# Patent

## Title
Selective reduction of allelic variants

## Abstract

Disclosed herein are antisense compounds and methods for selectively reducing expression of an allelic variant of a huntingtin gene containing a single nucleotide polymorphism (SNP). Such methods, compounds, and composition are useful to treat, prevent, or ameliorate Huntington's Disease (HD).

## Background
CROSS REFERENCED TO RELATED APPLICATIONS
This application is a U.S. National Phase filing under 35 U.S.C. §371 claiming priority to International Serial No. PCT/US2011/024104 filed Feb. 8, 2011, which claims priority to U.S. Provisional Application No. 61/371,640, filed Aug. 6, 2010, and U.S. Provisional Application No. 61/302,458, filed Feb. 8, 2010, each of which is incorporated herein by reference in its entirety.


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 BIOL0125USASEQ.TXT, created Aug. 6, 2012, which is 322 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
Embodiments of the present invention provide methods, compounds, and compositions for selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism (SNP). Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate Huntington's Disease.
BACKGROUND OF THE INVENTION
Genetic diseases are caused by abnormalities in genes or chromosomes. Such abnormalities may include insertions, deletions, and expansions. Huntington's Disease (HD) is one example of a genetic disease caused by an expansion. HD is a progressive neurodegenerative disorder that is inherited in a dominant fashion and results from a mutation that expands the polymorphic trinucleotide (CAG) tract in the huntingtin gene (HTT). The average CAG tract size in the general population is 17-26 repeats (wild type allele), however, in HD patients the CAG tract has expanded to 36 repeats or more (mutant allele) (Huntington's Disease Collaborative Research Group 1993. Cell 72(6):971-83). The HTT gene encodes the HTT protein and the expanded CAG tract results in a pathological increase in the polyglutamine repeats near the N-terminal of the protein. Individuals carry two copies of the HTT gene and one mutant allele is sufficient to result in HD.
HTT protein appears to have a role during development of the nervous system and a protective role in cells. In mouse models, constitutive knockout of the HTT gene is lethal during embryonic development (Nasir et al 1995. Cell 81(5):811-23), while adult inactivation of the HTT gene leads to progressive cell death in the brain and the testes (Dragatsis et al 2000. Nat. Genet. 26:300-306). Reduction of huntingtin expression from the wild type allele may, therefore, have negative consequences.
Like HD, there are disorders for which a strategy of selective reduction of a mutant allele would be beneficial. Thus, there remains an unmet need to selectively reduce expression of mutant allelic variants like that of HTT, which are causative of disease, over the wild type variant, which appears to be necessary for normal cellular processes.



BRIEF DESCRIPTION OF THE INVENTION
FIG. 1 provides the mRNA (SEQ ID NO: 2) and genomic (SEQ ID NO: 1) HTT sequence showing SNP positions.



SUMMARY OF THE INVENTION
Provided herein are methods, compounds, and compositions for selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism (SNP). Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate Huntington's Disease (HD). SNPs may be associated with a mutant allele, the expression of which causes HD.
In certain embodiments, selective reduction of mRNA and protein expression of a mutant huntingtin is achieved by targeting a SNP located on the mutant allele with an antisense compound. In certain embodiments, the antisense compound is an antisense oligonucleotide.
In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing a SNP are created based on potency and selectivity of the antisense compound as well as population genetics.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference 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 may 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 furosyl 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 the 5′ position. A 5-methylcytosine is a modified nucleobase.
“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 an allelic variant 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 a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
“Allele” is one member of a pair of genes or one member of a series of different forms of a DNA sequences that can exist at a single locus or marker on a specific chromosome. For a diploid organism or cell or for autosomal chromosomes, each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father. If these alleles are identical, the organism or cell is said to be ‘homozygous’ for that allele; if they differ, the organism or cell is said to be ‘heterozygous’ for that allele. “Major allele” refers to an allele containing the nucleotide present in a statistically significant proportion of individuals in the human population. “Minor allele” refers to an allele containing the nucleotide present in a relatively small proportion of individuals in the human population. “Wild type allele” refers to the genotype typically not associated with disease or dysfunction of the gene product. “Mutant allele” refers to the genotype associated with disease or dysfunction of the gene product.
“Allelic variant” refers to one of the pair of genes or DNA sequence existing at a single locus. For example, an allelic variant may refer to either the major allele or the minor allele.
“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may 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.
“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
“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.
“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 furosyl ring modified by the bridging of two ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleoside” means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH3)—O-2′.
“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 pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses 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.
“Differentiating polymorphism” means a variation in a nucleotide sequence that permits differentiation between a wild type and a mutant allele of a nucleic acid sequence. Differentiating polymorphisms may include insertions or deletions of one or a few nucleotides in a sequence, or changes in one or a few nucleotides in a sequence. A differentiating polymorphism or polymorphic allele can be in linkage disequilibrium with one or more other polymorphisms or polymorphic alleles.
“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 may be a liquid, e.g. saline solution.
“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 may 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 may 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 may be stated as the amount of pharmaceutical agent per hour, day, week, or month.
“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 may 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 first nucleic acid has a complementary nucleobase in 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 may be referred to as the “gap” and the external regions may be referred to as the “wings.”
“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.
“Gene product” refers to a biochemical material, such as RNA or protein, resulting from expression of a gene.
“Haplotype” means a set of alleles of closely linked loci on a chromosome that are generally inherited together. For example, a polymorphic allele at a first site in a nucleic acid sequence on the chromosome may be found to be associated with another polymorphic allele at a second site on the same chromosome, at a frequency other than would be expected for a random associate (e.g. “linkage equilibrium”). These two polymorphic alleles may be described as being in “linkage disequilibrium.” A haplotype may comprise two, three, four, or more alleles. The set of alleles in a haplotype along a given segment of a chromosome are generally transmitted to progeny together unless there has been a recombination event.
“High-affinity sugar modification” is a modified sugar moiety which when it is included in a nucleoside and said nucleoside is incorporated into an antisense oligonucleotide, the stability (as measured by Tm) of said antisense oligonucleotide: RNA duplex is increased as compared to the stability of a DNA:RNA duplex.
“High-affinity sugar-modified nucleoside” is a nucleoside comprising a modified sugar moiety that when said nucleoside is incorporated into an antisense compound, the binding affinity (as measured by Tm) of said antisense compound toward a complementary RNA molecule is increased. In certain embodiments of the invention at least one of said sugar-modified high-affinity nucleosides confers a ΔTm of at least 1 to 4 degrees per nucleoside against a complementary RNA as determined in accordance with the methodology described in Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443, which is incorporated by reference in its entirety. In another aspect, at least one of the high-affinity sugar modifications confers about 2 or more, 3 or more, or 4 or more degrees per modification. In the context of the present invention, examples of sugar-modified high affinity nucleosides include, but are not limited to, (i) certain 2′-modified nucleosides, including 2% subtstituted and 4′ to 2′ bicyclic nucleosides, and (ii) certain other non-ribofuranosyl nucleosides which provide a per modification increase in binding affinity such as modified tetrahydropyran and tricyclo DNA nucleosides. For other modifications that are sugar-modified high-affinity nucleosides see Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443.
“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.
“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 together.
“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 a modified internucleoside linkage, a modified sugar, or a modified nucleobase.
“Modified sugar” refers to a substitution or change from a natural sugar.
“Motif” means the pattern of chemically distinct regions in an antisense compound.
“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).
“Nuclease resistant modification” means a sugar modification or modified internucleoside linkage which, when incorporated into an oligonucleotide, makes said oligonucleotide more stable to degradation under cellular nucleases (e.g. exo- or endo-nucleases). Examples of nuclease resistant modifications include, but are not limited to, phosphorothioate internucleoside linkages, bicyclic sugar modifications, 2′-modified nucleotides, or neutral internucleoside linkages.
“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).
“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 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). 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.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“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.
“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 may comprise one or more active pharmaceutical 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 (P═S) 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.
“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.
“Selectively reducing expression of an allelic variant” means reducing expression of one allele more than the other, differing allele among a set of alleles. For example, a mutant allele containing a single nucleotide polymorphism (SNP) may be reduced more than a wild type allele not containing the SNP.
“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 may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
“Single nucleotide polymorphism” or “SNP” means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion. In general, SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. SNPs are thought to be mutationally more stable than other polymorphisms, lending their use in association studies in which linkage disequilibrium between markers and an unknown variant is used to map disease-causing mutations. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site. A heterozygous SNP allele can be a differentiating polymorphism. A SNP may be targeted with an antisense oligonucleotide, meaning that the SNP anneals to (or aligns with) position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the antisense oligonucleotide. The remainder of the antisense oligonucleotide bases must have sufficient complementarity to the SNP site to facilitate hybridization.
“Single nucleotide polymorphism position” or “SNP position” refers to the nucleotide position of the SNP on a reference sequence.
“Single nucleotide polymorphism site” or “SNP site” refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.
“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.
“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. For example, for the purposes of this patent application, the target segment may be within the SNP site. “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 a pharmaceutical 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.
“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
Embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a gene or DNA sequence. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, the SNP is a differentiating polymorphism. In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a SNP is in linkage disequilibrium with another polymorphism that is associated with or is causative of disease. In certain embodiments, a mutant allele is associated with disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.
In certain embodiments, the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease.
In certain embodiments, selective reduction of mRNA and protein expression of a mutant allele is achieved by targeting a SNP located on the mutant allele with an antisense compound. In certain embodiments, the antisense compound is an antisense oligonucleotide. In certain embodiments, the antisense compound is not a ribozyme, a double stranded siRNA, or an shRNA. In certain embodiments, the antisense oligonucleotide may have one or more modified sugar(s), nucleobase(s), or internucleoside linkage(s). In certain embodiments, the antisense oligonucleotide is complementary to the SNP site. In certain embodiments, the antisense oligonucleotide is complementary to the SNP site. In certain embodiments, the antisense oligonucleotide is at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the SNP site. In certain embodiments, the antisense oligonucleotide is 100% complementary to the SNP site. In certain embodiments, the SNP site is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length. In certain embodiments, the SNP anneals to position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the antisense oligonucleotide.
In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing a SNP are created based on potency and selectivity of the antisense compound as well as population genetics.
In certain embodiments, selective reduction of mRNA and protein expression of an allelic variant of a gene containing a SNP occurs in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human.
In certain embodiments, described is a method of treating Huntington's Disease, comprising selectively reducing expression of a mutant huntingtin allele in an animal in need thereof, comprising administering to the animal a compound, comprising a modified oligonucleotide consisting of 15 to 20 linked nucleosides and fully complementary to the mutant huntingtin allele, wherein position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with any of the SNP sites in the group consisting of rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325, rs35892913, rs1936032, rs2276881, rs363070, rs12502045, rs7685686, rs3733217, rs362331, rs2857936, rs12506200, rs762855, rs4690072, rs363081, rs363075, rs3025849, rs6855981, rs363144, rs3025838, rs2298969, rs362322, rs362307, rs362306, and rs1006798.
In certain embodiments, described is a compound comprising a modified oligonucleotide 15 to 20 linked nucleosides in length, targeted to a huntingtin single nucleotide polymorphism, comprising a 12 nucleobase portion of the nucleobase sequence of any one of SEQ ID NO: 6 to 285, wherein position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the huntingtin single nucleotide polymorphism.
In certain embodiments, the modified oligonucleotide comprises a wing-gap-wing motif of any one of the group consisting of 5-10-5, 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4-9-3, 4-9-4, 4-9-5, 4-10-5, 4-11-4, 4-11-5, 5-7-5, 5-8-6, 5-9-3, 5-9-5, 5-10-4, 5-10-5, 6-7-6, 6-8-5, and 6-9-2.
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 nucleobase. In certain embodiments, the modified nucleobase is a 5′-methylcytosine.
In certain embodiments, at least one nucleoside of at least one of the wing regions comprises a modified sugar or sugar surrogate. In certain embodiments, each of the nucleosides of each wing region comprises a modified sugar or sugar surrogate. In certain embodiments, the sugar or sugar surrogate is a 2′-O-methoxyethyl modified sugar. In certain embodiments, at least one of the wing regions comprises a 4′ to 2′ bicyclic nucleoside and at least one of the remaining wing nucleosides is a non-bicyclic 2′-modified nucleoside. In certain embodiments, the non-bicyclic 2′-modified nucleoside is a 2′-O-methoxyethyl nucleoside. In certain embodiments, the 4′ to 2′ bicyclic nucleoside is 4′-CH(CH3)-O-2′ bicyclic nucleoside.
In certain embodiments, described is a method of selectively reducing mutant huntingtin, comprising administering a compound comprising a modified oligonucleotide 15 to 20 linked nucleosides in length, targeted to a huntingtin single nucleotide polymorphism, comprising a 12 nucleobase portion of the nucleobase sequence of any one of SEQ ID NO: 6 to 285, wherein position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the huntingtin single nucleotide polymorphism.
In certain embodiments, described is a use of a compound comprising a modified oligonucleotide 15 to 20 linked nucleosides in length, targeted to a huntingtin single nucleotide polymorphism, comprising a 12 nucleobase portion of the nucleobase sequence of any one of SEQ ID NO: 6 to 285, wherein position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the huntingtin single nucleotide polymorphism for the treatment of Huntington's Disease.
Single Nucleotide Polymorphisms (SNPs)
Single-nucleotide polymorphisms (SNPs) are single base-pair alterations in the DNA sequence that represent a major source of genetic heterogeneity (Gene. 1999, 234:177). SNP genotyping is an important tool with which to investigate these genetic variants (Genome Res. 2000, 10:895; Trends Biotechnol. 2000, 18:77). In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing an SNP were selected based on potency, selectivity and population genetics coverage.
Potency
In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing a SNP are created based on potency of the antisense compound. Potency generally refers to how amenable the targeted sequence area is to antisense inhibition. In certain embodiments, specific SNP sites may be particularly amenable to antisense inhibition. Certain such highly amenable SNP sites may be targeted by antisense compounds for selectively reducing an allelic variant of a gene. Potency is demonstrated by the percent inhibition of mutant mRNA achieved by the antisense oligonucleotides targeting a SNP compared to the percent inhibition of mutant mRNA achieved by the benchmark oligonucleotide.
Selectivity
In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing a SNP are created based on selectivity of the antisense compound. Selectivity generally refers to antisense compounds comprising a particular sequence, motif, and chemical modification(s) that preferentially target the one or more differentiating polymorphisms (SNPs) in the RNA encoding a mutant HTT protein compared to the RNA encoding a wild type HTT protein. In certain embodiments, specific sequences, motifs, and chemical modification(s) are particularly selective in reducing an allelic variant of a gene containing a SNP. Certain such sequences, motifs, and chemical modification(s) are utilized to selectively reduce an allelic variant of a gene. Selectivity is demonstrated by the ability of the antisense oligonucleotide targeting a SNP to inhibit expression of the major allele or mutant allele preferentially compared to the minor allele or wild type allele.
Population Genetics
In certain embodiments, antisense compounds designed to selectively reduce an allelic variant of a gene containing an SNP are created based on the population genetics of a population afflicted with disease. Population genetics means the frequency at which the SNP appears in the disease chromosome of patients afflicted with a particular disease. In certain embodiments, the disease is Huntington disease. Where potency and selectivity amongst antisense compounds is equal, SNP targets that have higher population genetics coverage are favored over SNPs that have a weaker association with disease chromosomes.
Antisense Compounds
Oligomeric compounds may include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may 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 is an antisense oligonucleotide. In certain embodiments, the antisense compound is not a ribozyme, a double stranded siRNA, or an shRNA.
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, antisense compounds are 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain such embodiments, the antisense compounds are 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 subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleosides.
In certain embodiments antisense oligonucleotides targeted to a nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, 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 subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit 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.
However, selective reduction of expression of an allelic variant is optimized when the SNP contained in the target nucleic anneals to a complementary base in the antisense compound and not a mismatched base. Moreover, selectivity in general is increased when there are fewer mismatches between the SNP site and the antisense compound. However, a certain number of mismatches may be tolerated.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a 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 may 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 the case of an antisense oligonucleotide for selectively reducing expression of an allelic variant of a gene containing a SNP, the SNP anneals to a nucleobase within the gap segment.
In certain embodiments, the SNP anneals or is complementary to a nucleobase at position 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the antisense oligonucleotide, wherein position refers to the orientation of a nucleobase within the antisense oligonucleotide counting from the 5′ terminus of the antisense oligonucleotide. For example, the 5′ most nucleobase within the antisense oligonucleotide is in the first position of the antisense oligonucleotide. In certain embodiments, the SNP anneals or is complementary to a nucleobase at position 6, 7, 8, 9, or 10 of the antisense oligonucleotide (counting from the 5′ terminus). In certain embodiments, the SNP anneals or is complementary to a nucleobase at position 9 or 10 of the antisense oligonucleotide (counting from the 5′ terminus).
In certain embodiments, the SNP anneals to a nucleobase at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the gap segment, wherein position refers to the orientation of a nucleobase within the gap segment counting from the 5′ terminus of the gap segment. For example, the 5′ most nucleobase within the gap segment is in the first position of the gap segment. In certain embodiments, the SNP anneals to a nucleobase at position 4, 5, 6, or 7 counting from the 5′ terminus of the gap segment. In certain embodiments, the SNP anneals to a nucleobase at position 4 or 5 beginning from the 5′ terminus of the gap segment.
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 may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH2)n—O-2′ bridge, where n=1 or n=2). The bicyclic moiety may be a cEt having the formula 4′-CH(CH3)—O-2.′
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 to 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 certain embodiments, Y is between 8 and 15 nucleotides. In certain embodiments, Y is comprised of deoxynucleotides. 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 of the present invention include, but are not limited to, for example 1-10-1, 1-18-1, 2-8-2, 2-9-6, 2-10-2, 2-13-5, 2-16-2, 3-9-3, 3-9-5, 3-10-3, 3-14-3, 4-8-4, 4-9-5, 4-10-5, 4-11-4, 4-12-3, 4-12-4, 5-8-5, 5-9-5, 5-10-4, 5-10-5, or 6-8-6.
In certain embodiments, the antisense compound has 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 of the present invention 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, 5-13, 5-8, or 6-8.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 2-9-6 gapmer motif or a 6-9-2 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 3-9-3 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 3-9-5 gapmer motif or 5-9-3 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 4-9-5 gapmer motif or 5-9-4 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 4-10-5 gapmer motif or 5-10-4 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 4-11-4 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 5-9-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 5-8-6 gapmer motif or a 6-8-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 6-7-6 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 6-8-5 gapmer motif or a 5-8-6 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 3-9-4 gapmer motif or a 4-9-3 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 5-7-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 4-7-4 gapmer motif.
In certain embodiments, antisense compounds targeted to a nucleic acid possess a 5-10-5 gapmer motif.
In certain embodiments, an antisense compound targeted to a nucleic acid has a gap-widened motif.
Certain Mixed Wings
In certain embodiments, the invention provides gapmer compounds wherein at least one nucleoside of one wing is differently modified compared to at least one other nucleoside of the same wing. Such antisense compounds are referred to as mixed wing antisense compounds (see WO 2008/049085). In certain embodiments, the modifications (or no modification) of one or more nucleosides of the 3′ wing are different from those of one or more other nucleosides of the 3′ wing. Such antisense compounds may be referred to as 3′ mixed wing gapmers. In certain embodiments, the modifications (or no modification) of one or more nucleosides of the 5′ wing are different from those of one or more other nucleosides of the 5′ wing. Such antisense compounds may be referred to as 5′ mixed wing gapmers. In certain embodiments, the modifications (or no modification) of one or more nucleosides of the 3′ wing are different from those of one or more other nucleosides of the 3′ wing and the modifications (or no modification) of one or more nucleosides of the 5′ wing are different from those of one or more other nucleosides of the 5′ wing. Such antisense compounds may be referred to as 3′, 5′ mixed wing gapmers. In such embodiment, the modifications and combination of modifications at the 3′ wing and at the 5′ wing may be the same or they may be different.
In certain embodiments, mixed wing compounds have desirable properties. Certain nucleoside modifications confer on the antisense compound a desirable property, for example increased affinity for a target or nuclease resistance, but also confer an undesirable property, for example increased toxicity. Incorporation of certain other nucleoside modifications results in antisense compounds with different profiles of properties. In certain embodiments, one may combine modifications in one or both wings to optimize desirable characteristics and/or minimize undesirable characteristics. In certain embodiments, the wings of a mixed wing antisense compound comprise one or more nucleoside comprising a first modification that increases affinity of the antisense compound for a target nucleic acid compared to an antisense compound comprising unmodified nucleosides; and one or more nucleoside comprising a second modification that results in reduced toxicity compared to an antisense compound with wings comprising nucleosides that all comprise the first modification.
In certain embodiments, an antisense compound comprises at least one wing comprising at least one MOE substituted nucleoside and at least one high affinity modification. In certain such embodiments, the at least one MOE substituted nucleoside and the at least one high affinity are in the 3′ wing. In certain such embodiments, the at least one MOE substituted nucleoside and the at least one high affinity are in the 5′ wing.
In certain embodiments, an antisense compound comprises 1, 2 or 3 high affinity modifications in the 5′ and/or 3′ wings.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
In certain embodiments, an allelic variant of huntingtin is selectively reduced. Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT—006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT—006051), incorporated herein as SEQ ID NO: 1, and NM—002111.6, incorporated herein as SEQ ID NO: 2.
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 may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may 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 huntingtin 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 may 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 same 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 of a particular allelic variant. In certain embodiments, the desired effect is reduction of levels of the protein encoded by the target nucleic acid or a phenotypic change associated with a particular alleleic variant.
A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, 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 preceeding 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 may 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 may specifcally exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
Cell Lines
In certain embodiments, the GM04281, GM02171, and GM02173B cell lines are used in experiments described herein below. The GM04281 cell line has a wild-type HTT allele that contains 17 repeats and a mutant HTT allele that contains 69 repeats. The cell line was derived from a patient both of whose parents were also affected by the disease. The GM02171 cell line was chosen as a counter screen control to the GM04281. This cell line was derived from the daughter of parents, only one of whom had the disease. The daughter had not developed HD but was considered to be at risk. The GM02173B cell line was also patient-derived and was used as a haplotype test control.
Table 1 provides SNPs found in the GM04281, GM02171, and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C. The GM02171 cell line is homozygous CC, the GM02173 cell line is heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty percent of HD patients have a T at SNP position rs6446723.







