# Patent

## Title
Modulation of dystrophia myotonica-protein kinase (DMPK) expression

## Abstract

Provided herein are methods, compounds, and compositions for reducing expression of a DMPK mRNA and protein in an animal. Also provided herein are methods, compounds, and compositions for preferentially reducing CUGexp DMPK RNA, reducing myotonia or reducing spliceopathy in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate type 1 myotonic dystrophy, or a symptom thereof.

## Background
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under NS072323 awarded by the National Institutes of Health. The government has certain rights in the invention.


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 BIOL0134USC1SEQ.txt created Jul. 28, 2015, which is approximately 220 Mb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD
Provided herein are methods, compounds, and compositions for reducing expression of DMPK mRNA and protein in an animal. Also, provided herein are methods, compounds, and compositions comprising a DMPK inhibitor for preferentially reducing CUGexp DMPK RNA, reducing myotonia, or reducing spliceopathy in an animal. Such methods, compounds, and compositions are useful, for example, to treat, prevent, or ameliorate type 1 myotonic dystrophy (DM1) in an animal.
BACKGROUND
Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults with an estimated frequency of 1 in 7,500 (Harper P S., Myotonic Dystrophy. London: W.B. Saunders Company; 2001). DM1 is an autosomal dominant disorder caused by expansion of a non-coding CTG repeat in DMPK1. DMPK1 is a gene encoding a cytosolic serine/threonine kinase (Brook J D, et al., Cell., 1992, 68(4):799-808). The physiologic functions and substrates of this kinase have not been fully determined. The expanded CTG repeat is located in the 3′ untranslated region (UTR) of DMPK1. This mutation leads to RNA dominance, a process in which expression of RNA containing an expanded CUG repeat (CUGexp) induces cell dysfunction (Osborne R J and Thornton C A., Human Molecular Genetics., 2006, 15(2): R162-R169).
The DMPK gene normally has 5-37 CTG repeats in the 3′ untranslated region. In myotonic dystrophy type I, this number is significantly expanded and is, for example, in the range of 50 to greater than 3,500 (Harper, Myotonic Dystrophy (Saunders, London, ed.3, 2001); Annu. Rev. Neurosci. 29: 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007).
The CUGexp tract interacts with RNA binding proteins including muscleblind-like (MBNL) protein, a splicing factor, and causes the mutant transcript to be retained in nuclear foci. The toxicity of this RNA stems from sequestration of RNA binding proteins and activation of signaling pathways. Studies in animal models have shown that phenotypes of DM1 can be reversed if toxicity of CUGexp RNA is reduced (Wheeler T M, et al., Science., 2009, 325(5938):336-339; Mulders S A, et al., Proc Natl Acad Sci USA., 2009, 106(33):13915-13920).
In DM1, skeletal muscle is the most severely affected tissue, but the disease also has important effects on cardiac and smooth muscle, ocular lens, and brain. The cranial, distal limb, and diaphragm muscles are preferentially affected. Manual dexterity is compromised early, which causes several decades of severe disability. The median age at death is 55 years, usually from respiratory failure (de Die-Smulders C E, et al., Brain., 1998, 121(Pt 8):1557-1563).
Antisense technology is emerging as an effective means for modulating expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of DMPK1. Intramuscular injection of fully modified oligonucleotides targeting with the CAG-repeat were shown in mice to block formation of CUGexp-MBNL1 complexes, disperse nuclear foci of CUGexp transcripts, enhance the nucleocytoplasmic transport and translation of CUGexp transcripts, release MBNL proteins to the nucleoplasm, normalize alternative splicing of MBNL-dependent exons, and eliminate myotonia in CUGexp-expressing transgenic mice (Wheeler T M, et al., Science., 2009, 325(5938):336-339; WO2008/036406).
Presently there is no treatment that can modify the course of DM1. The burden of disease, therefore, is significant. It is, therefore, an object herein to provide compounds, compositions, and methods for treating DM1
SUMMARY
Provided herein are methods, compounds, and compositions for inhibiting expression of DMPK and treating, preventing, delaying or ameliorating a DMPK related disease and or a symptom thereof. In certain embodiments, the compounds and compositions inhibit mutant DMPK or CUGexp DMPK.
Certain embodiments provide a method of reducing DMPK expression in an animal comprising administering to the animal a compound comprising a modified oligonucleotide as further described herein targeted to DMPK.
Certain embodiments provide a method of preferentially reducing CUGexp DMPK, reducing myotonia, or reducing spliceopathy in an animal comprising administering to the animal a compound comprising a modified oligonucleotide, as further described herein, targeted to CUGexp DMPK. CUGexp DMPK transcripts are believed to be particularly sensitive to antisense knockdown via nuclear ribonucleases, because of their longer residence time in the nucleus, and this sensitivity is thought to permit effective antisense inhibition of CUGexp DMPK transcripts in relevant tissues such as muscle despite the biodistribution barriers to tissue uptake of antisense oligonucleotides. Antisense mechanisms that do not elicit cleavage via nuclear ribonucleases, such as the CAG-repeat ASOs described in, for example, Wheeler T M, et al., Science., 2009, 325(5938):336-339 and WO2008/036406, do not provide the same therapeutic advantage.
Certain embodiments provide a method of treating an animal with type 1 myotonic dystrophy. In certain embodiments, the method includes administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide as further described herein targeted to DMPK. In certain embodiments, the method includes identifying an animal with type 1 myotonic dystrophy.
Certain embodiments provide a method of treating, preventing, delaying, or ameliorating symptoms and outcomes associated with development of DM1 including muscle stiffness, myotonia, disabling distal weakness, weakness in face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts. Certain embodiments provide a method of treating, preventing, delaying, or ameliorating symptoms and outcomes associated with development of DM1 in children, including, developmental delays, learning problems, language and speech issues, and personality development issues.
Certain embodiments provide a method of administering an antisense oligonucleotide to counteract RNA dominance by directing the cleavage of pathogenic transcripts.
In certain embodiments, the DMPK has a sequence as set forth in GenBank Accession No. NM_001081560.1 (incorporated herein as SEQ ID NO: 1). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NT_011109.15 truncated from nucleotides 18540696 to Ser. No. 18/555,106 (incorporated herein as SEQ ID NO: 2). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NT_039413.7 truncated from nucleotides 16666001 to Ser. No. 16/681,000 (incorporated herein as SEQ ID NO: 3). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_032418.1 (incorporated herein as SEQ ID NO: 4). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. AI007148.1 (incorporated herein as SEQ ID NO: 5). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. AI304033.1 (incorporated herein as SEQ ID NO: 6). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC024150.1 (incorporated herein as SEQ ID NO: 7). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC056615.1 (incorporated herein as SEQ ID NO: 8). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC075715.1 (incorporated herein as SEQ ID NO: 793). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BU519245.1 (incorporated herein as SEQ ID NO: 794). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CB247909.1 (incorporated herein as SEQ ID NO: 795). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CX208906.1 (incorporated herein as SEQ ID NO: 796). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CX732022.1 (incorporated herein as SEQ ID NO: 797). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. 560315.1 (incorporated herein as SEQ ID NO: 798). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. 560316.1 (incorporated herein as SEQ ID NO: 799). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_001081562.1 (incorporated herein as SEQ ID NO: 800). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_001100.3 (incorporated herein as SEQ ID NO: 801).


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. 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 for the portions of the document discussed herein, as well as in their entirety.
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 can be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
“2′—O-methoxyethyl” (also 2′-MOE and 2′—O(CH2)2—OCH3) refers to an O-methoxy-ethyl modification of the 2′ position of a furanosyl ring. A 2′—O-methoxyethyl modified sugar is a modified sugar.
“2′—O-methoxyethyl nucleotide” means a nucleotide comprising a 2′—O-methoxyethyl modified sugar moiety.
“5-methylcytosine” means a cytosine modified with a methyl group attached to position 5. A 5-methylcytosine is a modified nucleobase.
“About” means within ±7% of a value. For example, if it is stated, “the compound affected at least 70% inhibition of DMPK”, it is implied that the DMPK levels are inhibited within a range of 63% and 77%.
“Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to DMPK is an active pharmaceutical agent.
“Active target region” or “target region” means a region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.
“Administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
“Administering” means providing an agent to an animal, and includes, but is not limited to, administering by a medical professional and self-administering.
“Agent” means an active substance that can provide a therapeutic benefit when administered to an animal. “First Agent” means a therapeutic compound of the invention. For example, a first agent can be an antisense oligonucleotide targeting DMPK. “Second agent” means a second therapeutic compound of the invention (e.g. a second antisense oligonucleotide targeting DMPK) and/or a non-DMPK therapeutic compound.
“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators can be determined by subjective or objective measures, which are known to those skilled in the art.
“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.
“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, snoRNAs, miRNAs, and satellite repeats.
“Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
“Bicyclic sugar” means a furanosyl ring modified by the bridging of two non-geminal carbon ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′—O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′—O-methoxyethyl modifications.
“Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions.
“Co-administration” means administration of two or more agents to an individual. The two or more agents can be in a single pharmaceutical composition, or can be in separate pharmaceutical compositions. Each of the two or more agents can be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
“Contiguous nucleobases” means nucleobases immediately adjacent to each other.
“CUGexp DMPK” means mutant DMPK RNA containing an expanded CUG repeat (CUGexp). The wild-type DMPK gene has 5-37 CTG repeats in the 3′ untranslated region. In a “CUGexp DMPK” (such as in a myotonic dystrophy type I patient) this number is significantly expanded and is, for example, in the range of 50 to greater than 3,500 (Harper, Myotonic Dystrophy (Saunders, London, ed.3, 2001); Annu Rev. Neurosci. 29: 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007).
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.
“DMPK” means any nucleic acid or protein of DMPK. DMPK can be a mutant DMPK including CUGexp DMPK nucleic acid.
“DMPK expression” means the level of mRNA transcribed from the gene encoding DMPK or the level of protein translated from the mRNA. DMPK expression can be determined by art known methods such as a Northern or Western blot.
“DMPK nucleic acid” means any nucleic acid encoding DMPK. For example, in certain embodiments, a DMPK nucleic acid includes a DNA sequence encoding DMPK, an RNA sequence transcribed from DNA encoding DMPK (including genomic DNA comprising introns and exons), and an mRNA or pre-mRNA sequence encoding DMPK. “DMPK mRNA” means an mRNA encoding a DMPK protein.
“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, 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, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month.
“Effective amount” or “therapeutically effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
“Fully complementary” or “100% complementary” means each nucleobase of a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region can be referred to as a “gap segment” and the external regions can be referred to as “wing segments.”
“Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from one to six nucleosides.
“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
“Identifying an animal with type 1 myotonic dystrophy” means identifying an animal having been diagnosed with a type 1 myotonic dystrophy, disorder or condition or identifying an animal predisposed to develop a type 1 myotonic dystrophy, disorder or condition. For example, individuals with a familial history can be predisposed to type 1 myotonic dystrophy, disorder or condition. Such identification can be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.
“Individual” means a human or non-human animal selected for treatment or therapy.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Linked nucleosides” means adjacent nucleosides which are bonded or linked together by an internucleoside linkage.
“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleotide.
“Modified sugar” refers to a substitution or change from a natural sugar. “Motif” means the pattern of chemically distinct regions in an antisense compound. “Myotonia” means an abnormally slow relaxation of a muscle after voluntary contraction or electrical stimulation.
“Nuclear ribonuclease” means a ribonuclease found in the nucleus. Nuclear ribonucleases include, but are not limited to, RNase H including RNase H1 and RNase H2, the double stranded RNase drosha and other double stranded RNases.
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Natural sugar moiety” means a sugar found in DNA (2′—H) or RNA (2′—OH).
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid can also comprise a combination of these elements in a single molecule.
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
“Nucleoside” means a nucleobase linked to a sugar.
“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Nucleotide mimetic” includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage).
“Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. Administration can be continuous, or chronic, or short or intermittent.
“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.
“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a sterile aqueous solution.
“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
“Preferentially reducing CUG exp DMPK RNA” refers to a preferential reduction of RNA transcripts from a CUGexp DMPK allele relative to RNA transcripts from a normal DMPK allele.
“Prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.
“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.
“Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum can indicate liver toxicity or liver function abnormality. For example, increased bilirubin can indicate liver toxicity or liver function abnormality.
“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.
“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.
“Spliceopathy” means a change in the alternative splicing of one or more RNAs that leads to the expression of altered splice products in a particular tissue.
“Subcutaneous administration” means administration just below the skin.
“Sugar surrogate” overlaps with the slightly broader term “nucleoside mimetic” but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.
“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
“Target nucleic acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.
“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5 ‘-most nucleotide of a target segment. “3’ target site” refers to the 3′-most nucleotide of a target segment.
“Therapeutically effective amount” means an amount of an agent that provides a therapeutic benefit to an individual.
“Treat” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.
“Type 1 myotonic dystrophy” or “DM1” means an autosomal dominant disorder caused by expansion of a non-coding CTG repeat in DMPK. This mutation leads to RNA dominance, a process in which expression of RNA containing an expanded CUG repeat (CUGexp) induced cell dysfunction. The CUGexp tract interacts with RNA binding proteins and causes the mutant transcript to be retained in nuclear foci. The toxicity of this RNA stems from sequestration of RNA binding proteins and activation of signaling pathways.
“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
Certain Embodiments
Certain embodiments provide methods, compounds, and compositions for inhibiting DMPK expression.
Certain embodiments provide a method of reducing DMPK expression in an animal comprising administering to the animal a compound comprising a modified oligonucleotide targeting DMPK.
Certain embodiments provide a method of preferentially reducing CUGexp DMPK RNA, reducing myotonia or reducing spliceopathy in an animal comprising administering to the animal a compound comprising a modified oligonucleotide targeted to DMPK, wherein the modified oligonucleotide preferentially reduces CUGexp DMPK RNA, reduces myotonia or reduces spliceopathy in the animal.
Certain embodiments provide a method of administering an antisense oligonucleotide to counteract RNA dominance by directing the cleavage of pathogenic transcripts.
Certain embodiments provide a method of reducing spliceopathy of Serca1. In certain embodiments, methods provided herein result in exon 22 inclusion. In certain embodiments, the corrective splicing occurs in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Certain embodiments provide a method of reducing spliceopathy of m-Titin. In certain embodiments, methods provided herein result in exon 5 inclusion. In certain embodiments, the corrective splicing occurs in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Certain embodiments provide a method of reducing spliceopathy of Clcn1. In certain embodiments, methods provided herein result in exon 7a inclusion. In certain embodiments, the corrective splicing occurs in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Certain embodiments provide a method of reducing spliceopathy of Zasp. In certain embodiments, methods provided herein result in exon 11 inclusion. In certain embodiments, the corrective splicing occurs in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Certain embodiments provide a method for treating an animal with type 1 myotonic dystrophy comprising: a) identifying said animal with type 1 myotonic dystrophy, and b) administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide targeted to DMPK. In certain embodiments, the therapeutically effective amount of the compound administered to the animal preferentially reduces CUGexp DMPK RNA, reduces myotonia or reduces spliceopathy in the animal.
Certain embodiments provide a method of achieving a preferential reduction of CUGexp DMPK RNA, including administering to the subject suspected of having type 1 myotonic dystrophy or having a CUGexp DMPK RNA a modified antisense oligonucleotide complementary to a non-repeat region of said CUGexp DMPK RNA. The modified antisense oligonucleotide, when bound to said CUGexp DMPK RNA, achieves a preferential reduction of the CUGexp DMPK RNA.
Certain embodiments provide a method of achieving a preferential reduction of CUGexp DMPK RNA, including selecting a subject having type 1 myotonic dystrophy or having a CUGexp DMPK RNA and administering to said subject a modified antisense oligonucleotide complementary to a non-repeat region of said CUGexp DMPK RNA. The modified antisense oligonucleotide, when bound to the CUGexp DMPK RNA, activates a ribonuclease or nuclear ribonuclease, thereby achieving a preferential reduction of the CUGexp DMPK RNA in the nucleus.
Certain embodiments provide a method of achieving a preferential reduction of CUGexp DMPK RNA, including selecting a subject having type 1 myotonic dystrophy or having a mutant or CUGexp DMPK RNA and systemically administering to said subject a modified antisense oligonucleotide complementary to a non-repeat region of said CUGexp DMPK RNA. The modified antisense oligonucleotide, when bound to the mutant or CUGexp DMPK RNA, achieves a preferential reduction of the mutant or CUGexp DMPK RNA.
Certain embodiments provide a method of reducing myotonia in a subject in need thereof. The method includes administering to the subject a modified antisense oligonucleotide complementary to a non-repeat region of a DMPK RNA, wherein the modified antisense oligonucleotide, when bound to the DMPK RNA, activates a ribonuclease or nuclear ribonuclease, thereby reducing myotonia. In certain embodiments, the subject has or is suspected of having type 1 myotonic dystrophy or having a mutant DMPK RNA or CUGexp DMPK RNA. In certain embodiments, the DMPK RNA is nuclear retained.
Certain embodiments provide a method of reducing spliceopathy in a subject in need thereof. The method includes administering to the subject a modified antisense oligonucleotide complementary to a non-repeat region of a DMPK RNA, wherein the modified antisense oligonucleotide, when bound to the DMPK RNA, activates a ribonuclease or nuclear ribonuclease, thereby reducing spliceopathy. In certain embodiments, the subject has or is suspected of having type 1 myotonic dystrophy or having a nuclear retained CUGexp DMPK RNA. In certain embodiments, the DMPK RNA is nuclear retained. In certain embodiments, the spliceopathy is MBNL dependent spliceopathy.
In certain embodiments, the modified antisense oligonucleotide of the methods is chimeric. In certain embodiments, the modified antisense oligonucleotide of the methods is a gapmer.
In certain embodiments of the methods provided herein, the administering is subcutaneous. In certain embodiments, the administering is intravenous.
In certain embodiments, the modified antisense oligonucleotide of the methods targets a non-coding sequence within the non-repeat region of a DMPK RNA. In certain embodiments, the oligonucleotide targets a coding region, an intron, a 5′UTR, or a 3′UTR of the mutant DMPK RNA.
In certain embodiments of the methods provided herein, the nuclear ribonuclease is RNase H1.
In certain embodiments of the methods, the DMPK RNA is reduced in muscle tissue. In certain embodiments, the mutant DMPK RNA CUGexp DMPK RNA is preferentially reduced.
In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_001081560.1 (incorporated herein as SEQ ID NO: 1). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NT_011109.15 truncated from nucleotides 18540696 to Ser. No. 18/555,106 (incorporated herein as SEQ ID NO: 2). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NT_039413.7 truncated from nucleotides 16666001 to Ser. No. 16/681,000 (incorporated herein as SEQ ID NO: 3). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_032418.1 (incorporated herein as SEQ ID NO: 4). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. AI007148.1 (incorporated herein as SEQ ID NO: 5). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. AI304033.1 (incorporated herein as SEQ ID NO: 6). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC024150.1 (incorporated herein as SEQ ID NO: 7). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC056615.1 (incorporated herein as SEQ ID NO: 8). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BC075715.1 (incorporated herein as SEQ ID NO: 793). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. BU519245.1 (incorporated herein as SEQ ID NO: 794). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CB247909.1 (incorporated herein as SEQ ID NO: 795). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CX208906.1 (incorporated herein as SEQ ID NO: 796). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. CX732022.1 (incorporated herein as SEQ ID NO: 797). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. 560315.1 (incorporated herein as SEQ ID NO: 798). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. 560316.1 (incorporated herein as SEQ ID NO: 799). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_001081562.1 (incorporated herein as SEQ ID NO: 800). In certain embodiments, the DMPK has the sequence as set forth in GenBank Accession No. NM_001100.3 (incorporated herein as SEQ ID NO: 801).
In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 9, at least 10, or at least 11, contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 13, or at least 14, contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 15 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 16, or at least 17, contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 18 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 19 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
In certain embodiments, the modified oligonucleotides provided herein are targeted to any one of the following regions of SEQ ID NO: 1: 1178-1206, 2159-2182, 2174-2196, 2426-2447, 2450-2518, 2679-2704, and 2697-2725.
In certain embodiments, the modified oligonucleotides provided herein are targeted to any one of the following regions of SEQ ID NO 1: 178-223, 232-253, 279-299, 366-399, 519-541, 923-975, 1073-1105, 1171-1196, 1215-1246, 1263-1324, 1706-1734, 1743-1763, 1932-1979, 1981-2003, 2077-2108, and 2152-2173.
In certain embodiments, the modified oligonucleotides provided herein are targeted to any one of the following regions of SEQ ID NO: 2: 1251-1303, 1305-1326, 1352-1372, 3762-3795, 4170-4192, 5800-5852, 6124-6149, 6168-6199, 6216-6277, 11979-12007, 12016-12036, 12993-13042, 13044-13066, 13140-13171, and 13215-13236.
In certain embodiments, the animal is a human.
In certain embodiments, the compounds or compositions of the invention are designated as a first agent and the methods of the invention further comprise administering a second agent. In certain embodiments, the first agent and the second agent are co-administered. In certain embodiments the first agent and the second agent are co-administered sequentially or concomitantly.
In certain embodiments, administration comprises parenteral administration.
In certain embodiments, the compound is a single-stranded modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to any one of SEQ ID NOs: 1-8 and 793-801 as measured over the entirety of said modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to any one of SEQ ID NOs: 1-8 and 793-801 as measured over the entirety of said modified oligonucleotide.
In certain embodiments, at least one internucleoside linkage of said modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar comprises a 2′—O-methoxyethyl or a 4′—(CH2)n—O-2′ bridge, wherein n is 1 or 2.
In certain embodiments, at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide comprises: a) a gap segment consisting of linked deoxynucleosides; b) a 5′ wing segment consisting of linked nucleosides; and c) a 3′ wing segment consisting of linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment and each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises: a) a gap segment consisting of ten linked deoxynucleosides; b) a 5′ wing segment consisting of five linked nucleosides; and c) a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar, each internucleoside linkage of said modified oligonucleotide is a phosphorothioate linkage, and each cytosine in said modified oligonucleotide is a 5′-methylcytosine.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
Certain embodiments provide a method of preferentially reducing CUGexp DMPK RNA, reducing myotonia or reducing spliceopathy in an animal comprising administering to the animal a compound comprising a modified oligonucleotide having a gap segment consisting of ten linked deoxynucleosides, a 5′ wing segment consisting of five linked nucleosides and a 3′ wing segment consisting of five linked nucleosides. The gap segment is positioned between the 5′ wing segment and the 3′ wing segment, each nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar, each internucleoside linkage of said modified oligonucleotide is a phosphorothioate linkage, each cytosine in said modified oligonucleotide is a 5′-methylcytosine.
Certain embodiments provide the use of any compound as described herein in the manufacture of a medicament for use in any of the therapeutic methods described herein. For example, certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for treating, ameliorating, or preventing type 1 myotonic dystrophy. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for inhibiting expression of DMPK and treating, preventing, delaying or ameliorating a DMPK related disease and or a symptom thereof. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for reducing DMPK expression in an animal. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for preferentially reducing CUGexp DMPK, reducing myotonia, or reducing spliceopathy in an animal. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for treating an animal with type 1 myotonic dystrophy. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for treating, preventing, delaying, or ameliorating symptoms and outcomes associated with development of DM1 including muscle stiffness, myotonia, disabling distal weakness, weakness in face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts. Certain embodiments provide the use of a compound as described herein in the manufacture of a medicament for counteracting RNA dominance by directing the cleavage of pathogenic transcripts.
Certain embodiments provide a kit for treating, preventing, or ameliorating type 1 myotonic dystrophy as described herein wherein the kit comprises: a) a compound as described herein; and optionally b) an additional agent or therapy as described herein. The kit can further include instructions or a label for using the kit to treat, prevent, or ameliorate type 1 myotonic dystrophy.
Certain embodiments provide any compound or composition as described herein, for use in any of the therapeutic methods described herein. For example, certain embodiments provide a compound or composition as described herein for inhibiting expression of DMPK and treating, preventing, delaying or ameliorating a DMPK related disease and or a symptom thereof. Certain embodiments provide a compound or composition as described herein for use in reducing DMPK expression in an animal. Certain embodiments provide a compound or composition as described herein for use in preferentially reducing CUGexp DMPK, reducing myotonia, or reducing spliceopathy in an animal. Certain embodiments provide a compound or composition as described herein for use in treating an animal with type 1 myotonic dystrophy. Certain embodiments provide a compound or composition as described herein for use in treating, preventing, delaying, or ameliorating symptoms and outcomes associated with development of DM1 including muscle stiffness, myotonia, disabling distal weakness, weakness in face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts. Certain embodiments provide a compound or composition as described herein for use in counteracting RNA dominance by directing the cleavage of pathogenic transcripts. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-156, 160-770, and 774-792.
Other compounds which can be used in the methods described herein are also provided.
For example, certain embodiments provide compounds comprising a modified oligonucleotide consisting of 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 41, 44, 76, 109, 153, 320, 321, 322, 325, 329, 335, and 657.
Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20, linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 15, 73, 77, 79, 83, 85, 130, 602, 648, 655, 674, and 680.
Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20, linked nucleosides having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, or more, contiguous nucleobases complementary to an equal length portion of nucleobases 664-683, 773-792, 926-945, 927-946, 928-947, 931-950, 935-954, 941-960, 2089-2108, 2163-2182, 2490-2509, 2499-2518, 2676-2695, 2685-2704, 2676-2695, 2688-2707, 2697-2716, 2764-2783, and 2770-2789 of SEQ ID NO: 1, wherein the nucleobase sequence is complementary to SEQ ID NO: 1.
Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 10 to 80, 12 to 50, 12 to 30, 15 to 30, 18 to 24, 19 to 22, or 20, linked nucleosides having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, or more, contiguous nucleobases complementary to an equal length portion of nucleobases 812-831, 3629-3648, 4447-4466, 4613-4632, 5803-5822, 5804-5823, 5805-5824, 5808-5827, 5818-5837, 6794-6813, 12463-12482, 13152-13171, and 13553-13572 of SEQ ID NO: 2, wherein the nucleobase sequence is complementary to SEQ ID NO: 2.
In certain embodiments, the modified oligonucleotide is a single-stranded oligonucleotide.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, complementary to any of SEQ ID NOs: 1-8 and 793-801.
In certain embodiments, at least one internucleoside linkage is a modified internucleoside linkage.
In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, at least one nucleoside comprises a modified sugar.
In certain embodiments, at least one modified sugar is a bicyclic sugar.
In certain embodiments, at least one modified sugar comprises a 2′—O-methoxyethyl.
In certain embodiments, at least one nucleoside comprises a modified nucleobase.
In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides; and
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides; and
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides.
In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
Antisense Compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound can be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to DMPK as described herein is 10 to 30 nucleotides in length. In other words, the antisense compounds are in some embodiments from 10 to 30 linked nucleobases. In other embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 30, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked nucleobases. In certain such embodiments, the antisense compound comprises a modified oligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked nucleobases in length, or a range defined by any two of the above values. In certain embodiments, antisense compounds of any of these lengths contain at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, contiguous nucleobases of the nucleobase sequence of any of the exemplary antisense compounds described herein (e.g., at least 8 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
In certain embodiments, the antisense compound comprises a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide can have a single nucleoside deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated oligonucleotide can have two nucleosides deleted from the 5′ end, or alternatively can have two subunits deleted from the 3′ end. Alternatively, the deleted nucleosides can be dispersed throughout the modified oligonucleotide, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.
When a single additional nucleoside is present in a lengthened oligonucleotide, the additional nucleoside can be located at the 5′ or 3′ end of the oligonucleotide. When two or more additional nucleosides are present, the added nucleosides can be adjacent to each other, for example, in an oligonucleotide having two nucleosides added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the oligonucleotide. Alternatively, the added nucleoside can be dispersed throughout the antisense compound, for example, in an oligonucleotide having one nucleoside added to the 5′ end and one subunit added to the 3′ end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a DMPK nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound can optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer can in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides can include 2′-MOE, and 2′—O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides can include those having a 4′—(CH2)n—O—2′ bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap segment is positioned immediately adjacent each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6, 5-8-5, 1-8-1, or 2-6-2.
In certain embodiments, the antisense compound as a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer configurations include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or 5-13.
In certain embodiments, antisense compounds targeted to a DMPK nucleic acid possess a 5-10-5 gapmer motif.
In certain embodiments, an antisense compound targeted to a DMPK nucleic acid has a gap-widened motif
In certain embodiments, antisense compounds of any of these gapmer or wingmer motifs contain at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, contiguous nucleobases of the nucleobase sequence of any of the exemplary antisense compounds described herein (e.g., at least 8 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode DMPK include, without limitation, the following sequences as set forth in GenBank Accession No. NM_001081560.1 (incorporated herein as SEQ ID NO: 1), GenBank Accession No. NT_011109.15 truncated from nucleotides 18540696 to Ser. No. 18/555,106 (incorporated herein as SEQ ID NO: 2), GenBank Accession No. NT_039413.7 truncated from nucleotides 16666001 to Ser. No. 16/681,000 (incorporated herein as SEQ ID NO: 3), GenBank Accession No. NM_032418.1 (incorporated herein as SEQ ID NO: 4), GenBank Accession No. AI007148.1 (incorporated herein as SEQ ID NO: 5), GenBank Accession No. AI304033.1 (incorporated herein as SEQ ID NO: 6), GenBank Accession No. BC024150.1 (incorporated herein as SEQ ID NO: 7), GenBank Accession No. BC056615.1 (incorporated herein as SEQ ID NO: 8), GenBank Accession No. BC075715.1 (incorporated herein as SEQ ID NO: 793), GenBank Accession No. BU519245.1 (incorporated herein as SEQ ID NO: 794), GenBank Accession No. CB247909.1 (incorporated herein as SEQ ID NO: 795), GenBank Accession No. CX208906.1 (incorporated herein as SEQ ID NO: 796), GenBank Accession No. CX732022.1 (incorporated herein as SEQ ID NO: 797), GenBank Accession No. 560315.1 (incorporated herein as SEQ ID NO: 798), GenBank Accession No. 560316.1 (incorporated herein as SEQ ID NO: 799), GenBank Accession No. NM_001081562.1 (incorporated herein as SEQ ID NO: 800), and GenBank Accession No. NM_001100.3 (incorporated herein as SEQ ID NO: 801). It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO can 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 motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region can encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for DMPK can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region can encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the target region.
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
A target region can contain one or more target segments. Multiple target segments within a target region can be overlapping. Alternatively, they can be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.
Suitable target segments can be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment can specifically exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments can include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that can hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
There can be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in DMPK mRNA levels are indicative of inhibition of DMPK protein expression. Reductions in levels of a DMPK protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes, such as a reducing myotonia or reducing spliceopathy, can be indicative of inhibition of DMPK mRNA and/or protein expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a DMPK nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., 2001). In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a DMPK nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a DMPK nucleic acid).
An antisense compound can hybridize over one or more segments of a DMPK nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a DMPK nucleic acid, a target region, target segment, or specified portion thereof. In certain embodiments, the antisense compounds are at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a DMPK nucleic acid, a target region, target segment, or specified portion thereof, and contain at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, contiguous nucleobases of the nucleobase sequence of any of the exemplary antisense compounds described herein (e.g., at least 8 contiguous nucleobases of a nucleobase sequence recited in any one of SEQ ID NOs: 12-156, 160-770, and 774-792). Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods, and is measured over the entirety of the antisense compound.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases can be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, antisense compound can be fully complementary to a DMPK nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound can be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase can be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases can be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they can be either contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a DMPK nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a DMPK nucleic acid, or specified portion thereof.
The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least an 8, at least a 9, at least a 10, at least an 11, at least a 12, at least a 13, at least a 14, at least a 15, at least a 16, at least a 17, at least an 18, at least a 19, at least a 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein can also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases can be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, at least 75%, 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 one or more of the exemplary antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides can also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a DMPK nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R, R1 and R2 are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′—S, 2′—F, 2′—OCH3, 2′—OCH2CH3, 2′—OCH2CH2F and 2′—O(CH2)2OCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), O—CH2—C(═O)—N(Rm)(Rn), and O—CH2—C(═O)—N(R1)—(CH2)2—N(Rm)(Rn), where each Rl, Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH2)—O—2′ (LNA); 4′—(CH2)—S—2; 4′—(CH2)2—O—2′ (ENA); 4′—CH(CH3)—O—2′ and 4′-CH(CH2OCH3)—O—2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′—C(CH3)(CH3)—O—2′ (and analogs thereof see PCT/US2008/068922 published as WO/2009/006478, published Jan. 8, 2009); 4′—CH2—N(OCH3)—2′ (and analogs thereof see PCT/US2008/064591 published as WO/2008/150729, published Dec. 11, 2008); 4′—CH2—O—N(CH3)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O—2′, wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ (and analogs thereof see PCT/US2008/066154 published as WO 2008/154401, published on Dec. 8, 2008).
Further bicyclic nucleosides have been reported in published literature (see for example: Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; U.S. Pat. Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication Nos.: US2008-0039618; US2007-0287831; US2004-0171570; U.S. patent application Ser. Nos. 12/129,154; 61/099,844; 61/097,787; 61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574; International applications WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226; and PCT International Applications Nos.: PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic nucleosides comprise a bridge between the 4′ and the 2′ carbon atoms of the pentofuranosyl sugar moiety including without limitation, bridges comprising 1 or from 1 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Ra and Rb 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 certain embodiments, the bridge of a bicyclic sugar moiety is, —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or —C(RaRb)—O—N(R)—. In certain embodiments, the bridge is 4′—CH2—2′, 4′—(CH2)2-2′, 4′—(CH2)3—2′, 4′—CH2—O—2′, 4′—(CH2)2—O—2′, 4′—CH2—O—N(R)—2′ and 4′—CH2—N(R)—O—2′-wherein each R is, independently, H, a protecting group or C1—C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′—(CH2)—O—2′ bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O—2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include those having a 4′ to 2′ bridge wherein such bridges include without limitation, α-L-4′—(CH2)—O—2′, β-D-4′—CH2—O—2′, 4′—(CH2)2—O—2′, 4′—CH2—O—N(R)-2′, 4′—CH2—N(R)—O—2′, 4′—CH(CH3)—O—2′, 4′—CH2—S—2′, 4′—CH2—N(R)-2′, 4′-CH2—CH(CH3)-2′, and 4′—(CH2)3-2′, wherein R is H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
-Qa-Qb-Qc-is —CH2—N(Rc)—CH2—, —C(═O)—N(Rc)—CH2—, —CH2—O—N(Rc)—, —CH2—N(Rc)—O— or —N(Rc)—O—CH2;
Rc is C1-C12 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za 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, substituted amide, thiol or substituted thiol.
In one embodiment, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJc, N3, OC(═X)Jc, and NJeC(═X)NJcJd, wherein each Jc, Jd and Je is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJc.
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(═O)—).
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl;
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(═O)OJj, C(═O)NJjJk, C(═O)Jj, O—C(═O)NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk or N(H)C(═S)NJjJk;
or qe and qf together are ═C(qg)(qh);
qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a 4′-CH2—O-2′ bridge, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides has also been described in WO 98/39352 and WO 99/14226.
Analogs of various bicyclic nucleosides that have 4′ to 2′ bridging groups such as 4′—CH2—O—2′ and 4′—CH2—S—2′, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide duplexes comprising bicyclic nucleosides for use 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.
In certain embodiments, bicyclic nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
each qi, qj, qk and ql is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C1-C12 alkoxyl, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(═O)OJj, C(═O)NJjJk, C(═O)Jj, O—C(═O)NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk or N(H)C(═S)NJjJk; and
qi and qj or ql and qk together are ═C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4′—(CH2)3-2′ bridge and the alkenyl analog bridge 4′—CH═CH—CH2-2′ have been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
In certain embodiments, bicyclic nucleosides 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, (E) oxyamino (4′—CH2—N(R)—O—-2′) BNA, (F) methyl(methyleneoxy) (4′—CH(CH3)—O—2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′—CH2—S-2′) BNA, (H) methylene-amino (4′—CH2—N(R)—2′) BNA, (I) methyl carbocyclic (4′—CH2—CH(CH3)-2′) BNA, (J) propylene carbocyclic (4′—(CH2)3-2′) BNA, and (K) vinyl BNA as depicted below.







