced PTEN mRNA in a similar in vivo study. A short antisense compound having 2-10-2 gapmer motifs, the sequence of SEQ ID NO: 1226, and 6-(R)—CH2—O—CH3—BNA modifications also reduced PTEN mRNA in a similar in vivo study.
Example 29
Single Close Administration of Short Antisense Compounds Comprising BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal injection of antisense compound at a dose of 8, 4, 2 or 1 μmol/kg. Each dose group consisted of four animals. The compounds tested were ISIS 392063, ISIS 392749, and ISIS 366006. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 48 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. ApoB mRNA levels were measured by real-time PCR as described herein. ApoB mRNA levels were normalized to RNA levels as determined by RIBOGREEN, and are presented in Table 93 as percent inhibition relative to ApoB mRNA levels in saline-treated control animals.







TABLE 93







Antisense inhibition of PTEN by short antisense


compounds comprising BNA modifications









Isis No
Dose (umol/kg)
% Inhib












116847
8
62


392063
8
92



4
82



2
58



1
38


396565
8
76



4
38



2
24



1
11


396006
8
94



4
82



2
48



1
18









Table 93 shows that PTEN mRNA levels were reduced in a dose-dependent manner following treatment with short antisense compounds having a 2-10-2 gapmer motif and BNA modifications in the wings. At the 8 umol/kg dose, PTEN inhibition by the short antisense compounds was greater than observed with a 5-10-5 MOE gapmer at an equivalent dose. The estimated ED50s were 7 mg/kg for ISIS 392063, 17.4 mg/kg for ISIS 396565, and 9.3 mg/kg for ISIS 396006.
With the exception of the highest dose of ISIS 392063, no significant increases in serum transaminases were observed. Overall, the short antisense compounds exhibited little to no adverse side effects.
Example 30
Antisense Inhibition of ApoB by Short Antisense Compounds Comprising Palmitic Acid Conjugates
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal injection of antisense compound at a dose of 2.5, 1.0. 0.4, and 0.16 umol/kg. Each dose group consisted of four animals. The compounds tested are shown in Table 94. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 48 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. ApoB mRNA levels were measured by real-time PCR as described herein. ApoB mRNA levels were normalized to RNA levels as determined by RIBOGREEN, and are presented in Table 95 as percent inhibition relative to ApoB mRNA levels in saline-treated control animals.







TABLE 94







Short antisense compounds comprising palmitic conjugates










ISIS


SEQ


NO
Sequence (5′-3′)
Gapmer Motif
ID NO





387462
GGTACATGGAAGTC
2-10-2 Methyleneoxy BNA
190


 


391871
GGTACATGGAAGTC
1-1-10-2 2′-(butylacetomido)-
190




palmitamide/MOE/MOE




Unmodified cytosines in gap




(i.e., 2-10-2 MOE with 2′-




(butylacetomido)-palmitamide




substituted at 5′ nucleotide


 


391872
GGTACATGGAAGTC
1-1-10-2 2′-(butylacetomido)-
190




palmitamide Methyleneoxy




BNA/Methyleneoxy BNA




Unmodified cytosines in gap




(i.e., 2-10-2 methyleneoxy BNA




with 2′-(butylacetomido)-




palmitamide substituted at 5′




nucleotide)
