TABLE 1







Allelic Variations for SNPs Associated with HD













SNP
Variation
GM0217
GM02173
GM04281
TargetPOP
allele





rs6446723
T/C
CC
TC
TT
0.50
T


rs3856973
A/G
AA
AG
GG
0.50
G


rs2285086
A/G
GG
AG
AA
0.50
A


rs363092
A/C
AA
AC
CC
0.49
C


rs916171
C/G
GG
GC
CC
0.49
C


rs6844859
T/C
CC
TC
TT
0.49
T


rs7691627
A/G
AA
AG
GG
0.49
G


rs4690073
A/G
AA
AG
GG
0.49
G


rs2024115
A/G
GG
AG
AA
0.48
A


rs11731237
T/C
CC
TC
TT
0.43
T


rs362296
A/C
AC
AC
AC
0.42
C


rs10015979
A/G
AA
AG
GG
0.42
G


rs7659144
C/G
CG
CG
CC
0.41
C


rs363096
T/C
CC
TC
TT
0.40
T


rs362273
A/G
AG
AG
AA
0.39
A


rs16843804
T/C
TC
TC
CC
0.38
C


rs362271
A/G
AG
AG
GG
0.38
G


rs362275
T/C
TC
TC
CC
0.38
C


rs3121419
T/C
TC
TC
CC
0.38
C


rs362272
A/G
—
AG
GG
0.38
G


rs3775061
A/G
AG
AG
AA
0.38
A


rs34315806
T/C
TC
TC
CC
0.38
C


rs363099
T/C
TC
TC
CC
0.38
C


rs2298967
T/C
TC
TC
TT
0.38
T


rs363088
A/T
TA
TA
AA
0.38
A


rs363064
T/C
TC
TC
CC
0.35
C


rs363102
A/G
AA
AA
AA
0.23
G


rs2798235
A/G
GG
GG
GG
0.21
A


rs363080
T/C
CC
CC
CC
0.21
T


rs363072
A/T
TA
AA
AA
0.13
A


rs363125
A/C
AC
CC
CC
0.12
C


rs362303
T/C
TC
CC
CC
0.12
C


rs362310
T/C
TC
CC
CC
0.12
C


rs10488840
A/G
AG
GG
GG
0.12
G


rs362325
T/C
TC
TT
TT
0.11
T


rs35892913
A/G
GG
GG
GG
0.10
A


rs363102
A/G
AA
AA
AA
0.09
A


rs363096
T/C
CC
TC
TT
0.09
C


rs11731237
T/C
CC
TC
TT
0.09
C


rs10015979
A/G
AA
AG
GG
0.08
A


rs363080
T/C
CC
CC
CC
0.07
C


rs2798235
A/G
GG
GG
GG
0.07
G


rs1936032
C/G
CC
CC
CC
0.06
C


rs2276881
A/G
GG
GG
GG
0.06
G


rs363070
A/G
AA
AA
AA
0.06
A


rs35892913
A/G
GG
GG
GG
0.04
G


rs12502045
T/C
CC
CC
CC
0.04
C


rs6446723
T/C
CC
TC
TT
0.04
C


rs7685686
A/G
GG
AG
AA
0.04
G


rs3733217
T/C
CC
CC
CC
0.03
C


rs6844859
T/C
CC
TC
TT
0.03
C


rs362331
T/C
CC
TC
TT
0.03
C









Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a SNP site. 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.
In certain embodiments, the antisense compounds provided herein are specifically hybridizable with the nucleic acid of a particular allelic variant.
Complementarily
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., selective reduction of a gene product of an allelic variant).
Non-complementary nucleobases between an antisense compound and a target nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a target 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 target nucleic acid, a target region, target segment, SNP site, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
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 may 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, a SNP site, target region, target segment, or specified portion 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 may or may not 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 may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be 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 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, SNP site, or specified portion thereof.
In certain embodiments, antisense oligonucleotides that are, or are up to 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, SNP site, 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 12 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 a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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 may 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 may 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%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
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 may 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.
Chemically modified nucleosides may also be employed to increase selectivity in reducing expression the gene product of an allelic variant.
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 phosphorothioate. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds 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, increased selectivity for an allelic variant, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a 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)(R)2 (R═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 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, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), and O—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides 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 bicyclic nucleosides wherein the bridge comprises a 4′ to 2′ bicyclic nucleoside. Examples of such 4′ to 2′ bicyclic nucleosides, include but are not limited to one of the formulae: 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 published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH2-β-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 published International Application WO 2008/154401, published on Dec. 8, 2008). See, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; and U.S. Pat. No. 6,670,461; International applications WO 2004/106356; WO 94/14226; WO 2005/021570; U.S. Patent Publication Nos. US2004-0171570; US2007-0287831; US2008-0039618; U.S. Pat. No. 7,399,845; U.S. patent Ser. Nos. 12/129,154; 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; 61/099,844; PCT International Applications Nos. PCT/US2008/064591; PCT/US2008/066154; PCT/US2008/068922; and Published PCT International Applications WO 2007/134181. 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 sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 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′-2′ methylene-oxy 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, 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, and (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA, (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) ethylene carbocyclic (4′-CH2—CH2-2′) (carba LNA or “cLNA”) as depicted below.







wherein Bx is the base moiety and R is independently H, a protecting group or C1-C12 alkyl.

In certain embodiments, bicyclic nucleoside having Formula I:




wherein:

Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is —CH2—N(Rc)—CH2—, —C(═O)—N(Rc)—CH2—, —CH2—O—N(Re)—, —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 nucleoside having Formula II:




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 thio.
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 NJcC(═X)NJcJd, wherein each Jc, Jd and Jc is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJc.
In certain embodiments, bicyclic nucleoside having Formula III:




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 nucleoside having Formula IV:




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, qa 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 nucleoside having Formula V:




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 gf 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)(qb);
qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4′-CH2—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4′-CH2—O-2′) BNA, methyleneoxy (4′-CH2—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally 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 nucleoside having Formula VI:




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 qj, 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, 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).
As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.
As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2C(═O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F—HNA) or those compounds having Formula X:




wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the 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;
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 alleynyl; 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,C(═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, the modified THP nucleosides of Formula X are provided wherein qm, qn, qp, qr, qs, qt and qn are each H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt and qu is other than H. In certain embodiments, at least one of qm, qn, qp, qr, qs, qt and is methyl. In certain embodiments, THP nucleosides of Formula X 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.
As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position.
As used herein, “2′-OMe” or “2′-OCH3” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH3 group at the 2′ position of the sugar ring.
As used herein, “MOE” or “2′-MOE” or “2′-OCH2CH2OCH3” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH2CH2OCH3 group at the 2′ position of the sugar ring.
As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
Many other bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 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.
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 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. In certain embodiments, the modified sugar moiety is a cEt. In certain embodiments, the cEt modified nucleotides are arranged throughout the wings of 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 may impart nuclease stability, binding affinity, increased selectivity for an allelic variant, 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 modified 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 may 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 comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides 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 may be admixed with pharmaceutically acceptable active or inert substances 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.
An antisense compound 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 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 may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution, increased selectivity for an allelic variant, 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 target nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and 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, and primary hepatocytes. Illustrative cell lines include GM04281, GM02171, and GM02173B 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% confluency 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 (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 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 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 LIPOFECTAMINE. 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
Reduction, inhibition, or expression of a target 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 quantitaive 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 may 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 (Invetrogen, 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 target nucleic acids. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).
Analysis of Protein Levels
Reduction, inhibition, or expression of target nucleic acids can be assessed by measuring target protein levels. Target protein levels 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. Antibodies useful for the detection of mouse, rat, monkey, and human proteins are commercially available.
In Vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to selectively reduce or inhibit expression of target gene product and produce phenotypic changes, such as, amelioration of a disease symptom. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA or protein is isolated from tissue and changes in target nucleic acid or protein expression are measured.
Administration
In certain embodiments, the compounds and compositions described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, pulmonary (including by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) or parenteral, for example, by intravenous drip, intravenous injection or subcutaneous, intraperitoneal, intraocular, intravitreal, or intramuscular injection.
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.
In certain embodiments, compounds and compositions are delivered to the CNS. In certain embodiments, compounds and compositions are delivered to the cerebrospinal fluid. In certain embodiments, compounds and compositions are administered to the brain parenchyma. In certain embodiments, compounds and compositions are delivered to an animal by intrathecal administration, or intracerebroventricular administration. Broad distribution of compounds and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.
In certain embodiments, parenteral administration is by injection. The injection may be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.
In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50. In certain embodiments, the pharmaceutical agent in an antisense compound as further described herein. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments the targeted tissue is striatal tissue. In certain embodiments, decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.
In certain embodiments, an antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.
Certain Compounds and Indications
Provided herein are compounds and methods that provide potent inhibition and increased selectivity for a mutant allele. Potency is demonstrated by the percent inhibition of mutant mRNA achieved by the antisense oligonucleotides targeting a SNP compared to the percent inhibition of mutant mRNA achieved by the benchmark oligonucleotide. Selectivity is demonstrated by the ability of the antisense oligonucleotide targeting a SNP to inhibit expression of the major allele or mutant allele preferentially compared to the minor allele or wild type allele. The usage of three cell lines with different genotypes at each SNP position have facilitated the determination of design rules that provide for potent and selective SNP targeting antisense oligonucleotides.
In certain embodiments, the compounds are antisense oligonucleotides as further described herein. The antisense oligonucleotides preferentially target a SNP or differentiating polymorphism. Oligonucleotides of various lengths were tested and certain lengths were determined to be beneficial for the targeting of SNPs.
In certain embodiments, the antisense oligonucleotides have a sequence that is 12-30 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 12-25 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 12-21 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 12-20 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 13-20 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 14-20 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 15-20 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 12-19 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 13-19 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 14-19 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 15-19, nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 16-19 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 17-19 nucleobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleobases in length.
For oligonucleotides of various lengths, the position of the nucleoside complementary to the SNP position was shifted within the gap and the wings and the effect was tested. Certain positions within the antisense oligonucleotide are shown to be beneficial for targeting SNPs.
In certain embodiments, the antisense oligonucleotide is 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 nucleobases in length and the SNP is complementary to positions 6-15 counting from the 5′ terminus of the antisense oligonucleotide and/or positions 1-9 counting from the 5′ end of the gap. In certain embodiments, the antisense oligonucleotide is 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 nucleobases in length and the SNP is complementary to positions 8-14 counting from the 5′ terminus of the antisense oligonucleotide and/or positions 1-9 counting from the 5′ end of the gap.
In certain embodiments, the antisense oligonucleotide is 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 nucleobases in length and the SNP is complementary to positions 8-14 counting from the 5′ terminus of the antisense oligonucleotide and/or positions 4-7 counting from the 5′ end of the gap. In certain embodiments, the antisense oligonucleotide is 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 nucleobases in length and the SNP is complementary to positions 8-10 counting from the 5′ terminus of the antisense oligonucleotide and/or positions 4-6 counting from the 5′ end of the gap.
In certain embodiments, the SNP is complementary to position 8, 9, or 10 counting from the 5′ terminus of the oligonucleotide or position 4, 5, or 6, counting from the 5′ end of the gap. For oligonucleotides of various lengths, the effect of the length of the gap, 5′ wing, and 3′ wing was tested.
Certain wing-gap-wing combinations were shown to be beneficial for a SNP targeting antisense oligonucleotide. In certain embodiments the gap is 7-11 nucleobases in length and each wing is independently 1-6 nucleobases in length. In certain embodiments the gap is 7-11 nucleobases in length and each wing is independently 2-6 nucleobases in length. In certain embodiments the gap is 8-11 nucleobases in length and each wing is independently 2-6 nucleobases in length. In certain embodiments the gap is 9-11 nucleobases in length and each wing is independently 2-6 nucleobases in length. In certain embodiments the gap is 9 nucleobases in length and each wing is independently 2-6 nucleobases in length. In certain embodiments the gap is 10 nucleobases in length and each wing is independently 2-6 or 4-5 nucleobases in length. In certain embodiments the gap is 11 nucleobases in length and each wing is independently 2-6, or 4-5 nucleobases in length. In certain embodiments, the wing-gap-wing configuration is one of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
For oligonucleotides of various lengths, the effect of certain chemistries was tested. Certain chemistry modifications were shown to be beneficial for a SNP targeting antisense oligonucleotide. In certain embodiments, each nucleoside of each wing of the modified antisense oligonucleotide has a 2′-MOE modification. In certain embodiments, each nucleoside of each wing of the modified antisense oligonucleotide has a high affinity modification. In certain embodiments, the antisense oligonucleotide is a mixed wing gapmer. In such embodiment, the modifications and combination of modifications at the 3′ wing and at the 5′ wing may be the same or they may be different. In certain embodiments, the antisense oligonucleotide has one or more 2′-MOE modifications in the wings and/or one or more high affinity modifications in the wings. In certain embodiments, the high affinity modification is a cEt modification. In certain embodiments, the antisense oligonucleotide has a high affinity modification at positions 2, 3, 13, and 14 of the antisense oligonucleotide (counting from the 5′ terminus). In certain embodiments, the antisense oligonucleotide has one, two, three, or four high affinity modifications in at least one of the wings. In certain embodiments, the antisense oligonucleotide has one, two, three, or four high affinity modifications in each of the 5′ and 3′ wings independently. In certain embodiments, the antisense oligonucleotide has a high affinity modification at positions 2 and 3 in one or both of the 5′ and 3′ wings (counting from the 5′ terminus of the 5′ wing and the 3′ terminus of the 3′ wing). In certain embodiments, the antisense oligonucleotide has a high affinity modification at positions 2, 3 and 4 in one or both of the 5′ and 3′ wings (counting from the 5′ terminus of the 5′ wing and the 3′ terminus of the 3′ wing,). In certain embodiments, the antisense oligonucleotide has a high affinity modification at positions 1 of the 5′ and/or 3′ wings (counting from the 5′ terminus of the 5′ wing and the 3′ terminus of the 3′ wing,). In certain embodiments, the antisense oligonucleotide has a high affinity modification at positions 1 of the 5′ and 3′ wings (counting from the 5′ terminus of the 5′ wing and the 3′ terminus of the 3′ wing,) and at least one other position in the wing. In certain embodiments, the antisense oligonucleotide has alternating 2′-MOE and high affinity modification in at least one of the 5′ and 3′ wings.
In certain embodiments, the compound comprises an antisense oligonucleotide incorporating one or more of the design rules provided above.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 30 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 6-15 beginning from the 5′ terminus of the antisense oligonucleotide or positions 1-9 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments the single nucleotide polymorphism site contains a differentiating polymorphism. In certain embodiments, the single nucleotide polymorphism site is on a mutant allele. In certain embodiments, the mutant allele is associated with disease. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 20 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 6-15 beginning from the 5′ terminus of the antisense oligonucleotide or positions 1-9 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 20 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-14 beginning from the 5′ terminus of the antisense oligonucleotide or positions 1-9 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 20 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-14 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-7 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 20 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-10 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-6 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 12 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-10 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-6 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 13 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-10 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-6 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 14 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-10 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-6 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 6-15 beginning from the 5′ terminus of the antisense oligonucleotide or positions 1-9 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein the single nucleotide polymorphism aligns with any one of positions 8-10 beginning from the 5′ terminus of the antisense oligonucleotide or positions 4-6 beginning from the 5′ end of the gap of the modified antisense oligonucleotide; and wherein each nucleoside of each wing has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 4-7-4, 5-8-6, 6-8-5, 6-7-6, 5-7-5, 6-8-5, 5-8-6, 3-9-4, 4-9-3, 2-9-6, 6,9,2, 3-9-3, 3-9-5, 5-9-3, 5-9-4, 4-9-5, 5-9-5, 4-11-4, 4-10-5 and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisnese oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 8, 9, 10, 11, or 14 beginning from the 5′ terminus of the modified antisense oligonucleotide aligns with the single nucleotide polymorphism; and wherein each nucleoside of each wing segment modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 1, 4, 5, 6, 7, or 9 of the gap segment aligns with the single nucleotide polymorphism; and wherein each nucleoside of each wing segment has a modified sugar or sugar surrogate. In certain embodiments, the wing-gap-wing-motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 7, 8, 9, 10, 11, or 12 of the modified antisense oligonucleotide aligns with the single nucleotide polymorphism; and positions 2 and 3 of the 5′ and 3′ wing segments comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 3, 4, 5, 6, 7, 8 or 9 of the gap segment aligns with the single nucleotide polymorphism; and positions 2 and 3 of the 5′ and 3′ wing segments comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
A compound comprising a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 7, 8, 9, 10, 11, or 12 of the modified antisense oligonucleotide aligns with the single nucleotide polymorphism; and positions 2, 3, 13, and 14 of the antisense oligonucleotide comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
A compound comprising a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 3, 4, 5, 6, 7, 8, or 9 of the gap segment aligns with the single nucleotide polymorphism; and positions 2, 3, 13, and 14 of the antisense antisense oligonucleotide comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprise a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 8, 9, or 10 of the modified antisense oligonucleotide aligns with the single nucleotide polymorphism; and wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 5, 6, or 7 of the gap segment aligns with the single nucleotide polymorphism; and wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides, fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 8, 9, or 10 of the modified antisense oligonucleotide aligns with the single nucleotide polymorphism; and positions 2 and 3 of the 5′ and 3′ wing segments comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In certain embodiments, the compound comprises a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 5, 6, or 7 of the gap segment aligns with the single nucleotide polymorphism; and positions 2 and 3 of the 5′ and 3′ wing segments comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
A compound comprising a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 8, 9, or 10 of the modified oligonucleotide aligns with the single nucleotide polymorphism; and positions 2, 3, 13, and 14 of the antisense antisense oligonucleotide comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
A compound comprising a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides and fully complementary to a single nucleotide polymorphism site, wherein the modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 5, 6, or 7 of the gap segment aligns with the single nucleotide polymorphism; and positions 2, 3, 13, and 14 of the antisense oligonucleotide comprise a 4′-CH(CH3)—O-2′ bridge. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-9-5, 4-9-5, 4-11-4, and 5-10-4.
In a certain embodiment, the antisense oligonucleotide is 11 to 20 linked nucleosides in length and has, independently, 2 to 5 linked nucleosides in the 5′ and 3′ wings and 7 to 11 linked nucleosides in the gap. The SNP is complementary to position 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the antisense oligonucleotide (counting from the 5′ terminus of the antisense oligonucleotide) or position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 counting from the 5′ terminus of the gap segment.
In a certain embodiment, the antisense oligonucleotide is 15 to 19 linked nucleosides in length and has, independently, 2 to 5 linked nucleosides in the 5′ and 3′ wings and 7 to 11 linked nucleosides in the gap. The SNP is complementary to position 6, 7, 8, 9, or 10 of the antisense oligonucleotide (counting from the 5′ terminus of the antisense oligonucleotide) or position 4, 5, 6, or 7 counting from the 5′ terminus of the gap segment.
In a certain embodiment, the antisense oligonucleotide is 17 linked nucleosides in length and has, independently, 2 to 5 linked nucleosides in the 5′ and 3′ wing segments and 9 to 11 linked nucleosides in the gap segment. The SNP is complementary to position 8, 9, or 10 of the antisense oligonucleotide (counting from the 5′ terminus of the antisense oligonucleotide) or position 5, 6, or 7 (counting from the 5′ terminus of the gap segment).
In a certain embodiment, the antisense oligonucleotide is 18 linked nucleosides in length and has, independently, 2 to 5 linked nucleosides in the 5′ and 3′ wing segments and 9 to 11 linked nucleosides in the gap segment. The SNP is complementary to position 8, 9, or 10 of the antisense oligonucleotide (counting from the 5′ terminus of the antisense oligonucleotide) or position 5, 6, or 7 (counting from the 5′ terminus of the gap segment).
In a certain embodiment, the antisense oligonucleotide is 19 linked nucleosides in length and has, independently, 2 to 5 linked nucleosides in the 5′ and 3′ wing segments and 9 to 11 linked nucleosides in the gap segment. The SNP is complementary to position 8, 9, or 10 of the antisense oligonucleotide (counting from the 5′ terminus of the antisense oligonucleotide) or position 5, 6, or 7 (counting from the 5′ terminus of the gap segment).
In certain, the antisense oligonucleotide is any of those listed in the examples. In certain embodiments, they are designated by sequence and chemistry as reflected by the ISIS number. In certain embodiments, the antisense oligonucleotide is any of those listed in the examples as having at least, about, greater than about or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% inhibition of a mutant Huntingtin allele.
In certain embodiments, the antisense oligonucleotide is any of those listed in the examples as selectively inhibiting the expression of the mutant huntingtin allele at least, about, greater than about or greater than 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold over the expression of a wild-type huntingtin allele or by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% compared to expression of a wild-type huntingtin allele.
In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions described herein. In certain embodiments, the individual has an allelic variant associated with a disease or disorder. The pharmaceutical compositions provided herein preferentially target a SNP. In certain embodiments, the SNP is a differentiating polymorphism.
Methods have been described for determining whether a SNP is specific to a disease associated allele and more specifically whether a SNP variant of an allele of a heterozygous patient is on the same allele as a disease-causing mutation that is at a remote region of the gene's mRNA (WO 2008/147930 and WO 2008/143774).
In certain embodiments, the disease is a neurodegenerative disorder. In certain embodiments, the neurodegenerative disorder is Huntington's Disease. In certain embodiments, the targeted SNP is one or more of: rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325, rs35892913, rs363102, rs363096, rs11731237, rs10015979, rs363080, rs2798235, rs1936032, rs2276881, rs363070, rs35892913, rs12502045, rs6446723, rs7685686, rs3733217, rs6844859, rs362331, rs1143646, rs2285086, rs2298969, rs4690072, rs916171, rs3025849, rs7691627, rs4690073, rs3856973, rs363092, rs362310, rs362325, rs363144, rs362303, rs34315806, rs363099, rs363081, rs3775061, rs2024115, rs10488840, rs363125, rs362296, rs2298967, rs363088, rs363064, rs362275, rs3121419, rs3025849, rs363070, rs362273, rs362272, rs362306, rs362271, rs363072, rs16843804, rs7659144, rs363120, and rs12502045. In certain embodiments the compounds are ISIS460065, ISIS 459978, ISIS 460028, ISIS 460209, ISIS 460208, and ISIS 460206.
In certain embodiments, described herein, are methods of selectively reducing the expression of a mutant huntingtin allele in a cell, tissue or animal by administering to the cell, tissue, or animal a compound as described herein. In certain embodiments, the compound comprises a modified oligonucleotide complementary to the mutant huntingtin allele. In certain embodiments, the modified oligonucleotide is complementary to the mutant huntingtin allele at a position comprising a single nucleotide polymorphism described herein. In certain embodiments, the single nucleotide polymorphism is selected from those described herein. In certain embodiments, the single nucleotide polymorphism is selected from rs362306, rs362331, rs2298969, rs7685686, rs4690072, rs2024115 or rs363088.
In certain embodiments, described herein, are methods of treating, ameliorating or slowing the onset or progression of Huntington's Disease by administering to an animal a compound described herein. In certain embodiment the compound comprises a modified oligonucleotide complementary to a mutant huntingtin allele at a position on the allele comprising a single nucleotide polymorphism described herein. In certain embodiments, the single nucleotide polymorphism is selected from rs362306, rs362331, rs2298969, rs7685686, rs4690072, rs2024115 or rs363088. In certain embodiments, the compound administered to the animal treats, ameliorates or slows the onset or progression Huntington's Disease by selectively reducing the mutant huntingtin allele.
In certain embodiments the expression of the mutant huntingtin allele is selectively reduced at least, about or greater than 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold over the expression of a wild-type huntingtin allele or by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% compared to expression of a wild-type huntingtin allele.
In certain embodiments, one or more additional compounds comprising a modified oligonucleotide complementary to a mutant huntingtin allele at a position comprising a single nucleotide polymorphism site selected from rs362306, rs362331, rs2298969, rs7685686, rs4690072, rs2024115 or rs363088 is administered.
In certain embodiments, the modified oligonucleotide has a specific length, gap, wing, chemistry and SNP position alignment as further described herein. In certain embodiments, the modified oligonucleotide consists of 15 to 20 linked nucleosides and position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the single nucleotide polymorphism.
In certain embodiments, the modified oligonucleotide is 90% complementary to the mutant huntingtin allele.
The method of claim 40, wherein the modified oligonucleotide is 95% complementary to the mutant huntingtin allele.
The method of claim 40, wherein the modified oligonucleotide is 100% complementary to the mutant huntingtin allele.
In certain embodiments, described herein are methods for determining whether an agent selectively inhibits expression of a first allelic variant of a gene relative to a second allelic variant of the gene wherein the first allelic variant comprises a first nucleotide at a SNP position and the second allelic variant comprises a second nucleotide at the SNP position, comprising:

    (i) contacting a first cell, tissue or animal with the agent at one or more concentrations, wherein the first cell, tissue or animal is homozygous for the first nucleotide at the SNP position;
    (ii) contacting a second cell, tissue or animal with the agent at one or more concentrations, wherein the second cell line is homozygous for the second nucleotide at the SNP position or is heterozygous for the first and the second nucleotides at the SNP position;
    (iii) measuring inhibition of expression of the allelic variant in each of the cell, tissue or animal for each of the one or more concentrations of the agent; and
    (iv) comparing the inhibition of expression in each of the cell, tissue or animal for each of the one or more concentrations of the agent to determine whether the agent selectively inhibits expression of the first allelic variant of the gene relative to the second allelic variant of the gene.


In certain embodiments, the methods described herein further comprise the step of contacting a third cell, tissue or animal with the agent at one or more concentrations, wherein the third cell, tissue or animal is homozygous for the second nucleotide at the SNP position or is heterozygous for the first and the second nucleotides at the SNP position and has a different genotype than the first cell, tissue or animal and the second cell, tissue or animal.

    
    
        In certain embodiments, the cell, tissue or animal is a a YAC18 cell, tissue or mouse, a BACHD cell, tissue or mouse, a cell line derived from a human Huntington's Disease patient or any combination thereof.
    
    


In certain embodiments, the first allelic variant is a mutant allele and the second allelic variant is a wild-type allele. In certain embodiments, the first allelic variant is a wild-type allele and the second allelic variant is a mutant allele.
In certain embodiments, the mutant allelic variant is associated with disease. In certain embodiments, the disease is the result of a gain of toxic function. In certain embodiments, the disease is Huntington's Disease.
In certain embodiments, the first nucleotide is in linkage disequilibrium with a disease associated mutation. In certain embodiments, the second nucleotide is in linkage disequilibrium with a disease associated mutation. In certain embodiments, the disease associated mutation is a tri-nucleotide repeat expansion. In certain embodiments, the tri-nucleotide repeat expansion is a CAG expansion. In certain embodiments, the CAG expansion is in a HTT gene.
In certain embodiments, the first nucleotide is A and the second nucleotide is any of C, G, or T. In certain embodiments, the first nucleotide is C and the second nucleotide is any of A, G, or T. In certain embodiments, the first nucleotide is G and the second nucleotide is any of A, C, or T. In certain embodiments, the first nucleotide is T and the second nucleotide is any of A, C, or G. In certain embodiments, the first nucleotide is in linkage disequilibrium with a disease associated mutation. In certain embodiments, the disease associated mutation is a CAG expansion. In certain embodiments, the CAG expansion is in a HTT gene.
In certain embodiments, the agent is an antisense compound. In certain embodiments, the antisense compound is an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide is a gapmer. In certain embodiments, the gapmer has a wing-gap-wing motif. In certain embodiments, the wing-gap-wing motif is any one of the group consisting of 5-10-5, 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4-9-3, 4-9-4, 4-9-5, 4-10-5, 4-11-4, 4-11-5, 5-7-5, 5-8-6, 5-9-3, 5-9-5, 5-10-4, 5-10-5, 6-7-6, 6-8-5, and 6-9-2. 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 nucleobase. In certain embodiments, the modified nucleobase is a 5% methylcytosine. In certain embodiments, at least one nucleoside of at least one of the wing regions comprises a modified sugar or sugar surrogate. In certain embodiments, each of the nucleosides of each wing region comprises a modified sugar or sugar surrogate. In certain embodiments, the sugar or sugar surrogate is a 2′-O-methoxyethyl modified sugar. In certain embodiments, at least one of the wing regions comprises a 4′ to 2′ bicyclic nucleoside and at least one of the remaining wing nucleosides is a non-bicyclic 2′-modified nucleoside. In certain embodiments, the non-bicyclic 2′-modified nucleoside is a 2′-O-methoxyethyl nucleoside. In certain embodiments, the 4′ to 2′ bicyclic nucleoside is 4′-CH(CH3)—O-2′ bicyclic nucleoside.
In certain embodiments, the first cell line is any of the group consisting of fibroblast cell line, neuronal cell line, or lymphoblast cell line. In certain embodiments, the second cell line is any of the group consisting of fibroblast cell line, neuronal cell line, or lymphoblast cell line. In certain embodiments, the third cell line is any of the group consisting of fibroblast cell line, neuronal cell line, or lymphoblast cell line. In certain embodiments, the fibroblast cell line is any of the group consisting of GM04281, GM02171, and GM02173B. In certain embodiments, the neuronal cell line comprises a cell line derived from a YAC18 mouse, a cell line derived from a BACHD mouse, or a cell line derived from a human Huntington's Disease patient.
In certain embodiments, the SNP position is any of the group consisting of rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325, rs35892913, rs363102, rs363096, rs11731237, rs10015979, rs363080, rs2798235, rs1936032, rs2276881, rs363070, rs35892913, rs12502045, rs6446723, rs7685686, rs3733217, rs6844859, rs362331, rs1143646, rs2285086, rs2298969, rs4690072, rs916171, rs3025849, rs7691627, rs4690073, rs3856973, rs363092, rs362310, rs362325, rs363144, rs362303, rs34315806, rs363099, rs363081, rs3775061, rs2024115, rs10488840, rs363125, rs362296, rs2298967, rs363088, rs363064, rs362275, rs3121419, rs3025849, rs363070, rs362273, rs362272, rs362306, rs362271, rs363072, rs16843804, rs7659144, rs363120, and rs12502045.
In certain embodiments, the one or more concentration is any of the group consisting of two concentrations, three concentrations, four concentrations, and five concentrations.
In certain embodiments, selectivity is determined by increased inhibition of the allelic variant in one cell line as compared to another cell line.
In certain embodiments, described herein are methods for determining whether an agent selectively inhibits expression of a first allelic variant of a gene relative to a second allelic variant of the gene wherein the first allelic variant comprises a first nucleotide at a SNP position and the second allelic variant comprises a second nucleotide at the SNP position, comprising:

    (i) contacting a first cell line with the agent at one or more concentrations, wherein the first cell line is homozygous for the first nucleotide at the SNP position;
    (ii) contacting a second cell line with the agent at one or more concentrations, wherein the second cell line is homozygous for the second nucleotide at the SNP position or is heterozygous for the first and the second nucleotides at the SNP position;
    (iii) measuring inhibition of expression of the allelic variant in each of the cell lines for each of the one or more concentrations of the agent; and
    (iv) comparing the inhibition of expression in each of the cell lines for each of the one or more concentrations of the agent to determine whether the agent selectively inhibits expression of the first allelic variant of the gene relative to the second allelic variant of the gene.
    (v) calculating IC50 values for the agent in each of the first cell line, the second cell line, and the third cell line; and
    (vi) comparing the IC50 values to determine whether the agent selectively inhibits expression of the first allelic variant or mutant allele.


In certain embodiments, the huntingtin SNP targeting oligonucleotides provided herein are selective for the mutant allele and demonstrate a potency of within at least 10-fold, at least 9-fold, at least 8-fold, at least 7-fold, at least 6-fold, at least 5-fold, at least 4-fold, at least 3-fold or at least 2-fold of the potency of the benchmark PAN ASO, ISIS 387916. In certain embodiments, the SNP targeting oligonucleotides provided herein have an IC50 in a cell line homozygous for the mutant allele of no more than 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.75. In certain embodiments, the SNP targeting oligonucleotides provided herein have an IC50 in a cell line that is homozygous for the wild-type allele that is at least 2 fold, 3 fold, 4 fold or 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold less than the IC50 in a cell line homozygous for the mutant allele. In certain embodiments, the SNP targeting oligonucleotides provided herein have an IC50 in a cell line that is heterozygous for the mutant and wild-type allele that is at least 1.4 fold, 1.7 fold, 2 fold, 2.2, 2.4, 2.5, 2.6, 2.8 or 3 fold less than the IC50 in a cell line homozygous for the mutant allele. In certain embodiments, the SNP targeting oligonucleotides include or are selected from ISIS Nos 435869, 460069, 460206, 460019, 460071, 460207, 460212, 460218, 460231, 474892, 460012, 460208, 435879, 460065, 460085, 460209, 474871, 474891, 474919, 474923, 476333, 476337, 435890, 460026, 460210, 463571, 476444.
Therapeutically Effective Dosages
In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to the mutant huntingtin allele is accompanied by monitoring of expression of a gene product in an individual, to determine an individual's response to administration of the antisense compound. In certain embodiments, the gene product is huntingtin mRNA or protein. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
In certain embodiments, administration of an antisense compound targeted to a mutant nucleic acid results in reduction of mRNA or protein expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, the mutant nucleic acid is huntingtin nucleic acid, the mRNA is huntingtin mRNA, and the protein is huntingtin protein.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to a mutant allele are used for the preparation of a medicament for treating a patient suffering or susceptible to Huntington's Disease.
Certain Combination Therapies
In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a synergistic effect.
In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
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 patents, applications, printed publications, and other published documents mentioned or referred to in this specification are herein incorporated by reference in their entirety.
Example 1
Single Nucleotide Polymorphisms (SNPs) in the Huntingtin (HTT) Gene Sequence
The HTT genomic sequence, designated herein as SEQ ID NO: 1 (NT—006081.18 truncated from nucleotides 1566000 to 1768000) was aligned with the HTT mRNA, designated herein as SEQ ID NO: 2 (NM—002111.6), using the EMBL-EBI sequence database (ClustalW2, http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the output is presented in FIG. 1. SNP positions (identified by Hayden et al, WO/2009/135322) associated with the HTT gene were mapped to the two sequences and have been demarcated in FIG. 1 by their reference SNP ID number from the Entrez SNP database at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp), incorporated herein by reference. Table 2 furnishes further details on each SNP. The ‘Reference SNP ID number’ or ‘RS number’ is the number designated to each SNP from the Entrez SNP database at NCBI, incorporated herein by reference. ‘SNP position’ refers to the nucleotide position of the SNP on SEQ ID NO: 1. ‘Polymorphism’ indicates the nucleotide variants at that SNP position. ‘Major allele’ indicates the nucleotide associated with the major allele, or the nucleotide present in a statistically significant proportion of individuals in the human population. ‘Minor allele’ indicates the nucleotide associated with the minor allele, or the nucleotide present in a relatively small proportion of individuals in the human population.







TABLE 2







Single Nuclear Polymorphisms (SNPs)


and their positions on SEQ ID NO: 1












SNP

Major
Minor


RS No.
position
Polymorphism
allele
allele














rs2857936
1963
C/T
C
T


rs12506200
3707
A/G
G
A


rs762855
14449
A/G
G
A


rs3856973
19826
G/A
G
A


rs2285086
28912
G/A
A
G


rs7659144
37974
C/G
C
G


rs16843804
44043
C/T
C
T


rs2024115
44221
G/A
A
G


rs10015979
49095
A/G
A
G


rs7691627
51063
A/G
G
A


rs2798235
54485
G/A
G
A


rs4690072
62160
G/T
T
G


rs6446723
66466
C/T
T
C


rs363081
73280
G/A
G
A


rs363080
73564
T/C
C
T


rs363075
77327
G/A
G
A


rs363064
81063
T/C
C
T


rs3025849
83420
A/G
A
G


rs6855981
87929
A/G
G
A


rs363102
88669
G/A
A
G


rs11731237
91466
C/T
C
T


rs4690073
99803
A/G
G
A


rs363144
100948
T/G
T
G


rs3025838
101099
C/T
C
T


rs34315806
101687
A/G
G
A


rs363099
101709
T/C
C
T


rs363096
119674
T/C
T
C


rs2298967
125400
C/T
T
C


rs2298969
125897
A/G
G
A


rs6844859
130139
C/T
T
C


rs363092
135682
C/A
C
A


rs7685686
146795
A/G
A
G


rs363088
149983
A/T
A
T


rs362331
155488
C/T
T
C


rs916171
156468
G/C
C
G


rs362322
161018
A/G
A
G


rs362275
164255
T/C
C
T


rs362273
167080
A/G
A
G


rs2276881
171314
G/A
G
A


rs3121419
171910
T/C
C
T


rs362272
174633
G/A
G
A


rs362271
175171
G/A
G
A


rs3775061
178407
C/T
C
T


rs362310
179429
A/G
G
A


rs362307
181498
T/C
C
T


rs362306
181753
G/A
G
A


rs362303
181960
T/C
C
T


rs362296
186660
C/A
C
A


rs1006798
198026
A/G
A
G









Example 2
Design of Antisense Oligonucleotides Targeting Huntingtin Gene SNPs and Inhibition of HTT mRNA in Coriell Fibroblast Cell Lines (GM04281, GM02171, and GM02173B)
Antisense oligonucleotides targeting nucleotides overlapping SNP positions presented in Table 1 were designed and tested for potency in three huntingtin patient-derived Coriell fibroblast cell lines, GM04281, GM02171, and GM02173B (from the Coriell Institute for Medical Research). Cultured GM04281 cells or GM02171 cells or GM02173B cells at a density of 20,000 cells per well were transfected using electroporation with 10,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real time PCR using primer probe set RTS2617 (forward sequence CTCCGTCCGGTAGACATGCT, designated herein as SEQ ID NO: 3; reverse sequence GGAAATCAGAACCCTCAAAATGG, designated herein as SEQ ID NO: 4; probe sequence TGAGCACTGTTCAACTGTGGATATCGGGAX, designated herein as SEQ ID NO: 5). HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells.
ISIS 387916 (TCTCTATTGCACATTCCAAG, 5-10-5 MOE (SEQ ID NO: 6)) and ISIS 388816 (GCCGTAGCCTGGGACCCGCC, 5-10-5 MOE (SEQ ID NO: 7)) were included in each study as benchmark oligonucleotides against which the potency of the antisense oligonucleotides targeting nucleotides overlapping each SNP position could be compared.
The chimeric antisense oligonucleotides in Tables 3 and 4 were designed as 5-9-5 MOE gapmers. The gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methylcytosines.
The oligonucleotides are further described in Table 3. The percent inhibition of HTT mRNA by the antisense oligonucleotides in each cell line is shown in Table 4. ‘Target allele’ indicates whether the gapmer is targeted to the major or the minor allele at the SNP position. The number in parentheses indicates the nucleotide position in the gapmer opposite to the SNP position, starting from the 5′-terminus of the oligonucleotide. ‘Start site’ indicates the 5′-most nucleotide to which the gapmer is targeted. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Tables 3 and 4 is targeted to human HTT pre-mRNA, which is SEQ ID NO: 1.