wherein Bx is the base moiety and R is, independently, H, a protecting group, C1-C6 alkyl or C1-C6 alkoxy.

In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:




In certain embodiments, sugar surrogates are selected having the formula:




wherein:

Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the oligomeric compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;
q1, q2, q3, q4, q5, q6 and q7 are each independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and
one of R1 and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein X is O, S or NJ1 and each J1, J2 and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
Such sugar surrogates include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10, 841-854).
In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have the formula:




wherein:

Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′-or 3′-terminal group; and q1, q2, q3, q4, q5, q6, q7, q8 and q9 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituent group.
Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, 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; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds targeted to a DMPK nucleic acid comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif.
Modified Nucleobases
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional unmodified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to a DMPK nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides targeted to a DMPK nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense oligonucleotides can be admixed with pharmaceutically acceptable active or inert substance for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Antisense compound targeted to a DMPK nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a DMPK nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide 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 pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
Conjugated Antisense Compounds
Antisense compounds can be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
Cell Culture and Antisense Compounds Treatment
The effects of antisense compounds on the level, activity or expression of DMPK nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commercial vendors (e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.
In Vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluence in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a Cytofectin® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
Inhibition of levels or expression of a DMPK nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
Quantitation of target RNA levels can be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carried out by methods well known to those skilled in the art.
Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN® fluorescence.
Probes and primers are designed to hybridize to a DMPK nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and can include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, Calif.).
Analysis of Protein Levels
Antisense inhibition of DMPK nucleic acids can be assessed by measuring DMPK protein levels. Protein levels of DMPK can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
In Vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of DMPK and produce phenotypic changes. Testing can be performed in normal animals, or in experimental disease models, for example, the HSALR mouse model of myotonic dystrophy (DM1).
The HSALR mouse model is an established model for DM1 (Mankodi, A. et al. Science. 289: 1769, 2000). The mice carry a human skeletal actin (hACTA1) transgene with 220 CTG repeats inserted in the 3′ UTR of the gene. The hACTA1-CUGexp transcript accumulates in nuclear foci in skeletal muscles and results in myotonia similar to that in human DM1 (Mankodi, A. et al. Mol. Cell 10: 35, 2002; Lin, X. et al. Hum. Mol. Genet. 15: 2087, 2006). Hence, it is expected that amelioration of DM1 symptoms in the HSALR mouse by antisense inhibition of the hACTA1 transgene would predict amelioration of similar symptoms in human patients by antisense inhibition of the DMPK transcript.
Expression of CUGexp RNA in mice causes extensive remodeling of the muscle transcriptome, much of which is reproduced by ablation of MBNL1. Hence, it is expected that normalization of the transcriptome in HSALR mice would predict normalization of the human transcriptome in DM1 patients by antisense inhibition of the DMPK transcript.
For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration. Following a period of treatment with antisense oligonucleotides, RNA is isolated from tissue and changes in DMPK nucleic acid expression are measured. Changes in DMPK protein levels are also measured.
Splicing
Myotonic dystrophy (DM1) is caused by CTG repeat expansions in the 3′ untranslated region of the DMPK gene (Brook, J. D. et al. Cell. 68: 799, 1992). This mutation leads to RNA dominance, a process in which expression of RNA containing an expanded CUG repeat (CUGexp) induces cell dysfunction (Osborne R J and Thornton C A., Human Molecular Genetics., 2006, 15(2): R162-R169). Such CUGexp are retained in the nuclear foci of skeletal muscles (Davis, B. M. et al. Proc. Natl. Acad. Sci. U.S.A. 94:7388, 1997). The accumulation of CUGexp in the nuclear foci leads to the sequestration of poly(CUG)-binding proteins, such as, Muscleblind-like 1 (MBLN1) (Miller, J. W. et al. EMBO J. 19: 4439, 2000). MBLN1 is a splicing factor and regulates the splicing of genes such as Serca1, CIC-1, Titin, and Zasp. Therefore, sequestration of MBLN1 by CUGexp triggers misregulated alternative splicing of the exons of genes that MBLN1 normally controls (Lin, X. et al. Hum. Mol. Genet. 15: 2087, 2006). Correction of alternative splicing in an animal displaying such disregulation, such as, for example, in a DM1 patient and the HSALR mouse model, is a useful indicator for the efficacy of a treatment, including treatment with an antisense oligonucleotide.
Certain Biomarkers
DM1 severity in mouse models is determined, at least in part, by the level of CUGexp transcript accumulation in the nucleus or nuclear foci. A useful physiological marker for DM1 severity is the development of high-frequency runs of involuntary action potentials (myotonia).
Certain Indications
In certain embodiments, provided herein are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has type 1 myotonic dystrophy (DM1).
Accordingly, provided herein are methods for ameliorating a symptom associated with type 1 myotonic dystrophy in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with type 1 myotonic dystrophy. In certain embodiments, provided is a method for reducing the severity of a symptom associated with type 1 myotonic dystrophy. In certain embodiments, symptoms associated with DM1 include muscle stiffness, myotonia, disabling distal weakness, weakness in face and jaw muscles, difficulty in swallowing, drooping of the eyelids (ptosis), weakness of neck muscles, weakness in arm and leg muscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia, respiratory insufficiency, irregular heartbeat, heart muscle damage, apathy, insulin resistance, and cataracts. In children, the symptoms may also be developmental delays, learning problems, language and speech issues, and personality development issues.
In certain embodiments, the methods comprise administering to an individual in need thereof a therapeutically effective amount of a compound targeted to a DMPK nucleic acid.
In certain embodiments, administration of an antisense compound targeted to a DMPK nucleic acid results in reduction of DMPK expression by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, by at least about 50%, by at least about 55%, by at least about 60%, by least about 65%, by least about 70%, by least about 75%, by least about 80%, by at least about 85%, by at least about 90%, by at least about 95% or by at least about 99%, or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to DMPK are used for the preparation of a medicament for treating a patient suffering or susceptible to type 1 myotonic dystrophy.
In certain embodiments, the methods described herein include administering a compound comprising a modified oligonucleotide having a contiguous nucleobases portion as described herein of a sequence recited in SEQ ID NO: 12-156, 160-770, and 774-792.
Administration
In certain embodiments, the compounds and compositions as described herein are administered parenterally.
In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection (e.g., bolus injection). The injection can be delivered with a syringe.
Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration. Administration can be continuous, or chronic, or short, or intermittent.
In certain embodiments, the administering is subcutaneous, intravenous, intracerebral, intracerebroventricular, intrathecal or another administration that results in a systemic effect of the oligonucleotide (systemic administration is characterized by a systemic effect, i.e., an effect in more than one tissue) or delivery to the CNS or to the CSF.
The duration of action as measured by inhibition of alpha 1 actin and reduction of myotonia in the HSALR mouse model of DM1 is prolonged in muscle tissue including quadriceps, gastrocnemius, and the tibialis anterior (see Examples, below). Subcutaneous injections of antisense oligonucleotide for 4 weeks results in inhibition of alpha 1 actin by at least 70% in quadriceps, gastrocnemius, and the tibialis anterior in HSALR mice for at least 11 weeks (77 days) after termination of dosing. Subcutaneous injections of antisense oligonucleotide for 4 weeks results in elimination of myotonia in quadriceps, gastrocnemius, and the tibialis anterior in HSALR mice for at least 11 weeks (77 days) after termination of dosing.
In certain embodiments, delivery of a compound of composition, as described herein, results in at least 70% down-regulation of a target mRNA and/or target protein for at least 77 days. In certain embodiments, delivery of a compound or composition, as described herein, results in 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% down-regulation of a target mRNA and/or target protein for at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 76 days, at least 77 days, at least 78 days, at least 79 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, at least 1 year.
In certain embodiments, an antisense oligonucleotide is delivered by injection or infusion once every 77 days. In certain embodiments, an antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every three months, every 6 months, twice a year or once a year.
Certain Combination Therapies
In certain embodiments, a first agent comprising the modified oligonucleotide of the invention is co-administered with one or more secondary agents. In certain embodiments, such second agents are designed to treat the same type 1 myotonic dystrophy as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder, or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, second agents are co-administered with the first agent to treat an undesired effect of the first agent. In certain embodiments, second agents are co-administered with the first agent to produce a combinational effect. In certain embodiments, second agents are co-administered with the first agent to produce a synergistic effect.
In certain embodiments, a first agent and one or more second agents are administered at the same time. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.
EXAMPLES
Non-Limiting 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 recited in the present application is incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of Human Dystrophia Myotonica Protein Kinase (DMPK) in Human Skeletal Muscle Cells (hSKMC)
Antisense oligonucleotides targeted to a human DMPK nucleic acid were tested for their effect on DMPK RNA transcript in vitro. Cultured hSKM cells at a density of 20,000 cells per well were transfected using electroporation with 100 nM antisense oligonucleotide. After approximately 24 hours, RNA was isolated from the cells and DMPK RNA transcript levels were measured by quantitative real-time PCR with human primer probe set RTS3164 (forward sequence AGCCTGAGCCGGGAGATG, designated herein as SEQ ID NO: 9; reverse sequence GCGTAGTTGACTGGCGAAGTT, designated herein as SEQ ID NO: 10; probe sequence AGGCCATCCGCACGGACAACCX, designated herein as SEQ ID NO: 11). DMPK RNA transcript levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of hDMPK, relative to untreated control cells.
The antisense oligonucleotides in Tables 1 and 2 are 5-10-5 gapmers, where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises five 2′-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. ‘Target start site’ indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted. ‘Target stop site’ indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted. All the antisense oligonucleotides listed in Table 1 target SEQ ID NO: 1 (GENBANK Accession No. NM_001081560.1). All the antisense oligonucleotides listed in Table 2 target SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_011109.15 truncated from nucleotides 18540696 to 18555106).
Several antisense oligonucleotides demonstrated significant inhibition of human DMPK mRNA levels under the conditions specified above.







TABLE 1







Inhibition of human DMPK RNA transcript in hSKMC by 5-10-5


gapmers targeting SEQ ID NO: 1












Target
Target






Start
Stop


%
SEQ ID


Site
Site
ISIS No
Sequence
inhibition
NO.















93
112
299476
CTGGCTGCATGTCTGCCTGT
81
12


 


277
296
299479
CCAGGAGAAGGTCGAGCAGG
57
13


 


737
756
299493
TCTATGGCCATGACAATCTC
57
14


 


773
792
299494
ATGTCCCTGTGCACGTAGCC
77
15


 


1194
1213
299501
ATGTGTCCGGAAGTCGCCTG
50
16


 


1628
1647
299511
CTCAGGCTCTGCCGGGTGAG
70
17


 


1855
1874
299517
GGCACTGGCCCACAGCCACG
78
18


 


2379
2398
299526
CCTGGCCGAAAGAAAGAAAT
31
19


 


2367
2386
444380
AAAGAAATGGTCTGTGATCC
56
20


 


2370
2389
444381
AAGAAAGAAATGGTCTGTGA
77
21


 


2376
2395
444382
GGCCGAAAGAAAGAAATGGT
61
22


 


2385
2404
444383
CCTCAGCCTGGCCGAAAGAA
57
23


 


2388
2407
444384
GGGCCTCAGCCTGGCCGAAA
65
24


 


2391
2410
444385
TCAGGGCCTCAGCCTGGCCG
61
25


 


2411
2430
444386
CTGCAGTTTGCCCATCCACG
68
26


 


2414
2433
444387
GGCCTGCAGTTTGCCCATCC
77
27


 


2417
2436
444388
CCAGGCCTGCAGTTTGCCCA
54
28


 


2423
2442
444389
GCCTTCCCAGGCCTGCAGTT
77
29


 


2426
2445
444390
GCTGCCTTCCCAGGCCTGCA
83
30


 


2429
2448
444391
CTTGCTGCCTTCCCAGGCCT
69
31


 


2435
2454
444392
GCCCGGCTTGCTGCCTTCCC
82
32


 


2438
2457
444393
ACGGCCCGGCTTGCTGCCTT
78
33


 


2441
2460
444394
CGGACGGCCCGGCTTGCTGC
57
34


 


2444
2463
444395
ACACGGACGGCCCGGCTTGC
73
35


 


2450
2469
444396
GATGGAACACGGACGGCCCG
80
36


 


2453
2472
444397
GAGGATGGAACACGGACGGC
86
37


 


2456
2475
444398
GTGGAGGATGGAACACGGAC
84
38


 


2481
2500
444399
GCGAACCAACGATAGGTGGG
80
39


 


2484
2503
444400
TTTGCGAACCAACGATAGGT
86
40


 


2490
2509
444401
TTGCACTTTGCGAACCAACG
89
41


 


2493
2512
444402
GCTTTGCACTTTGCGAACCA
89
42


 


2496
2515
444403
AAAGCTTTGCACTTTGCGAA
83
43


 


2499
2518
444404
AAGAAAGCTTTGCACTTTGC
91
44


 


2502
2521
444405
CACAAGAAAGCTTTGCACTT
70
45


 


2508
2527
444406
GTCATGCACAAGAAAGCTTT
34
46


 


2527
2546
444407
ACGCTCCCCAGAGCAGGGCG
39
47


 


2543
2562
444408
GCAGAGATCGCGCCAGACGC
85
48


 


2546
2565
444409
CAGGCAGAGATCGCGCCAGA
65
49


 


2549
2568
444410
AAGCAGGCAGAGATCGCGCC
84
50


 


2555
2574
444411
CCGAGTAAGCAGGCAGAGAT
58
51


 


2558
2577
444412
TTCCCGAGTAAGCAGGCAGA
70
52


 


2564
2583
444413
GCAAATTTCCCGAGTAAGCA
62
53


 


2567
2586
444414
AAAGCAAATTTCCCGAGTAA
53
54


 


2573
2592
444415
TTGGCAAAAGCAAATTTCCC
64
55


 


2576
2595
444416
GGTTTGGCAAAAGCAAATTT
23
56


 


2579
2598
444417
GCGGGTTTGGCAAAAGCAAA
70
57


 


2582
2601
444418
AAAGCGGGTTTGGCAAAAGC
43
58


 


2588
2607
444419
CCCGAAAAAGCGGGTTTGGC
71
59


 


2591
2610
444420
ATCCCCGAAAAAGCGGGTTT
53
60


 


2595
2614
444421
CGGGATCCCCGAAAAAGCGG
45
61


 


2598
2617
444422
GCGCGGGATCCCCGAAAAAG
48
62


 


2623
2642
444423
GAGAGCAGCGCAAGTGAGGA
77
63


 


2626
2645
444424
TCCGAGAGCAGCGCAAGTGA
62
64


 


2629
2648
444425
GGCTCCGAGAGCAGCGCAAG
79
65


 


2649
2668
444426
AAGCGGGCGGAGCCGGCTGG
20
66


 


2652
2671
444427
CCGAAGCGGGCGGAGCCGGC
0
67


 


2658
2677
444428
AAACCGCCGAAGCGGGCGGA
0
68


 


2661
2680
444429
TCCAAACCGCCGAAGCGGGC
45
69


 


2664
2683
444430
ATATCCAAACCGCCGAAGCG
31
70


 


2667
2686
444431
TAAATATCCAAACCGCCGAA
42
71


 


2670
2689
444432
CAATAAATATCCAAACCGCC
53
72


 


2676
2695
444433
CGAGGTCAATAAATATCCAA
63
73


 


2679
2698
444434
GGACGAGGTCAATAAATATC
83
74


 


2682
2701
444435
GGAGGACGAGGTCAATAAAT
82
75


 


2685
2704
444436
GTCGGAGGACGAGGTCAATA
86
76


 


2688
2707
444437
CGAGTCGGAGGACGAGGTCA
73
77


 


2694
2713
444438
TGTCAGCGAGTCGGAGGACG
79
78


 


2697
2716
444439
GCCTGTCAGCGAGTCGGAGG
83
79


 


2700
2719
444440
GTAGCCTGTCAGCGAGTCGG
94
80


 


2703
2722
444441
CCTGTAGCCTGTCAGCGAGT
90
81


 


2706
2725
444442
GGTCCTGTAGCCTGTCAGCG
90
82


 


2764
2783
444443
AAATACCGAGGAATGTCGGG
82
83


 


2767
2786
444444
AATAAATACCGAGGAATGTC
66
84


 


2770
2789
444445
GACAATAAATACCGAGGAAT
67
85


 


2093
2112
445546
CGGGGCCCCGGAGTCGAAGA
0
86


 


2097
2116
445547
CCAACGGGGCCCCGGAGTCG
38
87


 


2099
2118
445548
TTCCAACGGGGCCCCGGAGT
22
88


 


2102
2121
445549
GTCTTCCAACGGGGCCCCGG
50
89


 


2104
2123
445550
CAGTCTTCCAACGGGGCCCC
27
90


 


2106
2125
445551
CTCAGTCTTCCAACGGGGCC
57
91


 


2109
2128
445552
GCACTCAGTCTTCCAACGGG
69
92


 


2115
2134
445553
CCCCGGGCACTCAGTCTTCC
76
93


 


2117
2136
445554
TGCCCCGGGCACTCAGTCTT
59
94


 


2119
2138
445555
CGTGCCCCGGGCACTCAGTC
61
95


 


2123
2142
445556
GTGCCGTGCCCCGGGCACTC
26
96


 


2126
2145
445557
TCTGTGCCGTGCCCCGGGCA
50
97


 


2129
2148
445558
GCTTCTGTGCCGTGCCCCGG
57
98


 


2132
2151
445559
GCGGCTTCTGTGCCGTGCCC
27
99


 


2134
2153
445560
GCGCGGCTTCTGTGCCGTGC
0
100


 


2136
2155
445561
GGGCGCGGCTTCTGTGCCGT
8
101


 


2142
2161
445562
GGCGGTGGGCGCGGCTTCTG
62
102


 


2146
2165
445563
GGCAGGCGGTGGGCGCGGCT
49
103


 


2148
2167
445564
CTGGCAGGCGGTGGGCGCGG
51
104


 


2150
2169
445565
AACTGGCAGGCGGTGGGCGC
38
105


 


2153
2172
445566
GTGAACTGGCAGGCGGTGGG
64
106


 


2157
2176
445567
GGTTGTGAACTGGCAGGCGG
66
107


 


2159
2178
445568
GCGGTTGTGAACTGGCAGGC
85
108


 


2163
2182
445569
CGGAGCGGTTGTGAACTGGC
92
109


 


2167
2186
445570
CGCTCGGAGCGGTTGTGAAC
51
110


 


2171
2190
445571
CCCACGCTCGGAGCGGTTGT
74
111


 


2174
2193
445572
AGACCCACGCTCGGAGCGGT
80
112


 


2177
2196
445573
CGGAGACCCACGCTCGGAGC
83
113


 


2180
2199
445574
GGGCGGAGACCCACGCTCGG
62
114


 


2183
2202
445575
GCTGGGCGGAGACCCACGCT
11
115


 


2186
2205
445576
GGAGCTGGGCGGAGACCCAC
42
116


 


2188
2207
445577
CTGGAGCTGGGCGGAGACCC
17
117


 


2191
2210
445578
GGACTGGAGCTGGGCGGAGA
53
118


 


2193
2212
445579
CAGGACTGGAGCTGGGCGGA
46
119


 


2197
2216
445580
ATCACAGGACTGGAGCTGGG
66
120


 


2209
2228
445581
GGGCGGGCCCGGATCACAGG
85
121


 


2211
2230
445582
GGGGGCGGGCCCGGATCACA
96
122


 


179
198
445583
AGGCAGCACCATGGCCCCTC
88
123


 


235
254
445584
GGTCCAACACCAGCTGCTGG
84
124


 


418
437
445585
CGATCACCTTCAGAATCTCG
11
125


 


498
517
445586
CTTGTTCATGATCTTCATGG
0
126


 


565
584
445587
CCCCATTCACCAACACGTCC
83
127


 


583
602
445588
GCGTGATCCACCGCCGGTCC
59
128


 


639
658
445589
GTAATACTCCATGACCAGGT
86
129


 


664
683
445590
GCAGTGTCAGCAGGTCCCCG
83
130


 


744
763
445591
CACCGAGTCTATGGCCATGA
60
131


 


761
780
445592
ACGTAGCCAAGCCGGTGCAC
68
132


 


812
831
445593
ATGTGGCCACAGCGGTCCAG
56
133


 


1099
1118
445594
CTTCGTCCACCAGCGGCAGA
32
134


 


1104
1123
445595
GACCCCTTCGTCCACCAGCG
83
135


 


1178
1197
445596
CCTGCTCCACCCCGGCCCAG
82
136


 


1187
1206
445597
CGGAAGTCGCCTGCTCCACC
81
137


 


1229
1248
445598
CGGAGACCATCCCAGTCGAG
67
138


 


1402
1421
445599
TGAGGGCCATGCAGGAGTAG
26
139


 


1443
1462
445600
CTCCAGTTCCATGGGTGTGG
80
140


 


1477
1496
445601
GCGCTTGCACGTGTGGCTCA
94
141


 


1526
1545
445602
GCCACTTCAGCTGTTTCATC
54
142


 


1562
1581
445603
GCCTCAGCCTCTGCCGCAGG
71
143


 


1576
1595
445604
GCAGCGTCACCTCGGCCTCA
31
144


 


1630
1649
445605
GGCTCAGGCTCTGCCGGGTG
86
145


 


1700
1719
445606
TTCCGAGCCTCTGCCTCGCG
73
146


 


1708
1727
445607
GGTCCCGGTTCCGAGCCTCT
76
147


 


1742
1761
445608
ATCCGCTCCTGCAACTGCCG
93
148


 


1750
1769
445609
GCAACTCCATCCGCTCCTGC
60
149


 


1812
1831
445610
AGGTGGATCCGTGGCCCGGG
48
150


 


2133
2152
445611
CGCGGCTTCTGTGCCGTGCC
24
151


 


2428
2447
445612
TTGCTGCCTTCCCAGGCCTG
80
152
















TABLE 2







Inhibition of human DMPK RNA transcript in hSKMC by 5-10-5


gapmers targeting SEQ ID NO: 2












Target
Target






Start
Stop


%
SEQ ID


Site
Site
ISIS No
Sequence
inhibition
NO.