TABLE 95







Antisense inhibition by short antisense compounds


comprising palmitic acid conjugates









Isis No
Dose (umol/kg)
% Inhib












5-10-5
2.5
54


387462
2.5
99



1.0
91



0.4
65



0.16
16


391871
2.5
49



1.0
18



0.4
5



0.16
0


391872
2.5
99



1.0
92



0.4
50



0.16
18









Table 95 shows that ApoB mRNA levels were reduced in a dose-dependent manner following treatment with short antisense compounds having a palmitic acid (C16) conjugate. At the 2.5 umol/kg dose, ApoB inhibition by the short antisense compounds was greater than observed with a 5-10-5 MOE gapmer at an equivalent dose. In this study, the estimated ED50s were 1.5 mg/kg for ISIS 387462, 13.1 mg/kg for ISIS 391871, and 1.9 mg/kg for ISIS 391872. The estimated ED50 for the 5-10-5 MOE gapmer was 17.4 mg/kg. Triglycerides were reduced at the 2.5 and 1.0 mg/kg doses of ISIS 387462 and ISIS 391872. ISIS 387462 and ISIS 391872 markedly reduced total cholesterol, HDL-C and LDL-C in a dose-dependent manner; reduction in LDL-C was so marked that it fell below the limit of detection. Overall, the short antisense compounds exhibited little to no adverse effects.
Example 31
Antisense Inhibition of PCSK9 In Vivo by Short Antisense Compounds Comprising BNA Modifications
Six-week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were administered a single intraperitoneal injection of antisense compound at a dose of 15, 4.7, 1.5 and 0.47 umol/kg of ISIS 403739 or 403740. Each dose group consisted of four animals. A 5-10-5 MOE gapmer was used for a control treatment. Mice were sacrificed approximately 72 hours following the final dose. Liver tissue was collected for RNA isolation, and blood was collected for serum chemistry analyses. PCSK9 mRNA levels were measured by real-time PCR as described herein. PCSK9 mRNA levels were normalized to cyclophilin mRNA levels as determined by real-time PCR. ISIS 403739 reduced PCSK9 mRNA by approximately 70%, relative to saline controls. ISIS 403740 reduced PCSK9 by approximately 13% relative to saline controls, however, the reduction was not statistically significant. The lower doses did not significantly reduce PCSK9 mRNA. Overall, the short antisense compounds exhibited little to no adverse side effects.

## Claims

1. A compound comprising a short antisense oligonucleotide consisting of 12 to 14 linked nucleosides and having:
a gap region consisting of 8 to 12 linked 2′-deoxynucleosides;
a 5′-wing region adjacent to the gap region at the 5′side of the gap region and consisting of 1 or 2 linked modified nucleosides; and
a 3′-wing region adjacent to the gap region at the 3′ side of the gap region and consisting of 1 or 2 linked modified nucleosides; wherein
at least one modified nucleoside of one or both of the 5′-wing region and the 3′-wing region is a high-affinity modified monomer.

2. The compound of claim 1, wherein the short antisense oligonucleotide consists of 12 linked nucleosides.

3. The compound of claim 1, wherein the short antisense oligonucleotide consists of 13 linked nucleosides.

4. The compound of claim 1, wherein the short antisense oligonucleotide consists of 14 linked nucleosides.

5. The compound of claim 1, wherein the gap region consists of 8 linked 2′-deoxynucleosides.

6. The compound of claim 1, wherein the gap region consists of 9 linked 2′-deoxynucleosides.

7. The compound of claim 1, wherein the gap region consists of 10 linked 2′-deoxynucleosides.

8. The compound of claim 1, wherein the gap region consists of 11 linked 2′-deoxynucleosides.

9. The compound of claim 1, wherein the gap region consists of 12 linked 2′-deoxynucleosides.

10. The compound of claim 1, wherein the short antisense oligonucleotide has a motif selected from 1-10-1; 2-10-1; 1-10-2; 2-10-2; and 2-8-2 wherein, the first number represents the number of modified nucleosides in the 5′-wing region, the second number represents the number of linked 2′-deoxynucleosides in the gap region, and the third number represents the number of modified nucleosides in the 3′-wing region.

11. The compound of claim 10, wherein the motif is selected from 1-10-1; 2-10-2; and 2-8-2.

12. The compound of claim 1, wherein the short antisense compound comprises at least one modified internucleoside linkage.

13. The compound claim 12, wherein the short antisense compound comprises at least one phosphorothioate linkage.

14. The compound of claim 12, wherein each internucleoside linkage of the short antisense oligonucleotide is a modified internucleoside linkage.

15. The compound claim 14, wherein each modified internucleoside linkage is a phosphorothioate linkage.

16. The compound of claim 1, wherein each modified nucleoside of one or both of the 5′-wing region and the 3′-wing region comprises a 2′-O(CH2)2OCH3 substituent.

17. The compound of claim 1, wherein each modified nucleoside of both the 5′-wing region and the 3′-wing region comprises a 2′-O(CH2)2OCH3 substituent.

18. The compound of claim 1, wherein each high-affinity modified monomer is a bicyclic nucleotide.

19. The compound of claim 18, wherein each bicyclic nucleotide comprises a 4′-CH2-O-2′ bridge.

20. The compound of claim 1, wherein each high-affinity modified monomer is a bicyclic nucleotide.