TABLE 3







Chimeric oligonucleotides targeting SNP positions on the HTT gene



















SEQ


ISIS
SNP RS
Target

Start
Stop
ID


No
No.
allele
Sequence
Site
Site
NO
















387916
n/a
n/a
TCTCTATTGCACATTCCAAG
145466
145485
6


 


388816
n/a
n/a
GCCGTAGCCTGGGACCCGCC
16501
16520
7


 


435330
rs3856973
Major (8)
TAACACTCGATTAACCCTG
19815
19833
8


 


435348
rs3856973
Minor (8)
TAACACTTGATTAACCCTG
19815
19833
9


 


435294
rs3856973
Major (10)
GTTAACACTCGATTAACCC
19817
19835
10


 


435312
rs3856973
Minor (10)
GTTAACACTTGATTAACCC
19817
19835
11


 


435864
rs2285086
Major (10)
GCTAGTTCATCCCAGTGAG
28903
28921
12


 


435889
rs2285086
Minor (10)
GCTAGTTCACCCCAGTGAG
28903
28921
13


 


435878
rs7659144
Major (10)
TGGAAATGGGTTTTTCCAC
37965
37983
14


 


435903
rs7659144
Minor (10)
TGGAAATGGCTTTTTCCAC
37965
37983
15


 


435863
rs16843804
Major (10)
TTTAACCGTGGCATGGGCA
44034
44052
16


 


435888
rs16843804
Minor (10)
TTTAACCGTAGCATGGGCA
44034
44052
17


 


435331
rs2024115
Major (8)
TTCAAGCTAGTAACGATGC
44210
44228
18


 


435349
rs2024115
Minor (8)
TTCAAGCCAGTAACGATGC
44210
44228
19


 


435295
rs2024115
Major (10)
ACTTCAAGCTAGTAACGAT
44212
44230
20


 


435313
rs2024115
Minor (10)
ACTTCAAGCCAGTAACGAT
44212
44230
21


 


435862
rs10015979
Major (10)
GCAGCTAGGTTAAAGAGTC
49086
49104
22


 


435887
rs10015979
Minor (10)
GCAGCTAGGCTAAAGAGTC
49086
49104
23


 


435880
rs7691627
Major (10)
AATAAGAAACACAATCAAA
51054
51072
24


 


435905
rs7691627
Minor (10)
AATAAGAAATACAATCAAA
51054
51072
25


 


435885
rs2798235
Major (10)
CAGAGGAGGCATACTGTAT
54476
54494
26


 


435910
rs2798235
Minor (10)
CAGAGGAGGTATACTGTAT
54476
54494
27


 


435874
rs4690072
Major (10)
CACAGTGCTACCCAACCTT
62151
62169
28


 


435899
rs4690072
Minor (10)
CACAGTGCTCCCCAACCTT
62151
62169
29


 


435875
rs6446723
Major (10)
TAATTTTCTAGACTTTATG
66457
66475
30


 


435900
rs6446723
Minor (10)
TAATTTTCTGGACTTTATG
66457
66475
31


 


435332
rs363081
Major (8)
GCTACAACGCAGGTCAAAT
73269
73287
32


 


435350
rs363081
Minor (8)
GCTACAATGCAGGTCAAAT
73269
73287
33


 


435296
rs363081
Major (10)
GAGCTACAACGCAGGTCAA
73271
73289
34


 


435314
rs363081
Minor (10)
GAGCTACAATGCAGGTCAA
73271
73289
35


 


435886
rs363080
Major (10)
AGAGAGAACGAGAAGGCTC
73555
73573
36


 


435911
rs363080
Minor (10)
AGAGAGAACAAGAAGGCTC
73555
73573
37


 


435914
rs363075
Major (6)
AGCCCCTCTGTGTAAGTTT
77314
77332
38


 


435926
rs363075
Minor (6)
AGCCCTTCTGTGTAAGTTT
77314
77332
39


 


435916
rs363075
Major (7)
GAGCCCCTCTGTGTAAGTT
77315
77333
40


 


435928
rs363075
Minor (7)
GAGCCCTTCTGTGTAAGTT
77315
77333
41


 


435333
rs363075
Major (8)
TGAGCCCCTCTGTGTAAGT
77316
77334
42


 


435351
rs363075
Minor (8)
TGAGCCCTTCTGTGTAAGT
77316
77334
43


 


435918
rs363075
Major (9)
ATGAGCCCCTCTGTGTAAG
77317
77335
44


 


435930
rs363075
Minor (9)
ATGAGCCCTTCTGTGTAAG
77317
77335
45


 


435297
rs363075
Major (10)
GATGAGCCCCTCTGTGTAA
77318
77336
46


 


435315
rs363075
Minor (10)
GATGAGCCCTTCTGTGTAA
77318
77336
47


 


435920
rs363075
Major (11)
TGATGAGCCCCTCTGTGTA
77319
77337
48


 


435932
rs363075
Minor (11)
TGATGAGCCCTTCTGTGTA
77319
77337
49


 


435366
rs363075
Major (12)
ATGATGAGCCCCTCTGTGT
77320
77338
50


 


435924
rs363075
Minor (12)
ATGATGAGCCCTTCTGTGT
77320
77338
51


 


435922
rs363075
Major (14)
TAATGATGAGCCCCTCTGT
77322
77340
52


 


435934
rs363075
Minor (14)
TAATGATGAGCCCTTCTGT
77322
77340
53


 


435334
rs363064
Major (8)
AGAATACGGGTAACATTTT
81052
81070
54


 


435352
rs363064
Minor (8)
AGAATACAGGTAACATTTT
81052
81070
55


 


435298
rs363064
Major (10)
GGAGAATACGGGTAACATT
81054
81072
56


 


435316
rs363064
Minor (10)
GGAGAATACAGGTAACATT
81054
81072
57


 


435335
rs3025849
Major (8)
TTAGTAATCAATTTTAATG
83409
83427
58


 


435353
rs3025849
Minor (8)
TTAGTAACCAATTTTAATG
83409
83427
59


 


435299
rs3025849
Major (10)
AGTTAGTAATCAATTTTAA
83411
83429
60


 


435317
rs3025849
Minor (10)
AGTTAGTAACCAATTTTAA
83411
83429
61


 


435877
rs6855981
Major (10)
GAAGGAATGCTTTTACTAG
87920
87938
62


 


435902
rs6855981
Minor (10)
GAAGGAATGTTTTTACTAG
87920
87938
63


 


435336
rs363102
Major (8)
CTAAAACTAACTTGAGAAT
88658
88676
64


 


435354
rs363102
Minor (8)
CTAAAACCAACTTGAGAAT
88658
88676
65


 


435300
rs363102
Major (10)
ATCTAAAACTAACTTGAGA
88660
88678
66


 


435318
rs363102
Minor (10)
ATCTAAAACCAACTTGAGA
88660
88678
67


 


435884
rs11731237
Major (10)
GGTGGGCAGGAAGGACTGA
91457
91475
68


 


435909
rs11731237
Minor (10)
GGTGGGCAGAAAGGACTGA
91457
91475
69


 


435337
rs4690073
Major (8)
CCTAAATCAATCTACAAGT
99792
99810
70


 


435355
rs4690073
Minor (8)
CCTAAATTAATCTACAAGT
99792
99810
71


 


435301
rs4690073
Major (10)
TCCCTAAATCAATCTACAA
99794
99812
72


 


435319
rs4690073
Minor (10)
TCCCTAAATTAATCTACAA
99794
99812
73


 


435883
rs363144
Major (10)
GAAAATGTGAGTGGATCTA
100939
100957
74


 


435908
rs363144
Minor (10)
GAAAATGTGCGTGGATCTA
100939
100957
75


 


435338
rs3025838
Major (8)
GTAAGGCGAGACTGACTAG
101088
101106
76


 


435356
rs3025838
Minor (8)
GTAAGGCAAGACTGACTAG
101088
101106
77


 


435302
rs3025838
Major (10)
AGGTAAGGCGAGACTGACT
101090
101108
78


 


435320
rs3025838
Minor (10)
AGGTAAGGCAAGACTGACT
101090
101108
79


 


435339
rs363099
Major (8)
CTGAGCGGAGAAACCCTCC
101698
101716
80


 


435357
rs363099
Minor (8)
CTGAGCGAAGAAACCCTCC
101698
101716
81


 


435303
rs363099
Major (10)
GGCTGAGCGGAGAAACCCT
101700
101718
82


 


435321
rs363099
Minor (10)
GGCTGAGCGAAGAAACCCT
101700
101718
83


 


435367
rs363099
Major (12)
AAGGCTGAGCGGAGAAACC
101702
101720
84


 


435340
rs363096
Major (8)
TTCCCTAAAAACAAAAACA
119663
119681
85


 


435358
rs363096
Minor (8)
TTCCCTAGAAACAAAAACA
119663
119681
86


 


435304
rs363096
Major (10)
GATTCCCTAAAAACAAAAA
119665
119683
87


 


435322
rs363096
Minor (10)
GATTCCCTAGAAACAAAAA
119665
119683
88


 


435341
rs2298967
Major (8)
CTTTTCTATTGTCTGTCCC
125389
125407
89


 


435359
rs2298967
Minor (8)
CTTTTCTGTTGTCTGTCCC
125389
125407
90


 


435305
rs2298967
Major (10)
TGCTTTTCTATTGTCTGTC
125391
125409
91


 


435323
rs2298967
Minor (10)
TGCTTTTCTGTTGTCTGTC
125391
125409
92


 


435865
rs2298969
Major (10)
AAGGGATGCCGACTTGGGC
125888
125906
93


 


435890
rs2298969
Minor (10)
AAGGGATGCTGACTTGGGC
125888
125906
94


 


435876
rs6844859
Major (10)
ACCTTCCTCACTGAGGATG
130130
130148
95


 


435901
rs6844859
Minor (10)
ACCTTCCTCGCTGAGGATG
130130
130148
96


 


435872
rs363092
Major (10)
CAAACCACTGTGGGATGAA
135673
135691
97


 


435897
rs363092
Minor (10)
CAAACCACTTTGGGATGAA
135673
135691
98


 


435879
rs7685686
Major (10)
AATAAATTGTCATCACCAG
146786
146804
99


 


435904
rs7685686
Minor (10)
AATAAATTGCCATCACCAG
146786
146804
100


 


435871
rs363088
Major (10)
TCACAGCTATCTTCTCATC
149974
149992
101


 


435896
rs363088
Minor (10)
TCACAGCTAACTTCTCATC
149974
149992
102


 


435870
rs362331
Major (10)
GCACACAGTAGATGAGGGA
155479
155497
103


 


435895
rs362331
Minor (10)
GCACACAGTGGATGAGGGA
155479
155497
104


 


435881
rs916171
Major (10)
CAGAACAAAGAGAAGAATT
156459
156477
105


 


435906
rs916171
Minor (10)
CAGAACAAACAGAAGAATT
156459
156477
106


 


435342
rs362322
Major (8)
GCTTACATGCCTTCAGTGA
161007
161025
107


 


435360
rs362322
Minor (8)
GCTTACACGCCTTCAGTGA
161007
161025
108


 


435306
rs362322
Major (10)
CAGCTTACATGCCTTCAGT
161009
161027
109


 


435324
rs362322
Minor (10)
CAGCTTACACGCCTTCAGT
161009
161027
110


 


435868
rs362275
Major (10)
AAGAAGCCTGATAAAATCT
164246
164264
111


 


435893
rs362275
Minor (10)
AAGAAGCCTAATAAAATCT
164246
164264
112


 


435343
rs2276881
Major (8)
CATACATCAGCTCAAACTG
171303
171321
113


 


435361
rs2276881
Minor (8)
CATACATTAGCTCAAACTG
171303
171321
114


 


435307
rs2276881
Major (10)
CACATACATCAGCTCAAAC
171305
171323
115


 


435325
rs2276881
Minor (10)
CACATACATTAGCTCAAAC
171305
171323
116


 


435368
rs2276881
Major (12)
GTCACATACATCAGCTCAA
171307
171325
117


 


435866
rs3121419
Major (10)
GAGACTATAGCACCCAGAT
171901
171919
118


 


435891
rs3121419
Minor (10)
GAGACTATAACACCCAGAT
171901
171919
119


 


435344
rs362272
Major (8)
TAGAGGACGCCGTGCAGGG
174622
174640
120


 


435362
rs362272
Minor (8)
TAGAGGATGCCGTGCAGGG
174622
174640
121


 


435308
rs362272
Major (10)
CATAGAGGACGCCGTGCAG
174624
174642
122


 


435326
rs362272
Minor (10)
CATAGAGGATGCCGTGCAG
174624
174642
123


 


435369
rs362272
Major (12)
CACATAGAGGACGCCGTGC
174626
174644
124


 


435867
rs362271
Major (10)
ACGTGTGTACAGAACCTGC
175162
175180
125


 


435892
rs362271
Minor (10)
ACGTGTGTATAGAACCTGC
175162
175180
126


 


435873
rs3775061
Major (10)
TGTTCAGAATGCCTCATCT
178398
178416
127


 


435898
rs3775061
Minor (10)
TGTTCAGAACGCCTCATCT
178398
178416
128


 


435345
rs362310
Major (8)
AAACGGCGCAGCGGGAAGG
179418
179436
129


 


435363
rs362310
Minor (8)
AAACGGCACAGCGGGAAGG
179418
179436
130


 


435309
rs362310
Major (10)
AGAAACGGCGCAGCGGGAA
179420
179438
131


 


435327
rs362310
Minor (10)
AGAAACGGCACAGCGGGAA
179420
179438
132


 


435915
rs362307
Major (6)
AGGGCGCAGACTTCCAAAG
181485
181503
133


 


435927
rs362307
Minor (6)
AGGGCACAGACTTCCAAAG
181485
181503
134


 


435917
rs362307
Major (7)
AAGGGCGCAGACTTCCAAA
181486
181504
135


 


435929
rs362307
Minor (7)
AAGGGCACAGACTTCCAAA
181486
181504
136


 


435346
rs362307
Major (8)
CAAGGGCGCAGACTTCCAA
181487
181505
137


 


435364
rs362307
Minor (8)
CAAGGGCACAGACTTCCAA
181487
181505
138


 


435919
rs362307
Major (9)
ACAAGGGCGCAGACTTCCA
181488
181506
139


 


435931
rs362307
Minor (9)
ACAAGGGCACAGACTTCCA
181488
181506
140


 


435310
rs362307
Major (10)
CACAAGGGCGCAGACTTCC
181489
181507
141


 


435328
rs362307
Minor (10)
CACAAGGGCACAGACTTCC
181489
181507
142


 


435921
rs362307
Major (11)
GCACAAGGGCGCAGACTTC
181490
181508
143


 


435933
rs362307
Minor (11)
GCACAAGGGCACAGACTTC
181490
181508
144


 


435370
rs362307
Major (12)
GGCACAAGGGCGCAGACTT
181491
181509
145


 


435925
rs362307
Minor (12)
GGCACAAGGGCACAGACTT
181491
181509
146


 


435923
rs362307
Major (14)
AGGGCACAAGGGCGCAGAC
181493
181511
147


 


435935
rs362307
Minor (14)
AGGGCACAAGGGCACAGAC
181493
181511
148


 


435869
rs362306
Major (10)
GAGCAGCTGCAACCTGGCA
181744
181762
149


 


435894
rs362306
Minor (10)
GAGCAGCTGTAACCTGGCA
181744
181762
150


 


435347
rs362303
Major (8)
TGGTGCCGGGTGTCTAGCA
181949
181967
151


 


435365
rs362303
Minor (8)
TGGTGCCAGGTGTCTAGCA
181949
181967
152


 


435311
rs362303
Major (10)
AATGGTGCCGGGTGTCTAG
181951
181969
153


 


435329
rs362303
Minor (10)
AATGGTGCCAGGTGTCTAG
181951
181969
154


 


435882
rs362296
Major (10)
GGGGACAGGGTGTGCTCTC
186651
186669
155


 


435907
rs362296
Minor (10)
GGGGACAGGTTGTGCTCTC
186651
186669
156
















TABLE 4







Comparison of inhibition of HTT mRNA levels by ISIS 387916 and ISIS 388816


with that by chimeric oligonucleotides targeting SNP positions on the HTT gene


(SEQ ID NO: 1)












SNP RS
Target
% inhibition
SEQ













ISIS No
No.
allele
GM04281
GM02171
GM02173B
ID NO
















387916
n/a
n/a
96
96
98
6


388816
n/a
n/a
76
88
85
7


435330
rs3856973
Major (8)
64
51
36
8


435348
rs3856973
Minor (8)
50
88
80
9


435294
rs3856973
Major (10)
54
46
54
10


435312
rs3856973
Minor (10)
20
82
58
11


435864
rs2285086
Major (10)
54
28
26
12


435889
rs2285086
Minor (10)
17
43
41
13


435878
rs7659144
Major (10)
43
32
39
14


435903
rs7659144
Minor (10)
16
37
29
15


435863
rs16843804
Major (10)
63
78
81
16


435888
rs16843804
Minor (10)
58
75
77
17


435331
rs2024115
Major (8)
56
27
56
18


435349
rs2024115
Minor (8)
26
91
66
19


435295
rs2024115
Major (10)
53
57
62
20


435313
rs2024115
Minor (10)
25
87
53
21


435862
rs10015979
Major (10)
8
51
40
22


435887
rs10015979
Minor (10)
40
22
28
23


435880
rs7691627
Major (10)
43
17
21
24


435905
rs7691627
Minor (10)
13
27
15
25


435885
rs2798235
Major (10)
38
39
30
26


435910
rs2798235
Minor (10)
17
30
16
27


435874
rs4690072
Major (10)
61
34
48
28


435899
rs4690072
Minor (10)
50
41
45
29


435875
rs6446723
Major (10)
28
13
35
30


435900
rs6446723
Minor (10)
24
56
37
31


435332
rs363081
Major (8)
76
95
88
32


435350
rs363081
Minor (8)
27
61
43
33


435296
rs363081
Major (10)
59
77
66
34


435314
rs363081
Minor (10)
38
66
40
35


435886
rs363080
Major (10)
74
72
79
36


435911
rs363080
Minor (10)
57
58
54
37


435914
rs363075
Major (6)
95
92
95
38


435926
rs363075
Minor (6)
88
81
79
39


435916
rs363075
Major (7)
90
92
94
40


435928
rs363075
Minor (7)
83
79
85
41


435333
rs363075
Major (8)
86
97
91
42


435351
rs363075
Minor (8)
59
80
58
43


435918
rs363075
Major (9)
83
90
91
44


435930
rs363075
Minor (9)
29
49
49
45


435297
rs363075
Major (10)
74
84
83
46


435315
rs363075
Minor (10)
47
63
45
47


435920
rs363075
Major (11)
78
66
83
48


435932
rs363075
Minor (11)
39
20
19
49


435366
rs363075
Major (12)
80
91
85
50


435924
rs363075
Minor (12)
37
49
58
51


435922
rs363075
Major (14)
80
90
91
52


435934
rs363075
Minor (14)
63
70
80
53


435334
rs363064
Major (8)
50
59
44
54


435352
rs363064
Minor (8)
12
37
48
55


435298
rs363064
Major (10)
81
92
87
56


435316
rs363064
Minor (10)
69
90
80
57


435335
rs3025849
Major (8)
0
40
37
58


435353
rs3025849
Minor (8)
0
29
18
59


435299
rs3025849
Major (10)
0
34
67
60


435317
rs3025849
Minor (10)
0
38
34
61


435877
rs6855981
Major (10)
31
59
58
62


435902
rs6855981
Minor (10)
0
43
27
63


435336
rs363102
Major (8)
0
21
19
64


435354
rs363102
Minor (8)
0
36
33
65


435300
rs363102
Major (10)
0
34
24
66


435318
rs363102
Minor (10)
0
30
20
67


435884
rs11731237
Major (10)
7
46
51
68


435909
rs11731237
Minor (10)
30
47
41
69


435337
rs4690073
Major (8)
12
0
12
70


435355
rs4690073
Minor (8)
0
26
33
71


435301
rs4690073
Major (10)
23
0
10
72


435319
rs4690073
Minor (10)
0
45
53
73


435883
rs363144
Major (10)
24
23
39
74


435908
rs363144
Minor (10)
27
20
22
75


435338
rs3025838
Major (8)
31
46
69
76


435356
rs3025838
Minor (8)
3
25
17
77


435302
rs3025838
Major (10)
39
73
67
78


435320
rs3025838
Minor (10)
21
49
32
79


435339
rs363099
Major (8)
84
87
76
80


435357
rs363099
Minor (8)
71
91
90
81


435303
rs363099
Major (10)
83
92
85
82


435321
rs363099
Minor (10)
84
95
89
83


435367
rs363099
Major (12)
76
82
72
84


435340
rs363096
Major (8)
0
47
52
85


435358
rs363096
Minor (8)
0
25
35
86


435304
rs363096
Major (10)
5
33
36
87


435322
rs363096
Minor (10)
2
30
32
88


435341
rs2298967
Major (8)
54
72
56
89


435359
rs2298967
Minor (8)
25
59
63
90


435305
rs2298967
Major (10)
66
80
78
91


435323
rs2298967
Minor (10)
36
79
66
92


435865
rs2298969
Major (10)
53
72
79
93


435890
rs2298969
Minor (10)
65
46
54
94


435876
rs6844859
Major (10)
70
67
77
95


435901
rs6844859
Minor (10)
39
83
80
96


435872
rs363092
Major (10)
46
41
54
97


435897
rs363092
Minor (10)
37
69
57
98


435879
rs7685686
Major (10)
83
31
70
99


435904
rs7685686
Minor (10)
30
92
72
100


435871
rs363088
Major (10)
70
55
70
101


435896
rs363088
Minor (10)
66
74
80
102


435870
rs362331
Major (10)
88
74
88
103


435895
rs362331
Minor (10)
78
92
86
104


435881
rs916171
Major (10)
0
57
51
105


435906
rs916171
Minor (10)
14
26
17
106


435342
rs362322
Major (8)
47
74
67
107


435360
rs362322
Minor (8)
17
58
52
108


435306
rs362322
Major (10)
50
77
65
109


435324
rs362322
Minor (10)
42
61
64
110


435868
rs362275
Major (10)
54
35
43
111


435893
rs362275
Minor (10)
3
27
33
112


435343
rs2276881
Major (8)
59
76
65
113


435361
rs2276881
Minor (8)
58
44
20
114


435307
rs2276881
Major (10)
69
82
81
115


435325
rs2276881
Minor (10)
17
47
43
116


435368
rs2276881
Major (12)
84
96
92
117


435866
rs3121419
Major (10)
67
61
64
118


435891
rs3121419
Minor (10)
53
76
73
119


435344
rs362272
Major (8)
35
46
36
120


435362
rs362272
Minor (8)
34
68
57
121


435308
rs362272
Major (10)
26
30
35
122


435326
rs362272
Minor (10)
29
50
39
123


435369
rs362272
Major (12)
66
74
65
124


435867
rs362271
Major (10)
73
74
75
125


435892
rs362271
Minor (10)
52
74
79
126


435873
rs3775061
Major (10)
40
32
47
127


435898
rs3775061
Minor (10)
13
20
24
128


435345
rs362310
Major (8)
38
55
52
129


435363
rs362310
Minor (8)
45
67
60
130


435309
rs362310
Major (10)
33
44
56
131


435327
rs362310
Minor (10)
33
71
61
132


435915
rs362307
Major (6)
61
54
58
133


435927
rs362307
Minor (6)
31
35
44
134


435917
rs362307
Major (7)
67
76
66
135


435929
rs362307
Minor (7)
33
34
55
136


435346
rs362307
Major (8)
67
89
66
137


435364
rs362307
Minor (8)
46
72
66
138


435919
rs362307
Major (9)
84
79
70
139


435931
rs362307
Minor (9)
74
74
86
140


435310
rs362307
Major (10)
74
81
71
141


435328
rs362307
Minor (10)
47
69
75
142


435921
rs362307
Major (11)
74
77
69
143


435933
rs362307
Minor (11)
38
47
74
144


435370
rs362307
Major (12)
64
74
38
145


435925
rs362307
Minor (12)
60
66
80
146


435923
rs362307
Major (14)
73
66
71
147


435935
rs362307
Minor (14)
68
75
87
148


435869
rs362306
Major (10)
82
77
81
149


435894
rs362306
Minor (10)
28
79
72
150


435347
rs362303
Major (8)
68
74
71
151


435365
rs362303
Minor (8)
69
83
76
152


435311
rs362303
Major (10)
46
56
72
153


435329
rs362303
Minor (10)
49
62
39
154


435882
rs362296
Major (10)
29
48
56
155


435907
rs362296
Minor (10)
42
56
52
156









Example 3
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the study described in Example 2 were selected and tested at various doses in GM04281, GM02171, and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 750 nM, 1,500 nM, 3,000 nM, 6,000 nM, and 12,000 nM concentrations of antisense oligonucleotide, as specified in Table 5, 6, and 7. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 5, 6, and 7.