812
831
299471
TGCTCCCGACAAGCTCCAGA
95
153


 


876
895
299473
AGAACCTGCCCATTGCTGAA
68
154


 


2381
2400
299535
CACTGAGGGCCAGACATATG
68
155


 


3289
3308
299544
CTCTAGATTCAGATGCAGGT
88
156









The antisense oligonucleotides from Tables 1 and 2 were also tested in an assay with similar conditions as described above, and mRNA levels measured with the human primer probe RTS3162 (forward sequence CGGGCCGTCCGTGTT, designated herein as SEQ ID NO: 157; reverse sequence CTTTGCACTTTGCGAACCAA, designated herein as SEQ ID NO: 158; probe sequence CATCCTCCACGCACCCCCACCX, designated herein as SEQ ID NO: 159). The results are presented in Table 3. DMPK mRNA expression was also assessed by RTS3162 which targets the DMPK gene near the 3′UTR. The use of a second primer probe was employed to confirm that the expression of the entire DMPK gene had been inhibited







TABLE 3







Inhibition of human DMPK RNA transcript in 


hSKMC by 5-10-5 gapmers measured using


primer probe set RTS3162










ISIS
%



No
inhibition














299471
91



299473
65



299476
76



299479
53



299493
60



299494
66



299501
44



299511
39



299517
71



299526
39



299535
75



299544
84



444380
72



444381
82



444382
67



444383
63



444384
66



444385
66



444386
74



444387
85



444388
60



444389
81



444390
88



444391
79



444392
94



444393
88



444394
94



444395
96



444396
96



444397
95



444398
96



444399
95



444400
95



444401
95



444402
91



444403
84



444404
89



444405
71



444406
47



444407
42



444408
80



444409
56



444410
79



444411
66



444412
67



444413
55



444414
45



444415
57



444416
18



444417
64



444418
51



444419
66



444420 
0



444421
46



444422
33



444423
74



444424
73



444425
78



444426 
0



444427 
0



444428 
0



444429
75



444430
28



444431
58



444432
52



444433
60



444434
87



444435
76



444436
83



444437
71



444438
76



444439
73



444440
91



444441 
87



444442
93



444443
77



444444
64



444445
67



445546
0



445547
59



445548
49



445549 
77



445550 
62



445551 
74



445552 
84



445553 
70



445554 
63



445555 
75



445556 
52



445557 
78



445558 
81



445559 
58



445560 
12



445561 
42



445562 
70



445563 
76



445564 
69



445565 
60



445566 
86



445567 
84



445568 
92



445569 
93



445570 
59



445571 
84



445572 
88



445573 
84



445574 
74



445575 
26



445576 
56



445577 
38



445578 
69



445579 
70



445580 
75



445581 
85



445582 
95



445583 
88



445584 
87



445585
34



445586
0



445587
82



445588
66



445589
87



445590
82



445591
68



445592
64



445593
54



445594
52



445595
77



445596
84



445597
78



445598
73



445599
29



445600
68



445601
92



445602
53



445603
70



445604
32



445605
61



445606
84



445607
80



445608
91



445609
68



445610
63



445611
44



445612
91










Example 2
Design of Antisense Oligonucleotides Targeting CUG Repeats
Antisense oligonucleotides were designed targeting mRNA transcripts that contain multiple CUG repeats. The chemistry of these oligonucleotides as well as their sequence is shown in Table 4. The symbols designated to the sugar type are shown after the base in subscript and are as follows: b=2′—O—N-[2-(dimethylamino)ethyl]acetamido ribose; d=2′-deoxyribose; e=2′—O-methoxyethyl ribose; f=2′-alpha-fluoro-2′-deoxyribose; g=2′—O—2[2-(2-methoxyethoxy)ethoxy]ethyl ribose; h=3′-fluoro-HNA; k=(S)-cEt; 1=LNA (Locked Nucleic Acids); n=2′—O—(N-methylacetamide) ribose; o=2′—O—dimethylaminooxyethyl (DMAOE) ribose; p=PNA; r=propylribose; and x=amino acid core. The heterocycle names are defined with standard symbols for adenine, cytosine, thymine and guanine, ‘mC’ for 5-methylcytosine, and ‘K’ for Lysine Side Chain. Linkers are shown after the sugar type in subscript and designated with the following symbols: g=PNA-glycine full; a=amino acid; and s=thioate ester.







TABLE 4







Design of antisense oligonucleotides targeting CUG repeats















SEQ






ID


ISIS No
Sequence
Chemistry
Backbone
NO





431896
GdsCdsAlsGdsCdsAlsGdsCdsAls
Deoxy and LNA units
Phosphorothioate
802



GdsCdsAlsGdsCdsAlsGdsCdsAlsGd


 


433804
KxaGpgCpgApgGpgCpgApgGpgCpgApgGpgCpgApgGpg
PNA and Amino Acid
mixed
803



CpgApgGpgCpgApgGpgKxaKxaKxaKxaKxaKxaKxaKxa
Core units with a




Carboxy-amide endcap


 


444745
AesGesmCesAesGesmCesAesGesmCesAesGesmCes
Uniform MOE
Phosphorothioate
789



AesGesmCesAesGesmCesAesGesmCesAesGesmCesAe


 


444746
AesGesmCesAesGesmCesAesGesmCesAes
Uniform MOE
Phosphorothioate
804



GesmCesAesGesmCesAesGesmCesAesGe


 


444747
GesmCesAesGesmCesAesGesmCesAes
Uniform MOE
Phosphorothioate
802



GesmCesAesGesmCesAesGesmCesAesGes


 


444748
GesmCesAesGesmCesAesGesmCesAes
Uniform MOE
Phosphorothioate
805



GesmCesAesGesmCesAesGesmCesAe


 


444750
GksCksAdsGdsCksAdsGdsCksAds
Deoxy and (S)-cEt units
Phosphorothioate
805



GdsCksAdsGdsCksAdsGdsCksAk


 


444752
GksCksAesGesCksAesGesCksAes
MOE and (S)-cEt units
Phosphorothioate
805



GesCksAesGesCksAesGesCksAk


 


444754
GesmCesAfsGfsCfsAfsGfsCfsAfs
MOE and
Phosphorothioate
805



GfsCfsAfsGfsCfsAfsGfsmCesAes
2′-alpha-flouro units


 


444759
GhsmChsAhsGhsmChsAhsGhsmChsAhs
Uniform 3′-fluoro-HNA
Phosphorothioate
805



GhsmChsAhsGhsmChsAhsGhsmChsAh


 


444761
GrsmCrsArsGrsmCrsArsGrsmCrsArs
Uniform 2′-O-propylribose
Phosphorothioate
805



GrsmCrsArsGrsmCrsArsGrsmCrsAr


 


444762
GnsmCnsAnsGnsmCnsAnsGnsmCnsAns
Uniform 2′-O-(N-
Phosphorothioate
805



GnsmCnsAnsGnsmCnsAnsGnsmCnsAn
methylacetamide) ribose


 


444763
GosmCesAosGosmCesAosGosmCesAos
MOE and 2′-O-
Phosphorothioate
805



GosmCesAosGosmCesAosGosmCesAo
dimethylaminooxyethyl




(DMAOE) ribose units


 


444764
GgsmCesAesGgsmCesAesGgsmCesAes
MOE and 2′-O-2[2-(2-
Phosphorothioate
802



GgsmCesAesGgsmCesAesGgsmCesAesGg
methoxyethoxy)ethoxy]ethyl




ribose units


 


444765
GbsmCesAesGbsmCesAesGbsmCesAes
MOE and 2′-O-N-[2-
Phosphorothioate
802



GbsmCesAesGbsmCesAesGbsmCesAesGb
(dimethylamino)ethyl]acetamido




ribose units


 


473810
AksGdsmCdsAksGdsmCdsAksGasmCds
Deoxy and (S)-cEt units
Phosphorothioate
806



AksGdsmCdsAksGdsmCdsAksGdsmCdsAk


 


473811
AksGdsmCdsAksGdsmCdsAksGds
Deoxy and (S)-cEt units
Phosphorothioate
807



mCdsAksGdsmCdsAksGdsmCdsAk









Example 3
Dose-Dependent Antisense Inhibition of Human DMPK in Human Skeletal Muscle Cells
Several of the antisense oligonucleotides exhibiting in vitro inhibition of DMPK in hSKMC (see Example 1) were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and DMPK mRNA transcript levels were measured by quantitative real-time PCR using primer probe set RTS3164, described hereinabove. DMPK mRNA transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 5 as percent inhibition of DMPK, relative to untreated control cells.
The tested antisense oligonucleotides demonstrated dose-dependent inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 5







Dose-dependent antisense inhibition of human DMPK in hSKMC


tested with primer probe set RTS3164













ISIS


5,000

20,000
IC50


No.
1,250 nM
2,500 nM
nM
10,000 nM
nM
(μM)
















299471
34
65
87
91
94
1.60


299473
2
33
60
89
92
4.31


299476
15
17
49
81
91
4.89


299535
0
12
34
62
59
9.95


299535
20
33
47
67
80
5.11


299544
32
63
81
85
87
1.82


444397
10
30
58
85
82
4.51


444398
33
57
74
85
87
2.07


444400
52
46
63
82
88
1.76


444401
51
71
84
89
91
0.71


444402
53
79
83
87
84
<1.25


444404
48
68
77
86
90
0.95


444408
26
47
70
87
87
2.80


444410
22
47
67
83
87
3.12


444436
28
67
76
89
92
1.94


444440
70
77
83
89
85
<1.25


444441
33
55
81
87
86
1.99


444442
54
73
84
89
88
<1.25


445568
65
83
85
84
76
<1.25


445569
60
77
87
93
91
<1.25


445581
16
44
78
86
94
3.13


445582
0
7
26
96
99
5.60


445583
39
53
73
89
94
2.00


445584
20
26
61
81
93
4.02


445589
42
61
81
91
87
1.36


445601
49
79
87
93
94
0.66


445608
26
59
71
85
97
2.41


445612
46
59
72
88
93
1.51









The antisense oligonucleotides from Table 5 were also tested with primer probe set RTS3162, described hereinabove. The results are presented in Table 6. DMPK mRNA expression was also assessed by RTS3162 which targets the DMPK gene near the 3′UTR. The use of a second primer probe was employed to confirm that the expression of the entire DMPK gene had been inhibited.







TABLE 6







Dose-dependent antisense inhibition of human DMPK in hSKMC


tested with primer probe set RTS3164













ISIS


5,000

20,000
IC50


No.
1,250 nM
2,500 nM
nM
10,000 nM
nM
(μM)
















299471
40
72
86
91
93
1.17


299473
6
43
63
87
89
3.86


299476
3
21
48
74
86
5.58


299535
9
22
36
62
77
7.05


299535
6
19
49
68
70
6.70


299544
35
66
81
84
87
1.52


444397
88
90
95
97
96
<1.25


444398
91
97
97
97
98
<1.25


444400
72
87
93
96
96
<1.25


444401
86
92
97
98
97
<1.25


444402
83
91
94
95
95
<1.25


444404
49
69
81
90
93
0.92


444408
21
46
70
84
86
3.10


444410
35
55
77
89
91
2.02


444436
37
66
81
89
92
1.50


444440
66
79
89
92
89
<1.25


444441
40
62
85
89
89
1.40


444442
55
75
86
90
91
<1.25


445568
74
92
91
92
91
<1.25


445569
68
83
90
94
93
<1.25


445581
8
48
77
85
92
3.33


445582
15
22
44
97
99
4.29


445583
36
58
71
87
92
1.96


445584
25
43
66
86
94
3.05


445589
38
56
77
85
81
1.74


445601
55
76
84
93
93
<1.25


445608
22
56
72
86
94
2.66


445612
61
75
85
91
94
<1.25









Example 4
Dose-Dependent Antisense Inhibition of Human DMPK in Human Skeletal Muscle Cells
Several of the antisense oligonucleotides exhibiting in vitro inhibition of DMPK in hSKMC (see Example 3) were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and DMPK mRNA transcript levels were measured by quantitative real-time PCR using primer probe set RTS3164, described hereinabove. DMPK mRNA transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 7 as percent inhibition of DMPK, relative to untreated control cells.
The majority of the tested antisense oligonucleotides demonstrated dose-dependent inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 7







Dose-dependent antisense inhibition of human DMPK in hSKMC


tested with primer probe set RTS3164













ISIS


5,000

20,000
IC50


No.
1,250 nM
2,500 nM
nM
10,000 nM
nM
(μM)
















299471
34
65
87
91
94
1.59


299473
2
33
60
89
92
4.31


299476
15
17
49
81
91
4.89


299535
0
12
34
62
59
9.95


299535
20
33
47
67
80
5.11


299544
32
63
81
85
87
1.82


444397
10
30
58
85
82
4.51


444398
33
57
74
85
87
2.07


444400
52
46
63
82
88
1.76


444401
51
71
84
89
91
<1.25


444402
53
79
83
87
84
<1.25


444404
48
68
77
86
90
0.95


444408
26
47
70
87
87
2.80


444410
22
47
67
83
87
3.12


444436
28
67
76
89
92
1.94


444440
66
77
83
89
85
<1.25


444441
33
55
81
87
86
1.99


444442
54
73
84
89
88
<1.25


445568
65
83
85
84
76
<1.25


445569
60
77
87
93
91
<1.25


445581
16
44
78
86
94
3.13


445582
0
7
26
96
99
5.62


445583
39
53
73
89
94
1.97


445584
20
26
61
81
93
4.20


445589
42
61
81
91
87
1.36


445601
49
79
87
93
94
0.66


445608
26
59
71
85
97
2.41


445612
46
59
72
88
93
1.51









Example 5
Dose-Dependent Antisense Inhibition of Human DMPK in Human Skeletal Muscle Cells
Several antisense oligonucleotides were designed to target human DMPK mRNA and were tested in hSKMC at various doses. Several other antisense oligonucleotides were designed to target human actin mRNA and were also tested in hSKMC at various doses. The newly designed gapmers are 2-10-2 MOE or 3-10-3 MOE gapmers. The 2-10-2 MOE gapmers are 14 nucleosides in length and where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises two 2′-MOE nucleosides. The 3-10-3 MOE gapmers are 16 nucleosides in length and where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises three 2′-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. ‘Target start site’ indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted. ‘Target stop site’ indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted. The antisense oligonucleotides listed in Table 8 target either the human DMPK genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_011109.15 truncated from nucleotides 18540696 to Ser. No. 18/555,106) or the human actin sequence, designated herein as SEQ ID NO: 801 (GENBANK Accession No. NM_001100.3).
Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and DMPK mRNA transcript levels were measured by quantitative real-time PCR using primer probe set RTS3162, described hereinabove. DMPK mRNA transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 8 as percent inhibition of DMPK, relative to untreated control cells. The antisense oligonucleotides were also tested under similar conditions with RTS3164. The results are presented in Table 9.
Many of the tested antisense oligonucleotides demonstrated dose-dependent inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 8







Dose-dependent antisense inhibition of human DMPK and human actin


in hSKMC tested with primer probe set RTS3162





















Target







SEQ





SEQ ID
Start





IC50
ID


ISIS No
Sequence
Motif
NO
Site
1,250 nM
2,500 nM
5,000 nM
10,000 nM
20,000 nM
(nM)
NO





















468787
CTCCCGACAAGCTCCA
3-10-3
2
814
28
47
51
84
88
3.27
808


 


468772
TCCCGACAAGCTCC
2-10-2
2
815
17
39
67
72
80
4.04
809


 


468795
GCTTGCACGTGTGGCT
3-10-3
2
10935
32
58
77
85
75
1.94
810


 


468780
CTTGCACGTGTGGC
2-10-2
2
10936
22
17
43
66
77
6.23
811


 


468793
GGTTGTGAACTGGCAG
3-10-3
2
13224
69
77
93
96
96
<1.25
812


 


468778
GTTGTGAACTGGCA
2-10-2
2
13225
60
69
89
95
97
<1.25
813


 


468794
GAGCGGTTGTGAACTG
3-10-3
2
13228
21
32
61
70
86
4.27
814


 


468779
AGCGGTTGTGAACT
2-10-2
2
13229
40
45
72
91
97
2.20
815


 


468796
GCTGCCTTCCCAGGCC
3-10-3
2
13493
73
79
91
96
95
<1.25
816


 


468781
CTGCCTTCCCAGGC
2-10-2
2
13494
36
53
66
86
90
2.28
817


 


468788
GCACTTTGCGAACCAA
3-10-3
2
13555
55
80
84
94
96
<1.25
818


 


468773
CACTTTGCGAACCA
2-10-2
2
13556
31
52
82
91
93
2.16
819


 


468789
GAAAGCTTTGCACTTT
3-10-3
2
13564
42
66
83
91
98
1.31
820


 


468774
AAAGCTTTGCACTT
2-10-2
2
13565
21
0
31
41
55
1.87
821


 


468790
CGGAGGACGAGGTCAA
3-10-3
2
13750
43
57
79
87
89
1.51
822


 


468775
GGAGGACGAGGTCA
2-10-2
2
13751
27
51
58
78
81
3.18
823


 


468791
AGCCTGTCAGCGAGTC
3-10-3
2
13765
49
63
85
62
95
1.04
824


 


468776
GCCTGTCAGCGAGT
2-10-2
2
13766
65
47
81
88
93
<1.25
825


 


468792
TCCTGTAGCCTGTCAG
3-10-3
2
13771
38
57
73
85
93
1.91
826


 


468777
CCTGTAGCCTGTCA
2-10-2
2
13772
15
58
66
85
92
2.99
827


 


468783
GAAGCGAGGCTTCACT
3-10-3
801
22
0
20
5
0
0
>20.00
828


 


468768
AAGCGAGGCTTCAC
2-10-2
801
23
25
22
5
17
0
>20.00
829


 


468784
ACCTGCCCGTCTGGCA
3-10-3
801
836
15
25
32
18
25
>20.00
830


 


468769
CCTGCCCGTCTGGC
2-10-2
801
837
32
11
11
20
32
>20.00
831


 


468782
GGTCAGCGATCCCAGG
3-10-3
801
1030
0
0
0
0
0
>20.00
832


 


468767
GTCAGCGATCCCAG
2-10-2
801
1031
15
0
11
0
0
>20.00
833


 


468785
ATTTTCTTCCACAGGG
3-10-3
801
1432
12
0
0
0
0
>20.00
834


 


468770
TTTTCTTCCACAGG
2-10-2
801
1433
36
2
0
0
28
>20.00
835


 


468786
GAATGACTTTAATGCT
3-10-3
801
1462
0
0
0
4
0
>20.00
836


 


468771
AATGACTTTAATGC
2-10-2
801
1463
8
16
0
5
0
>20.00
837
















TABLE 9







Dose-dependent antisense inhibition of human DMPK in hSKMC


tested with primer probe set RTS3164
















5,000

20,000
IC50


ISIS No
1,250 nM
2,500 nM
nM
10,000 nM
nM
(μM)
















468777
20
66
72
87
96
2.41


468776
68
48
86
90
96
<1.25


468794
18
23
58
65
86
4.97


468787
36
50
51
88
92
2.69


468772
12
47
69
80
86
3.57


468773
33
48
82
91
96
2.21


468774
21
0
30
42
59
1.60


468790
50
57
77
91
91
1.26


468780
23
22
55
73
85
4.69


468775
29
52
55
79
84
3.03


468782
9
0
0
0
0
>20.00


468786
2
0
0
0
0
>20.00


468785
15
0
1
0
5
>20.00


468788
57
74
76
94
96
<1.25


468791
45
66
88
61
97
1.10


468789
26
65
82
90
97
2.02


468781
28
46
59
82
84
3.08


468779
26
31
66
90
97
3.29


468784
7
23
26
7
18
>20.00


468783
0
16
8
0
0
>20.00


468792
26
49
73
84
92
2.72


468795
30
53
83
86
85
2.14


468793
49
66
90
96
95
0.93


468768
23
3
5
9
0
>20.00


468767
0
0
14
0
0
>20.00


468769
31
0
0
16
25
>20.00


468771
4
0
0
0
0
>20.00


468770
33
0
0
0
32
>20.00


468796
62
72
84
96
95
<1.25


468778
44
58
86
96
98
1.44









Example 6
Dose Response Studies with Antisense Oligonucleotides Targeting Human Dystrophia Myotonica-Protein Kinase (DMPK) in DM1 Fibroblast Cells
The mutant form of the DMPK mRNA, harboring large CUG repeats, are fully transcribed and polyadenylated, but remain trapped in the nucleus (Davis et al, 1997, Proc. Natl. Acad. Sci. U.S.A 94, 7388-7393). These mutant nuclear-retained mRNAs are one of the most important pathological features of myotonic dystrophy 1 (DM1). Antisense inhibition of mutant DMPK mRNA in DM1 fibroblast cells was studied.
The DMPK gene normally has 5-37 CTG repeats in the 3′ untranslated region. In myotonic dystrophy type I, this number is significantly expanded and may be in the range of 50 to greater than 3,500 (Harper, Myotonic Dystrophy (Saunders, London, ed.3, 2001); Annu. Rev. Neurosci. 29: 259, 2006; EMBO J. 19: 4439, 2000; Curr Opin Neurol. 20: 572, 2007).DM1 fibroblast cells were plated at a density of 4,500 cells per well and transfected using Cytofectin reagent with 9.4 nM, 18.8 nM, 37.5 nM, 75.0 nM, 150.0 nM, and 300.0 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and DMPK RNA transcript levels were measured by quantitative real-time PCR using primer probe set RTS3164, described hereinabove. DMPK RNA transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 10 as percent inhibition of DMPK, relative to untreated control cells.
An assay with similar conditions was also performed with primer probe set RTS3162, described hereinabove, which targets the 3′-end of the DMPK transcript. Results are presented in Table 11 as percent inhibition of DMPK, relative to untreated control cells.
The tested antisense oligonucleotides demonstrated dose-dependent inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 10







Dose-dependent antisense inhibition of DMPK mRNA in DM1


fibroblast cells with RTS3164














ISIS

18.8

75.0

300.0
IC50


No.
9.4 nM
nM
37.5 nM
nM
150.0 nM
nM
(nM)

















299471
10
25
31
47
61
73
86.3


444401
8
27
41
60
67
74
64.3


444404
10
21
31
43
55
73
100.0


444436
7
17
36
64
68
70
72.3


445569
19
31
41
59
46
77
72.2
















TABLE 11







Dose-dependent antisense inhibition of DMPK mRNA in DM1


fibroblast cells with RTS3162
















18.8

75.0

300.0
IC50


ISIS No
9.4 nM
nM
37.5 nM
nM
150.0 nM
nM
(nM)

















299471
7
25
29
46
48
69
115.3


444401
20
34
52
72
83
89
35.8


444404
5
20
28
42
54
77
98.8


444436
12
15
27
61
68
75
74.3


445569
5
25
33
53
50
76
89.6









Example 7
Antisense Inhibition of Human DMPK in Human Skeletal Muscle Cells (hSKMc)
Antisense oligonucleotides targeted to a human DMPK nucleic acid were tested for their effect on DMPK RNA transcript in vitro. Cultured hSKMc at a density of 20,000 cells per well were transfected using electroporation with 10,000 nM antisense oligonucleotide. After approximately 24 hours, RNA was isolated from the cells and DMPK transcript levels were measured by quantitative real-time PCR. DMPK RNA transcript levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of DMPK, relative to untreated control cells.
The antisense oligonucleotides in Tables 12 and 13 are 5-10-5 gapmers, where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises five 2′-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytsoine residues throughout each gapmer are 5-methylcytosines. ‘Target start site’ indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted in the human genomic gene sequence. ‘Target stop site’ indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted in the human genomic sequence. All the antisense oligonucleotides listed in Table 12 target SEQ ID NO: 1 (GENBANK Accession No. NM_001081560.1). All the antisense oligonucleotides listed in Table 13 target SEQ ID NO: 2 (the complement of GENBANK Accession No. NT_011109.15 truncated from nucleotides 18540696 to Ser. No. 18/555,106).
Several of the antisense oligonucleotides demonstrated significant inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 12







Inhibition of human DMPK RNA transcript in hSKMc by 5-10-5


gapmers targeting SEQ ID NO: 1












Target
Target






Start
Stop
ISIS

%



Site
Site
No
Sequence
inhibition
SEQ ID NO.















124
143
502369
GCCTGGCAGCCCCTGTCCAG
16
160


 


125
144
502370
GGCCTGGCAGCCCCTGTCCA
58
161


 


126
145
502371
GGGCCTGGCAGCCCCTGTCC
62
162


 


169
188
502372
ATGGCCCCTCCCCGGGCCGG
41
163


 


170
189
502373
CATGGCCCCTCCCCGGGCCG
29
164


 


171
190
502374
CCATGGCCCCTCCCCGGGCC
34
165


 


172
191
502375
ACCATGGCCCCTCCCCGGGC
60
166


 


173
192
502376
CACCATGGCCCCTCCCCGGG
68
167


 


174
193
502377
GCACCATGGCCCCTCCCCGG
75
168


 


175
194
502378
AGCACCATGGCCCCTCCCCG
65
169


 


176
195
502379
CAGCACCATGGCCCCTCCCC
63
170


 


177
196
502380
GCAGCACCATGGCCCCTCCC
73
171


 


178
197
502381
GGCAGCACCATGGCCCCTCC
80
172


 


180
199
502382
CAGGCAGCACCATGGCCCCT
82
173


 


181
200
502383
ACAGGCAGCACCATGGCCCC
72
174


 


183
202
502384
GGACAGGCAGCACCATGGCC
70
175


 


184
203
502385
TGGACAGGCAGCACCATGGC
71
176


 


185
204
502386
TTGGACAGGCAGCACCATGG
73
177


 


186
205
502387
GTTGGACAGGCAGCACCATG
73
178


 


187
206
502388
TGTTGGACAGGCAGCACCAT
60
179


 


188
207
502389
ATGTTGGACAGGCAGCACCA
75
180


 


189
208
502390
CATGTTGGACAGGCAGCACC
81
181


 


190
209
502391
ACATGTTGGACAGGCAGCAC
67
182


 


191
210
502392
GACATGTTGGACAGGCAGCA
71
183


 


192
211
502393
TGACATGTTGGACAGGCAGC
81
184


 


193
212
502394
CTGACATGTTGGACAGGCAG
76
185


 


194
213
502395
GCTGACATGTTGGACAGGCA
70
186


 


195
214
502396
GGCTGACATGTTGGACAGGC
77
187


 


196
215
502397
CGGCTGACATGTTGGACAGG
74
188


 


197
216
502398
TCGGCTGACATGTTGGACAG
63
189


 


198
217
502399
CTCGGCTGACATGTTGGACA
80
190


 


199
218
502400
CCTCGGCTGACATGTTGGAC
71
191


 


200
219
502401
ACCTCGGCTGACATGTTGGA
64
192


 


201
220
502402
CACCTCGGCTGACATGTTGG
71
193


 


202
221
502403
GCACCTCGGCTGACATGTTG
77
194


 


203
222
502404
CGCACCTCGGCTGACATGTT
80
195


 


204
223
502405
CCGCACCTCGGCTGACATGT
80
196


 


205
224
502406
GCCGCACCTCGGCTGACATG
79
197


 


206
225
502407
AGCCGCACCTCGGCTGACAT
74
198


 


207
226
502408
CAGCCGCACCTCGGCTGACA
66
199


 


208
227
502409
TCAGCCGCACCTCGGCTGAC
15
200


 


209
228
502410
CTCAGCCGCACCTCGGCTGA
32
201


 


210
229
502411
CCTCAGCCGCACCTCGGCTG
65
202


 


211
230
502412
GCCTCAGCCGCACCTCGGCT
81
203


 


232
251
502413
CCAACACCAGCTGCTGGAGC
90
204


 


233
252
502414
TCCAACACCAGCTGCTGGAG
78
205


 


234
253
502415
GTCCAACACCAGCTGCTGGA
84
206


 


236
255
502416
GGGTCCAACACCAGCTGCTG
69
207


 


257
276
502417
GGCTCCAGCCCCAGGAAGCC
46
208


 


258
277
502418
GGGCTCCAGCCCCAGGAAGC
28
209


 


276
295
502419
CAGGAGAAGGTCGAGCAGGG
41
210


 


278
297
502420
CCCAGGAGAAGGTCGAGCAG
71
211


 


279
298
502421
GCCCAGGAGAAGGTCGAGCA
85
212


 


280
299
451363
CGCCCAGGAGAAGGTCGAGC
84
213


 


281
300
502422
ACGCCCAGGAGAAGGTCGAG
67
214


 


317
336
502423
TCCTGGGCCAGTTCGGAGGC
58
215


 


318
337
502424
GTCCTGGGCCAGTTCGGAGG
71
216


 


319
338
502425
TGTCCTGGGCCAGTTCGGAG
69
217


 


320
339
502426
TTGTCCTGGGCCAGTTCGGA
71
218


 


321
340
502427
CTTGTCCTGGGCCAGTTCGG
66
219


 


322
341
502428
ACTTGTCCTGGGCCAGTTCG
59
220


 


323
342
502429
TACTTGTCCTGGGCCAGTTC
75
221


 


324
343
502430
GTACTTGTCCTGGGCCAGTT
78
222


 


325
344
502431
CGTACTTGTCCTGGGCCAGT
74
223


 


343
362
502432
ACTGCAAGAAGTCGGCCACG
73
224


 


345
364
502433
CCACTGCAAGAAGTCGGCCA
65
225


 


346
365
451364
CCCACTGCAAGAAGTCGGCC
32
226


 


347
366
502434
GCCCACTGCAAGAAGTCGGC
70
227


 


348
367
502435
CGCCCACTGCAAGAAGTCGG
61
228


 


349
368
502436
CCGCCCACTGCAAGAAGTCG
54
229


 


350
369
502437
TCCGCCCACTGCAAGAAGTC
40
230


 


351
370
502438
CTCCGCCCACTGCAAGAAGT
33
231


 


352
371
502439
GCTCCGCCCACTGCAAGAAG
23
232


 


353
372
502440
GGCTCCGCCCACTGCAAGAA
23
233


 


354
373
502441
GGGCTCCGCCCACTGCAAGA
17
234


 


355
374
502442
TGGGCTCCGCCCACTGCAAG
22
235


 


356
375
502443
ATGGGCTCCGCCCACTGCAA
14
236


 


357
376
502444
GATGGGCTCCGCCCACTGCA
43
237


 


358
377
502445
CGATGGGCTCCGCCCACTGC
37
238


 


359
378
502446
ACGATGGGCTCCGCCCACTG
0
239


 


360
379
502447
CACGATGGGCTCCGCCCACT
59
240


 


361
380
502448
CCACGATGGGCTCCGCCCAC
69
241


 


362
381
502449
ACCACGATGGGCTCCGCCCA
63
242


 


363
382
502450
CACCACGATGGGCTCCGCCC
73
243


 


364
383
502451
TCACCACGATGGGCTCCGCC
77
244


 


365
384
502452
CTCACCACGATGGGCTCCGC
66
245


 


366
385
502453
CCTCACCACGATGGGCTCCG
81
246


 


367
386
502454
GCCTCACCACGATGGGCTCC
77
247


 


368
387
502455
AGCCTCACCACGATGGGCTC
63
248


 


369
388
502456
AAGCCTCACCACGATGGGCT
70
249


 


370
389
502457
TAAGCCTCACCACGATGGGC
78
250


 


371
390
502458
TTAAGCCTCACCACGATGGG
76
251


 


372
391
502459
CTTAAGCCTCACCACGATGG
78
252


 


373
392
502460
CCTTAAGCCTCACCACGATG
68
253


 


374
393
502461
TCCTTAAGCCTCACCACGAT
67
254


 


375
394
502462
CTCCTTAAGCCTCACCACGA
84
255


 


376
395
502463
CCTCCTTAAGCCTCACCACG
76
256


 


377
396
502464
ACCTCCTTAAGCCTCACCAC
64
257


 


378
397
502465
GACCTCCTTAAGCCTCACCA
72
258


 


379
398
502466
GGACCTCCTTAAGCCTCACC
69
259


 


380
399
502467
CGGACCTCCTTAAGCCTCAC
81
260


 


381
400
502468
TCGGACCTCCTTAAGCCTCA
78
261


 


382
401
502469
GTCGGACCTCCTTAAGCCTC
57
262


 


384
403
502470
CAGTCGGACCTCCTTAAGCC
62
263


 


385
404
502471
GCAGTCGGACCTCCTTAAGC
45
264


 


386
405
502472
TGCAGTCGGACCTCCTTAAG
60
265


 


412
431
502473
CCTTCAGAATCTCGAAGTCG
67
266


 


413
432
502474
ACCTTCAGAATCTCGAAGTC
50
267


 


415
434
502475
TCACCTTCAGAATCTCGAAG
54
268


 


416
435
502476
ATCACCTTCAGAATCTCGAA
38
269


 


417
436
502477
GATCACCTTCAGAATCTCGA
35
270


 


419
438
502478
CCGATCACCTTCAGAATCTC
52
271


 


420
439
502479
TCCGATCACCTTCAGAATCT
50
272


 


421
440
502480
GTCCGATCACCTTCAGAATC
44
273


 


422
441
502481
CGTCCGATCACCTTCAGAAT
41
274


 


467
486
502482
CCCGTCTGCTTCATCTTCAC
67
275


 


468
487
502483
GCCCGTCTGCTTCATCTTCA
76
276


 


469
488
502484
GGCCCGTCTGCTTCATCTTC
57
277


 


470
489
502485
TGGCCCGTCTGCTTCATCTT
64
278


 


471
490
502486
CTGGCCCGTCTGCTTCATCT
64
279


 


472
491
502487
CCTGGCCCGTCTGCTTCATC
73
280


 


473
492
502488
ACCTGGCCCGTCTGCTTCAT
64
281


 


474
493
502489
CACCTGGCCCGTCTGCTTCA
80
282


 


475
494
502490
ACACCTGGCCCGTCTGCTTC
71
283


 


476
495
502491
TACACCTGGCCCGTCTGCTT
74
284


 


497
516
502492
TTGTTCATGATCTTCATGGC
56
285


 


499
518
502493
ACTTGTTCATGATCTTCATG
23
286


 


500
519
502494
CACTTGTTCATGATCTTCAT
43
287


 


501
520
502495
CCACTTGTTCATGATCTTCA
43
288


 


502
521
502496
CCCACTTGTTCATGATCTTC
47
289


 


503
522
502497
TCCCACTTGTTCATGATCTT
34
290


 


504
523
502498
GTCCCACTTGTTCATGATCT
34
291


 


505
524
502499
TGTCCCACTTGTTCATGATC
27
292


 


506
525
502500
ATGTCCCACTTGTTCATGAT
23
293


 


507
526
502501
CATGTCCCACTTGTTCATGA
51
294


 


508
527
502502
GCATGTCCCACTTGTTCATG
20
295


 


509
528
502503
AGCATGTCCCACTTGTTCAT
52
296


 


510
529
502504
CAGCATGTCCCACTTGTTCA
72
297


 


511
530
502505
TCAGCATGTCCCACTTGTTC
70
298


 


512
531
502506
TTCAGCATGTCCCACTTGTT
53
299


 


513
532
502507
CTTCAGCATGTCCCACTTGT
52
300


 


514
533
502508
TCTTCAGCATGTCCCACTTG
45
301


 


516
535
502509
CCTCTTCAGCATGTCCCACT
68
302


 


517
536
502510
CCCTCTTCAGCATGTCCCAC
68
303


 


518
537
502511
CCCCTCTTCAGCATGTCCCA
79
304


 


519
538
502512
GCCCCTCTTCAGCATGTCCC
85
305


 


520
539
502513
CGCCCCTCTTCAGCATGTCC
84
306


 


521
540
502514
TCGCCCCTCTTCAGCATGTC
80
307


 


522
541
502515
CTCGCCCCTCTTCAGCATGT
82
308


 


523
542
502516
CCTCGCCCCTCTTCAGCATG
78
309


 


524
543
502517
ACCTCGCCCCTCTTCAGCAT
73
310


 


525
544
502518
CACCTCGCCCCTCTTCAGCA
76
311


 


526
545
502519
ACACCTCGCCCCTCTTCAGC
79
312


 


527
546
502520
GACACCTCGCCCCTCTTCAG
73
313


 


821
840
502521
GCCAGGCGGATGTGGCCACA
57
314


 


868
887
502522
ACCGCACCGTTCCATCTGCC
62
315


 


869
888
502523
GACCGCACCGTTCCATCTGC
29
316


 


923
942
502524
ACAGCCTGCAGGATCTCGGG
86
317


 


924
943
502525
CACAGCCTGCAGGATCTCGG
81
318


 


925
944
502526
CCACAGCCTGCAGGATCTCG
83
319


 


926
945
502527
CCCACAGCCTGCAGGATCTC
84
320


 


927
946
502528
GCCCACAGCCTGCAGGATCT
91
321


 


928
947
502529
CGCCCACAGCCTGCAGGATC
90
322


 


929
948
502530
CCGCCCACAGCCTGCAGGAT
82
323


 


930
949
502531
ACCGCCCACAGCCTGCAGGA
83
324


 


931
950
502532
CACCGCCCACAGCCTGCAGG
85
325


 


932
951
502533
CCACCGCCCACAGCCTGCAG
84
326


 


933
952
502534
CCCACCGCCCACAGCCTGCA
80
327


 


934
953
502535
GCCCACCGCCCACAGCCTGC
90
328


 


935
954
502536
GGCCCACCGCCCACAGCCTG
94
329


 


936
955
502537
AGGCCCACCGCCCACAGCCT
88
330


 


937
956
502538
CAGGCCCACCGCCCACAGCC
91
331


 


938
957
502539
CCAGGCCCACCGCCCACAGC
73
332


 


939
958
502540
CCCAGGCCCACCGCCCACAG
86
333


 


940
959
502541
TCCCAGGCCCACCGCCCACA
88
334


 


941
960
502542
GTCCCAGGCCCACCGCCCAC
84
335


 


942
961
502543
TGTCCCAGGCCCACCGCCCA
85
336


 


943
962
502544
CTGTCCCAGGCCCACCGCCC
65
337


 


944
963
502545
CCTGTCCCAGGCCCACCGCC
81
338


 


945
964
502546
GCCTGTCCCAGGCCCACCGC
90
339


 


946
965
502547
TGCCTGTCCCAGGCCCACCG
85
340


 


947
966
502548
CTGCCTGTCCCAGGCCCACC
89
341


 


948
967
502549
GCTGCCTGTCCCAGGCCCAC
91
342


 


949
968
502550
AGCTGCCTGTCCCAGGCCCA
94
343


 


950
969
502551
TAGCTGCCTGTCCCAGGCCC
92
344


 


951
970
502552
GTAGCTGCCTGTCCCAGGCC
88
345


 


952
971
502553
CGTAGCTGCCTGTCCCAGGC
85
346


 


953
972
502554
CCGTAGCTGCCTGTCCCAGG
83
347


 


954
973
502555
CCCGTAGCTGCCTGTCCCAG
64
348


 


955
974
502556
GCCCGTAGCTGCCTGTCCCA
83
349


 


956
975
502557
GGCCCGTAGCTGCCTGTCCC
89
350


 


1004
1023
502558
TAGAACATTTCATAGGCGAA
68
351


 


1042
1061
502559
TCTCCGCCGTGGAATCCGCG
75
352


 


1043
1062
502560
GTCTCCGCCGTGGAATCCGC
79
353


 


1044
1063
502561
GGTCTCCGCCGTGGAATCCG
66
354


 


1045
1064
502562
AGGTCTCCGCCGTGGAATCC
50
355


 


1046
1065
502563
TAGGTCTCCGCCGTGGAATC
71
356


 


1067
1086
502564
TTGTAGTGGACGATCTTGCC
68
357


 


1068
1087
502565
CTTGTAGTGGACGATCTTGC
70
358


 


1069
1088
502566
CCTTGTAGTGGACGATCTTG
61
359


 


1070
1089
502567
TCCTTGTAGTGGACGATCTT
72
360


 


1071
1090
502568
CTCCTTGTAGTGGACGATCT
75
361


 


1072
1091
502569
GCTCCTTGTAGTGGACGATC
75
362


 


1073
1092
502570
TGCTCCTTGTAGTGGACGAT
83
363


 


1074
1093
502571
GTGCTCCTTGTAGTGGACGA
72
364


 


1075
1094
502572
GGTGCTCCTTGTAGTGGACG
66
365


 


1076
1095
502573
AGGTGCTCCTTGTAGTGGAC
51
366


 


1077
1096
502574
GAGGTGCTCCTTGTAGTGGA
46
367


 


1078
1097
502575
AGAGGTGCTCCTTGTAGTGG
70
368


 


1079
1098
502576
GAGAGGTGCTCCTTGTAGTG
47
369


 


1080
1099
502577
AGAGAGGTGCTCCTTGTAGT
65
370


 


1081
1100
502578
GAGAGAGGTGCTCCTTGTAG
45
371


 


1082
1101
502579
AGAGAGAGGTGCTCCTTGTA
63
372


 


1083
1102
502580
CAGAGAGAGGTGCTCCTTGT
77
373


 


1085
1104
502581
GGCAGAGAGAGGTGCTCCTT
70
374


 


1086
1105
502582
CGGCAGAGAGAGGTGCTCCT
80
375


 


1087
1106
502583
GCGGCAGAGAGAGGTGCTCC
62
376


 


1088
1107
502584
AGCGGCAGAGAGAGGTGCTC
44
377


 


1089
1108
502585
CAGCGGCAGAGAGAGGTGCT
78
378


 


1090
1109
502586
CCAGCGGCAGAGAGAGGTGC
71
379


 


1165
1184
502587
GGCCCAGCCGTGTCTCCGGG
77
380


 


1166
1185
502588
CGGCCCAGCCGTGTCTCCGG
69
381


 


1167
1186
502589
CCGGCCCAGCCGTGTCTCCG
70
382


 


1168
1187
502590
CCCGGCCCAGCCGTGTCTCC
75
383


 


1169
1188
502591
CCCCGGCCCAGCCGTGTCTC
77
384


 


1170
1189
502592
ACCCCGGCCCAGCCGTGTCT
73
385


 


1171
1190
502593
CACCCCGGCCCAGCCGTGTC
84
386


 


1172
1191
502594
CCACCCCGGCCCAGCCGTGT
78
387


 


1173
1192
502595
TCCACCCCGGCCCAGCCGTG
71
388


 


1174
1193
502596
CTCCACCCCGGCCCAGCCGT
81
389


 


1175
1194
502597
GCTCCACCCCGGCCCAGCCG
86
390


 


1176
1195
502598
TGCTCCACCCCGGCCCAGCC
83
391


 


1177
1196
502599
CTGCTCCACCCCGGCCCAGC
88
392


 


1199
1218
502600
AAGGGATGTGTCCGGAAGTC
60
393


 


1200
1219
502601
GAAGGGATGTGTCCGGAAGT
58
394


 


1201
1220
502602
AGAAGGGATGTGTCCGGAAG
63
395


 


1202
1221
502603
AAGAAGGGATGTGTCCGGAA
62
396


 


1203
1222
502604
GAAGAAGGGATGTGTCCGGA
61
397


 


1204
1223
502605
AGAAGAAGGGATGTGTCCGG
62
398


 


1205
1224
502606
AAGAAGAAGGGATGTGTCCG
56
399


 


1206
1225
502607
AAAGAAGAAGGGATGTGTCC
58
400


 


1207
1226
502608
CAAAGAAGAAGGGATGTGTC
50
401


 


1208
1227
502609
CCAAAGAAGAAGGGATGTGT
61
402


 


1210
1229
502610
GGCCAAAGAAGAAGGGATGT
73
403


 


1211
1230
502611
AGGCCAAAGAAGAAGGGATG
56
404


 


1212
1231
502612
GAGGCCAAAGAAGAAGGGAT
73
405


 


1213
1232
502613
CGAGGCCAAAGAAGAAGGGA
75
406


 


1214
1233
502614
TCGAGGCCAAAGAAGAAGGG
75
407


 


1215
1234
502615
GTCGAGGCCAAAGAAGAAGG
83
408


 


1216
1235
502616
AGTCGAGGCCAAAGAAGAAG
58
409


 


1217
1236
502617
CAGTCGAGGCCAAAGAAGAA
52
410


 


1218
1237
502618
CCAGTCGAGGCCAAAGAAGA
68
411


 


1219
1238
502619
CCCAGTCGAGGCCAAAGAAG
78
412


 


1220
1239
502620
TCCCAGTCGAGGCCAAAGAA
66
413


 


1221
1240
502621
ATCCCAGTCGAGGCCAAAGA
75
414


 


1222
1241
502622
CATCCCAGTCGAGGCCAAAG
70
415


 


1223
1242
502623
CCATCCCAGTCGAGGCCAAA
81
416


 


1224
1243
502624
ACCATCCCAGTCGAGGCCAA
82
417


 


1225
1244
502625
GACCATCCCAGTCGAGGCCA
88
418


 


1226
1245
502626
AGACCATCCCAGTCGAGGCC
79
419


 


1227
1246
502627
GAGACCATCCCAGTCGAGGC
82
420


 


1228
1247
502628
GGAGACCATCCCAGTCGAGG
60
421


 


1263
1282
502629
TTCGAAATCCGGTGTAAAGG
84
422


 


1264
1283
502630
CTTCGAAATCCGGTGTAAAG
57
423


 


1265
1284
502631
CCTTCGAAATCCGGTGTAAA
64
424


 


1266
1285
502632
ACCTTCGAAATCCGGTGTAA
73
425


 


1267
1286
502633
CACCTTCGAAATCCGGTGTA
77
426


 


1268
1287
502634
GCACCTTCGAAATCCGGTGT
59
427


 


1269
1288
502635
GGCACCTTCGAAATCCGGTG
85
428


 


1270
1289
502636
TGGCACCTTCGAAATCCGGT
86
429


 


1271
1290
502637
GTGGCACCTTCGAAATCCGG
74
430


 


1272
1291
502638
GGTGGCACCTTCGAAATCCG
79
431


 


1273
1292
502639
CGGTGGCACCTTCGAAATCC
85
432


 


1274
1293
502640
TCGGTGGCACCTTCGAAATC
71
433


 


1275
1294
502641
GTCGGTGGCACCTTCGAAAT
88
434


 


1276
1295
502642
TGTCGGTGGCACCTTCGAAA
89
435


 


1277
1296
502643
GTGTCGGTGGCACCTTCGAA
88
436


 


1278
1297
502644
TGTGTCGGTGGCACCTTCGA
87
437


 


1279
1298
502645
ATGTGTCGGTGGCACCTTCG
88
438


 


1280
1299
502646
CATGTGTCGGTGGCACCTTC
88
439


 


1281
1300
502647
GCATGTGTCGGTGGCACCTT
91
440


 


1282
1301
502648
TGCATGTGTCGGTGGCACCT
87
441


 


1283
1302
502649
TTGCATGTGTCGGTGGCACC
86
442


 


1284
1303
502650
GTTGCATGTGTCGGTGGCAC
83
443


 


1285
1304
502651
AGTTGCATGTGTCGGTGGCA
81
444


 


1286
1305
502652
AAGTTGCATGTGTCGGTGGC
79
445


 


1287
1306
502653
GAAGTTGCATGTGTCGGTGG
58
446


 


1288
1307
502654
CGAAGTTGCATGTGTCGGTG
85
447


 


1290
1309
502655
GTCGAAGTTGCATGTGTCGG
77
448


 


1291
1310
502656
AGTCGAAGTTGCATGTGTCG
79
449


 


1292
1311
502657
AAGTCGAAGTTGCATGTGTC
74
450


 


1293
1312
502658
CAAGTCGAAGTTGCATGTGT
82
451


 


1294
1313
502659
CCAAGTCGAAGTTGCATGTG
82
452


 


1295
1314
502660
ACCAAGTCGAAGTTGCATGT
70
453


 


1296
1315
502661
CACCAAGTCGAAGTTGCATG
76
454


 


1297
1316
502662
CCACCAAGTCGAAGTTGCAT
79
455


 


1298
1317
502663
TCCACCAAGTCGAAGTTGCA
68
456


 


1299
1318
502664
CTCCACCAAGTCGAAGTTGC
71
457


 


1300
1319
502665
CCTCCACCAAGTCGAAGTTG
67
458


 


1301
1320
502666
TCCTCCACCAAGTCGAAGTT
70
459


 


1302
1321
502667
GTCCTCCACCAAGTCGAAGT
80
460


 


1303
1322
502668
CGTCCTCCACCAAGTCGAAG
76
461


 


1304
1323
502669
CCGTCCTCCACCAAGTCGAA
78
462


 


1305
1324
502670
CCCGTCCTCCACCAAGTCGA
83
463


 


1306
1325
502671
GCCCGTCCTCCACCAAGTCG
76
464


 


1307
1326
502672
AGCCCGTCCTCCACCAAGTC
72
465


 


1308
1327
502673
GAGCCCGTCCTCCACCAAGT
71
466


 


1309
1328
502674
TGAGCCCGTCCTCCACCAAG
60
467


 


1702
1721
502675
GGTTCCGAGCCTCTGCCTCG
44
468


 


1703
1722
502676
CGGTTCCGAGCCTCTGCCTC
74
469


 


1704
1723
502677
CCGGTTCCGAGCCTCTGCCT
72
470


 


1705
1724
502678
CCCGGTTCCGAGCCTCTGCC
73
471


 


1706
1725
502679
TCCCGGTTCCGAGCCTCTGC
84
472


 


1707
1726
502680
GTCCCGGTTCCGAGCCTCTG
66
473


 


1709
1728
502681
AGGTCCCGGTTCCGAGCCTC
82
474


 


1710
1729
502682
TAGGTCCCGGTTCCGAGCCT
83
475


 


1711
1730
502683
CTAGGTCCCGGTTCCGAGCC
81
476


 


1712
1731
502684
TCTAGGTCCCGGTTCCGAGC
74
477


 


1713
1732
502685
CTCTAGGTCCCGGTTCCGAG
78
478


 


1714
1733
502686
CCTCTAGGTCCCGGTTCCGA
75
479


 


1715
1734
502687
GCCTCTAGGTCCCGGTTCCG
80
480


 


1743
1762
502688
CATCCGCTCCTGCAACTGCC
89
481


 


1744
1763
502689
CCATCCGCTCCTGCAACTGC
81
482


 


1745
1764
502690
TCCATCCGCTCCTGCAACTG
71
483


 


1746
1765
502691
CTCCATCCGCTCCTGCAACT
75
484


 


1747
1766
502692
ACTCCATCCGCTCCTGCAAC
64
485


 


1748
1767
502693
AACTCCATCCGCTCCTGCAA
52
486


 


1749
1768
502694
CAACTCCATCCGCTCCTGCA
45
487


 


1751
1770
502695
AGCAACTCCATCCGCTCCTG
78
488


 


1752
1771
502696
CAGCAACTCCATCCGCTCCT
64
489


 


1753
1772
502697
GCAGCAACTCCATCCGCTCC
56
490


 


1774
1793
502698
CAGCTGTGGCTCCCTCTGCC
60
491


 


1775
1794
502699
ACAGCTGTGGCTCCCTCTGC
45
492


 


1776
1795
502700
GACAGCTGTGGCTCCCTCTG
49
493


 


1777
1796
502701
TGACAGCTGTGGCTCCCTCT
26
494


 


1778
1797
502702
GTGACAGCTGTGGCTCCCTC
32
495


 


1779
1798
502703
CGTGACAGCTGTGGCTCCCT
28
496


 


1780
1799
502704
CCGTGACAGCTGTGGCTCCC
35
497


 


1781
1800
502705
CCCGTGACAGCTGTGGCTCC
33
498


 


1782
1801
502706
CCCCGTGACAGCTGTGGCTC
53
499


 


1783
1802
502707
CCCCCGTGACAGCTGTGGCT
39
500


 


1784
1803
502708
ACCCCCGTGACAGCTGTGGC
53
501


 


1785
1804
502709
GACCCCCGTGACAGCTGTGG
51
502


 


1786
1805
502710
GGACCCCCGTGACAGCTGTG
58
503


 


1787
1806
502711
GGGACCCCCGTGACAGCTGT
71
504


 


1814
1833
502712
GAAGGTGGATCCGTGGCCCG
73
505


 


1815
1834
502713
GGAAGGTGGATCCGTGGCCC
70
506


 


1816
1835
502714
GGGAAGGTGGATCCGTGGCC
72
507


 


1817
1836
502715
TGGGAAGGTGGATCCGTGGC
50
508


 


1818
1837
502716
ATGGGAAGGTGGATCCGTGG
62
509


 


1819
1838
502717
GATGGGAAGGTGGATCCGTG
75
510


 


1821
1840
502718
TAGATGGGAAGGTGGATCCG
52
511


 


1822
1841
502719
CTAGATGGGAAGGTGGATCC
56
512


 


1823
1842
502720
TCTAGATGGGAAGGTGGATC
21
513


 


1824
1843
502721
ATCTAGATGGGAAGGTGGAT
34
514


 


1826
1845
502722
CCATCTAGATGGGAAGGTGG
43
515


 


1827
1846
502723
GCCATCTAGATGGGAAGGTG
17
516


 


1828
1847
451383
GGCCATCTAGATGGGAAGGT
0
517


 


1863
1882
502724
CACCAGCGGGCACTGGCCCA
51
518


 


1864
1883
502725
CCACCAGCGGGCACTGGCCC
55
519


 


1865
1884
502726
CCCACCAGCGGGCACTGGCC
61
520


 


1866
1885
502727
CCCCACCAGCGGGCACTGGC
43
521


 


1868
1887
502728
GGCCCCACCAGCGGGCACTG
16
522


 


1869
1888
502729
TGGCCCCACCAGCGGGCACT
43
523


 


1870
1889
502730
CTGGCCCCACCAGCGGGCAC
43
524


 


1871
1890
502731
CCTGGCCCCACCAGCGGGCA
41
525


 


1872
1891
502732
GCCTGGCCCCACCAGCGGGC
30
526


 


1874
1893
502733
GGGCCTGGCCCCACCAGCGG
66
527


 


1892
1911
502734
AGGTGGCGGCGGTGCATGGG
31
528


 


1893
1912
502735
CAGGTGGCGGCGGTGCATGG
23
529


 


1894
1913
502736
GCAGGTGGCGGCGGTGCATG
57
530


 


1895
1914
502737
AGCAGGTGGCGGCGGTGCAT
54
531


 


1896
1915
502738
CAGCAGGTGGCGGCGGTGCA
61
532


 


1897
1916
502739
GCAGCAGGTGGCGGCGGTGC
57
533


 


1898
1917
502740
AGCAGCAGGTGGCGGCGGTG
36
534


 


1899
1918
502741
GAGCAGCAGGTGGCGGCGGT
53
535


 


1900
1919
502742
GGAGCAGCAGGTGGCGGCGG
39
536


 


1901
1920
502743
GGGAGCAGCAGGTGGCGGCG
36
537


 


1902
1921
502744
AGGGAGCAGCAGGTGGCGGC
62
538


 


1903
1922
502745
CAGGGAGCAGCAGGTGGCGG
56
539


 


1904
1923
502746
GCAGGGAGCAGCAGGTGGCG
58
540


 


1905
1924
502747
GGCAGGGAGCAGCAGGTGGC
65
541


 


1906
1925
502748
TGGCAGGGAGCAGCAGGTGG
47
542


 


1907
1926
502749
CTGGCAGGGAGCAGCAGGTG
41
543


 


1909
1928
451432
CCCTGGCAGGGAGCAGCAGG
53
544


 


1910
1929
502750
ACCCTGGCAGGGAGCAGCAG
52
545


 


1911
1930
502751
GACCCTGGCAGGGAGCAGCA
77
546


 


1912
1931
502752
GGACCCTGGCAGGGAGCAGC
0
547


 


1919
1938
502753
GGCCTAGGGACCCTGGCAGG
39
548


 


1920
1939
502754
AGGCCTAGGGACCCTGGCAG
35
549


 


1922
1941
502755
CCAGGCCTAGGGACCCTGGC
44
550


 


1923
1942
502756
GCCAGGCCTAGGGACCCTGG
60
551


 


1924
1943
502757
GGCCAGGCCTAGGGACCCTG
58
552


 


1925
1944
502758
AGGCCAGGCCTAGGGACCCT
57
553


 


1926
1945
502759
TAGGCCAGGCCTAGGGACCC
52
554


 


1927
1946
502760
ATAGGCCAGGCCTAGGGACC
51
555


 


1928
1947
502761
GATAGGCCAGGCCTAGGGAC
41
556


 


1929
1948
502762
CGATAGGCCAGGCCTAGGGA
69
557


 


1930
1949
502763
CCGATAGGCCAGGCCTAGGG
80
558


 


1931
1950
502764
TCCGATAGGCCAGGCCTAGG
78
559


 


1932
1951
502765
CTCCGATAGGCCAGGCCTAG
89
560


 


1933
1952
502766
CCTCCGATAGGCCAGGCCTA
79
561


 


1934
1953
502767
GCCTCCGATAGGCCAGGCCT
73
562


 


1936
1955
502768
GCGCCTCCGATAGGCCAGGC
83
563


 


1952
1971
502769
AACAGGAGCAGGGAAAGCGC
83
564


 


1953
1972
502770
GAACAGGAGCAGGGAAAGCG
70
565


 


1954
1973
502771
CGAACAGGAGCAGGGAAAGC
43
566


 


1955
1974
502772
GCGAACAGGAGCAGGGAAAG
47
567


 


1956
1975
502773
GGCGAACAGGAGCAGGGAAA
61
568


 