TABLE 5







Dose-dependent antisense inhibition of human HTT in GM04281 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
51
81
80
91
97
0.6


435330
24
49
50
73
85
2.5


435331
23
38
64
72
74
2.4


435868
3
17
7
29
63
6.7


435870
53
73
77
86
93
0.6


435871
28
51
52
78
89
1.7


435874
14
21
28
64
82
3.3


435879
42
57
57
81
91
1.1


435890
48
56
62
76
91
0.9


435929
10
0
5
12
48
13.8


435931
20
17
53
62
81
2.9


435933
0
7
24
43
49
10.7


435935
0
38
38
62
29
4.2
















TABLE 6







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
57
73
81
93
98
0.4


435330
27
37
0
44
63
4.4


435331
35
34
19
41
63
3.5


435868
21
21
39
24
12
>12.0


435870
50
53
57
70
79
0.9


435871
32
46
45
58
62
3.9


435874
1
0
4
11
6
>12.0


435879
32
14
17
45
38
>12.0


435890
34
33
40
51
62
5.4


435929
25
22
31
5
29
>12.0


435931
15
28
27
60
79
3.7


435933
13
36
21
43
48
12.2


435935
25
42
27
61
68
3.2
















TABLE 7







Dose-dependent antisense inhibition of human HTT in GM02173B cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
43
67
80
86
97
1.1


435330
22
21
0
52
62
5.3


435331
19
17
32
50
55
9.4


435868
17
25
41
13
26
>12.0


435870
24
57
70
78
75
1.8


435871
8
30
42
50
48
5.0


435874
31
35
28
35
42
>12.0


435879
39
44
42
60
64
2.5


435890
38
36
50
65
73
3.1


435929
19
17
19
42
35
7.7


435931
40
19
31
48
71
5.8


435933
35
24
47
52
59
4.4


435935
25
23
40
73
77
3.7









Example 4
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the study described in Example 2 were selected and tested at various doses in GM04281, GM02171, and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 750 nM, 1,500 nM, 3,000 nM, 6,000 nM, and 12,000 nM concentrations of antisense oligonucleotide, as specified in Table 8, 9, and 10. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA relative to untreated control cells. IC50 values are also provided in Tables 8, 9, and 10.







TABLE 8







Dose-dependent antisense inhibition of human HTT in GM04281 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
61
78
90
94
97
<0.8


435303
33
39
69
79
91
1.5


435328
0
12
16
51
75
5.3


435331
27
48
48
70
82
2.1


435339
46
37
61
73
89
2.3


435869
17
35
44
66
80
3.3


435870
44
60
64
84
84
1.1


435871
41
50
71
78
87
1.2


435874
24
36
35
65
73
3.1


435879
46
52
78
81
92
0.9


435890
41
53
63
80
86
1.3


435925
0
14
39
60
87
4.2


435926
20
28
67
81
89
2.0


435928
32
49
73
86
86
1.8


435931
22
24
40
59
90
3.8
















TABLE 9







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
50
64
90
95
96
0.7


435303
14
32
68
79
85
2.8


435328
0
12
20
38
55
10.3


435331
0
13
5
30
36
>12.0


435339
30
40
58
63
49
2.5


435869
13
25
31
47
87
4.0


435870
18
31
44
66
74
3.5


435871
1
20
29
49
64
6.5


435874
3
6
12
17
31
>12.0


435879
0
2
12
35
44
>12.0


435890
15
16
30
48
72
5.8


435925
0
0
22
48
29
6.3


435926
25
28
58
74
85
2.3


435928
18
53
61
86
83
2.5


435931
0
4
25
46
68
6.7
















TABLE 10







Dose-dependent antisense inhibition of human HTT in GM02173B cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
27
65
84
81
96
1.9


435303
23
48
52
76
76
2.9


435328
8
14
19
34
50
15.7


435331
10
17
16
27
32
>12.0


435339
28
26
38
67
82
3.8


435869
12
24
37
45
79
4.2


435870
20
26
58
53
78
2.7


435871
15
16
32
45
71
6.0


435874
13
8
28
36
31
>12.0


435879
22
20
36
53
60
6.0


435890
21
28
34
54
71
4.3


435925
2
10
28
43
78
5.9


435926
7
25
37
73
79
3.5


435928
15
39
60
73
87
2.5


435931
13
13
32
61
62
6.7









Example 5
Antisense Inhibition of Human HTT in GM04281 Cells
Additional antisense oligonucleotides were designed based on the gapmers selected from studies described in Example 4. These oligonucleotides were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Tables 8, 9, and 10. Antisense oligonucleotides were also created with uniform MOE, as well as with various motifs, 2-9-6 MOE, 3-9-3 MOE, 3-9-4 MOE, 3-9-5 MOE, 4-10-5 MOE, 4-11-4 MOE, 4-7-4 MOE, 4-9-4 MOE, 4-9-5 MOE, 5-10-4 MOE, 5-7-5 MOE, 5-8-6 MOE, 5-9-3 MOE, 5-9-5 MOE, 6-7-6 MOE, 6-9-2 MOE, and 6-8-5 MOE.
In addition, antisense oligonucleotides were designed targeting SNP RS Nos. rs2857936, rs12506200, rs762855, and rs1006798 (refer to Table 2). The oligonucleotides were designed targeting either the major allele or the minor allele, and with the SNP position opposite either position 8 or position 10 of the gapmer.
These gapmers were tested in vitro. Cultured GM04281 cells at a density of 25,000 cells per well were transfected using electroporation with 10,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented in Tables 11-19 as percent inhibition of HTT mRNA, relative to untreated control cells.
The gapmers, ISIS 435869, ISIS 435870, ISIS 435874, ISIS 435879, and ISIS 435890, from which some of the newly designed gapmers were derived are marked with an asterisk (*) in the table. ISIS 387916 was included in the study as a benchmark oligonucleotide against which the potency of the antisense oligonucleotides targeting nucleotides overlapping each SNP position could be compared.
The uniform MOE oligonucleotides are 15 nucleotides in length.
The 2-9-6 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 2 nucleotides and on the 3′ direction by a wing comprising 6 nucleotides.
The 3-9-3 gapmers are 15 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 3 nucleotides each.
The 3-9-4 gapmers are 16 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 3 nucleotides and on the 3′ direction by a wing comprising 4 nucleotides.
The 3-9-5 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 3 nucleotides and on the 3′ direction by a wing comprising 5 nucleotides.
The 4-10-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 4 nucleotides and on the 3′ direction by a wing comprising 5 nucleotides.
The 4-11-4 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of eleven 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 4 nucleotides each.
The 4-7-4 gapmers are 15 nucleotides in length, wherein the central gap segment is comprised of seven 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 4 nucleotides each.
The 4-9-4 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 4 nucleotides each.
The 4-9-5 gapmers are 18 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 4 nucleotides and on the 3′ direction by a wing comprising 5 nucleotides.
The 5-10-4 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 5 nucleotides and on the 3′ direction by a wing comprising 4 nucleotides.
The 5-7-5 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of seven 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 5 nucleotides each.
The 5-8-6 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 5 nucleotides and on the 3′ direction by a wing comprising 6 nucleotides.
The 5-9-3 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 5 nucleotides and on the 3′ direction by a wing comprising 3 nucleotides.
The 5-9-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 5 nucleotides each.
The 6-7-6 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of seven 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 6 nucleotides each.
The 6-9-2 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 6 nucleotides and on the 3′ direction by a wing comprising 2 nucleotides.
The 6-8-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on the 5′ direction by a wing comprising 6 nucleotides and on the 3′ direction by a wing comprising 5 nucleotides.
For each of the motifs, each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methylcytosines.
The oligonucleotides are organized in tables according to the SNP they target. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. ‘Target allele’ indicates whether the gapmer is targeted to the major or the minor allele. The number in parentheses indicates the position on the oligonucleotide opposite to the SNP position.







TABLE 11







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs2857936 (nucleobases


1952 to 1972 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


1952
1970
Minor (8)
459908
GCTTTTCATTGAAAAGAAA
5-9-5
26
157


 


1952
1970
Major (8)
459916
GCTTTTCGTTGAAAAGAAA
5-9-5
8
158


 


1954
1972
Minor (10)
459904
CTGCTTTTCATTGAAAAGA
5-9-5
23
159


 


1954
1972
Major (10)
459912
CTGCTTTTCGTTGAAAAGA
5-9-5
8
160
















TABLE 12







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs12506200


(nucleobases 3695 to 3715 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


3695
3713
Major (8)
459909
ACTAGGCCGGGCATGCTGG
5-9-5
48
161


 


3695
3713
Minor (8)
459917
ACTAGGCTGGGCATGCTGG
5-9-5
35
162


 


3697
3715
Major (10)
459905
AGACTAGGCCGGGCATGCT
5-9-5
33
163


 


3697
3715
Minor (10)
459913
AGACTAGGCTGGGCATGCT
5-9-5
45
164
















TABLE 13







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs762855


(nucleobases 14437 to 14457 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


14437
14455
Minor (8)
459910
AAACAGCTGTTAGTTCCCA
5-9-5
27
165


 


14437
14455
Major (8)
459918
AAACAGCCGTTAGTTCCCA
5-9-5
39
166


 


14439
14457
Minor (10)
459906
AGAAACAGCTGTTAGTTCC
5-9-5
24
167


 


14439
14457
Major (10)
459914
AGAAACAGCCGTTAGTTCC
5-9-5
28
168
















TABLE 14







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs4690072


(nucleobases 62147 to 62173 of SEQ ID NO: 1)





















SEQ


Start
Stop




%
ID


Site
Site
Target allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


62147
62165
Major (6)
460145
GTGCTACCCAACCTTTCTG
5-9-5
62
169


 


62148
62166
Major (7)
460144
AGTGCTACCCAACCTTTCT
5-9-5
61
170


 


62149
62167
Major (8)
460143
CAGTGCTACCCAACCTTTC
5-9-5
65
171


 


62150
62168
Major (9)
460142
ACAGTGCTACCCAACCTTT
5-9-5
83
172


 


62151
62169
Major (10)
*435874 
CACAGTGCTACCCAACCTT
5-9-5
76
28


 


62151
62169
Major (10)
460022
CACAGTGCTACCCAACCTT
4-10-5
75
28


 


62151
62169
Major (10)
460033
CACAGTGCTACCCAACCTT
4-11-4
89
28


 


62151
62168
Major (9)
460063
ACAGTGCTACCCAACCTT
4-9-5
77
173


 


62151
62169
Major (10)
460073
CACAGTGCTACCCAACCTT
5-10-4
86
28


 


62151
62169
Major (10)
460093
CACAGTGCTACCCAACCTT
5-8-6
61
28


 


62151
62169
Major (10)
460169
CACAGTGCTACCCAACCTT
6-7-6
16
28


 


62151
62169
Major (10)
460188
CACAGTGCTACCCAACCTT
6-8-5
53
28


 


62152
62168
Major (9)
459978
ACAGTGCTACCCAACCT
2-9-6
87
174


 


62152
62167
Major (8)
459999
CAGTGCTACCCAACCT
3-9-4
48
175


 


62152
62168
Major (9)
460012
ACAGTGCTACCCAACCT
3-9-5
84
174


 


62152
62168
Major (9)
460052
ACAGTGCTACCCAACCT
4-9-4
51
174


 


62152
62168
Major (9)
460083
ACAGTGCTACCCAACCT
5-7-5
37
174


 


62152
62168
Major (9)
460103
ACAGTGCTACCCAACCT
5-9-3
80
174


 


62152
62170
Major (11)
460137
TCACAGTGCTACCCAACCT
5-9-5
65
176


 


62152
62168
Major (9)
460179
ACAGTGCTACCCAACCT
6-9-2
67
174


 


62153
62167
Major (8)
459989
CAGTGCTACCCAACC
3-9-3
60
177


 


62153
62167
Major (8)
460043
CAGTGCTACCCAACC
4-7-4
24
177


 


62153
62171
Major (12)
460138
ATCACAGTGCTACCCAACC
5-9-5
76
178


 


62154
62172
Major (13)
460139
TATCACAGTGCTACCCAAC
5-9-5
68
179


 


62155
62173
Major (14)
460140
ATATCACAGTGCTACCCAA
5-9-5
79
180
















TABLE 15







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs2298969


(nucleobases 125883 to 125911 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


125883
125901
Minor (5)
460166
ATGCTGACTTGGGCCATTC
5-9-5
83
181


 


125884
125902
Minor (6)
460165
GATGCTGACTTGGGCCATT
5-9-5
88
182


 


125885
125903
Minor (7)
460164
GGATGCTGACTTGGGCCAT
5-9-5
68
183


 


125886
125904
Minor (8)
460163
GGGATGCTGACTTGGGCCA
5-9-5
73
184


 


125887
125905
Minor (9)
460162
AGGGATGCTGACTTGGGCC
5-9-5
88
185


 


125888
125906
Minor (10)
*435890 
AAGGGATGCTGACTTGGGC
5-9-5
83
94


 


125888
125906
Minor (10)
460026
AAGGGATGCTGACTTGGGC
4-10-5
90
94


 


125888
125906
Minor (10)
460037
AAGGGATGCTGACTTGGGC
4-11-4
86
94


 


125888
125905
Minor (9)
460068
AGGGATGCTGACTTGGGC
4-9-5
90
186


 


125888
125906
Minor (10)
460076
AAGGGATGCTGACTTGGGC
5-10-4
90
94


 


125888
125906
Minor (10)
460096
AAGGGATGCTGACTTGGGC
5-8-6
88
94


 


125888
125906
Minor (10)
460171
AAGGGATGCTGACTTGGGC
6-7-6
87
94


 


125888
125906
Minor (10)
460190
AAGGGATGCTGACTTGGGC
6-8-5
69
94


 


125889
125905
Minor (9)
459983
AGGGATGCTGACTTGGG
2-9-6
80
187


 


125889
125904
Minor (8)
460005
GGGATGCTGACTTGGG
3-9-4
80
284


 


125889
125905
Minor (9)
460016
AGGGATGCTGACTTGGG
3-9-5
90
187


 


125889
125905
Minor (9)
460057
AGGGATGCTGACTTGGG
4-9-4
86
187


 


125889
125905
Minor (9)
460087
AGGGATGCTGACTTGGG
5-7-5
86
187


 


125889
125905
Minor (9)
460107
AGGGATGCTGACTTGGG
5-9-3
79
187


 


125889
125907
Major (11)
460157
CAAGGGATGCTGACTTGGG
5-9-5
88
188


 


125889
125905
Minor (9)
460181
AGGGATGCTGACTTGGG
6-9-2
62
187


 


125890
125904
Minor (8)
459972
GGGATGCTGACTTGG
Uniform
18
189


 


125890
125904
Minor (8)
459992
GGGATGCTGACTTGG
3-9-3
90
189


 


125890
125904
Minor (8)
460046
GGGATGCTGACTTGG
4-7-4
59
189


 


125890
125908
Major (12)
460158
CCAAGGGATGCTGACTTGG
5-9-5
79
190


 


125891
125909
Major (13)
460159
GCCAAGGGATGCTGACTTG
5-9-5
82
191


 


125892
125910
Major (14)
460160
TGCCAAGGGATGCTGACTT
5-9-5
87
192


 


125893
125911
Major (15)
460161
CTGCCAAGGGATGCTGACT
5-9-5
78
193
















TABLE 16







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs7685686


(nucleobases 146781 to 146809 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target
ISIS


%
ID


Site
Site
allele
No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


146781
146799
Major (5)
460156
ATTGTCATCACCAGAAAAA
5-9-5
88
194


 


146782
146800
Major (6)
460155
AATTGTCATCACCAGAAAA
5-9-5
89
195


 


146783
146801
Major (7)
460154
AAATTGTCATCACCAGAAA
5-9-5
89
196


 


146784
146802
Major (8)
460153
TAAATTGTCATCACCAGAA
5-9-5
93
197


 


146785
146803
Major (9)
460152
ATAAATTGTCATCACCAGA
5-9-5
95
198


 


146786
146804
Major (10)
*435879 
AATAAATTGTCATCACCAG
5-9-5
94
99


 


146786
146804
Major (10)
460024
AATAAATTGTCATCACCAG
4-10-5
88
99


 


146786
146804
Major (10)
460035
AATAAATTGTCATCACCAG
4-11-4
91
99


 


146786
146803
Major (9)
460065
ATAAATTGTCATCACCAG
4-9-5
96
199


 


146786
146804
Major (10)
460074
AATAAATTGTCATCACCAG
5-10-4
94
99


 


146786
146804
Major (10)
460095
AATAAATTGTCATCACCAG
5-8-6
92
99


 


146786
146804
Major (10)
460170
AATAAATTGTCATCACCAG
6-7-6
91
99


 


146786
146804
Major (10)
460189
AATAAATTGTCATCACCAG
6-8-5
94
99


 


146787
146803
Major (9)
459981
ATAAATTGTCATCACCA
2-9-6
85
200


 


146787
146802
Major (8)
460002
TAAATTGTCATCACCA
3-9-4
86
201


 


146787
146803
Major (9)
460014
ATAAATTGTCATCACCA
3-9-5
91
200


 


146787
146803
Major (9)
460055
ATAAATTGTCATCACCA
4-9-4
90
200


 


146787
146803
Major (9)
460085
ATAAATTGTCATCACCA
5-7-5
94
200


 


146787
146803
Major (9)
460104
ATAAATTGTCATCACCA
5-9-3
93
200


 


146787
146805
Major (11)
460147
TAATAAATTGTCATCACCA
5-9-5
91
202


 


146787
146803
Major (9)
460180
ATAAATTGTCATCACCA
6-9-2
91
200


 


146788
146802
Major (8)
459970
TAAATTGTCATCACC
Uniform
9
203


 


146788
146802
Major (8)
459990
TAAATTGTCATCACC
3-9-3
67
203


 


146788
146802
Major (8)
460045
TAAATTGTCATCACC
4-7-4
84
203


 


146788
146806
Major (12)
460148
TTAATAAATTGTCATCACC
5-9-5
88
204


 


146789
146807
Major (13)
460149
ATTAATAAATTGTCATCAC
5-9-5
32
205


 


146790
146808
Major (14)
460150
TATTAATAAATTGTCATCA
5-9-5
29
206


 


146791
146809
Major (15)
460151
CTATTAATAAATTGTCATC
5-9-5
33
207
















TABLE 17







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362331


(nucleobases 155474 to 155502 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


155474
155492
Major (5)
460136
CAGTAGATGAGGGAGCAGG
5-9-5
81
208


 


155475
155493
Major (6)
460135
ACAGTAGATGAGGGAGCAG
5-9-5
84
209


 


155476
155494
Major (7)
460134
CACAGTAGATGAGGGAGCA
5-9-5
87
210


 


155477
155495
Major (8)
460133
ACACAGTAGATGAGGGAGC
5-9-5
85
211


 


155478
155496
Major (9)
460132
CACACAGTAGATGAGGGAG
5-9-5
86
212


 


155479
155497
Major (10)
*435870 
GCACACAGTAGATGAGGGA
5-9-5
91
103


 


155479
155497
Major (10)
460019
GCACACAGTAGATGAGGGA
4-10-5
92
103


 


155479
155497
Major (10)
460031
GCACACAGTAGATGAGGGA
4-11-4
95
103


 


155479
155496
Major (9)
460061
CACACAGTAGATGAGGGA
4-9-5
87
213


 


155479
155497
Major (10)
460071
GCACACAGTAGATGAGGGA
5-10-4
94
103


 


155479
155497
Major (10)
460090
GCACACAGTAGATGAGGGA
5-8-6
86
103


 


155479
155497
Major (10)
460168
GCACACAGTAGATGAGGGA
6-7-6
84
103


 


155479
155497
Major (10)
460187
GCACACAGTAGATGAGGGA
6-8-5
89
103


 


155480
155496
Major (9)
459977
CACACAGTAGATGAGGG
2-9-6
90
214


 


155480
155495
Major (8)
459996
ACACAGTAGATGAGGG
3-9-4
37
215


 