1957
1976
502774
CGGCGAACAGGAGCAGGGAA
74
569


 


1958
1977
502775
ACGGCGAACAGGAGCAGGGA
60
570


 


1959
1978
502776
AACGGCGAACAGGAGCAGGG
86
571


 


1960
1979
502777
CAACGGCGAACAGGAGCAGG
84
572


 


1981
2000
502778
GGGCGGCGGCACGAGACAGA
80
573


 


1982
2001
502779
AGGGCGGCGGCACGAGACAG
76
574


 


1983
2002
502780
CAGGGCGGCGGCACGAGACA
58
575


 


1984
2003
502781
CCAGGGCGGCGGCACGAGAC
80
576


 


1985
2004
502782
CCCAGGGCGGCGGCACGAGA
59
577


 


1986
2005
502783
GCCCAGGGCGGCGGCACGAG
68
578


 


1987
2006
502784
AGCCCAGGGCGGCGGCACGA
75
579


 


1988
2007
502785
CAGCCCAGGGCGGCGGCACG
76
580


 


1989
2008
502786
GCAGCCCAGGGCGGCGGCAC
70
581


 


2026
2045
502787
CTGCGGTGAGTTGGCCGGCG
68
582


 


2027
2046
502788
ACTGCGGTGAGTTGGCCGGC
67
583


 


2028
2047
502789
GACTGCGGTGAGTTGGCCGG
58
584


 


2029
2048
502790
AGACTGCGGTGAGTTGGCCG
71
585


 


2030
2049
502791
CAGACTGCGGTGAGTTGGCC
70
586


 


2031
2050
502792
CCAGACTGCGGTGAGTTGGC
79
587


 


2032
2051
502793
GCCAGACTGCGGTGAGTTGG
76
588


 


2033
2052
502794
CGCCAGACTGCGGTGAGTTG
66
589


 


2077
2096
502795
AAGACAGTTCTAGGGTTCAG
87
590


 


2078
2097
502796
GAAGACAGTTCTAGGGTTCA
78
591


 


2079
2098
502797
CGAAGACAGTTCTAGGGTTC
85
592


 


2080
2099
502798
TCGAAGACAGTTCTAGGGTT
78
593


 


2081
2100
502799
GTCGAAGACAGTTCTAGGGT
92
594


 


2082
2101
502800
AGTCGAAGACAGTTCTAGGG
85
595


 


2083
2102
502801
GAGTCGAAGACAGTTCTAGG
83
596


 


2084
2103
502802
GGAGTCGAAGACAGTTCTAG
86
597


 


2085
2104
502803
CGGAGTCGAAGACAGTTCTA
91
598


 


2086
2105
502804
CCGGAGTCGAAGACAGTTCT
76
599


 


2087
2106
502805
CCCGGAGTCGAAGACAGTTC
90
600


 


2088
2107
502806
CCCCGGAGTCGAAGACAGTT
83
601


 


2089
2108
502807
GCCCCGGAGTCGAAGACAGT
82
602


 


2090
2109
502808
GGCCCCGGAGTCGAAGACAG
73
603


 


2091
2110
502809
GGGCCCCGGAGTCGAAGACA
67
604


 


2143
2162
502810
AGGCGGTGGGCGCGGCTTCT
73
605


 


2144
2163
502811
CAGGCGGTGGGCGCGGCTTC
57
606


 


2145
2164
502812
GCAGGCGGTGGGCGCGGCTT
69
607


 


2147
2166
502813
TGGCAGGCGGTGGGCGCGGC
73
608


 


2149
2168
502814
ACTGGCAGGCGGTGGGCGCG
56
609


 


2151
2170
502815
GAACTGGCAGGCGGTGGGCG
71
610


 


2152
2171
502816
TGAACTGGCAGGCGGTGGGC
80
611


 


2154
2173
502817
TGTGAACTGGCAGGCGGTGG
85
612


 


2187
2206
502818
TGGAGCTGGGCGGAGACCCA
55
613


 


2189
2208
502819
ACTGGAGCTGGGCGGAGACC
53
614


 


2190
2209
502820
GACTGGAGCTGGGCGGAGAC
55
615


 


2192
2211
502821
AGGACTGGAGCTGGGCGGAG
76
616


 


2194
2213
502822
ACAGGACTGGAGCTGGGCGG
77
617


 


2195
2214
502823
CACAGGACTGGAGCTGGGCG
74
618


 


2196
2215
502824
TCACAGGACTGGAGCTGGGC
90
619


 


2386
2405
502825
GCCTCAGCCTGGCCGAAAGA
80
620


 


2387
2406
502826
GGCCTCAGCCTGGCCGAAAG
72
621


 


2490
2509
444401
TTGCACTTTGCGAACCAACG
97
41
















TABLE 13







Inhibition of human DMPK RNA transcript in hSKMc by


5-10-5 gapmers targeting SEQ ID NO: 2












Target
Target






Start
Stop
ISIS


SEQ ID


Site
Site
No
Sequence
% inhibition
NO.















503
522
502983
TGGTGGAGCCAAGCCCTCCC
83
622


 


561
580
502984
GGGCACCCTCAGAGCCTGAA
82
623


 


1197
1216
502369
GCCTGGCAGCCCCTGTCCAG
16
160


 


1198
1217
502370
GGCCTGGCAGCCCCTGTCCA
58
161


 


1199
1218
502371
GGGCCTGGCAGCCCCTGTCC
62
162


 


1242
1261
502372
ATGGCCCCTCCCCGGGCCGG
41
163


 


1243
1262
502373
CATGGCCCCTCCCCGGGCCG
29
164


 


1244
1263
502374
CCATGGCCCCTCCCCGGGCC
34
165


 


1245
1264
502375
ACCATGGCCCCTCCCCGGGC
60
166


 


1246
1265
502376
CACCATGGCCCCTCCCCGGG
68
167


 


1247
1266
502377
GCACCATGGCCCCTCCCCGG
75
168


 


1248
1267
502378
AGCACCATGGCCCCTCCCCG
65
169


 


1249
1268
502379
CAGCACCATGGCCCCTCCCC
63
170


 


1250
1269
502380
GCAGCACCATGGCCCCTCCC
73
171


 


1251
1270
502381
GGCAGCACCATGGCCCCTCC
80
172


 


1253
1272
502382
CAGGCAGCACCATGGCCCCT
82
173


 


1254
1273
502383
ACAGGCAGCACCATGGCCCC
72
174


 


1256
1275
502384
GGACAGGCAGCACCATGGCC
70
175


 


1257
1276
502385
TGGACAGGCAGCACCATGGC
71
176


 


1258
1277
502386
TTGGACAGGCAGCACCATGG
73
177


 


1259
1278
502387
GTTGGACAGGCAGCACCATG
73
178


 


1260
1279
502388
TGTTGGACAGGCAGCACCAT
60
179


 


1261
1280
502389
ATGTTGGACAGGCAGCACCA
75
180


 


1262
1281
502390
CATGTTGGACAGGCAGCACC
81
181


 


1263
1282
502391
ACATGTTGGACAGGCAGCAC
67
182


 


1264
1283
502392
GACATGTTGGACAGGCAGCA
71
183


 


1265
1284
502393
TGACATGTTGGACAGGCAGC
81
184


 


1266
1285
502394
CTGACATGTTGGACAGGCAG
76
185


 


1267
1286
502395
GCTGACATGTTGGACAGGCA
70
186


 


1268
1287
502396
GGCTGACATGTTGGACAGGC
77
187


 


1269
1288
502397
CGGCTGACATGTTGGACAGG
74
188


 


1270
1289
502398
TCGGCTGACATGTTGGACAG
63
189


 


1271
1290
502399
CTCGGCTGACATGTTGGACA
80
190


 


1272
1291
502400
CCTCGGCTGACATGTTGGAC
71
191


 


1273
1292
502401
ACCTCGGCTGACATGTTGGA
64
192


 


1274
1293
502402
CACCTCGGCTGACATGTTGG
71
193


 


1275
1294
502403
GCACCTCGGCTGACATGTTG
77
194


 


1276
1295
502404
CGCACCTCGGCTGACATGTT
80
195


 


1277
1296
502405
CCGCACCTCGGCTGACATGT
80
196


 


1278
1297
502406
GCCGCACCTCGGCTGACATG
79
197


 


1279
1298
502407
AGCCGCACCTCGGCTGACAT
74
198


 


1280
1299
502408
CAGCCGCACCTCGGCTGACA
66
199


 


1281
1300
502409
TCAGCCGCACCTCGGCTGAC
15
200


 


1282
1301
502410
CTCAGCCGCACCTCGGCTGA
32
201


 


1283
1302
502411
CCTCAGCCGCACCTCGGCTG
65
202


 


1284
1303
502412
GCCTCAGCCGCACCTCGGCT
81
203


 


1305
1324
502413
CCAACACCAGCTGCTGGAGC
90
204


 


1306
1325
502414
TCCAACACCAGCTGCTGGAG
78
205


 


1307
1326
502415
GTCCAACACCAGCTGCTGGA
84
206


 


1309
1328
502416
GGGTCCAACACCAGCTGCTG
69
207


 


1330
1349
502417
GGCTCCAGCCCCAGGAAGCC
46
208


 


1331
1350
502418
GGGCTCCAGCCCCAGGAAGC
28
209


 


1349
1368
502419
CAGGAGAAGGTCGAGCAGGG
41
210


 


1351
1370
502420
CCCAGGAGAAGGTCGAGCAG
71
211


 


1352
1371
502421
GCCCAGGAGAAGGTCGAGCA
85
212


 


1353
1372
451363
CGCCCAGGAGAAGGTCGAGC
84
213


 


1354
1373
502422
ACGCCCAGGAGAAGGTCGAG
67
214


 


1390
1409
502423
TCCTGGGCCAGTTCGGAGGC
58
215


 


1391
1410
502424
GTCCTGGGCCAGTTCGGAGG
71
216


 


1392
1411
502425
TGTCCTGGGCCAGTTCGGAG
69
217


 


1393
1412
502426
TTGTCCTGGGCCAGTTCGGA
71
218


 


1394
1413
502427
CTTGTCCTGGGCCAGTTCGG
66
219


 


1395
1414
502428
ACTTGTCCTGGGCCAGTTCG
59
220


 


1396
1415
502429
TACTTGTCCTGGGCCAGTTC
75
221


 


1397
1416
502430
GTACTTGTCCTGGGCCAGTT
78
222


 


1398
1417
502431
CGTACTTGTCCTGGGCCAGT
74
223


 


1416
1435
502432
ACTGCAAGAAGTCGGCCACG
73
224


 


1418
1437
502433
CCACTGCAAGAAGTCGGCCA
65
225


 


1419
1438
451364
CCCACTGCAAGAAGTCGGCC
32
226


 


1421
1440
502985
ACCCCACTGCAAGAAGTCGG
60
624


 


1551
1570
502986
GCCCCAGGATGGGAGGATCT
58
625


 


1597
1616
502987
CATAGGACAGAGAAATGTTG
70
626


 


1630
1649
502988
TGCTGACCTTACTCTGCCCC
86
627


 


1666
1685
502989
TAAGCCATGGCTCTGAGTCA
51
628


 


1712
1731
502990
AGAGAGGCCATGGGAGGCTG
42
629


 


1841
1860
502991
CTGGCCCTCCTGGCTTGCCC
72
630


 


1853
1872
502992
AGCTGCCCCATGCTGGCCCT
76
631


 


1862
1881
502993
GCCCCTGGCAGCTGCCCCAT
70
632


 


1873
1892
502994
CTGTCGGCTGCGCCCCTGGC
78
633


 


1887
1906
502995
CGCCGAACACCTGCCTGTCG
68
634


 


1931
1950
502996
CCTCCCAGTGCCTGGGCACC
52
635


 


1981
2000
502998
GCGCCTGTCTGCAAAGCTGG
84
636


 


2025
2044
502999
CCCAAAGTTGTCCCTCCTGG
83
637


 


2038
2057
503000
ACACCCAGAAGAACCCAAAG
75
638


 


2117
2136
503001
CTGACCCACACGGCTCATAG
65
639


 


2235
2254
503002
TGGCCCCAGGCCCTGGAAAG
67
640


 


2278
2297
503003
GACAAGGCAGCTGGCAGAAG
79
641


 


2331
2350
503004
AAGAAACCAGTGACCAGTGA
85
642


 


2523
2542
503005
CTGTGAAATGGGAGGAGGAG
0
643


 


2578
2597
503006
GAAGGTTTTTCCAGAGGCTG
88
644


 


2615
2634
503007
GGCCAGGAGAGTCATTAGGG
84
645


 


2710
2729
503008
CCACAAAAGGAGTGCTCCTC
79
646


 


2789
2808
503009
CCTTTTAAGGCAGCAGGAAC
78
647


 


3629
3648
503010
CTAGGACTGTCTGCTTCCCA
88
648


 


3761
3780
502452
CTCACCACGATGGGCTCCGC
66
245


 


3762
3781
502453
CCTCACCACGATGGGCTCCG
81
246


 


3763
3782
502454
GCCTCACCACGATGGGCTCC
77
247


 


3764
3783
502455
AGCCTCACCACGATGGGCTC
63
248


 


3765
3784
502456
AAGCCTCACCACGATGGGCT
70
249


 


3766
3785
502457
TAAGCCTCACCACGATGGGC
78
250


 


3767
3786
502458
TTAAGCCTCACCACGATGGG
76
251


 


3768
3787
502459
CTTAAGCCTCACCACGATGG
78
252


 


3769
3788
502460
CCTTAAGCCTCACCACGATG
68
253


 


3770
3789
502461
TCCTTAAGCCTCACCACGAT
67
254


 


3771
3790
502462
CTCCTTAAGCCTCACCACGA
84
255


 


3772
3791
502463
CCTCCTTAAGCCTCACCACG
76
256


 


3773
3792
502464
ACCTCCTTAAGCCTCACCAC
64
257


 


3774
3793
502465
GACCTCCTTAAGCCTCACCA
72
258


 


3775
3794
502466
GGACCTCCTTAAGCCTCACC
69
259


 


3776
3795
502467
CGGACCTCCTTAAGCCTCAC
81
260


 


3777
3796
502468
TCGGACCTCCTTAAGCCTCA
78
261


 


3778
3797
502469
GTCGGACCTCCTTAAGCCTC
57
262


 


3780
3799
502470
CAGTCGGACCTCCTTAAGCC
62
263


 


3781
3800
502471
GCAGTCGGACCTCCTTAAGC
45
264


 


3782
3801
502472
TGCAGTCGGACCTCCTTAAG
60
265


 


3808
3827
502473
CCTTCAGAATCTCGAAGTCG
67
266


 


3809
3828
502474
ACCTTCAGAATCTCGAAGTC
50
267


 


3811
3830
502475
TCACCTTCAGAATCTCGAAG
54
268


 


3812
3831
502476
ATCACCTTCAGAATCTCGAA
38
269


 


3813
3832
502477
GATCACCTTCAGAATCTCGA
35
270


 


3815
3834
502478
CCGATCACCTTCAGAATCTC
52
271


 


3816
3835
502479
TCCGATCACCTTCAGAATCT
50
272


 


3817
3836
502480
GTCCGATCACCTTCAGAATC
44
273


 


3818
3837
502481
CGTCCGATCACCTTCAGAAT
41
274


 


3921
3940
503011
GTCATTCATCAATTTCTAAG
44
649


 


4118
4137
502482
CCCGTCTGCTTCATCTTCAC
67
275


 


4119
4138
502483
GCCCGTCTGCTTCATCTTCA
76
276


 


4120
4139
502484
GGCCCGTCTGCTTCATCTTC
57
277


 


4121
4140
502485
TGGCCCGTCTGCTTCATCTT
64
278


 


4122
4141
502486
CTGGCCCGTCTGCTTCATCT
64
279


 


4123
4142
502487
CCTGGCCCGTCTGCTTCATC
73
280


 


4124
4143
502488
ACCTGGCCCGTCTGCTTCAT
64
281


 


4125
4144
502489
CACCTGGCCCGTCTGCTTCA
80
282


 


4126
4145
502490
ACACCTGGCCCGTCTGCTTC
71
283


 


4127
4146
502491
TACACCTGGCCCGTCTGCTT
74
284


 


4148
4167
502492
TTGTTCATGATCTTCATGGC
56
285


 


4150
4169
502493
ACTTGTTCATGATCTTCATG
23
286


 


4151
4170
502494
CACTTGTTCATGATCTTCAT
43
287


 


4152
4171
502495
CCACTTGTTCATGATCTTCA
43
288


 


4153
4172
502496
CCCACTTGTTCATGATCTTC
47
289


 


4154
4173
502497
TCCCACTTGTTCATGATCTT
34
290


 


4155
4174
502498
GTCCCACTTGTTCATGATCT
34
291


 


4156
4175
502499
TGTCCCACTTGTTCATGATC
27
292


 


4157
4176
502500
ATGTCCCACTTGTTCATGAT
23
293


 


4158
4177
502501
CATGTCCCACTTGTTCATGA
51
294


 


4159
4178
502502
GCATGTCCCACTTGTTCATG
20
295


 


4160
4179
502503
AGCATGTCCCACTTGTTCAT
52
296


 


4161
4180
502504
CAGCATGTCCCACTTGTTCA
72
297


 


4162
4181
502505
TCAGCATGTCCCACTTGTTC
70
298


 


4163
4182
502506
TTCAGCATGTCCCACTTGTT
53
299


 


4164
4183
502507
CTTCAGCATGTCCCACTTGT
52
300


 


4165
4184
502508
TCTTCAGCATGTCCCACTTG
45
301


 


4167
4186
502509
CCTCTTCAGCATGTCCCACT
68
302


 


4168
4187
502510
CCCTCTTCAGCATGTCCCAC
68
303


 


4169
4188
502511
CCCCTCTTCAGCATGTCCCA
79
304


 


4170
4189
502512
GCCCCTCTTCAGCATGTCCC
85
305


 


4171
4190
502513
CGCCCCTCTTCAGCATGTCC
84
306


 


4172
4191
502514
TCGCCCCTCTTCAGCATGTC
80
307


 


4173
4192
502515
CTCGCCCCTCTTCAGCATGT
82
308


 


4174
4193
502516
CCTCGCCCCTCTTCAGCATG
78
309


 


4175
4194
502517
ACCTCGCCCCTCTTCAGCAT
73
310


 


4176
4195
502518
CACCTCGCCCCTCTTCAGCA
76
311


 


4239
4258
503012
GGAGGAGCTGCAGCCGGAGA
7
650


 


4245
4264
503013
GCACCCGGAGGAGCTGCAGC
0
651


 


4261
4280
503014
GCACGACACCTGCAGGGCAC
23
652


 


4355
4374
503015
AGCTCACCAGGTAGTTCTCA
49
653


 


4427
4446
503016
GCTTCCTCTCCCCACCTCCT
65
654


 


4447
4466
503017
GCAGCACCCCCAATCCTAGA
67
655


 


4508
4527
503018
GCCCCTCATCCACCTGACAC
62
656


 


4613
4632
503019
TTCCAGGTAAGAGACCCCCC
87
657


 


4679
4698
503020
AGAATAGGTCCCAGACACTC
81
658


 


4731
4750
503021
CTCCCCCTGAGATGTTCTGG
53
659


 


4858
4877
503022
CCCCAGCCCAGAGATAACCA
74
660


 


4927
4946
503023
CCTGATCCATCACGGATGGC
69
661


 


4987
5006
503024
TACTCCATGACCAGGTACTG
81
662


 


5185
5204
503025
GCTCTGACCTTCCAAGAACC
56
663


 


5354
5373
503026
CTCCCTTCTGTGGTCCCACC
0
664


 


5407
5426
503027
GTCGGGTTTGATGTCCCTGC
75
665


 


5445
5464
502521
GCCAGGCGGATGTGGCCACA
57
314


 


5500
5519
503028
AGGGCACTGGCTCACCGTTC
45
666


 


5681
5700
503029
GGGCCCTCCTTCCAACCACT
28
667


 


5708
5727
503030
GCCCACCCCTCTGGGCCCAC
45
668


 


5728
5747
503031
AGGAGCAGAGCGAGGCTTGG
38
669


 


5800
5819
502524
ACAGCCTGCAGGATCTCGGG
86
317


 


5801
5820
502525
CACAGCCTGCAGGATCTCGG
81
318


 


5802
5821
502526
CCACAGCCTGCAGGATCTCG
83
319


 


5803
5822
502527
CCCACAGCCTGCAGGATCTC
84
320


 


5804
5823
502528
GCCCACAGCCTGCAGGATCT
91
321


 


5805
5824
502529
CGCCCACAGCCTGCAGGATC
90
322


 


5806
5825
502530
CCGCCCACAGCCTGCAGGAT
82
323


 


5807
5826
502531
ACCGCCCACAGCCTGCAGGA
83
324


 


5808
5827
502532
CACCGCCCACAGCCTGCAGG
85
325


 


5809
5828
502533
CCACCGCCCACAGCCTGCAG
84
326


 


5810
5829
502534
CCCACCGCCCACAGCCTGCA
80
327


 


5811
5830
502535
GCCCACCGCCCACAGCCTGC
90
328


 


5812
5831
502536
GGCCCACCGCCCACAGCCTG
94
329


 


5813
5832
502537
AGGCCCACCGCCCACAGCCT
88
330


 


5814
5833
502538
CAGGCCCACCGCCCACAGCC
91
331


 


5815
5834
502539
CCAGGCCCACCGCCCACAGC
73
332


 


5816
5835
502540
CCCAGGCCCACCGCCCACAG
86
333


 


5817
5836
502541
TCCCAGGCCCACCGCCCACA
88
334


 


5818
5837
502542
GTCCCAGGCCCACCGCCCAC
84
335


 


5819
5838
502543
TGTCCCAGGCCCACCGCCCA
85
336


 


5820
5839
502544
CTGTCCCAGGCCCACCGCCC
65
337


 


5821
5840
502545
CCTGTCCCAGGCCCACCGCC
81
338


 


5822
5841
502546
GCCTGTCCCAGGCCCACCGC
90
339


 


5823
5842
502547
TGCCTGTCCCAGGCCCACCG
85
340


 


5824
5843
502548
CTGCCTGTCCCAGGCCCACC
89
341


 


5825
5844
502549
GCTGCCTGTCCCAGGCCCAC
91
342


 


5826
5845
502550
AGCTGCCTGTCCCAGGCCCA
94
343


 


5827
5846
502551
TAGCTGCCTGTCCCAGGCCC
92
344


 


5828
5847
502552
GTAGCTGCCTGTCCCAGGCC
88
345


 


5829
5848
502553
CGTAGCTGCCTGTCCCAGGC
85
346


 


5830
5849
502554
CCGTAGCTGCCTGTCCCAGG
83
347


 


5831
5850
502555
CCCGTAGCTGCCTGTCCCAG
64
348


 


5832
5851
502556
GCCCGTAGCTGCCTGTCCCA
83
349


 


5833
5852
502557
GGCCCGTAGCTGCCTGTCCC
89
350


 


5881
5900
502558
TAGAACATTTCATAGGCGAA
68
351


 


5919
5938
502559
TCTCCGCCGTGGAATCCGCG
75
352


 


5920
5939
502560
GTCTCCGCCGTGGAATCCGC
79
353


 


5921
5940
502561
GGTCTCCGCCGTGGAATCCG
66
354


 


5922
5941
502562
AGGTCTCCGCCGTGGAATCC
50
355


 


5923
5942
502563
TAGGTCTCCGCCGTGGAATC
71
356


 


5944
5963
502564
TTGTAGTGGACGATCTTGCC
68
357


 


5945
5964
502565
CTTGTAGTGGACGATCTTGC
70
358


 


5946
5965
502566
CCTTGTAGTGGACGATCTTG
61
359


 


5948
5967
503032
CACCTTGTAGTGGACGATCT
62
670


 


6039
6058
502582
CGGCAGAGAGAGGTGCTCCT
80
375


 


6040
6059
502583
GCGGCAGAGAGAGGTGCTCC
62
376


 


6041
6060
502584
AGCGGCAGAGAGAGGTGCTC
44
377


 


6042
6061
502585
CAGCGGCAGAGAGAGGTGCT
78
378


 


6043
6062
502586
CCAGCGGCAGAGAGAGGTGC
71
379


 


6118
6137
502587
GGCCCAGCCGTGTCTCCGGG
77
380


 


6119
6138
502588
CGGCCCAGCCGTGTCTCCGG
69
381


 


6120
6139
502589
CCGGCCCAGCCGTGTCTCCG
70
382


 


6121
6140
502590
CCCGGCCCAGCCGTGTCTCC
75
383


 


6122
6141
502591
CCCCGGCCCAGCCGTGTCTC
77
384


 


6123
6142
502592
ACCCCGGCCCAGCCGTGTCT
73
385


 


6124
6143
502593
CACCCCGGCCCAGCCGTGTC
84
386


 


6125
6144
502594
CCACCCCGGCCCAGCCGTGT
78
387


 


6126
6145
502595
TCCACCCCGGCCCAGCCGTG
71
388


 


6127
6146
502596
CTCCACCCCGGCCCAGCCGT
81
389


 


6128
6147
502597
GCTCCACCCCGGCCCAGCCG
86
390


 


6129
6148
502598
TGCTCCACCCCGGCCCAGCC
83
391


 


6130
6149
502599
CTGCTCCACCCCGGCCCAGC
88
392


 


6152
6171
502600
AAGGGATGTGTCCGGAAGTC
60
393


 


6153
6172
502601
GAAGGGATGTGTCCGGAAGT
58
394


 


6154
6173
502602
AGAAGGGATGTGTCCGGAAG
63
395


 


6155
6174
502603
AAGAAGGGATGTGTCCGGAA
62
396


 


6156
6175
502604
GAAGAAGGGATGTGTCCGGA
61
397


 


6157
6176
502605
AGAAGAAGGGATGTGTCCGG
62
398


 


6158
6177
502606
AAGAAGAAGGGATGTGTCCG
56
399


 


6159
6178
502607
AAAGAAGAAGGGATGTGTCC
58
400


 


6160
6179
502608
CAAAGAAGAAGGGATGTGTC
50
401


 


6161
6180
502609
CCAAAGAAGAAGGGATGTGT
61
402


 


6163
6182
502610
GGCCAAAGAAGAAGGGATGT
73
403


 


6164
6183
502611
AGGCCAAAGAAGAAGGGATG
56
404


 


6165
6184
502612
GAGGCCAAAGAAGAAGGGAT
73
405


 


6166
6185
502613
CGAGGCCAAAGAAGAAGGGA
75
406


 


6167
6186
502614
TCGAGGCCAAAGAAGAAGGG
75
407


 


6168
6187
502615
GTCGAGGCCAAAGAAGAAGG
83
408


 


6169
6188
502616
AGTCGAGGCCAAAGAAGAAG
58
409


 


6170
6189
502617
CAGTCGAGGCCAAAGAAGAA
52
410


 


6171
6190
502618
CCAGTCGAGGCCAAAGAAGA
68
411


 


6172
6191
502619
CCCAGTCGAGGCCAAAGAAG
78
412


 


6173
6192
502620
TCCCAGTCGAGGCCAAAGAA
66
413


 


6174
6193
502621
ATCCCAGTCGAGGCCAAAGA
75
414


 


6175
6194
502622
CATCCCAGTCGAGGCCAAAG
70
415


 


6176
6195
502623
CCATCCCAGTCGAGGCCAAA
81
416


 


6177
6196
502624
ACCATCCCAGTCGAGGCCAA
82
417


 


6178
6197
502625
GACCATCCCAGTCGAGGCCA
88
418


 


6179
6198
502626
AGACCATCCCAGTCGAGGCC
79
419


 


6180
6199
502627
GAGACCATCCCAGTCGAGGC
82
420


 


6181
6200
502628
GGAGACCATCCCAGTCGAGG
60
421


 


6216
6235
502629
TTCGAAATCCGGTGTAAAGG
84
422


 


6217
6236
502630
CTTCGAAATCCGGTGTAAAG
57
423


 


6218
6237
502631
CCTTCGAAATCCGGTGTAAA
64
424


 


6219
6238
502632
ACCTTCGAAATCCGGTGTAA
73
425


 


6220
6239
502633
CACCTTCGAAATCCGGTGTA
77
426


 


6221
6240
502634
GCACCTTCGAAATCCGGTGT
59
427


 


6222
6241
502635
GGCACCTTCGAAATCCGGTG
85
428


 


6223
6242
502636
TGGCACCTTCGAAATCCGGT
86
429


 


6224
6243
502637
GTGGCACCTTCGAAATCCGG
74
430


 


6225
6244
502638
GGTGGCACCTTCGAAATCCG
79
431


 


6226
6245
502639
CGGTGGCACCTTCGAAATCC
85
432


 


6227
6246
502640
TCGGTGGCACCTTCGAAATC
71
433


 


6228
6247
502641
GTCGGTGGCACCTTCGAAAT
88
434


 


6229
6248
502642
TGTCGGTGGCACCTTCGAAA
89
435


 


6230
6249
502643
GTGTCGGTGGCACCTTCGAA
88
436


 