155480
155496
Major (9)
460009
CACACAGTAGATGAGGG
3-9-5
90
214


 


155480
155496
Major (9)
460051
CACACAGTAGATGAGGG
4-9-4
73
214


 


155480
155496
Major (9)
460081
CACACAGTAGATGAGGG
5-7-5
77
214


 


155480
155496
Major (9)
460101
CACACAGTAGATGAGGG
5-9-3
84
214


 


155480
155498
Major (11)
460127
TGCACACAGTAGATGAGGG
5-9-5
89
216


 


155480
155496
Major (9)
460178
CACACAGTAGATGAGGG
6-9-2
92
214


 


155481
155495
Major (8)
459967
ACACAGTAGATGAGG
Uniform
81
217


 


155481
155495
Major (8)
459987
ACACAGTAGATGAGG
3-9-3
18
217


 


155481
155495
Major (8)
460041
ACACAGTAGATGAGG
4-7-4
54
217


 


155481
155499
Major (12)
460128
GTGCACACAGTAGATGAGG
5-9-5
73
218


 


155482
155500
Major (13)
460129
AGTGCACACAGTAGATGAG
5-9-5
86
219


 


155483
155501
Major (14)
460130
AAGTGCACACAGTAGATGA
5-9-5
60
220


 


155484
155502
Major (15)
460131
GAAGTGCACACAGTAGATG
5-9-5
73
221
















TABLE 18







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362306


(nucleobases 181739 to 181767 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


181739
181757
Major (5)
460126
GCTGCAACCTGGCAACAAC
5-9-5
87
222


 


181740
181758
Major (6)
460125
AGCTGCAACCTGGCAACAA
5-9-5
70
223


 


181741
181759
Major (7)
460123
CAGCTGCAACCTGGCAACA
5-9-5
83
224


 


181742
181760
Major (8)
460121
GCAGCTGCAACCTGGCAAC
5-9-5
47
225


 


181743
181761
Major (9)
460118
AGCAGCTGCAACCTGGCAA
5-9-5
75
226


 


181744
181762
Major (10)
*435869 
GAGCAGCTGCAACCTGGCA
5-9-5
91
149


 


181744
181762
Major (10)
460018
GAGCAGCTGCAACCTGGCA
4-10-5
86
149


 


181744
181762
Major (10)
460028
GAGCAGCTGCAACCTGGCA
4-11-4
89
149


 


181744
181761
Major (9)
460058
AGCAGCTGCAACCTGGCA
4-9-5
85
227


 


181744
181762
Major (10)
460069
GAGCAGCTGCAACCTGGCA
5-10-4
91
149


 


181744
181762
Major (10)
460089
GAGCAGCTGCAACCTGGCA
5-8-6
54
149


 


181744
181762
Major (10)
460167
GAGCAGCTGCAACCTGGCA
6-7-6
85
149


 


181744
181762
Major (10)
460186
GAGCAGCTGCAACCTGGCA
6-8-5
84
149


 


181745
181761
Major (9)
459975
AGCAGCTGCAACCTGGC
2-9-6
86
228


 


181745
181760
Major (8)
459995
GCAGCTGCAACCTGGC
3-9-4
87
229


 


181745
181761
Major (9)
460008
AGCAGCTGCAACCTGGC
3-9-5
83
228


 


181745
181761
Major (9)
460049
AGCAGCTGCAACCTGGC
4-9-4
88
228


 


181745
181761
Major (9)
460079
AGCAGCTGCAACCTGGC
5-7-5
46
228


 


181745
181761
Major (9)
460099
AGCAGCTGCAACCTGGC
5-9-3
44
228


 


181745
181763
Major (11)
460108
AGAGCAGCTGCAACCTGGC
5-9-5
50
230


 


181745
181761
Major (9)
460177
AGCAGCTGCAACCTGGC
6-9-2
67
228


 


181746
181760
Major (8)
459966
GCAGCTGCAACCTGG
Uniform
26
231


 


181746
181760
Major (8)
459985
GCAGCTGCAACCTGG
3-9-3
69
231


 


181746
181760
Major (8)
460039
GCAGCTGCAACCTGG
4-7-4
56
231


 


181746
181764
Major (12)
460110
AAGAGCAGCTGCAACCTGG
5-9-5
75
232


 


181747
181765
Major (13)
460113
CAAGAGCAGCTGCAACCTG
5-9-5
36
233


 


181748
181766
Major (14)
460115
GCAAGAGCAGCTGCAACCT
5-9-5
78
234


 


181749
181767
Major (15)
460117
TGCAAGAGCAGCTGCAACC
5-9-5
73
235
















TABLE 19







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs1006798


(nucleobases 198015 to 198035 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
98
6


 


198015
198033
Minor (8)
459911
ACCATGATATCTCCAGCAC
5-9-5
33
236


 


198015
198033
Minor (8)
459919
ACCATGACATCTCCAGCAC
5-9-5
26
237


 


198017
198035
Major (10)
459907
CCACCATGATATCTCCAGC
5-9-5
32
238


 


198017
198035
Minor (10)
459915
CCACCATGACATCTCCAGC
5-9-5
51
239









Example 6
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the studies described in Example 5 were selected and tested at various doses in GM04281, GM02171, and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 750 nM, 1,500 nM, 3,000 nM, 6,000 nM, and 12,000 nM concentrations of antisense oligonucleotide, as specified in Tables 20, 21, and 22. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 20, 21, and 22.







TABLE 20







Dose-dependent antisense inhibition of human HTT in GM04281 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
56
81
89
96
98
0.6


435869
38
49
66
86
91
1.4


435874
33
27
37
49
62
8.4


435879
42
55
73
86
96
1.1


435890
39
51
74
83
89
1.3


459978
29
33
51
69
86
2.5


459992
14
27
51
54
84
3.2


460012
15
24
54
70
81
3.1


460016
3
36
48
71
77
3.3


460019
54
59
74
87
94
0.7


460026
48
47
71
79
88
0.8


460028
39
38
73
77
87
1.4


460031
44
62
72
87
92
0.9


460033
11
38
52
64
87
3.0


460065
43
54
74
89
96
1.1


460068
47
28
63
76
90
2.6


460069
38
50
65
77
91
1.4


460071
53
61
80
89
93
0.6


460073
16
39
42
58
75
4.0


460076
26
47
54
70
86
2.1


460085
48
60
79
89
94
0.8


460140
6
24
44
44
64
6.6


460142
2
38
46
46
68
4.8


460152
35
61
76
92
94
1.2


460157
51
36
53
74
89
2.6


460162
64
41
71
76
85
2.1


460165
41
50
56
76
84
1.5
















TABLE 21







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
53
66
88
96
98
0.7


435869
4
20
36
63
86
3.9


435870
25
39
48
62
83
2.8


435874
12
20
18
27
37
>12.0


435879
10
7
11
42
51
10.6


435890
10
23
29
29
55
9.2


459978
15
7
6
29
52
12.7


459992
11
19
26
39
62
8.7


460012
3
3
10
19
41
>12.0


460016
0
14
12
22
48
>12.0


460019
27
21
41
60
73
4.4


460026
9
25
30
46
58
7.8


460028
24
8
32
54
77
5.3


460031
8
25
42
60
83
3.8


460033
11
25
30
40
75
4.1


460065
11
16
11
31
53
10.3


460068
15
13
39
44
53
8.8


460069
17
28
37
60
79
3.9


460071
16
36
58
70
88
2.6


460073
5
19
24
33
56
8.7


460076
19
29
44
54
83
3.3


460085
10
15
17
28
31
>12.0


460140
8
22
22
28
47
>12.0


460142
11
24
28
36
38
>12.0


460152
14
21
8
25
44
22


460157
22
21
29
44
66
6.7


460162
24
55
52
62
82
2.8


460165
14
34
50
69
81
3.1
















TABLE 22







Dose-dependent antisense inhibition of human HTT in GM02173B cells













ISIS
750




IC50


No.
nM
1,500 nM
3,000 nM
6,000 nM
12,000 nM
(μM)
















387916
37
63
86
88
98
1.0


435869
10
20
43
70
85
3.5


435870
24
24
56
72
87
2.3


435874
0
11
12
30
44
>12.0


435879
4
17
43
64
74
4.3


435890
31
29
54
57
69
4.4


459978
7
13
17
35
64
8.4


459992
18
15
30
51
71
5.7


460012
0
10
24
37
72
7.1


460016
15
5
30
38
59
9.5


460019
10
32
51
65
87
3.1


460026
0
34
21
55
65
6.4


460028
0
14
31
51
77
5.2


460031
0
31
53
71
88
3.2


460033
11
8
6
52
84
5.0


460065
19
37
53
58
74
3.6


460068
17
11
31
59
69
5.5


460069
11
21
37
55
75
4.6


460071
6
42
61
83
88
2.6


460073
7
13
19
49
66
6.3


460076
27
31
49
43
81
2.9


460085
17
34
51
54
68
4.4


460140
0
2
28
18
46
>12.0


460142
2
32
37
42
59
7.6


460152
17
32
35
51
66
5.5


460157
9
34
38
52
74
4.5


460162
22
45
57
65
79
2.5


460165
5
45
52
72
84
3.2









Example 7
Antisense Inhibition of Human HTT in GM04281 Cells and GM02171 Cells
Additional antisense oligonucleotides were designed based on the gapmers selected from studies described in Example 2. These oligonucleotides were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Table 4.
The gapmers were tested in the GM04281 and the GM02171 cell lines. Cultured GM04281 or GM02171 cells at a density of 25,000 cells per well were transfected using electroporation with 10,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR using primer probe set RTS2617. HIT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells.
The gapmers, from which the newly designed oligonucleotides were derived, were also included in the assay. These parent gapmers, ISIS 435294, ISIS 435295, ISIS 435301, ISIS 435303, ISIS 435304, ISIS 435305, ISIS 435308, ISIS 435330, ISIS 435331, ISIS 435337, ISIS 435339, ISIS 435340, ISIS 435341, ISIS 435344, ISIS 435862, ISIS 435863, ISIS 435864, ISIS 435866, ISIS 435867, ISIS 435868, ISIS 435871, ISIS 435873, ISIS 435875, ISIS 435876, ISIS 435878, ISIS 435880, ISIS 435881, ISIS 435882, ISIS 435884, ISIS 435890, and ISIS 435897 are marked with an asterisk (*) in the table. ISIS 387916 was included in the study as a benchmark oligonucleotide against which the potency of the antisense oligonucleotides targeting nucleotides overlapping each SNP position could be compared.
The chimeric antisense oligonucleotides in Tables 23-48 were designed as 5-9-5 MOE gapmers. The 5-9-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 5 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methylcytosines.
The gapmers are organized in Tables 23-48, according to the SNP site they target. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. ‘Target allele’ indicates whether the gapmer is targeted to the major or the minor allele. The number in parentheses indicates the position on the oligonucleotide opposite to the SNP position.







TABLE 23







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs3856973


(nucleobases 19815 to 19835 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


19815
19833
*435330 
Major (8)
TAACACTCGATTAACCCTG
88
31
8


 


19816
19834
476441
Major (9)
TTAACACTCGATTAACCCT
88
0
240


 


19817
19835
*435294 
Major (10)
GTTAACACTCGATTAACCC
72
30
10
















TABLE 24







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs2285086


(nucleobases 28901 to 28921 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


28901
28919
463570
Major (8)
TAGTTCATCCCAGTGAGAA
66
12
241


 


28902
28920
463573
Major (9)
CTAGTTCATCCCAGTGAGA
66
36
242


 


28903
28921
*435864 
Major (10)
GCTAGTTCATCCCAGTGAG
40
18
12
















TABLE 25







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs7659144


(nucleobases 37963 to 37983 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


37963
37981
476462
Major (8)
GAAATGGGTTTTTCCACAT
38
0
243


 


37964
37982
476439
Major (9)
GGAAATGGGTTTTTCCACA
80
45
244


 


37965
37983
*435878 
Major (10)
TGGAAATGGGTTTTTCCAC
76
3
14
















TABLE 26







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs16843804


(nucleobases 44032 to 44052 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


44032
44050
476471
Major (8)
TAACCGTGGCATGGGCAGT
82
53
245


 


44033
44051
476452
Major (9)
TTAACCGTGGCATGGGCAG
84
44
246


 


44034
44052
*435863 
Major (10)
TTTAACCGTGGCATGGGCA
89
89
16
















TABLE 27







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs2024115


(nucleobases 44210 to 44230 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


44210
44228
*435331
Major (8)
TTCAAGCTAGTAACGATGC
84
20
18


 


44211
44229
476447
Major (9)
CTTCAAGCTAGTAACGATG
87
57
247


 


44212
44230
*435295
Major (10)
ACTTCAAGCTAGTAACGAT
85
67
20
















TABLE 28







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs10015979


(nucleobases 49084 to 49104 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


49084
49102
476470
Major (8)
AGCTAGGTTAAAGAGTCAC
55
74
248


 


49085
49103
476450
Major (9)
CAGCTAGGTTAAAGAGTCA
44
5
249


 


49086
49104
*435862
Major (10)
GCAGCTAGGTTAAAGAGTC
56
49
22
















TABLE 29







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs7691627


(nucleobases 51052 to 51072 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


51052
51070
476467
Major (8)
TAAGAAACACAATCAAAGA
45
21
250


 


51053
51071
476445
Major (9)
ATAAGAAACACAATCAAAG
34
1
251


 


51054
51072
*435880
Major (10)
AATAAGAAACACAATCAAA
68
7
24
















TABLE 30







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs6446723


(nucleobases 66455 to 66475 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


66455
66473
476463
Major (8)
ATTTTCTAGACTTTATGAT
37
7
252


 


66456
66474
476440
Major (9)
AATTTTCTAGACTTTATGA
57
0
253


 


66457
66475
*435875
Major (10)
TAATTTTCTAGACTTTATG
42
0
30
















TABLE 31







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


a chimeric antisense oligonucleotide targeted to SNP rs363064


(nucleobases 81053 to 81071 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


81053
81071
476461
Major (9)
GAGAATACGGGTAACATTT
87
62
254
















TABLE 32







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs11731237


(nucleobases 91455 to 91475 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


91455
91473
476468
Major (8)
TGGGCAGGAAGGACTGAAC
58
56
255


 


91456
91474
476448
Major (9)
GTGGGCAGGAAGGACTGAA
61
69
256


 


91457
91475
*435884
Major (10)
GGTGGGCAGGAAGGACTGA
59
49
68
















TABLE 33







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs4690073


(nucleobases 99792 to 99812 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


99792
99810
*435337
Major (8)
CCTAAATCAATCTACAAGT
69
7
70


 


99793
99811
476446
Major (9)
CCCTAAATCAATCTACAAG
61
0
257


 


99794
99812
*435301
Major (10)
TCCCTAAATCAATCTACAA
63
1
72
















TABLE 34







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs34315806


(nucleobases 101676 to 101696 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


101676
101694
463569
Major (8)
CTTTTCCGTGCTGTTCTGA
96
95
258


 


101677
101695
463572
Major (9)
ACTTTTCCGTGCTGTTCTG
93
91
259


 


101678
101696
463567
Major (10)
AACTTTTCCGTGCTGTTCT
98
97
260
















TABLE 35







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs363099


(nucleobases 101698 to 101718 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


101698
101716
*435339
Major (8)
CTGAGCGGAGAAACCCTCC
94
85
80


 


101699
101717
476458
Major (9)
GCTGAGCGGAGAAACCCTC
92
79
261


 


101700
101718
*435303
Major (10)
GGCTGAGCGGAGAAACCCT
96
93
82
















TABLE 36







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs363096


(nucleobases 119663 to 119683 of SEQ ID NO: 1)



















%
%








inhibition
inhibition


Start
Stop

Target

in
in
SEQ


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
ID NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


119663
119681
*435340
Major (8)
TTCCCTAAAAACAAAAACA
42
21
85


 


119664
119682
476451
Major (9)
ATTCCCTAAAAACAAAAAC
0
0
262


 


119665
119683
*435304
Major (10)
GATTCCCTAAAAACAAAAA
41
27
87
















TABLE 37







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs2298967


(nucleobases 125389 to 125409 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ



Stop

Target

in
in
ID


Start Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


125389
125407
*435341
Major (8)
CTTTTCTATTGTCTGTCCC
83
65
89


 


125390
125408
476459
Major (9)
GCTTTTCTATTGTCTGTCC
89
82
263


 


125391
125409
*435305
Major (10)
TGCTTTTCTATTGTCTGTC
92
85
91
















TABLE 38







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


a chimeric antisense oligonucleotide targeted to SNP rs2298969


(nucleobases 125888 to 125906 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


125888
125906
*435890
Minor (10)
AAGGGATGCTGACTTGGGC
91
64
94
















TABLE 39







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs6844859


(nucleobases 130128 to 130148 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


130128
130146
476466
Major (8)
CTTCCTCACTGAGGATGAA
87
64
264


 


130129
130147
476444
Major (9)
CCTTCCTCACTGAGGATGA
92
77
265


 


130130
130148
*435876
Major (10)
ACCTTCCTCACTGAGGATG
94
87
95
















TABLE 40







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs363092


(nucleobases 135671 to 135691 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop



in
in
ID


Site
Site
ISIS No
Target allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


135671
135689
476464
Major (8)
AACCACTTTGGGATGAATA
51
71
266


 


135672
135690
476442
Major (9)
AAACCACTTTGGGATGAAT
58
59
267


 


135673
135691
*435897
Minor (10)
CAAACCACTTTGGGATGAA
48
78
98
















TABLE 41







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs363088


(nucleobases 149972 to 149992 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


149972
149990
476476
Major (8)
ACAGCTATCTTCTCATCAA
90
65
268


 


149973
149991
476460
Major (9)
CACAGCTATCTTCTCATCA
86
39
269


 


149974
149992
*435871
Major (10)
TCACAGCTATCTTCTCATC
91
54
101
















TABLE 42







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs916171


(nucleobases 156457 to 156477 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop



in
in
ID


Site
Site
ISIS No
Target allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


156457
156475
476465
Major (8)
GAACAAAGAGAAGAATTTC
38
0
270


 


156458
156476
476443
Major (9)
AGAACAAAGAGAAGAATTT
58
0
271


 


156459
156477
*435881 
Major (10)
CAGAACAAAGAGAAGAATT
59
16
105
















TABLE 43







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362275


(nucleobases 164244 to 164264 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop



in
in
ID


Site
Site
ISIS No
Target allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


164244
164262
476473
Major (8)
GAAGCCTGATAAAATCTCT
83
51
272


 


164245
164263
476454
Major (9)
AGAAGCCTGATAAAATCTC
79
61
273


 


164246
164264
*435868 
Major (10)
AAGAAGCCTGATAAAATCT
69
56
111
















TABLE 44







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362273


(nucleobases 167061 to 167081 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


167061
167079
463568
Major (8)
TGATCTGTAGCAGCAGCTT
96
78
274


 


167062
167080
463571
Major (9)
TTGATCTGTAGCAGCAGCT
95
86
275


 


167063
167081
463566
Major (10)
GTTGATCTGTAGCAGCAGC
94
78
276
















TABLE 45







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362272


(nucleobases 174622 to 174642 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


174622
174640
*435344 
Major (8)
TAGAGGACGCCGTGCAGGG
78
63
120


 


174623
174641
476456
Major (9)
ATAGAGGACGCCGTGCAGG
87
60
277


 


174624
174642
*435308 
Major (10)
CATAGAGGACGCCGTGCAG
76
48
122
















TABLE 46







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362271


(nucleobases 175160 to 175180 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop



in
in
ID


Site
Site
ISIS No
Target allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


175160
175178
476472
Major (8)
GTGTGTACAGAACCTGCCG
85
52
278


 


175161
175179
476453
Major (9)
CGTGTGTACAGAACCTGCC
88
69
279


 


175162
175180
*435867 
Major (10)
ACGTGTGTACAGAACCTGC
91
80
125
















TABLE 47







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs3775061


(nucleobases 178396 to 178416 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


178396
178414
476475
Major (8)
TTCAGAATGCCTCATCTGG
61
1
280


 


178397
178415
476457
Major (9)
GTTCAGAATGCCTCATCTG
80
50
281


 


178398
178416
*435873 
Major (10)
TGTTCAGAATGCCTCATCT
80
43
127
















TABLE 48







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362296


(nucleobases 186649 to 1786669 of SEQ ID NO: 1)



















%
%








inhibition
inhibition
SEQ


Start
Stop

Target

in
in
ID


Site
Site
ISIS No
allele
Sequence
GM04281
GM02171
NO

















145466
145485
387916
n/a
TCTCTATTGCACATTCCAAG
100
99
6


 


186649
186667
476469
Major (8)
GGACAGGGTGTGCTCTCCG
80
58
282


 


186650
186668
476449
Major (9)
GGGACAGGGTGTGCTCTCC
80
64
283


 


186651
186669
*435882 
Major (10)
GGGGACAGGGTGTGCTCTC
61
61
155









Example 8
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the studies described in Example 7 were selected and tested at various doses in GM04281, GM02171, and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 750 nM, 1,500 nM, 3,000 nM, 6,000 nM, and 12,000 nM concentrations of antisense oligonucleotide, as specified in Tables 49, 50, and 51. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 49, 50, and 51.