6231
6250
502644
TGTGTCGGTGGCACCTTCGA
87
437


 


6232
6251
502645
ATGTGTCGGTGGCACCTTCG
88
438


 


6233
6252
502646
CATGTGTCGGTGGCACCTTC
88
439


 


6234
6253
502647
GCATGTGTCGGTGGCACCTT
91
440


 


6235
6254
502648
TGCATGTGTCGGTGGCACCT
87
441


 


6236
6255
502649
TTGCATGTGTCGGTGGCACC
86
442


 


6237
6256
502650
GTTGCATGTGTCGGTGGCAC
83
443


 


6238
6257
502651
AGTTGCATGTGTCGGTGGCA
81
444


 


6239
6258
502652
AAGTTGCATGTGTCGGTGGC
79
445


 


6240
6259
502653
GAAGTTGCATGTGTCGGTGG
58
446


 


6241
6260
502654
CGAAGTTGCATGTGTCGGTG
85
447


 


6243
6262
502655
GTCGAAGTTGCATGTGTCGG
77
448


 


6244
6263
502656
AGTCGAAGTTGCATGTGTCG
79
449


 


6245
6264
502657
AAGTCGAAGTTGCATGTGTC
74
450


 


6246
6265
502658
CAAGTCGAAGTTGCATGTGT
82
451


 


6247
6266
502659
CCAAGTCGAAGTTGCATGTG
82
452


 


6248
6267
502660
ACCAAGTCGAAGTTGCATGT
70
453


 


6249
6268
502661
CACCAAGTCGAAGTTGCATG
76
454


 


6250
6269
502662
CCACCAAGTCGAAGTTGCAT
79
455


 


6251
6270
502663
TCCACCAAGTCGAAGTTGCA
68
456


 


6252
6271
502664
CTCCACCAAGTCGAAGTTGC
71
457


 


6253
6272
502665
CCTCCACCAAGTCGAAGTTG
67
458


 


6254
6273
502666
TCCTCCACCAAGTCGAAGTT
70
459


 


6255
6274
502667
GTCCTCCACCAAGTCGAAGT
80
460


 


6256
6275
502668
CGTCCTCCACCAAGTCGAAG
76
461


 


6257
6276
502669
CCGTCCTCCACCAAGTCGAA
78
462


 


6258
6277
502670
CCCGTCCTCCACCAAGTCGA
83
463


 


6259
6278
502671
GCCCGTCCTCCACCAAGTCG
76
464


 


6260
6279
502672
AGCCCGTCCTCCACCAAGTC
72
465


 


6261
6280
502673
GAGCCCGTCCTCCACCAAGT
71
466


 


6262
6281
502674
TGAGCCCGTCCTCCACCAAG
60
467


 


6289
6308
503033
CTACCCCGCCCCCGCTCACC
60
671


 


6445
6464
503034
CTAGGTCACTGCTGGGTCCT
86
672


 


6596
6615
503035
CTCAGATAGCTCCCCACTCC
55
673


 


6794
6813
503036
AATTCTCTAATTCTCTAGAC
19
674


 


8666
8685
503037
TACCTGAGGGCCATGCAGGA
51
675


 


8765
8784
503038
GTTCCAAGACTGATCCTGCA
69
676


 


11975
11994
502675
GGTTCCGAGCCTCTGCCTCG
44
468


 


11976
11995
502676
CGGTTCCGAGCCTCTGCCTC
74
469


 


11977
11996
502677
CCGGTTCCGAGCCTCTGCCT
72
470


 


11978
11997
502678
CCCGGTTCCGAGCCTCTGCC
73
471


 


11979
11998
502679
TCCCGGTTCCGAGCCTCTGC
84
472


 


11980
11999
502680
GTCCCGGTTCCGAGCCTCTG
66
473


 


11982
12001
502681
AGGTCCCGGTTCCGAGCCTC
82
474


 


11983
12002
502682
TAGGTCCCGGTTCCGAGCCT
83
475


 


11984
12003
502683
CTAGGTCCCGGTTCCGAGCC
81
476


 


11985
12004
502684
TCTAGGTCCCGGTTCCGAGC
74
477


 


11986
12005
502685
CTCTAGGTCCCGGTTCCGAG
78
478


 


11987
12006
502686
CCTCTAGGTCCCGGTTCCGA
75
479


 


11988
12007
502687
GCCTCTAGGTCCCGGTTCCG
80
480


 


12016
12035
502688
CATCCGCTCCTGCAACTGCC
89
481


 


12017
12036
502689
CCATCCGCTCCTGCAACTGC
81
482


 


12018
12037
502690
TCCATCCGCTCCTGCAACTG
71
483


 


12019
12038
502691
CTCCATCCGCTCCTGCAACT
75
484


 


12020
12039
502692
ACTCCATCCGCTCCTGCAAC
64
485


 


12021
12040
502693
AACTCCATCCGCTCCTGCAA
52
486


 


12022
12041
502694
CAACTCCATCCGCTCCTGCA
45
487


 


12024
12043
502695
AGCAACTCCATCCGCTCCTG
78
488


 


12025
12044
502696
CAGCAACTCCATCCGCTCCT
64
489


 


12026
12045
502697
GCAGCAACTCCATCCGCTCC
56
490


 


12173
12192
503039
AGGAGGGCGGTGGCGCGGCG
0
677


 


12221
12240
503040
TGACAGCTGGAAGGAGAAGA
41
678


 


12258
12277
502712
GAAGGTGGATCCGTGGCCCG
73
505


 


12259
12278
502713
GGAAGGTGGATCCGTGGCCC
70
506


 


12260
12279
502714
GGGAAGGTGGATCCGTGGCC
72
507


 


12261
12280
502715
TGGGAAGGTGGATCCGTGGC
50
508


 


12262
12281
502716
ATGGGAAGGTGGATCCGTGG
62
509


 


12263
12282
451417
CATGGGAAGGTGGATCCGTG
77
679


 


12463
12482
503041
GGAGGTTATCTAGGGAGATC
42
680


 


12542
12561
503042
GAAGGGACAGGTGACCCGAT
69
681


 


12596
12615
502724
CACCAGCGGGCACTGGCCCA
51
518


 


12597
12616
502725
CCACCAGCGGGCACTGGCCC
55
519


 


12598
12617
502726
CCCACCAGCGGGCACTGGCC
61
520


 


12599
12618
502727
CCCCACCAGCGGGCACTGGC
43
521


 


12601
12620
502728
GGCCCCACCAGCGGGCACTG
16
522


 


12602
12621
502729
TGGCCCCACCAGCGGGCACT
43
523


 


12603
12622
502730
CTGGCCCCACCAGCGGGCAC
43
524


 


12604
12623
502731
CCTGGCCCCACCAGCGGGCA
41
525


 


12605
12624
502732
GCCTGGCCCCACCAGCGGGC
30
526


 


12607
12626
502733
GGGCCTGGCCCCACCAGCGG
66
527


 


12625
12644
502734
AGGTGGCGGCGGTGCATGGG
31
528


 


12626
12645
502735
CAGGTGGCGGCGGTGCATGG
23
529


 


12627
12646
502736
GCAGGTGGCGGCGGTGCATG
57
530


 


12628
12647
502737
AGCAGGTGGCGGCGGTGCAT
54
531


 


12629
12648
502738
CAGCAGGTGGCGGCGGTGCA
61
532


 


12630
12649
502739
GCAGCAGGTGGCGGCGGTGC
57
533


 


12631
12650
502740
AGCAGCAGGTGGCGGCGGTG
36
534


 


12632
12651
502741
GAGCAGCAGGTGGCGGCGGT
53
535


 


12633
12652
502742
GGAGCAGCAGGTGGCGGCGG
39
536


 


12634
12653
502743
GGGAGCAGCAGGTGGCGGCG
36
537


 


12635
12654
502744
AGGGAGCAGCAGGTGGCGGC
62
538


 


12636
12655
502745
CAGGGAGCAGCAGGTGGCGG
56
539


 


12637
12656
502746
GCAGGGAGCAGCAGGTGGCG
58
540


 


12638
12657
502747
GGCAGGGAGCAGCAGGTGGC
65
541


 


12639
12658
502748
TGGCAGGGAGCAGCAGGTGG
47
542


 


12640
12659
502749
CTGGCAGGGAGCAGCAGGTG
41
543


 


12642
12661
451432
CCCTGGCAGGGAGCAGCAGG
53
544


 


12643
12662
502750
ACCCTGGCAGGGAGCAGCAG
52
545


 


12646
12665
503043
CGTACCCTGGCAGGGAGCAG
59
682


 


12918
12937
502977
GGACTCGCCCCGCCTACGCC
71
683


 


12924
12943
502978
CTCCTGGGACTCGCCCCGCC
67
684


 


12925
12944
503044
GCTCCTGGGACTCGCCCCGC
66
685


 


12929
12948
503045
ATTGGCTCCTGGGACTCGCC
77
686


 


12930
12949
502979
GATTGGCTCCTGGGACTCGC
70
687


 


12936
12955
502980
GCCTCTGATTGGCTCCTGGG
56
688


 


12942
12961
502981
GCATGGGCCTCTGATTGGCT
20
689


 


12948
12967
502982
CACCCGGCATGGGCCTCTGA
20
690


 


12986
13005
503046
GCCAGGCCTAGGGACCTGCG
58
691


 


12990
13009
502760
ATAGGCCAGGCCTAGGGACC
51
555


 


12991
13010
502761
GATAGGCCAGGCCTAGGGAC
41
556


 


12992
13011
502762
CGATAGGCCAGGCCTAGGGA
69
557


 


12993
13012
502763
CCGATAGGCCAGGCCTAGGG
80
558


 


12994
13013
502764
TCCGATAGGCCAGGCCTAGG
78
559


 


12995
13014
502765
CTCCGATAGGCCAGGCCTAG
89
560


 


12996
13015
502766
CCTCCGATAGGCCAGGCCTA
79
561


 


12997
13016
502767
GCCTCCGATAGGCCAGGCCT
73
562


 


12999
13018
502768
GCGCCTCCGATAGGCCAGGC
83
563


 


13015
13034
502769
AACAGGAGCAGGGAAAGCGC
83
564


 


13016
13035
502770
GAACAGGAGCAGGGAAAGCG
70
565


 


13017
13036
502771
CGAACAGGAGCAGGGAAAGC
43
566


 


13018
13037
502772
GCGAACAGGAGCAGGGAAAG
47
567


 


13019
13038
502773
GGCGAACAGGAGCAGGGAAA
61
568


 


13020
13039
502774
CGGCGAACAGGAGCAGGGAA
74
569


 


13021
13040
502775
ACGGCGAACAGGAGCAGGGA
60
570


 


13022
13041
502776
AACGGCGAACAGGAGCAGGG
86
571


 


13023
13042
502777
CAACGGCGAACAGGAGCAGG
84
572


 


13044
13063
502778
GGGCGGCGGCACGAGACAGA
80
573


 


13045
13064
502779
AGGGCGGCGGCACGAGACAG
76
574


 


13046
13065
502780
CAGGGCGGCGGCACGAGACA
58
575


 


13047
13066
502781
CCAGGGCGGCGGCACGAGAC
80
576


 


13048
13067
502782
CCCAGGGCGGCGGCACGAGA
59
577


 


13049
13068
502783
GCCCAGGGCGGCGGCACGAG
68
578


 


13050
13069
502784
AGCCCAGGGCGGCGGCACGA
75
579


 


13051
13070
502785
CAGCCCAGGGCGGCGGCACG
76
580


 


13052
13071
502786
GCAGCCCAGGGCGGCGGCAC
70
581


 


13089
13108
502787
CTGCGGTGAGTTGGCCGGCG
68
582


 


13090
13109
502788
ACTGCGGTGAGTTGGCCGGC
67
583


 


13091
13110
502789
GACTGCGGTGAGTTGGCCGG
58
584


 


13092
13111
502790
AGACTGCGGTGAGTTGGCCG
71
585


 


13093
13112
502791
CAGACTGCGGTGAGTTGGCC
70
586


 


13094
13113
502792
CCAGACTGCGGTGAGTTGGC
79
587


 


13095
13114
502793
GCCAGACTGCGGTGAGTTGG
76
588


 


13096
13115
502794
CGCCAGACTGCGGTGAGTTG
66
589


 


13140
13159
502795
AAGACAGTTCTAGGGTTCAG
87
590


 


13141
13160
502796
GAAGACAGTTCTAGGGTTCA
78
591


 


13142
13161
502797
CGAAGACAGTTCTAGGGTTC
85
592


 


13143
13162
502798
TCGAAGACAGTTCTAGGGTT
78
593


 


13144
13163
502799
GTCGAAGACAGTTCTAGGGT
92
594


 


13145
13164
502800
AGTCGAAGACAGTTCTAGGG
85
595


 


13146
13165
502801
GAGTCGAAGACAGTTCTAGG
83
596


 


13147
13166
502802
GGAGTCGAAGACAGTTCTAG
86
597


 


13148
13167
502803
CGGAGTCGAAGACAGTTCTA
91
598


 


13149
13168
502804
CCGGAGTCGAAGACAGTTCT
76
599


 


13150
13169
502805
CCCGGAGTCGAAGACAGTTC
90
600


 


13151
13170
502806
CCCCGGAGTCGAAGACAGTT
83
601


 


13152
13171
502807
GCCCCGGAGTCGAAGACAGT
82
602


 


13153
13172
502808
GGCCCCGGAGTCGAAGACAG
73
603


 


13154
13173
502809
GGGCCCCGGAGTCGAAGACA
67
604


 


13206
13225
502810
AGGCGGTGGGCGCGGCTTCT
73
605


 


13207
13226
502811
CAGGCGGTGGGCGCGGCTTC
57
606


 


13208
13227
502812
GCAGGCGGTGGGCGCGGCTT
69
607


 


13210
13229
502813
TGGCAGGCGGTGGGCGCGGC
73
608


 


13212
13231
502814
ACTGGCAGGCGGTGGGCGCG
56
609


 


13214
13233
502815
GAACTGGCAGGCGGTGGGCG
71
610


 


13215
13234
502816
TGAACTGGCAGGCGGTGGGC
80
611


 


13217
13236
502817
TGTGAACTGGCAGGCGGTGG
85
612


 


13250
13269
502818
TGGAGCTGGGCGGAGACCCA
55
613


 


13252
13271
502819
ACTGGAGCTGGGCGGAGACC
53
614


 


13253
13272
502820
GACTGGAGCTGGGCGGAGAC
55
615


 


13255
13274
502821
AGGACTGGAGCTGGGCGGAG
76
616


 


13257
13276
502822
ACAGGACTGGAGCTGGGCGG
77
617


 


13258
13277
502823
CACAGGACTGGAGCTGGGCG
74
618


 


13259
13278
502824
TCACAGGACTGGAGCTGGGC
90
619


 


13449
13468
502825
GCCTCAGCCTGGCCGAAAGA
80
620


 


13450
13469
502826
GGCCTCAGCCTGGCCGAAAG
72
621


 


13553
13572
444401
TTGCACTTTGCGAACCAACG
97
41


 


14037
14056
503047
TTCCTCCCCCAACCCTGATT
34
692


 


14255
14274
503048
AAGTTTGCAGCAACTTTTCT
0
693


 


14325
14344
503049
GCCCCTCGGAATTCCCGGCT
0
694


 


14343
14362
503050
CATCTCGGCCTGCGCTCCGC
39
695


 


14361
14380
503051
GCAGGCCCCCACATTCCCCA
0
696


 


14392
14411
503052
CTTCTGCACGCCTCCGTCTC
30
697









Example 8
Antisense Inhibition of Murine DMPK in Mouse Primary Hepatocytes
Antisense oligonucleotides targeted to a murine DMPK nucleic acid were tested for their effect on DMPK RNA transcript in vitro. Cultured mouse primary hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 8,000 nM antisense oligonucleotide. After approximately 24 hours, RNA was isolated from the cells and DMPK transcript levels were measured by quantitative real-time PCR. DMPK RNA transcript levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of DMPK, relative to untreated control cells.
The antisense oligonucleotides in Tables 14, 15, and 16 are 5-10-5 gapmers, where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises five 2′-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. ‘Murine Target start site’ indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted in the murine gene sequence. ‘Murine Target stop site’ indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted in the murine gene sequence. All the antisense oligonucleotides listed in Table 12 target SEQ ID NO: 3 (GENBANK Accession No. NT_039413.7 truncated from nucleotides 16666001 to Ser. No. 16/681,000). All the antisense oligonucleotides listed in Table 13 target SEQ ID NO: 4 (GENBANK Accession No. NM_032418.1). The antisense oligonucleotides of Table 14 target SEQ ID NO: 5 (GENBANK Accession No. AI007148.1), SEQ ID NO: 6 (GENBANK Accession No. AI304033.1), SEQ ID NO: 7 (GENBANK Accession No. BC024150.1), SEQ ID NO: 8 (GENBANK Accession No. BC056615.1), SEQ ID NO: 793 (GENBANK Accession No. BC075715.1), SEQ ID NO: 794 (GENBANK Accession No. BU519245.1), SEQ ID NO: 795 (GENBANK Accession No. CB247909.1), SEQ ID NO: 796 (GENBANK Accession No. CX208906.1), SEQ ID NO: 797 (GENBANK Accession No. CX732022.1), SEQ ID NO: 798 (GENBANK Accession No. S60315.1), or SEQ ID NO: 799 (GENBANK Accession No. S60316.1). In addition, the human antisense oligonucleotide ISIS 451421 targeting SEQ ID NO: 800(GENBANK Accession No. NM_001081562.1) was also included in this assay and is listed in Table 14.
The murine oligonucleotides of Tables 14, 15, and 16 may also be cross-reactive with human gene sequences. ‘Mismatches’ indicate the number of nucleobases by which the murine oligonucleotide is mismatched with a human gene sequence. The greater the complementarity between the murine oligonucleotide and the human sequence, the more likely the murine oligonucleotide can cross-react with the human sequence. The murine oligonucleotides in Tables 14, 15, and 16 were compared to SEQ ID NO: 800 (GENBANK Accession No. NM_001081562.1). “Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence.
Several of the tested antisense oligonucleotides demonstrated significant inhibition of DMPK mRNA levels under the conditions specified above. Certain of the tested antisense oligonucleotides are cross-reactive with human gene sequences.







TABLE 14







Inhibition of murine DMPK RNA transcript in mouse primary hepatocytes by 5-10-5


gapmers targeting SEQ ID NO: 800















Murine
Murine




Human
Human



Target
Target



SEQ
Target
Target



Start
Stop


%
ID
Start
Stop



Site
Site
ISIS No
Sequence
inhibition
NO.
Site
Site
Mismatches


















11904
11923
299516
TGGCCCACAGCCACGGCCGG
47
698
1850
1869
0


 


11927
11946
299520
GGCCTGGCCCCACCAGCGGG
58
699
1873
1892
0


 


11962
11981
299521
CCTGGCAGGGAGCAGCAGGT
44
700
1908
1927
0


 


3345
3364
451360
CAGCCGCACTTCGGCTGACA
29
701
207
226
1


 


3378
3397
451361
GCCTGGGTCCAGCACCAGCT
67
702
240
259
2


 


3388
3407
451362
GTCCCAGGAAGCCTGGGTCC
62
703
250
269
2


 


3418
3437
451363
CGCCCAGGAGAAGGTCGAGC
69
213
280
299
0


 


3484
3503
451364
CCCACTGCAAGAAGTCGGCC
69
226
346
365
0


 


6264
6283
451366
CGTTAGCAGGTCCCCGCCCA
73
704
660
679
2


 


6342
6361
451367
GTCTATGGCCATGACAATCT
61
705
738
757
0


 


6363
6382
451368
GTAGCCCAGCCGGTGCACGG
54
706
759
778
2


 


6851
6870
451370
GGGTGCCCACAGCCACCAGC
72
707
889
908
0


 


6919
6938
451371
TGGCCCGTAGCTGCCTGCCC
80
708
957
976
2


 


7448
7467
451373
GGAAATCACCTGCCCCACCT
80
709
n/a
n/a
n/a


 


7458
7477
451374
GGATGTTTCTGGAAATCACC
84
710
n/a
n/a
n/a


 


7533
7552
451375
GTGGCACCCTCGAAGTCTGG
77
711
1271
1290
3


 


7589
7608
451376
CCCCGCTCACCATGGCAGTG
31
712
n/a
n/a
n/a


 


10278
10297
451378
GGTCCGGGACCTGATTGTCT
85
713
n/a
n/a
n/a


 


3229
3248
451385
GCTGCATGTCTGCCCGTCCC
74
714
90
109
1


 


3244
3263
451386
GGCCCCAGAACCCTAGCTGC
73
715
n/a
n/a
n/a


 


3270
3289
451387
TCACAGGGCCTGGCTGCCCC
62
716
131
150
1


 


3333
3352
451388
GGCTGACATGTTGGGCAGGC
60
717
195
214
1


 


3250
3269
451389
TGTCCAGGCCCCAGAACCCT
68
718
111
130
3


 


12295
12314
451391
GGCCAGGCCTAGGGATCTGC
51
719
n/a
n/a
n/a


 


12306
12325
451392
CGCCTCGGATAGGCCAGGCC
52
720
1935
1954
1


 


12450
12469
451393
GGCTTGGAGTCTTAGGGTTC
85
721
n/a
n/a
n/a


 


12623
12642
451394
TCCCCGGCCGCCAGGTGGCA
43
722
2224
2243
3


 


12651
12670
451395
GGTGCTGGGCACGAGCCCTG
62
723
n/a
n/a
n/a


 


12698
12717
451396
GCCCAGCTGCTGCAGCAGCG
66
724
n/a
n/a
n/a


 


12876
12895
451397
CCGTGTGTGCTGGCAGAGGT
76
725
n/a
n/a
n/a


 


13084
13103
451398
ATAAATACCGAGGAATGTCG
77
726
2766
2785
0


 


13094
13113
451399
GGGACAGACAATAAATACCG
80
727
2776
2795
0


 


12362
12381
451405
GTGCAGCCCAGTGTGGCGGC
69
728
1991
2010
3


 


11175
11194
451415
CCTGGAGAAGTTCTGGTTGG
48
729
1674
1693
3


 


11585
11604
451417
CATGGGAAGGTGGATCCGTG
65
679
1819
1838
1


 


11854
11873
451419
GGTGACCCGATCGGAGCCCA
11
730
n/a
n/a
n/a


 


11874
11893
451420
AGCTGGAGAGAGAAGGGACA
37
731
n/a
n/a
n/a


 


11379
11398
451422
GTGAGGGACTCGCCTGCGGC
36
732
n/a
n/a
n/a


 


11479
11498
451423
GCGGCTGCGGTGCCCCAGCC
50
733
n/a
n/a
n/a


 


11883
11902
451424
GGGCCATCTAGCTGGAGAGA
45
734
n/a
n/a
n/a


 


3485
3504
451427
CCCCACTGCAAGAAGTCGGC
57
735
347
366
1


 


4621
4640
451428
TTGAGCCCTTTTAAGGCAGC
43
736
n/a
n/a
n/a


 


6232
6251
451429
TGACCAGGTACTGGGAGCGG
47
737
n/a
n/a
n/a


 


10985
11004
451430
CCTGGAGCTGGATCAGTCCC
6
738
n/a
n/a
n/a


 


11586
11605
451431
ACATGGGAAGGTGGATCCGT
70
739
1820
1839
1


 


11963
11982
451432
CCCTGGCAGGGAGCAGCAGG
42
544
1909
1928
0


 


11973
11992
451433
GTGGGACATACCCTGGCAGG
34
740
n/a
n/a
n/a


 


12294
12313
451434
GCCAGGCCTAGGGATCTGCA
35
741
n/a
n/a
n/a
















TABLE 15







Inhibition of murine DMPK RNA transcript in mouse primary


hepatocytes by 5-10-5 gapmers targeting SEQ ID NO: 800















Murine
Murine




Human
Human



Target
Target



SEQ
Target
Target



Start
Stop
ISIS

%
ID
Start
Stop



Site
Site
No
Sequence
inhibition
NO.
Site
Site
Mismatches


















330
349
451365
GGAAGCACGACACCTCGCCT
67
742
 535
 554
1


 


662
681
451369
CCTCACCATTCCATCAGGCT
81
743
n/a
n/a
n/a


 


881
900
451372
CGGCAGCGACAAGTGTTCCC
90
744
n/a
n/a
n/a


 


1217
1236
451377
GTCTCTGAAGGCCATGCAGC
69
745
1407
1426
3


 


1329
1348
451379
CAGCCACTTGATCCGGTGGG
62
746
n/a
n/a
n/a


 


1342
1361
451380
AGGTCGGCCTCTTCAGCCAC
74
747
n/a
n/a
n/a


 


1494
1513
451381
GTTGGCTGGAGAAGTTCTGG
39
748
1678
1697
2


 


1598
1617
451382
CCCCGTGATGGCTGCGGCTC
54
749
1782
1801
3


 


1644
1663
451383
GGCCATCTAGATGGGAAGGT
21
517
1828
1847
0


 


1741
1760
451384
AGGCCAGGCCTAGGGATCCT
39
750
1925
1944
1
















TABLE 16







Inhibition of murine DMPK RNA transcript in mouse primary hepatocytes by 5-10-5


gapmers targeting SEQ ID NOs: 5-8 and 793-799
















Murine
Murine
Murine




Human
Human



Target
Target
Target



SEQ
Target
Target



Start
Stop
SEQ
ISIS

%
ID
Start
Stop



Site
Site
ID NO
No
Sequence
inhibition
NO.
Site
Site
Mismatches



















324
343
5
451410
GGCGCGGTGCCCCAGCCTGG
67
751
n/a
n/a
n/a


 


485
504
5
451411
GTCCTGGCCCCACCAGCGGG
66
752
1873
1892
1


 


534
553
5
451412
CCAGGCCTAGGAATCCTGGC
17
753
1922
1941
2


 


547
566
5
451413
GCGCCTCGGATAGCCAGGCC
51
754
n/a
n/a
n/a


 


594
613
5
451414
CCCAGTGTGGCGCAGCAGCC
65
755
n/a
n/a
n/a


 


393
412
6
451402
GTGTTTCATCTTCACCACCG
80
756
 462
 481
3


 


1475
1494
7
451390
AGGTCAGCCTCTTCAGCCAC
60
757
n/a
n/a
n/a


 


n/a
n/a
n/a
451425
GGCCATATGGGAAGGTGGAT
48
758
1824
1843
0


 


1763
1782
8
451418
GGAGGATTTGGCGAGAAGCA
48
759
n/a
n/a
n/a


 


1032
1051
793
451403
CGAAGTCTGCCCCACCTCGA
58
760
n/a
n/a
n/a


 


1042
1061
793
451404
GTGGCACCCTCGAAGTCTGC
72
761
n/a
n/a
n/a


 


217
236
794
451400
GGGTCCATTGTAAGGAAGCT
4
762
n/a
n/a
n/a


 


754
773
794
451401
GGTGCCCACAGCCACCAGGG
82
763
 888
 907
1


 


322
341
795
451406
TCCATGGCAGTGAGCCGGTC
55
764
1319
1338
1


 


523
542
795
451407
GGGACCACTTGATCCGGTGG
63
765
n/a
n/a
n/a


 


534
553
795
451408
GGATCAGAGTTGGGACCACT
0
766
n/a
n/a
n/a


 


492
511
796
451416
CCCCGTGATGGCTGCGGTTC
49
767
n/a
n/a
n/a


 


469
488
797
451409
GTGTGTCCTCATACCCCGCC
60
768
n/a
n/a
n/a


 


629
648
798
451421
GCACCCTCGAAGTCTCGACC
72
769
n/a
n/a
n/a


 


854
873
799
451426
GCTCTGAAGGCCATGCAGCA
52
770
n/a
n/a
n/a









Example 9
Dose-Dependent Antisense Inhibition of Murine DMPK in Mouse Primary Hepatocytes
Several of the antisense oligonucleotides exhibiting in vitro inhibition of DMPK in mouse primary hepatocytes (see Example 8) were tested at various doses. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 1,000 nM, 2,000 nM, 4,000 nM, 8,000 nM, and 16,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and DMPK transcript levels were measured by quantitative real-time PCR using primer probe set RTS3181 (forward sequence GACATATGCCAAGATTGTGCACTAC, designated herein as SEQ ID NO: 771; reverse sequence CACGAATGAGGTCCTGAGCTT, designated herein as SEQ ID NO: 772; probe sequence AACACTTGTCGCTGCCGCTGGCX, designated herein as SEQ ID NO: 773). DMPK transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 17 as percent inhibition of DMPK, relative to untreated control cells.
The majority of the tested antisense oligonucleotides demonstrated dose-dependent inhibition of DMPK mRNA levels under the conditions specified above.