TABLE 49







Dose-dependent antisense inhibition of human HTT in GM04281 cells













ISIS





IC50


No.
750 nM
1500 nM
3000 nM
6000 nM
12000 nM
(μM)
















387916
67
88
95
97
99
<0.8


463566
25
65
79
88
95
1.5


463567
34
73
90
93
98
1.1


463568
33
56
75
87
92
1.3


463571
32
21
70
90
93
1.4


476441
11
27
50
70
87
3.1


476444
20
31
68
49
93
2.3


476449
4
28
34
47
77
4.9


476453
21
21
48
73
85
2.7


476455
5
19
34
56
80
4.6


476458
36
72
83
93
96
1.1


476459
23
59
75
85
91
1.5


476469
17
27
47
47
67
5.5


476473
0
6
32
50
68
6.2


476476
3
7
32
53
86
4.9
















TABLE 50







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS





IC50


No.
750 nM
1500 nM
3000 nM
6000 nM
12000 nM
(μM)
















387916
59
79
93
98
98
<0.8


463566
4
33
42
62
79
3.8


463567
38
41
69
85
94
1.5


463568
21
26
41
58
64
4.8


463571
8
23
56
63
75
3.7


476441
0
13
7
0
12
>12.0


476444
11
0
0
67
59
8.8


476449
4
27
37
51
63
5.8


476453
6
40
40
51
73
4.9


476455
32
15
18
47
61
7.8


476458
42
54
71
86
84
1.2


476459
22
38
70
44
73
4.3


476469
7
24
30
56
58
7.8


476473
4
10
15
33
43
>12.0


476476
5
16
18
23
41
>12.0
















TABLE 51







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS





IC50


No.
750 nM
1500 nM
3000 nM
6000 nM
12000 nM
(μM)
















387916
66
89
95
97
99
<0.8


463566
32
55
76
77
93
1.3


463567
51
61
87
94
97
0.7


463568
26
23
72
87
94
1.6


463571
32
34
60
86
94
1.9


476441
18
18
27
47
44
>12.0


476444
15
0
31
51
58
7.1


476449
27
33
56
80
81
2.6


476453
24
28
55
75
83
2.7


476455
24
26
52
55
73
3.7


476458
63
77
87
89
94
0.2


476459
37
55
56
62
86
1.5


476469
22
41
40
63
76
2.9


476473
7
28
33
51
73
5.0


476476
11
29
26
55
69
4.6









Example 9
Antisense Inhibition of Human HTT in GM04281 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on the gapmers selected from studies described in Example 4. These gapmers were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Tables 8, 9, and 10. Gapmers were also created with 3-9-3 or 5-9-5 motifs, and with constrained 6(S)—CH3-bicyclic nucleic acid (BNA) molecules at various nucleoside positions.
These gapmers were tested in vitro. Cultured GM04281 cells at a density of 25,000 cells per well were transfected using electroporation with 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. HIT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells.
The chimeric antisense oligonucleotides in Tables 52-56 were designed as 3-9-3 or 5-9-5 gapmers. The parent gapmers, ISIS 435869, ISIS 435870, ISIS 435874, ISIS 435879, and ISIS 435890, from which the newly designed gapmers were derived are marked with an asterisk (*) in the table. ISIS 387916 was included in the study as a benchmark oligonucleotide against which the potency of the antisense oligonucleotides targeting nucleotides overlapping each SNP position could be compared.
The 3-9-3 gapmers are 15 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleosides and is flanked on both 5′ and 3′ directions by wings comprising 3 sugar modified nucleosides each.
The 5-9-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleosides and is flanked on both 5′ and 3′ directions by wings comprising 5 sugar modified nucleosides each.
The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methylcytosines. Bolded and underlined nucleotides in Tables 52-56 indicate the positions of the 6(S)—CH3-BNA molecules (e.g. cEt molecules) in each gapmer. Italicized nucleotides are MOE subunits.
“Start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. ‘Target allele’ indicates whether the gapmer is targeted to the major or the minor allele. The number in parentheses indicates the position on the oligonucleotide opposite to the SNP position.







TABLE 52







Comparison of inhibition of human HTT mRNA levels by ISIS 387916 and


chimeric antisense oligonucleotides targeted to SNP rs4690072 (nucleobases


62147 to 62173 of SEQ ID NO: 1)














Start
Stop
Target



%
SEQ


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
ID NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
97
6


 


62147
62165
Major (6)
460266
GTGCTACCCAACCTTTCTG
5-9-5
63
169


 


62151
62169
Major (10)
*435874 
CACAGTGCTACCCAACCTT
5-9-5
50
28


 


62151
62169
Major (10)
460213
CACAGTGCTACCCAACCTT
5-9-5
22
28


 


62151
62169
Major (10)
460220
CACAGTGCTACCCAACCTT
5-9-5
24
28


 


62151
62169
Major (10)
460221
CACAGTGCTACCCAACCTT
5-9-5
28
28


 


62153
62167
Major (8)
460208
CAGTGCTACCCAACC
3-9-3
81
177


 


62155
62173
Major (14)
460267
ATATCACAGTGCTACCCAA
5-9-5
37
180
















TABLE 53







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs2298969


(nucleobases 125884 to 125910 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
97
6


 


125884
125902
Minor (6)
460233
GATGCTGACTTGGGCCATT
5-9-5
76
182


 


125888
125906
Minor (10)
*435890 
AAGGGATGCTGACTTGGGC
5-9-5
75
94


 


125888
125906
Minor (10)
460215
AAGGGATGCTGACTTGGGC
5-9-5
26
94


 


125888
125906
Minor (10)
460224
AAGGGATGCTGACTTGGGC
5-9-5
38
94


 


125888
125906
Minor (10)
460225
AAGGGATGCTGACTTGGGC
5-9-5
49
94


 


125890
125904
Minor (8)
460210
GGGATGCTGACTTGG
3-9-3
97
189


 


125892
125910
Minor (14)
460229
TGCCAAGGGATGCTGACTT
5-9-5
60
192
















TABLE 54







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs7685686


(nucleobases 146782 to 146808 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
97
6


 


146782
146800
Major (6)
460232
AATTGTCATCACCAGAAAA
5-9-5
82
195


 


146786
146804
Major (10)
*435879 
AATAAATTGTCATCACCAG
5-9-5
84
99


 


146786
146804
Major (10)
460214
AATAAATTGTCATCACCAG
5-9-5
33
99


 


146786
146804
Major (10)
460222
AATAAATTGTCATCACCAG
5-9-5
87
99


 


146786
146804
Major (10)
460223
AATAAATTGTCATCACCAG
5-9-5
75
99


 


146788
146802
Major (8)
460209
TAAATTGTCATCACC
3-9-3
96
203


 


146790
146808
Major (14)
460228
TATTAATAAATTGTCATCA
5-9-5
0
206
















TABLE 55







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362331


(nucleobases 155475 to 155501 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
97
6


 


155475
155493
Major (6)
460231
ACAGTAGATGAGGGAGCAG
5-9-5
88
209


 


155479
155497
Major (10)
*435870 
GCACACAGTAGATGAGGGA
5-9-5
86
103


 


155479
155497
Major (10)
460212
GCACACAGTAGATGAGGGA
5-9-5
89
103


 


155479
155497
Major (10)
460218
GCACACAGTAGATGAGGGA
5-9-5
90
103


 


155479
155497
Major (10)
460219
GCACACAGTAGATGAGGGA
5-9-5
88
103


 


155481
155495
Major (8)
460207
ACACAGTAGATGAGG
3-9-3
89
217


 


155483
155501
Major (14)
460227
AAGTGCACACAGTAGATGA
5-9-5
45
220
















TABLE 56







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362306


(nucleobases 181740 to 181766 of SEQ ID NO: 1)





















SEQ


Start
Stop
Target



%
ID


Site
Site
allele
ISIS No.
Sequence
Motif
inhibition
NO

















145466
145485
n/a
387916
TCTCTATTGCACATTCCAAG
5-10-5
97
6


 


181740
181758
Major (6)
460230
AGCTGCAACCTGGCAACAA
5-9-5
66
223


 


181744
181762
Major (10)
*435869 
GAGCAGCTGCAACCTGGCA
5-9-5
69
149


 


181744
181762
Major (10)
460211
GAGCAGCTGCAACCTGGCA
5-9-5
22
149


 


181744
181762
Major (10)
460216
GAGCAGCTGCAACCTGGCA
5-9-5
18
149


 


181744
181762
Major (10)
460217
GAGCAGCTGCAACCTGGCA
5-9-5
56
149


 


181746
181760
Major (8)
460206
GCAGCTGCAACCTGG
3-9-3
83
231


 


181748
181766
Major (14)
460226
GCAAGAGCAGCTGCAACCT
5-9-5
51
234









Example 10
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Conch Fibroblast Cell Lines
Gapmers from studies described in Example 9 were selected and tested at various doses in GM04281, GM02171 and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 312.5 nM, 625 nM, 1,250 nM, 2,500 nM, 5,000 nM and 10,000 nM concentrations of antisense oligonucleotide, as specified in Tables 75, 58, and 59. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 57, 58, and 59.







TABLE 57







Dose-dependent antisense inhibition of human HTT in GM04281 cells














ISIS
312.5
625
1,250
2,500


IC50


No.
nM
nM
nM
nM
5,000 nM
10,000 nM
(μM)

















387916
26
49
68
86
94
97
0.7


435869
0
0
23
48
62
82
3.2


435870
15
38
50
65
85
88
1.3


435874
14
22
32
49
65
73
2.7


435879
0
17
40
61
83
94
1.8


435890
5
13
37
56
70
82
2.3


460206
10
18
37
52
66
85
2.3


460207
20
27
50
65
80
91
1.4


460208
21
34
51
63
70
79
1.5


460209
52
74
89
94
94
95
0.2


460210
34
61
84
91
97
98
0.5


460212
13
31
50
62
75
82
1.6


460218
14
27
50
63
78
86
1.8


460219
9
32
42
64
77
87
1.6


460222
19
21
42
57
73
78
1.7


460231
12
24
41
57
71
84
1.9


460233
16
28
59
66
72
74
1.8


460266
4
17
32
48
60
75
2.9
















TABLE 58







Dose-dependent antisense inhibition of human HTT in GM02171 cells














ISIS
312.5



5,000
10,000
IC50


No.
nM
625 nM
1,250 nM
2,500 nM
nM
nM
(μM)

















387916
32
56
77
89
95
97
0.7


435869
0
6
22
40
69
84
2.9


435870
15
19
32
51
68
77
2.4


435874
0
5
1
17
17
30
>10.0


435879
0
8
0
16
36
47
15.3


435890
14
16
19
19
39
57
9.3


460206
5
13
33
41
68
80
2.7


460207
13
10
22
22
33
39
45.6


460208
13
15
11
11
15
53
10.8


460209
8
27
46
70
80
86
1.6


460210
19
37
55
75
88
96
1.1


460212
8
23
30
43
57
74
2.2


460218
15
26
27
36
52
78
3.2


460219
16
17
32
44
69
76
2.5


460222
14
3
0
0
13
0
>10.0


460231
6
8
13
16
33
56
10.4


460233
27
30
39
46
61
73
2.4


460266
0
15
20
15
18
34
>10.0
















TABLE 59







Dose-dependent antisense inhibition of human HTT in GM02173B cells














ISIS



2,500
5,000
10,000
IC50


No.
312.5 nM
625 nM
1,250 nM
nM
nM
nM
(μM)

















387916
22
47
76
88
96
98
0.7


435869
10
0
16
38
59
76
3.9


435870
22
36
44
58
69
81
2.0


435874
11
6
25
23
32
42
>10.0


435879
0
9
21
30
52
68
4.8


435890
12
16
30
31
48
66
4.5


460206
11
13
18
35
59
74
3.5


460207
15
25
30
37
42
66
4.3


460208
5
14
27
32
52
51
9.0


460209
27
49
61
79
81
74
0.8


460210
19
40
61
77
89
95
1.0


460212
0
19
32
32
61
78
2.9


460218
4
17
26
38
64
82
3.0


460219
5
6
26
47
68
84
2.9


460222
13
19
23
30
35
50
16.1


460231
7
33
25
35
54
77
3.7


460233
11
20
37
52
68
69
2.3


460266
12
6
10
21
25
47
>10.0









Example 11
Dose-Dependent Antisense Inhibition of Human HTT in GM04281 and GM02171 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on the gapmers selected from studies described in Example 10. These gapmers were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Tables 57, 58, and 59. Gapmers were also created with 4-9-4 MOE or 5-9-5 MOE motifs, and with constrained 6(S)—CH3-bicyclic nucleic acid (BNA) molecules at various nucleotide positions.
These gapmers were tested in the GM04281 and GM02171 cell lines. Cultured GM04281 or GM02171 cells at a density of 25,000 cells per well were transfected using electroporation with 2,500 nM or 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells.
The chimeric antisense oligonucleotides in Tables 60, 61, and 62 were designed as 3-9-3, 4-9-4, or 5-9-5 MOE gapmers. The parent gapmers, ISIS 435890, ISIS 460210, ISIS 435879, ISIS 460209, ISIS 435870, and ISIS 460207, from which the newly designed gapmers were derived are marked with an asterisk (*) in the table. ISIS 387916 was included in the study as a benchmark oligonucleotide against which the potency of the antisense oligonucleotides targeting nucleotides overlapping each SNP position could be compared.
The 3-9-3 gapmers are 15 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 3 nucleotides each.
The 4-9-4 gapmers are 17 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 4 nucleotides each.
The 5-9-5 gapmers are 19 nucleotides in length, wherein the central gap segment is comprised of nine 2′-deoxynucleotides and is flanked on both 5′ and 3′ directions by wings comprising 5 nucleotides each.
The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methylcytosines. Bolded and underlined nucleotides in Tables 60, 61, and 62 indicate the positions of the 6(S)—CH3-BNA (e.g. cEt molecules) molecules in each gapmer. Italicized nucleotides are MOE subunits.
The gapmers are organized in Tables 60, 61, and 62, according to the SNP site they target. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. ‘Target allele’ indicates whether the gapmer is targeted to the major or the minor allele. The number in parentheses indicates the position on the oligonucleotide opposite to the SNP position.







TABLE 60







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs2298969


(nucleobases 125888 to 125907 of SEQ ID NO: 1)





















%
%









inhibition
inhibition
SEQ


Start
Stop



Concentration
in
in
ID


position
position
ISIS No.
Sequence
Motif
(nM)
GM04281
GM02171
NO


















145466
145485
387916
TCTCTATTGCA
5-10-5
5000
57
24
6





CATTCCAAG


 


125888
125907
*435890 
AAGGGATGCTG
5-9-5
2500
22
0
94





ACTTGGGC

5000
41
23


 


125890
125904
*460210 
GGGATGCTGAC
3-9-3
2500
59
24
189





TTGG

5000
81
33


 


125889
125905
474870
AGGGATGCTG
4-9-4
2500
23
3
187





ACTTGGG

5000
44
34


 


125889
125905
474890
AGGGATGCTG
4-9-4
2500
38
6
187





ACTTGGG

5000
49
25


 


125889
125905
474910
AGGGATGCTGA
4-9-4
2500
34
8
187





CTTGGG

5000
49
41


 


125889
125905
474914
AGGGATGCTGA
4-9-4
2500
44
14
187





CTTGGG

5000
44
21


 


125888
125907
474918
AAGGGATGCT
5-9-5
2500
31
0
94





GACTTGGGC

5000
26
25


 


125888
125907
474922
AAGGGATGCT
5-9-5
2500
33
14
94





GACTTGGGC

5000
65
24


 


125889
125905
476332
AGGGATGCTG
4-9-4
2500
23
13
187





ACTTGGG

5000
51
42


 


125888
125907
476336
AAGGGATGCTG
5-9-5
2500
5
0
94





ACTTGGGC

5000
43
9
















TABLE 61







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs7685686


(nucleobases 146786 to 146805 of SEQ ID NO: 1)





















%
%









inhibition
inhibition
SEQ


Start
Stop



Concentration
in
in
ID


position
position
ISIS No.
Sequence
Motif
(nM)
GM04281
GM02171
NO


















145466
145485
387916
TCTCTATTGCA
5-10-5
5000
57
24
6





CATTCCAAG


 


146786
146805
*435879 
AATAAATTGTC
5-9-5
2500
39
0
99





ATCACCAG

5000
59
19


 


146788
146802
*460209 
TAAATTGTCAT
3-9-3
2500
3
0
203





CACC

5000
13
5


 


146787
146803
474871
ATAAATTGTCA
4-9-4
2500
82
32
200





TCACCA

5000
83
58


 


146787
146803
474891
ATAAATTGTCA
4-9-4
2500
84
29
200





TCACCA

5000
89
56


 


146787
146803
474911
ATAAATTGTCA
4-9-4
2500
70
18
200





TCACCA

5000
83
40


 


146787
146803
474915
ATAAATTGTCA
4-9-4
2500
38
9
200





TCACCA

5000
74
14


 


146786
146805
474919
AATAAATTGTC
5-9-5
2500
80
7
99





ATCACCAG

5000
84
37


 


146786
146805
474923
AATAAATTGTC
5-9-5
2500
74
32
99





ATCACCAG

5000
83
51


 


146787
146803
476333
ATAAATTGTCA
4-9-4
2500
75
28
200





TCACCA

5000
86
21


 


146786
146805
476337
AATAAATTGTC
5-9-5
2500
71
6
99





ATCACCAG

5000
83
31
















TABLE 62







Comparison of inhibition of human HTT mRNA levels by ISIS 387916


and chimeric antisense oligonucleotides targeted to SNP rs362331


(nucleobases 155478 to 155498 of SEQ ID NO: 1)





















%
%









inhibition
inhibition
SEQ


Start
Stop



Concentration
in
in
ID


position
position
ISIS No.
Sequence
Motif
(nM)
GM04281
GM02171
NO


















145466
145485
387916
TCTCTATTGCAC
5-10-5
5000
57
24
6





ATTCCAAG


 


155479
155498
*435870 
GCACACAGTAG
5-9-5
2500
19
1
103





ATGAGGGA

5000
49
34


 


155481
155495
*460207 
ACACAGTAGAT
3-9-3
2500
0
0
217





GAGG

5000
7
8


 


155480
155496
474872
CACACAGTAGA
4-9-4
2500
35
9
214





TGAGGG

5000
63
37


 


155480
155496
474892
CACACAGTAGA
4-9-4
2500
43
16
214





TGAGGG

5000
69
31


 


155480
155496
474912
CACACAGTAGA
4-9-4
2500
16
9
214





TGAGGG

5000
36
6


 


155480
155496
474916
CACACAGTAGA
4-9-4
2500
22
5
214





TGAGGG

5000
47
7


 


155479
155498
474920
GCACACAGTAG
5-9-5
2500
19
0
103





ATGAGGGA

5000
43
23


 


155479
155498
474924
GCACACAGTAG
5-9-5
2500
29
8
103





ATGAGGGA

5000
48
22


 


155480
155496
476334
CACACAGTAGA
4-9-4
2500
35
7
214





TGAGGG

5000
62
32


 


155479
155498
476338
GCACACAGTAG
5-9-5
2500
26
9
103





ATGAGGGA

5000
40
4


 


155479
155495
474873
ACACAGTAGAT
4-9-4
2500
53
9
285





GAGGGA

5000
61
29


 


155479
155495
474893
ACACAGTAGAT
4-9-4
2500
47
5
285





GAGGGA

5000
59
30


 


155479
155495
474913
ACACAGTAGAT
4-9-4
2500
30
16
285





GAGGGA

5000
29
17


 


155479
155495
474917
ACACAGTAGAT
4-9-4
2500
23
12
285





GAGGGA

5000
40
5


 


155478
155497
474921
CACACAGTAGA
5-9-5
2500
28
0
212





TGAGGGAG

5000
43
23


 


155478
155497
474925
CACACAGTAGA
5-9-5
2500
30
9
212





TGAGGGAG

5000
61
34


 


155479
155495
476335
ACACAGTAGAT
4-9-4
2500
35
2
285





GAGGGA

5000
53
31


 


155478
155497
476339
CACACAGTAGA
5-9-5
2500
15
0
212





TGAGGGAG

5000
34
13









Example 12
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the studies described in Example 11 were selected and tested at various doses in GM04281, GM02171 and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 625 nM, 1,250 nM, 2,500 nM, 5,000 nM and 10,000 nM concentrations of antisense oligonucleotide, as specified in Tables 63, 64, and 65. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 63, 64, and 65.