TABLE 17







Dose-dependent antisense inhibition of murine DMPK


in mouse primary hepatocytes


















16,000
IC50


ISIS No
1,000 nM
2,000 nM
4,000 nM
8,000 nM
nM
(μM)
















451369
33
59
78
87
94
1.57


451371
60
77
84
90
91
0.24


451373
53
62
82
89
92
0.74


451374
33
42
76
88
94
2.00


451375
43
62
81
89
88
1.05


451378
39
79
80
87
94
0.87


451385
22
57
80
78
93
2.01


451393
49
63
86
80
80
0.59


451397
63
75
74
81
92
0.22


451398
29
72
84
83
90
1.29


451399
27
53
81
68
80
2.07


451401
34
71
87
86
92
1.12


451402
34
69
75
86
74
1.14









Example 10
Antisense Inhibition of Human Alpha1 Skeletal Actin in HepG2 Cells
Antisense oligonucleotides targeted to a human alpha1 skeletal actin nucleic acid, a gene which may carry an expanded CTG repeat capable of causing symptoms of DM1 when inserted into mouse models, were tested for their effect on alpha1 actin RNA transcript in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 10,000 nM antisense oligonucleotide. After approximately 24 hours, RNA was isolated from the cells and alpha1 actin RNA transcript levels were measured by quantitative real-time PCR. Alpha1 actin RNA transcript levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of alpha1 actin, relative to untreated control cells.
The antisense oligonucleotides in Table 18 are 5-10-5 gapmers, where the gap segment comprises ten 2′-deoxynucleosides and each wing segment comprises five 2′-MOE nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. ‘Target start site’ indicates the 5′-most nucleoside to which the antisense oligonucleotide is targeted. ‘Target stop site’ indicates the 3′-most nucleoside to which the antisense oligonucleotide is targeted. All the antisense oligonucleotides listed in Table 18 target SEQ ID NO: 801 (GENBANK Accession No. NM_001100.3).
The tested antisense oligonucleotide sequences demonstrated dose-dependent inhibition of alpha 1 actin mRNA levels under the conditions specified above.







TABLE 18







Inhibition of human alpha 1 actin RNA transcript in


HepG2 cells by 5-10-5 gapmers targeting SEQ ID NO: 801












Target
Target






Start
Stop


%
SEQ ID


Site
Site
ISIS No
Sequence
inhibition
NO.















16
35
445205
AGCGAGGCTTCACTTGGCGC
74
774


 


20
39
190403
GGGAAGCGAGGCTTCACTTG
75
775


 


1028
1047
190401
GCGGTCAGCGATCCCAGGGT
78
776


 


1058
1077
445225
GGGTGCCAGCGCGGTGATCT
73
777


 


1320
1339
445231
TGTTACAAAGAAAGTGACTG
74
778


 


1339
1358
445232
CGATGGCAGCAACGGAAGTT
96
779


 


1348
1367
445233
GTCAGTTTACGATGGCAGCA
100
780


 


1417
1436
445235
CAGGGCTTTGTTTCGAAAAA
91
781


 


1430
1449
445236
CCATTTTCTTCCACAGGGCT
99
782


 


1447
1466
445237
ATGCTTCTTCAAGTTTTCCA
97
783


 


1460
1479
445238
CAGAATGACTTTAATGCTTC
95
784









Example 11
Dose-Dependent Antisense Inhibition of Human Alpha1 Actin in HepG2 Cells
Several of the antisense oligonucleotides exhibiting in vitro inhibition of alpha1 actin in HepG2 cells (see Example 8) were tested at various doses. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 625 nM, 1,250 nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations of each antisense oligonucleotide. After approximately 16 hours, RNA was isolated from the cells and alpha1 actin RNA transcript levels were measured by quantitative real-time PCR using primer probe set RTS3154 (forward sequence CCACCGCAAATGCTTCTAGAC, designated herein as SEQ ID NO: 785; reverse sequence CCCCCCCATTGAGAAGATTC, designated herein as SEQ ID NO: 786; probe sequence CTCCACCTCCAGCACGCGACTTCTX, designated herein as SEQ ID NO: 787). Alpha1 actin RNA transcript levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in Table 19 as percent inhibition of alpha1 actin, relative to untreated control cells.
Several of the antisense oligonucleotides demonstrated dose-dependent inhibition of alpha 1 actin mRNA levels under the conditions specified above.







TABLE 19







Dose-dependent antisense inhibition of human alpha1 actin in HepG2 cells














ISIS
625
1,250

5,000

20,000
IC50


No.
nM
nM
2,500 nM
nM
10,000 nM
nM
(μM)

















445233
21
72
63
82
96
83
1.1


445236
26
68
82
91
90
91
0.8


445237
36
59
76
84
83
90
0.8


445232
14
42
54
59
80
91
2.6


445238
27
43
54
73
76
90
2.0


445235
26
52
29
58
59
24
0.7


190403
25
29
36
25
61
54
11.9


190401
17
14
40
68
76
72
3.9


445225
25
23
49
28
52
50
15.8


445205
26
31
34
28
55
36
7.6


445231
30
25
39
26
42
36
>20.0









Example 12
In vivo Antisense Inhibition of Human Alpha1 Actin by Intramuscular Administration in Transgenic Mice
To test the effect of antisense inhibition for the treatment of myotonic dystrophy, an appropriate mouse model was required. The HSALR mouse model is an established model for DM1 (Mankodi, A. et al. Science. 289: 1769, 2000). The mice carry a human skeletal actin (hACTA1) transgene with 220 CTG repeats inserted in the 3′ UTR of the gene. The hACTA1-CUGexp transcript accumulates in nuclear foci in skeletal muscles and results in myotonia similar to that in human DM1 (Mankodi, A. et al. Mol. Cell 10: 35, 2002; Lin, X. et al. Hum. Mol. Genet. 15: 2087, 2006). Hence, it was expected that amelioration of DM1 symptoms in the HSALR mouse by antisense inhibition of the hACTA1 transgene would predict amelioration of similar symptoms in human patients by antisense inhibition of the DMPK transcript.
HSA (human skeletal actin)LR (long repeat) DM1 mice were generated by insertion in FVB/N mice of a transgene with 250 CUG repeats in the 3′ UTR of human skeletal actin. The transgene is expressed in the mice as a CUG repeat RNA, which is retained in the nucleus, forming nuclear inclusions or foci, similar to that seen in human tissue samples of patients with myotonic dystrophy (DM1).
ISIS 190403 and ISIS 445238, which demonstrated statistically significant dose-dependent inhibition in vitro (see Example 11), were evaluated for their ability to reduce human alpha1 actin RNA transcript in vivo.
Treatment
HSALR mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into two treatment groups. The two groups received direct intramuscular injections of ISIS 190403 or ISIS 445238 at a dose of 0.8 nM into the tibialis anterior muscle on one side. The contralateral tibialis anterior muscle in each mouse received a single dose intramuscular injection of PBS. The PBS-injected muscle acted as the control.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the tibialis anterior muscles of both sides was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 20, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
The results indicate that treatment with ISIS 190403 and ISIS 445238 resulted in inhibition of alpha 1 actin RNA levels in the mice.







TABLE 20







Percent inhibition of human alpha1 


actin RNA transcript in HSALR mice










ISIS
%



No.
inhibition














190403
38



445238
40










Example 13
Dose Dependent Antisense Inhibition of Human Alpha1 Actin by Intramuscular Administration in Transgenic Mice
ISIS 445236, which demonstrated statistically significant dose-dependent inhibition in vitro (see Example 11), was evaluated for its ability to reduce human alpha1 actin RNA transcript in vivo.
Treatment
HSALR mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into three treatment groups. The groups received direct intramuscular injections of ISIS 445236 at doses of 0.2 nM, 0.4 nM or 0.8 nM into the tibialis anterior muscle of one side. The contralateral tibialis anterior muscle in each mouse received a single dose intramuscular injection of PBS. The PBS-injected muscle acted as the control.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the tibialis anterior muscles of both sides was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 21, treatment with ISIS 445236 reduced human alpha1 actin RNA transcript expression at all dosages. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the control.
The results indicate that treatment with ISIS 445236 resulted in significant inhibition of alpha 1 actin mRNA levels under the conditions specified above.







TABLE 21







Inhibition of human alpha1 actin RNA


transcriptby ISIS 445236 in HSALR mice










Dose
%



(nM)
inhibition














0.2
70



0.4
54



0.8
78










Assessment of Myotonia by Electromyography

Myotonia refers to repetitive action potential that is due to delayed relaxation of muscle fibers. This phenomenon is observed in patients of myotonic dystrophy as well as in the HSALR mice. When the EMG needle is inserted into a myotonic muscle, the electrical activity is prolonged for up to several seconds past when the insertional activity should normally cease. The frequency of myotonic discharges ranges from 50 to 100 impulses per second.
Myotonia was measured via electromyography and graded in the following manner: grade 0 refers to no myotonia elicited by any needle insertion (0%); grade 1 refers to myotonia elicited by less than 50% needle insertions; grade 2 refers to myotonia elicited by more than 50% needle insertions; and grade 3 refers to mytonia elicited by 100% needle insertions.
Before electromyography, mice were anesthetized by using i.p. a cocktail of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine. Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 22 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 445236.







TABLE 22







Average reduction of myotonia in various muscles 


of antisense oligonucleotide-treated HSALR mice










Dose
Myotonia


Treatment
(nM)
grade












PBS

2.7


ISIS
0.2
1.3


455236
0.4
1.0



0.8
1.0









Correction of Alternative Splicing

In DM1/HSALR mouse model, the accumulation of expanded CUG RNA in the nucleus leads to the sequestration of poly(CUG)-binding proteins, such as Muscleblind-like 1 (MBLN1) (Miller, J. W. et al. EMBO J. 19: 4439, 2000). The splicing factor MBNL1, which controls alternative splicing of the Serca1 gene is sequestered in expanded CUG foci. This triggers dysregulation of the alternative splicing of this gene. To evaluate the effect of antisense inhibition of human alpha 1 actin on such alternative splicing, total RNA was purified from the tibialis anterior, gastrocnemius, and quadriceps muscle using RNeasy Lipid Tissue Mini Kit (Qiagen), according to the manufacturer's instructions. RT-PCR was performed with the SuperScript III One-Step RT-PCR System and Platinum Taq Polymerase (Invitrogen), using gene-specific primers for cDNA synthesis and PCR amplification. The forward and reverse primers for Serca-1 have been described in Bennett and Swayze (Annu Rev. Pharmacol. 2010; 50: 259-93). PCR products were separated on agarose gels, stained with SybrGreen I Nucleic Acid Gel Stain (Invitrogen), and imaged using a Fujifilm LAS-3000 Intelligent Dark Box.
The PCR products of Serca1 splicing in the PBS control demonstrated exon 22 exclusion as a result of dysregulation of MBLN1. Treatment with ISIS 445236 resulted in exon 22 inclusion and normalization of alternative splicing of the Serca1 gene in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Therefore, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects MBLN1 sequestration thereby allowing normal splicing to occur.
Example 14
In vivo Antisense Inhibition of Human Alpha1 Actin by Subcutaneous Administration in Transgenic Mice
ISIS 190403, ISIS 445236 and ISIS 445238 were evaluated for their ability to reduce human alpha1 actin RNA transcript in vivo.
Treatment
HSALR mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into four treatment groups. The first three groups received subcutaneous injections of ISIS 190403, ISIS 445236 or ISIS 445238 at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group received subcutaneous injections of PBS twice weekly for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles (left and right), gastrocnemius muscles (left and right), and tibialis anterior muscles (left and right) was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 23, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the control.
Both ISIS 445236 and ISIS 445238 demonstrated significant inhibition of alpha1 actin mRNA levels under the conditions specified above.







TABLE 23







Percent inhibition of human alpha1 actin 


RNA transcript in HSALR mice













ISIS
ISIS
ISIS



Muscle Type
190403
445236
445238
















Quadriceps
16
83
72



Gastrocnemius
0
85
73



Tibialis anterior
2
81
71










Fluorescence In Situ Hybridization of Alpha1 Actin in Muscles

Frozen muscle tissue sections were fixed in fresh 3% paraformaldehyde in PBS solution for 15-20 minutes, after which they were rinsed twice with PBS for 5 minutes. The nuclei were permeabilized with 0.5% Triton X-100 for 5 minutes after which the tissue was blocked with normal goat serum for 30 minutes. The sections were incubated a 2′-O-methyl RNA targeted to alpha1 actin that is 5′-labeled with Texas Red (Integrated DNA Technologies). The sections were counter-stained with DAPI to label the nuclei. The sections were mounted and viewed with a standard fluorescence microscope. Image acquisition was by Metavue software and deconvolution was achieved by Autoquant software.
All muscle tissue sections from mice treated with ISIS 445236 and ISIS 445238 displayed reduced fluorescent intensity of alpha1 actin signal at the ribonuclear foci, indicating antisense inhibition of human alpha1 actin mRNA and reduction of the RNA in the nuclear foci.
Assessment of Myotonia by Electromyography
Myotonia refers to repetitive action potential that is due to delayed relaxation of muscle fibers. This phenomenon is observed in patients of myotonic dystrophy as well as in the HSALR mice. When the EMG needle is inserted into a myotonic muscle, the electrical activity is prolonged for up to several seconds past when the insertional activity should normally cease. The frequency of myotonic discharges ranges from 50 to 100 impulses per second.
Myotonia may be measured via electromyography and is graded in the following manner: grade 0 refers to no myotonia elicited by any needle insertion (0%); grade 1 refers to myotonia elicited by less than 50% needle insertions; grade 2 refers to myotonia elicited by more than 50% needle insertions; and grade 3 refers to mytonia elicited by 100% needle insertions.
Before electromyography, mice were anesthetized by using i.p. 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine or 250 mg/kg 2,2,2-tribromoethanol. Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 24 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 445236 and ISIS 445238.







TABLE 24







Average reduction of myotonia in various muscles of 


antisense oligonucleotide-treated HSALR mice















ISIS
ISIS
ISIS




PBS
190403
445236
445238

















Left quadriceps
3.00
3.00
0.00
0.25



Right quadriceps
3.00
3.00
0.00
0.00



Left gastrocnemius
3.00
3.00
0.00
0.25



Right gastrocnemius
3.00
3.00
0.00
0.25



Left Tibialis anterior
2.75
2.50
0.00
0.00



Right Tibialis anterior
2.75
2.50
0.00
0.00



Lumbar paraspinals
3.00
3.00
0.00
0.75










Correction of Alternative Splicing

The splicing factor MBNL1, which controls Serca1 splicing, m-Titin splicing, CIC-1 chloride channel gene (Clcn1) splicing, and Zasp splicing, is sequestered in expanded CUG foci. MBNL1 sequestration triggers dysregulated splicing in each of these genes. To evaluate the effect of antisense inhibition of human alpha 1 actin on splicing, total RNA was purified from the tibialis anterior, gastrocnemius, and quadriceps muscle and RT-PCR was performed, as described in Example 13. The forward and reverse primers for Serca-1, m-Titin, Clcn1, and ZASP have been described in Bennett and Swayze, Annu Rev. Pharmacol. 2010; 50: 259-93.
In PBS treated HSALR mice, Serca1 splicing is dysregulated as demonstrated by exon 22 exclusion. Treatment with each of ISIS 445236 and ISIS 445238 resulted in exon 22 inclusion and normalization of alternative splicing of the Serca1 gene in the tibialis anterior, gastrocnemius, and quadriceps muscles.
In PBS treated HSALR mice, m-Titin splicing is dysregulated as demonstrated by exon 5 inclusion. Treatment with each of ISIS 445236 and ISIS 445238 resulted in skipping of exon 5 and normalization of alternative splicing of the m-Titin gene in the tibialis anterior, gastrocnemius, and quadriceps muscles.
In PBS treated HSALR mice, Clcn1 splicing is dysregulated as demonstrated by exon 7a inclusion. Treatment with each of ISIS 445236 and ISIS 445238 resulted in skipping of exon 7a and normalization of alternative splicing of the Clcn1 gene in the tibialis anterior, gastrocnemius, and quadriceps muscles.
In PBS treated HSALR mice, Zasp splicing is dysregulated as demonstrated by exon 11 inclusion. Treatment with each of ISIS 445236 and ISIS 445238 resulted in skipping of exon 11 and normalization of alternative splicing of the Zasp gene in the tibialis anterior, gastrocnemius, and quadriceps muscles.
Therefore, antisense inhibition of alpha1 actin corrected Serca1, m-Titin, Clcn1, and Zasp splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci correct MBLN1 sequestration thereby allowing normal splicing to occur.
Example 15
In vivo Antisense Inhibition of Human Alpha1 Actin in Transgenic Mice
Antisense inhibition of human alpha1 actin RNA transcript by ISIS 445236 and ISIS 445238 on myotonia in HSALR mice was further evaluated.
Treatment
HSALR mice were divided into three treatment groups. The first two groups received subcutaneous injections of ISIS 445236 or ISIS 445238 at a dose of 25 mg/kg twice per week for 2 weeks. The third group received subcutaneous injections of PBS twice per week for 2 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles, gastrocnemius muscles, and tibialis anterior muscles was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 25, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
Both ISIS 445236 and ISIS 445238 demonstrated significant inhibition of alpha1 actin mRNA levels under the conditions specified above.







TABLE 25







Percent inhibition of human alpha1 


actin RNA transcriptin HSALR mice











Muscle Type
ISIS 445236
ISIS 445238















Quadriceps
61
64



Gastrocnemius
68
37



Tibialis anterior
68
41










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 26 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 445236 and ISIS 445238.







TABLE 26







Average reduction of myotonia in various muscles 


of antisense oligonucleotide-treated HSALR mice














ISIS
ISIS




PBS
445236
445238
















Left quadriceps
3.00
0.00
1.75



Right quadriceps
3.00
0.00
1.75



Left gastrocnemius
3.00
0.25
1.5



Right gastrocnemius
3.00
0.25
1.00



Left Tibialis anterior
2.75
0.00
0.00



Right Tibialis anterior
2.75
0.00
0.00



Lumbar paraspinals
3.00
0.50
2.00










Correction of Alternative Splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1, total RNA purified from the tibialis anterior gastrocnemius, and quadriceps muscle was analyzed in a procedure similar to that described in Example 13.
In PBS treated HSALR mice, Serca1 splicing is dysregulated as demonstrated by exon 22 exclusion, as a result of MBLN1 dysregulation. Treatment with each of ISIS 445236 and ISIS 445238 resulted in near-complete inclusion and normalization of alternative splicing of exon 22 of the Serca1 gene in the tibialis anterior and quadriceps muscles.
Therefore, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci correct MBLN1 sequestration thereby allowing normal splicing to occur.
Example 16
Dose-Dependent Antisense Inhibition of Human Alpha1 Actin in Transgenic Mice
Dose-dependent inhibition of human alpha1 actin RNA transcript by ISIS 445236 and ISIS 445238 on myotonia in HSALR mice was evaluated.
Treatment
HSALR mice were subcutaneously injected with ISIS 445236 or ISIS 445238 at doses of 2.5 mg/kg, 8.5 mg/kg or 25.0 mg/kg twice per week for 4 weeks. The control group received subcutaneous injections of PBS twice per week for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles (Quad), gastrocnemius muscles (Gastroc), and tibialis anterior muscles (TA) was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 27, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
Both the antisense oligonucleotides demonstrated dose-dependent inhibition of alpha1 actin mRNA levels in quadriceps muscles, gastrocnemius muscles, and tibialis anterior muscles under the conditions specified above.







TABLE 27







Dose-dependent inhibition of human alpha1 actin RNA 


transcript in HSALR mice












mg/kg/wk
Quad
Gastroc
TA














ISIS 445236
5
24
36
46



17
53
57
59



50
86
86
90


ISIS 445238
5
21
37
3



17
30
39
60



50
59
81
70









Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps (Quad), left and right gastrocnemius muscles (Gastroc), left and right tibialis anterior (TA) muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 28 as the average myotonia grade observed in four mice of each group and demonstrates significant dose-dependent reduction of myotonia in mice treated with ISIS 445236 and ISIS 445238.







TABLE 28







Average reduction of myotonia in various muscles of antisense oligonucleotide-treated


HSALR mice


















Right
Left
Right
Left
Right
Lumbar



mg/kg/wk
Left Quad
Quad
Gastroc
Gastroc
TA
TA
paraspinals



















PBS
—
3.00
3.00
3.00
3.00
2.75
2.75
3.00


ISIS
5
3.00
3.00
3.00
3.00
2.25
2.25
3.00


445236
17
0.75
0.75
0.75
1.00
0.00
0.00
1.75



50
0.00
0.00
0.00
0.00
0.00
0.00
0.00


ISIS
5
2.75
2.75
2.50
2.50
2.00
1.75
2.75


445238
17
3.00
3.00
2.00
2.25
0.00
0.00
2.75



50
0.75
0.75
0.25
0.25
0.00
0.00
1.00









Correction of Alternative Splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1, total RNA purified from the tibialis anterior gastrocnemius, and quadriceps muscle was analyzed in a procedure similar to that described in Example 13.
In PBS treated HSALR mice, Serca1 splicing is dysregulated as demonstrated by exon 22 exclusion, as a result of MBLN1 dysregulation. Treatment with either ISIS 445236 or ISIS 445238 at doses of 8.5 mg/kg or 25.0 mg/kg twice a week (or 17.0 mg/kg/week and 50.0 mg/kg/week) resulted in complete inclusion and normalization of alternative splicing of exon 22 of the Serca1 gene in all three muscle types.
Therefore, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci correct MBLN1 sequestration thereby allowing normal splicing to occur.
Example 17
In vivo Antisense Inhibition by an Oligonucleotide Targeting the HSA Coding Region of Human Alpha1 Actin in Transgenic Mice
Antisense inhibition of human alpha1 actin RNA transcript by ISIS 190401 (5′-GCGGTCAGCGATCCCAGGGT-3′ (SEQ ID NO: 788), target start site 1028 of SEQ ID NO: 1) on myotonia in HSALR mice was evaluated.
Treatment
HSALR mice received subcutaneous injections of ISIS 190401 at a dose of 25 mg/kg twice per week for 4 weeks. A control group received subcutaneous injections of PBS twice per week for 2 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles, gastrocnemius muscles, and tibialis anterior muscles was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 29, treatment with antisense oligonucleotides reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
Treatment with ISIS 190401 resulted in significant inhibition of alpha1 actin mRNA levels in quadriceps muscle, gastrocnemius muscle, and tibialis anterior muscle under the conditions specified above.







TABLE 29







Antisense inhibition of human alpha1 actin RNA 


transcript in HSALR mice











%



Muscle Type
inhibition







Quadriceps
85



Gastrocnemius
86



Tibialis anterior
89










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 30 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 190401.







TABLE 30







Average reduction of myotonia in various muscles 


of antisense oligonucleotide-treated HSALR mice













ISIS




PBS
190401







Left quadriceps
3.00
0.00



Right quadriceps
3.00
0.00



Left gastrocnemius
3.00
0.00



Right gastrocnemius
3.00
0.00



Left Tibialis anterior
2.50
0.00



Right Tibialis anterior
2.50
0.00



Lumbar paraspinals
3.00
0.50










Correction of Alternative Splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1, total RNA purified from the tibialis anterior gastrocnemius, and quadriceps muscle was analyzed in a procedure similar to that described in Example 13.
In PBS treated HSALR mice, Serca1 splicing is dysregulated as demonstrated by exon 22 exclusion, as a result of MBLN1 dysregulation. Treatment with ISIS 190401 resulted in complete inclusion and normalization of alternative splicing of exon 22 of the Serca1 gene in all three muscle types.
Therefore, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects MBLN1 sequestration thereby allowing normal splicing to occur.
Example 18
Duration of Action of Antisense Inhibition by an Oligonucleotide Targeting Human Alpha1 Actin in Transgenic Mice
The duration of action of antisense inhibition of human alpha1 actin RNA transcript by ISIS 445236 in HSALR mice was evaluated.
Treatment
HSALR mice received subcutaneous injections of ISIS 445236 at a dose of 25 mg/kg twice per week for 4 weeks. A control group received subcutaneous injections of PBS twice per week for 2 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared. The mice were analyzed 6 weeks after administration of the last dose.
Inhibition of Alpha1 Actin RNA
Six weeks after the final dose, the animals were sacrificed and tissue from the quadriceps muscles, gastrocnemius muscles, and tibialis anterior muscles was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 31, treatment with ISIS 445236 reduced human alpha1 actin RNA transcript expression, and this effect was sustained at least for 6 weeks. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
Treatment with ISIS 445236 resulted in significant inhibition of alpha1 actin mRNA levels in quadriceps muscle, gastrocnemius muscle, and tibialis anterior muscle under the conditions specified above.







TABLE 31







Antisense inhibition of human alpha1 actin RNA 


transcript in HSALR mice











%



Muscle Type
inhibition







Quadriceps
88



Gastrocnemius
76



Tibialis anterior
67










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 32 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 445236. Therefore, the effect of antisense inhibition of alpha actin by ISIS 445236 was sustained at least for 6 weeks.







TABLE 32







Average reduction of myotonia in various muscles 


of antisense oligonucleotide-treated HSALR mice













ISIS




PBS
445236







Left quadriceps
3.00
0.00



Right quadriceps
3.00
0.00



Left gastrocnemius
3.00
0.00



Right gastrocnemius
3.00
0.00



Left Tibialis anterior
2.50
0.00



Right Tibialis anterior
2.50
0.00



Lumbar paraspinals
3.00
0.00










Example 19
In vivo Effect of Antisense Inhibition of mRNA with CUG Repeats by Intramuscular Administration in Transgenic Mice
The effect of antisense inhibition of mRNA transcripts containing multiple CUG repeats on myotonia in HSALR mice was evaluated. Three antisense oligonucleotides targeting the CUG repeats and with varying lengths were assayed for their effectiveness in inhibiting myotonia in the mice. ISIS 444745 (AGCAGCAGCAGCAGCAGCAGCAGCA (SEQ ID NO: 789) is a uniform 2′—O-methoxyethyl oligonucleotide, 25 nucleotides in length and with a phosphorothioate backbone. ISIS 444746 (AGCAGCAGCAGCAGCAGCAG (SEQ ID NO: 790) is a uniform 2′—O-methoxyethyl oligonucleotide, 20 nucleotides in length and with a phosphorothioate backbone. ISIS 444749 (GCAGCAGCAGCAGCA (SEQ ID NO: 791) is a uniform 2′—O-methoxyethyl oligonucleotide, 15 nucleotides in length and with a phosphorothioate backbone. ISIS 445236 was included in the assay as a positive control.
Treatment
HSALR mice were divided into three treatment groups. The groups received direct intramuscular injections of ISIS 444745, ISIS 444746 or ISIS 444749 at a dose of 0.4 nM into the tibialis anterior muscle. The contralateral tibialis anterior muscle in each mouse received a single dose intramuscular injection of PBS. The PBS-injected muscle acted as the control.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the tibialis anterior (left and right) was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 33, only treatment with ISIS 444745 reduced human alpha1 actin RNA transcript expression. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.







TABLE 33







Percent inhibition of human alpha1 actin RNA 


transcript in HSALR mice











%



ISIS No.
inhibition














444745
51



444746
0



444749
12










Example 20
In vivo Dose Dependent Inhibition of mRNA with CUG Repeats by Intramuscular Administration in Transgenic Mice
ISIS 444745 and ISIS 444746 were further evaluated for their ability to reduce human alpha 1 actin mRNA in vivo.
Treatment
HSALR mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into 6 treatment groups. Three of the groups received direct intramuscular injections of ISIS 444745 at doses of 0.2 nM, 0.5 nM, or 1.0 nM into the tibialis anterior muscle on one side. Another three groups direct intramuscular injections of ISIS 444746 at doses of 0.2 nM, 0.5 nM, or 1.0 nM into the tibialis anterior muscle on one side. The contralateral tibialis anterior muscle in each mouse received a single dose intramuscular injection of PBS. The PBS-injected muscle acted as the control for the corresponding muscle treated with ISIS oligonucleotide.
Assessment of Myotonia by Electromyography
Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 34 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with either ISIS 444745 or ISIS 444746. The effect of antisense inhibition of alpha actin by ISIS 444745 and 444746 was sustained at least for 6 weeks.







TABLE 34







Dose-dependent reduction of myotonia in muscles 


of antisense oligonucleotide-treated HSALR mice











0.2 nM
0.5 nM
1.0 nM





PBS
3.00
3.00
2.33


ISIS 444745
1.67
1.00
0.33


PBS
2.50
2.00
3.00


ISIS 444746
2.00
0.00
1.00









Example 21
In vivo Effect of Antisense Inhibition of mRNA with CUG Repeats by Subcutaneous Administration in Transgenic Mice
The effect of antisense inhibition of mRNA transcripts containing multiple CUG repeats on myotonia in HSALR mice was evaluated. ISIS 445236 was included in the assay as a positive control.
Treatment
HSALR mice were divided into five treatment groups. The first three groups received subcutaneous injections of ISIS 444745, ISIS 444746 or ISIS 444749 at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group received subcutaneous injections of PBS twice per week for 4 weeks. The fifth group received subcutaneous injections of ISIS 445236 at a dose of 25 mg/kg twice per week for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Assessment of Myotonia by Electromyography
Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 35 as the average myotonia grade observed in four mice of each group.
Treatment with ISIS 445236 led to significant reduction in myotonia. Treatment with ISIS 444745 and ISIS 444746 also resulted in reduced myotonia in some of the tissues tested.







TABLE 35







Average reduction of myotonia in various muscles of antisense 


oligonucleotide-treated HSALR mice














ISIS
ISIS
ISIS
ISIS



PBS
444745
444746
444749
445236





Left quadriceps
3.00
3.00
3.00
3.00
0.00


Right quadriceps
3.00
3.00
3.00
3.00
0.00


Left gastrocnemius
3.00
2.75
3.00
3.00
0.00


Right gastrocnemius
3.00
2.75
2.75
3.00
0.00


Left Tibialis anterior
3.00
2.25
2.75
2.75
0.00


Right Tibialis anterior
3.00
2.25
2.50
2.75
0.00


Lumbar paraspinals
3.00
3.00
3.00
3.00
0.00









Example 22
Dose-Dependent Inhibition of Long CUG Repeat mRNA (HSALR Mice) and a Short CUG Repeat (HSASR Mice) by Subcutaneous Administration in Transgenic Mice
Dose-dependent inhibition of mRNA transcripts containing a long CUG repeat (HSALR mice) and a short CUG repeat (HSASR mice), was evaluated. HSA-short repeat (HSASR) mice express the identical transgene as the HSALR mice, except that 5 instead of 250 CUG repeats are inserted in the 3′ UTR. HSASR mice do not have myotonia, splicing changes, or any other observable myotonia phenotype. ISIS 445236 was used in this assay.
Treatment
HSALR mice were divided into four treatment groups. The first three groups received subcutaneous injections of ISIS 445236 at doses of 2.5 mg/kg, 8.5 mg/kg or 25.0 mg/kg twice per week for 4 weeks. The fourth group received subcutaneous injections of PBS twice per week for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared. HSASR mice were also divided into four groups and similarly treated.
Inhibition of Alpha1 Actin RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles (left and right), gastrocnemius muscles (left and right), and tibialis anterior muscles (left and right) was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. The results are presented in Tables 36 and 37 and are expressed as percent inhibition of alpha1 actin transcript, relative to the control. Greater inhibition of the nuclear-retained long repeat in the muscle of HSALR mice was achieved compared with the non-nuclear-retained short repeat in the muscle of HSASR mice.







TABLE 36







Percent inhibition of human alpha1 actin RNA transcript in HSALR mice










Dose





(mg/kg)
Quadriceps
Gastrocnemius
Tibialis anterior













2.5
24
36
46


8.5
53
66
59


25
86
86
90
















TABLE 37







Percent inhibition of human alpha1 actin RNA transcript inHSASR mice










Dose





(mg/kg)
Quadriceps
Gastrocnemius
Tibialis anterior













2.5
15
14
0


8.5
30
11
0


25
59
48
54









Example 23
In vivo Antisense Inhibition of Human DMPK in Transgenic Mice
LC15 mice, Line A, are transgenic mice containing the entire human DMPK 3′UTR (developed by Wheeler et al, University of Rochester). The mice are the second generation of mice backcrossed to an FVB background. The transgene is expressed in the mice as a CUG repeat RNA, which is retained in the nucleus, forming nuclear inclusions or foci, similar to that seen in human tissue samples of patients with myotonic dystrophy (DM1). There are 350-400 CUG repeats in the DMPK transgene. These mice display early signs of DM1 and do not display any myotonia in their muscle tissues.
ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810, which demonstrated statistically significant dose-dependent inhibition in vitro (see Example 5), were evaluated for their ability to reduce human DMPK RNA transcript in vivo.
Treatment
LC15, Line A mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into five treatment groups. The first three groups received subcutaneous injections of ISIS 445569, ISIS 444404 or ISIS 444436 at a dose of 25 mg/kg twice per week for 4 weeks. The fourth group received subcutaneous injections of ISIS 473810 at a dose of 12.5 mg/kg twice per week for 4 weeks. The fifth group received subcutaneous injections of PBS twice weekly for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of DMPK RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles was isolated. RNA was isolated for real-time PCR analysis of DMPK and normalized to 18s RNA. As presented in Table 38, treatment with antisense oligonucleotides reduced human DMPK RNA transcript expression. The results are expressed as percent inhibition of DMPK transcript, relative to the PBS control.







TABLE 38







Antisense inhibition of human DMPK RNA transcript 


in LC15 mice













%



ISIS No
mg/kg/wk
inhibition







444404
50
20



444404
50
55



444436
50
41



473810
25
56










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. Since LC15 mice do not have myotonia, neither the control group nor the treatment groups displayed any myotonia in any muscle tested.
Example 24
In vivo Antisense Inhibition of Human DMPK in Transgenic Mice
LC15 mice, Line D, are transgenic mice containing the entire human DMPK 3′UTR (developed by Wheeler et al, University of Rochester). The mice are the third generation of mice backcrossed to an FVB background. The transgene is expressed in the mice as a CUG repeat RNA, which is retained in the nucleus, forming nuclear inclusions or foci, similar to that seen in human tissue samples of patients with myotonic dystrophy (DM1). There are 350-400 CUG repeats in the DMPK transgene. These mice display early signs of DM1 and do not display any myotonia in their muscle tissues.
ISIS 445569, ISIS 444404, ISIS 444436 and ISIS 473810 were further evaluated for their ability to reduce human DMPK RNA transcript in vivo.
Treatment
LC15, Line D mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The mice were divided into six treatment groups. The first three groups received subcutaneous injections of ISIS 445569, ISIS 444404 or ISIS 444436 at a dose of 25.00 mg/kg twice per week for 4 weeks. The fourth group received subcutaneous injections of ISIS 473810 at a dose of 12.50 mg/kg twice per week for 4 weeks. The fifth group received subcutaneous injections of ISIS 473810 at a dose of 6.25 mg/kg twice per week for 4 weeks. The sixth group received subcutaneous injections of PBS twice weekly for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Inhibition of DMPK RNA
Twenty four hours after the final dose, the animals were sacrificed and tissue from the quadriceps muscles was isolated. RNA was isolated for real-time PCR analysis of DMPK and normalized to 18s RNA. As presented in Table 39, treatment with antisense oligonucleotides reduced human DMPK RNA transcript expression. The results are expressed as percent inhibition of DMPK transcript, relative to the PBS control.
The results indicate that treatment with the antisense oligonucleotides resulted in inhibition of DMPK mRNA in the mice.







TABLE 39







Antisense inhibition of human DMPK RNA transcript 


in LC15 mice













%



ISIS No
mg/kg/wk
inhibition















444404
50.00
24



444404
50.00
30



444436
50.00
17



473810
25.00
7



473810
12.50
18










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. Since LC15 mice do not have myotonia, neither the control group nor the treatment groups displayed any myotonia in any muscle tested.
Example 25
In vivo Antisense Inhibition of Human DMPK in SXL Transgenic Mouse Model
Using hDMPK-targeting ASOs 444401 and 299471 target knockdown in soleus muscle was measured in SXL mice. The SXL mouse is transgenic for the entire DMPK gene and promoter and contains a 1000 CUG repeat sequence in the 3′UTR of DMPK gene. Mice were dosed 50 mg/kg twice weekly for 4 weeks (n=3 mice per group, except n=2 for saline-injected controls). Results of Taqman assays demonstrated that treatment with either ISISI 444401 or ISIS 299471 significantly reduced mut-hDMPK mRNA levels but had negligible effect on endogenous mouse Dmpk mRNA levels.
Therefore, ISIS 444401 and ISIS 299471 selectively target human DMPK mRNA transcript.
Example 26
Duration of Action of Antisense Inhibition by an Oligonucleotide Targeting Human Alpha1 Actin in Transgenic Mice
The duration of action of antisense inhibition of human alpha1 actin RNA transcript by ISIS 190401 in HSALR mice was evaluated.
Treatment
HSALR mice received subcutaneous injections of ISIS 190401 at a dose of 25 mg/kg twice per week for 4 weeks. A control group received subcutaneous injections of PBS twice per week for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared. The mice were analyzed 15 weeks after administration of the last dose.
Inhibition of Alpha1 Actin RNA
Fifteen weeks after the final dose, the animals were sacrificed and tissue from the quadriceps muscles, gastrocnemius muscles, and tibialis anterior muscles was isolated. RNA was isolated for real-time PCR analysis of alpha1 actin and normalized to 18s RNA. As presented in Table 40, treatment with ISIS 190401 reduced human alpha1 actin RNA transcript expression, and this effect was sustained at least for 15 weeks. The results are expressed as percent inhibition of alpha1 actin transcript, relative to the PBS control.
Treatment with ISIS 190401 resulted in significant inhibition of alpha1 actin mRNA levels under the conditions specified above.







TABLE 40







Antisense inhibition of human alpha1 actin 


RNA transcript in HSALR mice











%



Muscle Type
inhibition







Quadriceps
74



Gastrocnemius
81



Tibialis anterior
75










Assessment of Myotonia by Electromyography

Electromyography on left and right quadriceps, left and right gastrocnemius muscles, left and right tibialis anterior muscles and lumbar paraspinals muscles was performed as previously described (Kanadia et al, 2003, Science, 302: 1978-1980) by using 30 gauge concentric needle electrodes and a minimum of 10 needle insertions for each muscle. The data is presented in Table 41 as the average myotonia grade observed in four mice of each group and demonstrates significant reduction of myotonia in mice treated with ISIS 190401. Therefore, the effect of antisense inhibition of alpha actin by ISIS 190401 was sustained at least for 15 weeks.







TABLE 41







Average reduction of myotonia in various muscles 


of antisense oligonucleotide-treated HSALR mice













ISIS




PBS
190401







Left quadriceps
3.0
0.0



Right quadriceps
3.0
0.0



Left gastrocnemius
2.5
0.0



Right gastrocnemius
2.5
0.0



Left Tibialis anterior
2.5
0.0



Right Tibialis anterior
2.5
0.0



Lumbar paraspinals
2.5
0.0










Correction of Alternative Splicing

To evaluate the effect of ISIS 190401 on alternative splicing of Serca1, total RNA purified from the tibialis anterior gastrocnemius, and quadriceps muscle was analyzed in a procedure similar to that described in Example 13.
In PBS treated HSALR mice, Serca1 splicing is dysregulated as demonstrated by exon 22 exclusion. Treatment with ISIS 190401 resulted in complete inclusion and normalization of alternative splicing of exon 22 of the Serca1 gene in all three muscle types, which was sustained even after 15 weeks.
Therefore, antisense inhibition of alpha1 actin corrected Serca1 splicing dysregulation, which indicates that treatment with antisense oligonucleotide reduced accumulation of CUGexp in the nuclear foci. Reduced accumulation of CUGexp in the nuclear foci corrects MBLN1 sequestration thereby allowing normal splicing to occur.
Example 27
Microarray Analysis of Transcriptomic Effect of Antisense Inhibition of Human Actin
Expression of actin mRNA with expanded CUG repeats causes extensive remodeling of the muscle transcriptome. To evaluate the overall transcriptomic effects of ISIS 190401 and ISIS 445236, microarray analyses was utilized in HSALR mice.
Treatment
HSALR mice received subcutaneous injections of ISIS 190401 or ISIS 445236 at a dose of 25 mg/kg twice per week for 4 weeks. A control group received subcutaneous injections of PBS twice per week for 4 weeks. The PBS-injected group served as the control group to which the oligonucleotide-treated group was compared.
Transcriptome Analysis by Microarray
RNA was isolated from the quadriceps muscle of wild-type or HSALR mice. RNA integrity was verified using an Agilent Bioanalyzer (RNA integrity number >7.5). RNA was processed to complementary RNA (cRNA) and hybridized on microbeads using MouseRef-8 v2.0 Expression BeadChip Kits (Illumina, San Diego), according to the manufacturer's recommendations. Image data were quantified using BeadStudio software (Illumina). Signal intensities were quantile normalized. Row-specific offsets were used to avoid any values of less than 2 prior to normalization. Data from all probe sets with 6 or more nucleotides of CUG, UGC, or GCU repeats was suppressed to eliminate the possibility that expanded repeats in the hybridization mixture (CAG repeats in cRNA originating from CUG repeats in the mRNA) could cross-hybridize with repeat sequences in the probes. To eliminate genes whose expression was not readily quantified on the arrays, probes showing a P value for detection probability of <0.1 were suppressed in all samples. Comparisons between groups were summarized and rank-ordered by fold-changes of mean expression level and t tests. The software package R (Butler et al. Diabetes. 2002; 51: 1028-34) was used to perform principal components analysis (Levin et al. In Antisense Drug Technology: Principles, Strategies, and Applications, S. T. Crooke, Ed. (CRC Press, Boca Raton, 2008), pp 183-215; Geary et al. Drug Metab. Dispos. 2003; 31: 1419-28) on wild-type, ISIS oligonucleotide-treated, and PBS-treated microarray samples. The principle components allowed the capture of the majority of the expression variation in each sample within 3 dimensions. The first three principal components of each sample were plotted.
The principle component analysis of untreated wild-type and HSALR mice demonstrated segregation of HSALR away from wild-type mice, in widely separated clusters. In contrast, antisense oligonucleotide-treated HSALR mice clustered more closely to wild-type mice, suggesting an overall trend for transcriptome normalization. Comparisons of HSALR transgenic mice with wild-type mice identified 93 transcripts whose expression levels were altered more than two-fold (P<0.0001), as presented in Table 42, below. The extent of dysregulation for these transcripts was reduced or normalized for antisense oligonucleotides (88% dysregulated transcripts responded to ISIS 445236, P<0.05 for ISIS 445236 vs. PBS control, whereas 90% responded to ISIS 190401).
In order to consider transcripts that have off-target knockdown, all transcripts whose expression was reduced in antisense oligonucleotide-treated HSALR mice were identified (>two-fold reduction by either oligonucleotide, P<0.0001, n=41 transcripts). All transcripts that were down-regulated by these criteria demonstrated upregulation in HSALR mice. The only exception, collagen 6 alpha2, is unlikely to result from off-target cleavage because it was down-regulated by the two antisense oligonucleotides with non-overlapping sequences.
These results indicate that treatment with antisense oligonucleotides for 4 weeks resulted in a general improvement of the muscle transcriptome without any evidence for off-target effects.







TABLE 42







Comparisons of HSALR transgenic mice with wild-type mice identified 93 transcripts




















Fold-



Fold-
t test
Fold-




Fold-

change
t test
Fold-
t test
change
HSALR-
change
t test



change
t test
HSALR-
HASLR
change
HSALR-
HSALR-
445236
HSALR-
HSALR-



HSALR-
HSALR-
190104 vs.
190401 vs.
HSALR-
190401
445236 vs.
vs.
445236
445236



saline vs.
Saline vs.
HSALR-
HSALR-
190401 vs.
vs.
HSALR-
HSALR-
vs.
vs.


Transcript
WT
WT
saline
saline
WT
WT
saline
saline
WT
WT




















OSBPL10
15.11
0.0000
0.46
0.0023
6.95
0.0008
0.39
0.0007
5.92
0.0002


FBXL13
12.12
0.0000
0.49
0.0159
5.91
0.0385
0.65
0.0255
7.93
0.0026


NGFR
11.57
0.0000
0.23
0.0001
2.66
0.0314
0.16
0.0000
1.84
0.0133


SLC1A1
9.39
0.0000
0.39
0.0001
3.66
0.0001
0.30
0.0001
2.85
0.0116


CXADR
9.13
0.0000
0.14
0.0000
1.30
0.6119
0.21
0.0001
1.94
0.2244


NFATC2
8.48
0.0000
0.32
0.0002
2.67
0.0043
0.22
0.0001
1.84
0.0394


ATP1B4
7.02
0.0000
0.24
0.0000
1.68
0.0021
0.24
0.0000
1.70
0.0091


UCHL1
6.80
0.0000
0.71
0.0168
4.86
0.0005
0.72
0.1187
4.91
0.0090


TEAD4
6.76
0.0000
0.50
0.0030
3.39
0.0085
0.30
0.0004
2.06
0.1213


TAS1R1
6.72
0.0000
0.28
0.0003
1.91
0.1857
0.43
0.0002
2.88
0.0047


MUSTN1
6.52
0.0000
0.31
0.0000
2.01
0.0006
0.33
0.0000
2.15
0.0115


IRF5
6.01
0.0000
0.21
0.0000
1.28
0.0556
0.33
0.0001
1.96
0.0035


CRIP3
5.82
0.0000
0.33
0.0000
1.92
0.0151
0.29
0.0001
1.67
0.1470


TAL2
5.75
0.0000
0.20
0.0001
1.13
0.7717
0.36
0.0002
2.08
0.0274


ORF63
5.39
0.0000
0.27
0.0001
1.45
0.0206
0.47
0.0018
2.51
0.0066


COPG
5.05
0.0000
0.30
0.0000
1.53
0.0218
0.25
0.0001
1.25
0.3617


CAMK1D
4.92
0.0000
0.23
0.0002
1.12
0.8157
0.27
0.0000
1.32
0.2449


HSPA2
4.76
0.0000
0.43
0.0000
2.02
0.0079
0.42
0.0000
2.02
0.0197


CAMK2D
4.70
0.0000
0.36
0.0001
1.70
0.0493
0.45
0.0004
2.12
0.0095


CNTNAP2
4.49
0.0000
0.58
0.0001
2.59
0.0000
0.67
0.0007
3.02
0.0000


TTC7
4.33
0.0000
0.38
0.0000
1.63
0.0085
0.68
0.0468
2.96
0.0126


CD276
4.08
0.0001
0.36
0.0001
1.47
0.1613
0.59
0.0029
2.39
0.0072


USH1C
4.07
0.0000
0.50
0.0011
2.04
0.0077
0.38
0.0029
1.55
0.2881


LRP11
4.03
0.0000
0.55
0.0017
2.24
0.0011
0.55
0.0006
2.23
0.0000


PHLDA3
3.96
0.0000
0.40
0.0001
1.60
0.0019
0.36
0.0001
1.42
0.0609


HSPB7
3.80
0.0000
0.30
0.0000
1.14
0.5358
0.30
0.0000
1.15
0.4474


TRIT1
3.74
0.0000
0.43
0.0000
1.62
0.0003
0.31
0.0000
1.16
0.1043


PCNX
3.66
0.0000
0.37
0.0002
1.34
0.1628
0.42
0.0001
1.53
0.0105


3632451O06RIK
3.51
0.0000
0.81
0.1094
2.83
0.0025
0.71
0.0015
2.51
0.0002


AMHR2
3.46
0.0000
0.45
0.0001
1.56
0.0037
0.52
0.0003
1.79
0.0016


SNX13
3.27
0.0000
0.47
0.0000
1.55
0.0007
0.44
0.0000
1.42
0.0003


ATP9A
3.26
0.0000
0.60
0.0001
1.96
0.0024
0.42
0.0002
1.38
0.2009


D030028O16RIK
3.22
0.0000
0.53
0.0011
1.70
0.0104
0.48
0.0001
1.56
0.0007


RPS6KA3
3.09
0.0000
0.38
0.0000
1.17
0.1845
0.44
0.0001
1.37
0.0321


GCA
3.00
0.0000
0.70
0.0031
2.09
0.0005
0.74
0.0103
2.22
0.0006


PACRG
2.89
0.0001
0.51
0.0002
1.46
0.0063
0.46
0.0001
1.34
0.0229


SPSB2
2.88
0.0001
0.33
0.0000
0.95
0.6599
0.37
0.0000
1.07
0.6216


POU4F1
2.83
0.0000
0.42
0.0000
1.19
0.2046
0.60
0.0007
1.68
0.0074


STRN4
2.72
0.0000
0.38
0.0000
1.03
0.8900
0.46
0.0000
1.25
0.2128


NCAM1
2.67
0.0001
0.70
0.0259
1.87
0.0135
0.54
0.0006
1.43
0.0343


A930018M24Rik
2.65
0.0001
0.58
0.0058
1.53
0.0727
0.43
0.0002
1.13
0.3919


TUBA4A
2.60
0.0000
0.42
0.0000
1.09
0.1806
0.50
0.0000
1.31
0.0041


1AP
2.57
0.0000
0.57
0.0002
1.46
0.0108
0.59
0.0016
1.52
0.0333


ANKRD40
2.56
0.0000
0.63
0.0155
1.60
0.0683
0.57
0.0002
1.46
0.0047


UVRAG
2.48
0.0000
0.59
0.0000
1.48
0.0005
0.52
0.0000
1.28
0.0165


HIST1H4H
2.46
0.0001
0.55
0.0001
1.34
0.0474
0.65
0.0014
1.60
0.0125


EPS15
2.44
0.0000
0.61
0.0001
1.50
0.0057
0.77
0.0043
1.87
0.0007


PANX1
2.41
0.0001
0.46
0.0004
1.11
0.4311
0.36
0.0000
0.87
0.0561


CALML4
2.41
0.0001
0.45
0.0008
1.10
0.6994
0.67
0.0154
1.62
0.0538


ASPH
2.40
0.0000
0.40
0.0000
0.95
0.6969
0.44
0.0000
1.05
0.7267


CREB3L2
2.37
0.0001
0.71
0.0287
1.67
0.0416
0.65
0.0051
1.54
0.0410


TRAF3
2.32
0.0001
0.50
0.0001
1.16
0.2851
0.57
0.0001
1.32
0.0481


CMYA1
2.30
0.0000
0.44
0.0007
1.02
0.9450
0.44
0.0000
1.01
0.9265


ADAMTSL5
2.30
0.0001
0.48
0.0000
1.11
0.3365
0.53
0.0004
1.22
0.1827


HS2ST1
2.27
0.0001
0.64
0.0002
1.44
0.0223
0.74
0.0041
1.68
0.0062


HIST1H4J
2.21
0.0000
0.59
0.0000
1.31
0.0283
0.72
0.0002
1.60
0.0023


SPSB1
2.20
0.0000
0.53
0.0005
1.16
0.2409
0.48
0.0000
1.05
0.3088


LANCL1
2.20
0.0000
0.63
0.0002
1.39
0.0002
0.66
0.0006
1.46
0.0005


KCNC4
2.16
0.0000
0.91
0.3892
1.96
0.0036
0.98
0.8712
2.12
0.0029


PRRC1
2.16
0.0000
0.57
0.0001
1.23
0.0324
0.59
0.0000
1.26
0.0070


MID1IP1
2.13
0.0001
1.27
0.0161
2.70
0.0001
1.09
0.4336
2.32
0.0014


DICER1
2.13
0.0000
0.65
0.0006
1.39
0.0051
0.69
0.0018
1.47
0.0035


IKBKB
2.10
0.0001
0.74
0.0240
1.56
0.0262
0.78
0.0039
1.64
0.0015


D5WSU178E
2.10
0.0000
0.86
0.1447
1.80
0.0049
0.88
0.0352
1.84
0.0002


ZFP106
2.08
0.0000
0.53
0.0000
1.11
0.1324
0.58
0.0002
1.20
0.0706


B930041F14RIK
2.06
0.0000
0.71
0.0002
1.47
0.0000
0.72
0.0030
1.49
0.0025


FHL1
2.04
0.0000
0.58
0.0000
1.17
0.1332
0.40
0.0000
0.81
0.0815


UHRF1BP1L
2.04
0.0001
0.78
0.0315
1.59
0.0071
0.68
0.0024
1.38
0.0151


PHCA
2.02
0.0000
0.64
0.0001
1.29
0.0354
0.74
0.0070
1.50
0.0145


B230312A22RIK
2.02
0.0000
0.79
0.0022
1.59
0.0004
0.77
0.0019
1.56
0.0007


PPP2R5C
2.01
0.0000
0.59
0.0001
1.16
0.0161
0.66
0.0017
1.32
0.0177


UCK2
2.01
0.0001
0.70
0.0004
1.41
0.0129
0.64
0.0001
1.28
0.0510


LEPROTL1
0.50
0.0000
1.45
0.0013
0.72
0.0004
1.47
0.0011
0.73
0.0005


COPS7A
0.49
0.0000
1.35
0.0645
0.66
0.0039
1.49
0.0026
0.73
0.0016


PRM17
0.48
0.0001
1.51
0.2023
0.73
0.1585
1.34
0.0445
0.65
0.0002


LDB3
0.47
0.0000
1.55
0.0550
0.73
0.0607
1.57
0.0010
0.74
0.0055


LOC100046120
0.47
0.0000
1.31
0.0077
0.61
0.0000
1.27
0.0381
0.60
0.0002


LOC677317
0.45
0.0001
1.49
0.0004
0.68
0.0012
1.93
0.0011
0.88
0.2082


LDB2
0.45
0.0000
1.73
0.0424
0.78
0.1234
1.23
0.0817
0.56
0.0000


SUM03
0.44
0.0000
1.70
0.0123
0.74
0.0223
1.37
0.0960
0.60
0.0023


LRRC24
0.43
0.0001
1.89
0.0009
0.82
0.0212
1.42
0.0898
0.61
0.0041


HNRPH1
0.42
0.0000
1.64
0.0077
0.69
0.0094
1.70
0.0057
0.71
0.0144


ARMETL1
0.38
0.0000
2.58
0.0000
0.98
0.7666
2.70
0.0000
1.02
0.7109


LOC100041504
0.37
0.0000
2.02
0.0001
0.75
0.0061
1.84
0.0040
0.68
0.0094


MMP9
0.32
0.0000
2.40
0.0006
0.77
0.0340
1.37
0.1834
0.44
0.0009


CBFB
0.28
0.0000
2.66
0.0304
0.75
0.1852
1.94
0.0056
0.55
0.0004


MDH2
0.24
0.0000
1.20
0.0473
0.29
0.0000
1.12
0.1037
0.27
0.0000


APCDD1
0.20
0.0000
1.98
0.2157
0.39
0.0059
4.55
0.0001
0.90
0.2873


LOC654842
0.19
0.0000
1.28
0.1712
0.24
0.0000
1.07
0.8807
0.20
0.0001


F2RL3
0.15
0.0000
5.78
0.0001
0.86
0.1901
4.92
0.0004
0.73
0.0310


EIF3H
0.13
0.0000
1.99
0.2185
0.26
0.0001
1.86
0.1997
0.24
0.0000


AVIL
0.12
0.0000
4.22
0.0156
0.52
0.0081
1.88
0.2270
0.23
0.0001


ACTC1
0.08
0.0000
1.42
0.0346
0.11
0.0000
6.07
0.0098
0.48
0.0087

## Claims

1. A method of treating an animal having type 1 myotonic dystrophy comprising administering to the animal a therapeutically effective amount of a compound comprising an oligonucleotide consisting of 10 to 30 linked nucleosides targeted to a DMPK nucleic acid, wherein the oligonucleotide has a nucleobase sequence that is at least 90% complementary to a non-CUG repeat region of SEQ ID NO: 1 or SEQ ID NO: 2 as measured over the entirety of the oligonucleotide, wherein the compound reduces myotonia or reduces spliceopathy in the animal.

2. The method of claim 1, wherein the animal is a human.

3. The method of claim 2, wherein myotonia is reduced by at least 25%.

4. The method of claim 3, wherein myotonia is reduced in any of the quadriceps muscle, the gastrocnemius muscle, or the tibialis anterior muscle.

5. The method of claim 2, wherein the oligonucleotide has a nucleobase sequence that is 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2.

6. The method of claim 1, wherein the oligonucleotide is a modified oligonucleotide.

7. The method of claim 6, wherein the modified oligonucleotide is a single-stranded oligonucleotide.

8. The method of claim 7, wherein the single-stranded modified oligonucleotide is a gapmer.

9. The method of claim 7, wherein at least one internucleoside linkage of the modified oligonucleotide is a modified linkage.

10. The method of claim 9, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

11. The method of claim 9, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.

12. The method of claim 11, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

13. The method of claim 9, wherein at least one internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage and at least one internucleoside linkage of the modified oligonucleotide is a phosphodiester internucleoside linkage.

14. The method of claim 7, wherein at least one nucleobase of the modified oligonucleotide is a modified nucleobase.

15. The method of claim 14, wherein the modified nucleobase is a 5-methylcytosine.

16. The method of claim 7, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar.

17. The method of claim 16, wherein the modified sugar is a bicyclic sugar.

18. The method of claim 17, wherein the bicyclic sugar comprises a chemical bridge between the 4′ and 2′ positions of the sugar, wherein the chemical bridge is selected from: 4′—CH(R)—O—2′ and 4′—(CH2)2—O—2′, wherein R is independently H, C1-C6 alkyl, and C1-C6 alkoxy.

19. The method of claim 18, wherein the chemical bridge is 4′—CH(R)—O—2′ and wherein R is methyl.

20. The method of claim 18, wherein the chemical bridge is 4′—CH(R)—O—2′ and wherein R is H.

21. The method of claim 18, wherein the chemical bridge is 4′—CH(R)—O—2′ and wherein R is —CH2—O—CH3.

22. The method of claim 16, wherein the modified sugar comprises a 2′—O-methoxyethyl group.

23. The method of claim 1, wherein the compound comprises the oligonucleotide covalently linked to a conjugate group.

24. The method of claim 1, wherein the oligonucleotide is a salt.