TABLE 63







Dose-dependent antisense inhibition of human HTT in GM04281 cells













ISIS




10000
IC50


No
625 nM
1250 nM
2500 nM
5000 nM
nM
(μM)
















387916
70
83
94
96
98
<0.6


460207
51
63
83
91
93
0.5


460209
83
93
96
97
97
<0.6


460210
70
89
94
97
98
0.6


474871
94
97
96
96
95
<0.6


474873
51
73
89
94
95
0.5


474891
93
95
97
96
95
<0.6


474892
48
72
89
93
95
0.6


474911
85
92
96
95
94
<0.6


474919
89
94
95
94
96
<0.6


474922
21
47
73
86
96
1.5


474923
86
94
96
95
94
<0.6


476333
92
94
95
95
96
<0.6


476334
45
70
87
92
95
0.6


476337
83
92
95
96
96
<0.6
















TABLE 64







Dose-dependent antisense inhibition of human HTT in GM02171 cells













ISIS





IC50


No
625 nM
1250 nM
2500 nM
5000 nM
10000 nM
(μM)
















387916
28
38
63
82
99
1.6


460207
16
0
20
22
55
10.0


460209
27
50
61
87
94
9.9


460210
34
60
80
86
97
0.9


474871
62
74
84
87
90
0.1


474873
13
29
61
77
89
2.2


474891
57
72
80
83
88
0.2


474892
23
26
51
68
81
2.5


474911
47
58
68
72
82
0.7


474919
44
48
65
71
83
1.1


474922
15
27
49
74
79
2.6


474923
27
53
74
79
84
1.5


476333
42
53
75
76
84
1.0


476334
20
23
58
71
87
2.3


476337
23
34
60
62
75
2.7
















TABLE 65







Dose-dependent antisense inhibition of human HTT in GM02173B cells



















IC50


ISIS No
625 nM
1250 nM
2500 nM
5000 nM
10000 nM
(μM)
















387916
38
75
89
95
99
0.9


460207
13
27
52
46
63
6.5


460209
79
68
84
90
92
<0.6


460210
37
62
79
92
97
0.9


474871
74
83
87
92
89
<0.6


474873
22
32
67
72
92
1.9


474891
69
78
84
89
89
<0.6


474892
26
50
75
83
91
1.3


474911
50
66
76
86
86
0.6


474919
57
67
74
87
82
<0.6


474922
15
32
61
71
90
2.2


474923
49
67
78
83
85
0.5


476333
58
71
78
87
89
<0.6


476334
20
42
63
76
91
1.8


476337
48
63
71
79
80
0.6









Example 13
Strategy for Selection of Antisense Oligonucleotides Based on Potency and Selectivity
Gapmers from each of the studies described above were selected for further analysis based on potency and selectivity.
Potency was based on the percent inhibition of HTT mRNA achieved by the antisense oligonucleotides targeting a SNP compared to the percent inhibition of HTT mRNA achieved by the benchmark oligonucleotide, ISIS 387916.
Selectivity was based on the ability of the antisense oligonucleotides targeting a SNP to inhibit expression of the major allele and not of the minor allele. The usage of the three cell lines with different genotypes at each SNP position facilitated this process.
ISIS 460065 (5′-ATAAATTGTCATCACCAG-3′ (SEQ ID NO: 199)) is a 4-9-5 MOE gapmer targeted to SNP rs7685686 (major allele A, minor allele G) at position 9 of the oligonucleotide. The GM04281 cell line is homozygous AA at SNP position rs7685686. The GM02173B cell line is heterozygous AG at SNP position rs7685686. The GM02171 cell line is homozygous GG at SNP position rs7685686. Therefore, selectivity is shown if ISIS 460065 causes potent inhibition of HTT mRNA in GM04281, less potent inhibition of HTT mRNA in GM02173, and little to no significant inhibition of HTT mRNA in GM02171. IC50 values taken from Table 20, 21, and 22, and presented below in Table 66, confirm varying degrees of inhibition in the three cell lines, wherein expression was most reduced in the homozygous AA cell line, moderately reduced in the heterozygous AG cell line, and less reduced in the homozygous GG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 66







Genotype of the Coriell cell lines for SNP rs7685686 and comparison


of inhibition of HTT mRNA by ISIS 460065 in each cell line











GM04281
GM02173B
GM02171
















Genotype
AA
AG
GG



IC50 with ISIS
1.1
3.6
10.3



460065










ISIS 459978 (5′-ACAGTGCTACCCAACCT-3′ (SEQ ID NO: 174)) is a 2-9-6 MOE gapmer targeted to SNP rs4690072 (major allele T, minor allele G) at position 9 of the oligonucleotide. The GM04281 cell line is homozygous TT at SNP position rs4690072. The GM02173B cell line is heterozygous TG at SNP position rs4690072. The GM02171 cell line is homozygous GG at SNP position rs4690072. Therefore, selectivity is shown if ISIS 459978 causes potent inhibition of HTT mRNA in GM04281, less potent inhibition of HTT mRNA in GM02173, and little to no significant inhibition of HTT mRNA in GM02171. IC50 values taken from Table 20, 21, and 22, and presented below in Table 67, confirm varying degrees of inhibition in the three cell lines, wherein expression was most reduced in the homozygous TT cell line, moderately reduced in the heterozygous TG cell line, and less reduced in the homozygous GG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 67







Genotype of the Coriell cell lines for SNP rs4690072 and comparison


of inhibition of HTT mRNA by ISIS 459978 in each cell line











GM04281
GM02173B
GM02171
















Genotype
TT
TG
GG



IC50 with ISIS
2.5
8.4
12.7



459978










ISIS 460028 (5′-GAGCAGCTGCAACCTGGCA-3′ (SEQ ID NO: 149)) is a 4-11-4 MOE gapmer targeted to SNP rs362306 (major allele G, minor allele A) at position 10 of the oligonucleotide. The GM04281 cell line is homozygous GG at SNP position rs362306. The GM02173B and GM02171 cell lines are heterozygous GA at SNP position rs362306. Therefore, selectivity is shown if ISIS 460028 causes potent inhibition of HTT mRNA in GM04281 and less potent inhibition of HTT mRNA in GM02173 and GM02171. IC50 values taken from Table 20, 21, and 22, and presented below in Table 68, confirm varying degrees of inhibition between the GM04281 cell line and the GM02173B and GM02171 cell lines, wherein expression was most reduced in the homozygous GG cell line and less reduced in the heterozygous AG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 68







Genotype of the Coriell cell lines for SNP rs362306 and comparison


of inhibition of HTT mRNA by ISIS 460028 in each cell line











GM04281
GM02173B
GM02171
















Genotype
GG
AG
AG



IC50 with ISIS
1.4
5.2
5.3



460028










Example 14
Strategy for Selection of Antisense Oligonucleotides with cEt Motifs Based on Potency and Selectivity
Gapmers from each of the studies described above were selected for further analysis based on potency and selectivity.
Potency was based on the percent inhibition of HTT mRNA achieved by the antisense oligonucleotides targeting a SNP compared to the percent inhibition of HTT mRNA achieved by the benchmark oligonucleotide, ISIS 387916.
Selectivity was based on the ability of the antisense oligonucleotides targeting a SNP to inhibit expression of the major allele and not of the minor allele. The usage of the three cell lines with different genotypes at each SNP position facilitated this process.
ISIS 460209 (5′-TAAATTGTCATCACC-3′ (SEQ ID NO: 203)) is a 3-9-3 gapmer with cEt subunits at positions 2, 3, 13, and 14, targeted to SNP rs7685686 (major allele A, minor allele G) at position 8 of the oligonucleotide. The GM04281 cell line is homozygous AA at SNP position rs7685686. The GM02173B cell line is heterozygous AG at SNP position rs7685686. The GM02171 cell line is homozygous GG at SNP position rs7685686. Therefore, selectivity is shown if ISIS 460209 causes potent inhibition of HTT mRNA in GM04281, less potent inhibition of HTT mRNA in GM02173, and little to no significant inhibition of HTT mRNA in GM02171. IC50 values taken from Table 57, 58, and 59, and presented below in Table 69, confirm varying degrees of inhibition in the three cell lines, wherein expression was most reduced in the homozygous AA cell line, moderately reduced in the heterozygous AG cell line, and less reduced in the homozygous GG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 69







Genotype of the Coriell cell lines for SNP rs7685686 and comparison


of inhibition of HTT mRNA by ISIS 460209 in each cell line











GM04281
GM02173B
GM02171
















Genotype
AA
AG
GG



IC50 with ISIS
0.2
0.8
1.6



460209










ISIS 460208 (5′-CAGTGCTACCCAACC-3′ (SEQ ID NO: 177)) is a 3-9-3 gapmer with cEt subunits at positions 2, 3, 13, and 14, targeted to SNP rs4690072 (major allele T, minor allele G) at position 8 of the oligonucleotide. The GM04281 cell line is homozygous TT at SNP position rs4690072. The GM02173B cell line is heterozygous TG at SNP position rs4690072. The GM02171 cell line is homozygous GG at SNP position rs4690072. Therefore, selectivity is shown if ISIS 460208 causes potent inhibition of HTT mRNA in GM04281, less potent inhibition of HTT mRNA in GM02173, and little to no significant inhibition of HTT mRNA in GM02171. IC50 values taken from Table 57, 58, and 59, and presented below in Table 70, confirm varying degrees of inhibition in the three cell lines, wherein expression was most reduced in the homozygous TT cell line, moderately reduced in the heterozygous TG cell line, and less reduced in the homozygous GG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 70







Genotype of the Coriell cell lines for SNP rs4690072 and comparison


of inhibition of HTT mRNA by ISIS 460208 in each cell line











GM04281
GM02173B
GM02171
















Genotype
TT
TG
GG



IC50 with ISIS
1.5
9.0
10.8



460208










ISIS 460206 (5′-GCAGCTGCAACCTGG-3′ (SEQ ID NO: 231)) is a 3-9-3 gapmer with cEt subunits at positions 2, 3, 13, and 14, targeted to SNP rs362306 (major allele G, minor allele A) at position 8 of the oligonucleotide. The GM04281 cell line is homozygous GG at SNP position rs362306. The GM02173B and GM02171 cell lines are heterozygous GA at SNP position rs362306. Therefore, selectivity is shown if ISIS 460206 causes potent inhibition of HTT mRNA in GM04281 and less potent inhibition of HTT mRNA in GM02173 and GM02171. IC50 values taken from Table 57, 58, and 59, and presented below in Table 71, confirm varying degrees of inhibition between the GM04281 cell line and the GM02173B and GM02171 cell lines, wherein expression was most reduced in the homozygous GG cell line and less reduced in the heterozygous AG cell line. IC50 is the concentration of antisense oligonucleotide required for 50 percent inhibition HTT mRNA. IC50 values are in μM.







TABLE 71







Genotype of the Coriell cell lines for SNP rs362306 and comparison


of inhibition of HTT mRNA by ISIS 460206 in each cell line











GM04281
GM02173B
GM02171
















Genotype
GG
AG
AG



IC50 with ISIS
2.3
2.7
2.7



460206










Example 15
Comparison of SNPs in Various Cell Lines and Mouse Models Associated with Huntington's Disease
The genotype at various SNP positions associated with Huntington's disease was compared amongst the three Corriell cell lines, used in the above Examples, as well as with the GM04022 fibroblast, the BACHD mouse model and the YAC 18 mouse model.
The donor patient of the GM04022 fibroblast cell line was heterozygous at SNP position rs363125 (NCBI Entrez SNP database), harboring an A allele (adenine) and a C allele (cytosine) at nucleotide 5310 of SEQ ID NO: 2 (van Bilsen, P. H. J. et al., Human Gene Therapy. 19: 710-718, 2008). YAC18 mice were developed with a YAC transgene containing human huntingtin gene (Hodgson, et al. Hum. Mol. Genet. 5: 1875-85, 1996). BACHD mice were developed expressing a full-length mutant huntingtin gene with 97 glutamine repeats under the control of a bacterial artificial chromosome (Gray, M. et al., J. Neurosc. 28: 6182-95, 2008). The comparative genotype at the indicated SNP positions in all four cell lines and mouse models is presented in Table 72.







TABLE 72







Genotypes of the Coriell cell lines and Huntington mouse models













SNP
GM02171
GM02173
GM04281
GM04022
BACHD
YAC18





rs3856973
AA
AG
GG
AG
GG
AA


rs2285086
GG
AG
AA
AG
AA
GG


rs7659144
CG
CG
CC
CG
CC
GG


rs16843804
TC
TC
CC
CC
CC
TT


rs2024115
GG
AG
AA
AG
AA
GG


rs3733217
CC
CC
CC
CC
CC
CC


rs10015979
AA
AG
GG
AA
AA
AA


rs7691627
AA
AG
GG
AG
GG
AA


rs2798235
GG
GG
GG
AG
GG
GG


rs4690072
GG
TG
TT
TG
TT
GG


rs6446723
CC
TC
TT
TC
TT
CC


rs363081
GG
GG
GG
GG
GG
GG


rs363080
CC
CC
CC
TC
CC
CC


rs363075
GG
GG
GG
GG
GG
GG


rs363064
TC
TC
CC
CC
CC
TT


rs3025849
AA
AA
AA
AA
AA
AA


rs363102
AA
AA
AA
AG
AA
AA


rs11731237
CC
TC
TT
CC
CC
CC


rs4690073
AA
AG
GG
AG
GG
AA


rs363144
TT
TT
TT
TT
TT
TT


rs3025838
CC
CC
CC
CC
CC
CC


rs34315806
TC
TC
CC
CC
CC
TT


rs363099
TC
TC
CC
CC
CC
TT


rs363096
CC
TC
TT
CC
TT
CC


rs2298967
TC
TC
TT
TT
TT
CC


rs2298969
GG
AG
AA
AG
AA
GG


rs6844859
CC
TC
TT
TC
TT
CC


rs363092
AA
AC
CC
AC
AA
AA


rs7685686
GG
AG
AA
AG
AA
GG


rs363088
TA
TA
AA
AA
AA
TT


rs362331
CC
TC
TT
TC
TT
CC


rs916171
GG
GC
CC
GC
CC
GG


rs362322
AA
AA
AA
AA
AA
AA


rs362275
TC
TC
CC
CC
CC
TT


rs362273
AG
AG
AA
AA
AA
GG


rs2276881
GG
GG
GG
GG
GG
GG


rs3121419
TC
TC
CC
CC
CC
TT


rs362272
—
AG
GG
GG
GG
AA


rs362271
AG
AG
GG
GG
GG
AA


rs3775061
AG
AG
AA
AA
AA
GG


rs362310
TC
CC
CC
TC
CC
CC


rs362307
CC
TC
CC
CC
CC
CC


rs362306
AG
AG
GG
GG
GG
AA


rs362303
TC
CC
CC
TC
CC
CC


rs362296
AC
AC
AC
CC
CC
AA









Example 16
Allele-Specific Inhibition Measured in BacHD Cortical Neurons
Antisense oligonucleotides, ISIS 460209 (5′-TAAATTGTCATCACC-3′ (SEQ ID NO: 203)), targeting SNP rs7685686 of human HTT, and ISIS 387916 (TCTCTATTGCACATTCCAAG (SEQ ID NO: 6)), and with no human or murine SNP target site, were tested for their effect on Htt protein levels in vitro. ISIS 387916 is cross-reactive with murine Htt mRNA (GENBANK Accession No. NM—010414.1, designated herein as SEQ ID NO: 286) at target start site 5763 with one mismatch. ISIS 460209 is cross-reactive with murine Htt mRNA at target start site 6866 with three mismatches.
Primary BacHD cortical neurons, which express human Htt and murine Htt, were isolated in the following way: Embryos were dissected from E15.5-E17.5 pregnant females. Cortices were dissected into ice-cold divalent-free Hank's Balanced Salt Solution (Invitrogen, 14025-134). The cortices were chopped into pieces and digested with 0.05% Trypsin-EDTA (Invitrogen, 25300-120) at 37° C. for 8 minutes. The digestion was halted by addition of complete neurobasal media (Invitrogen, 10888-022). Cells were resuspended in media and treated with DNAse I (Invitrogen, 18047-019). After titration through a 100 ul pipette tip, cells are resuspended in neurobasal media with B27 supplement (Invitrogen, 17504-044), and counted. 1.7×105 cells/well were plated in 24-well plates precoated with poly-D-lysine (BD Biosciences, 354210). Neurons were fed with 200 μl neurobasal media with B27 on the second day in vitro.
ISIS 460209 or ISIS 387916 was added to the supplementary media fed to neurons on division 2 at 0.7 μM, 1.4 μM or 1.5 μM final concentrations. Cells were harvested after 8 days with into 1 mL of media using a cell scraper. Cells were centrifuged at 2,500 rpm for 5 min at 4° C. and the pellets were resuspended in a buffer of 50 mM Tris, pH=8.0, 150 mM NaCl, 1% Igepal, 40 mM β-glycerophosphate, 10 mM NaF, 1× Roche complete protease inhibitor, 1 mM Sodium Orthovanadate and 800 μM PMSF. The lysates were centrifuged after 15 min incubation and protein concentration was measured with the DC assay (BioRad).
Protein lysates were run on low-bis gels to separate huntingtin alleles (resolving gel—2001:Acrylamide:BIS (10% acrylamide, 0.5% BIS, 375 mMTris pH 8.8; stacking gel—4% Acrylamide-BIS(29:1), 156 mM Tris pH6.8; Running buffer—25 mM Tris, 190 mM Glycine, 0.1% SDS+10 μM beta-mercaptoethanol added fresh). After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Hybond-C Extra; GE Healthcare Bio-Sciences) at 90V for 40′ to allow samples to penetrate the stacking gel and then at 190V for 2.5 h to resolve proteins.
Primary antibodies specific for human Htt and murine calnexin protein were used at 1:10,000 dilutions. HRP-conjugated anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories) was used for visualizing proteins using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Protein bands were quantified using ImageJ software and normalized to calnexin levels. Protein bands were quantified using ImageJ software. Table 73 provides an estimate of the percentage inhibition relative to the negative control sample. The comparative percent inhibitions of the human Htt protein and the murine Htt protein are presented.







TABLE 73







Effect of antisense inhibition on mutant human and wild-type murine


Htt protein (percent inhibition normalized to PBS control)











Dose





(μM)
Human
Murine
















ISIS 387916
0.7
54
38




1.4
75
58




1.5
92
88



ISIS 460209
0.2
71
35




0.4
82
41




1.5
94
56










Example 17
Dose-Dependent Antisense Inhibition of Human Huntingtin mRNA Levels in Coriell Fibroblast Cell Lines
Gapmers from the studies described in Examples, 3, 4, 10, and 12 were selected and tested at various doses in GM04281, GM02171 and GM02173B cell lines. Each cell line was plated at a density of 25,000 cells per well and transfected using electroporation with 0.4747 nM, 1.5011 nM, 4.7463 nM, 15.0079 nM 45.455 nM, 150.0527 nM, 474.4673 nM, 1,500.27 nM, 4,743.833 nM, and 15,000 nM concentrations of antisense oligonucleotide, as specified in Tables 72, 73, and 74. After a treatment period of approximately 16 hours, RNA was isolated from the cells and HTT mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of HTT mRNA, relative to untreated control cells. IC50 values are also provided in Tables 72, 73, and 74.







TABLE 74







Dose-dependent antisense inhibition of human HTT in GM04281 cells


















ISIS
0.4747
1.5011
4.7463
15.0079
47.455
150.0527
474.4673
1500.27
4743.833
15000.0
IC50


No
nM
nM
nM
nM
nM
nM
nM
nM
nM
nM
(μM)





















387916
15
12
4
5
7
26
70
89
98
99
0.33


435879
0
8
19
13
24
23
45
53
84
93
0.25


435890
16
1
8
12
25
23
32
52
61
91
0.82


460209
2
9
21
17
36
46
80
89
94
93
0.09


460210
4
7
5
19
20
35
69
85
98
98
0.21


476333
7
10
8
11
42
65
86
93
93
95
0.05
















TABLE 75







Dose-dependent antisense inhibition of human HTT in GM02171 cells



















0.4747
1.5011
4.7463
15.0079
47.455
150.0527
474.4673
1500.27
4743.833
15000.0
IC50


ISIS No
nM
nM
nM
nM
nM
nM
nM
nM
nM
nM
(μM)





















387916
22
8
0
9
0
32
60
90
96
97
0.27


435879
0
1
6
2
0
0
8
9
46
57
7.62


435890
0
0
0
6
0
0
0
31
27
71
4.37


460209
11
5
15
0
0
7
30
69
82
88
0.96


460210
0
0
0
2
17
18
38
70
93
95
0.56


476333
0
0
0
0
13
18
44
69
72
91
0.75
















TABLE 76







Dose-dependent antisense inhibition of human HTT in GM02173B cells


















ISIS
0.4747
1.5011
4.7463
15.0079
47.455
150.0527
474.4673
1500.27
4743.833
15000.0
IC50


No
nM
nM
nM
nM
nM
nM
nM
nM
nM
nM
(μM)





















387916
3
17
7
25
27
33
65
88
98
99
0.19


435879
0
6
0
8
3
10
16
24
50
68
3.72


435890
0
13
0
1
2
12
16
23
49
82
4.60


460209
0
7
29
2
9
32
52
71
82
86
0.27


460210
0
13
0
5
16
18
49
74
93
97
0.27


476333
11
13
20
7
23
36
63
75
83
90
0.13









Example 18
Validation of the Specificity of ISIS Oligonucleotides Targeting SNPs of Human Huntingtin by the Molecular Beacon Assay
Some of the gapmers from the study described in Example 17 were tested in GM04022 fibroblasts (from the Coriell Institute for Medical Research).
To verify allele-specific suppression of HTT mRNA in GM04022 fibroblasts by ISIS 435879, ISIS 460209, and ISIS 476333, the Molecular Beacon assay, as described in the van Bilsen at el publication (van Bilsen, P. H. J. et al., Human Gene Therapy. 19: 710-718, 2008), was conducted using ‘molecular beacon’ synthetic oligonucleotides linked with a fluorophore and quencher. GM04022 fibroblasts were transfected by electroporation with ISIS 435879, ISIS 460209, or ISIS 476333 at 0.06 μM, 0.19 μM, 0.56 μM, 1.67 μM, 5 μM and 15 μM concentrations of antisense oligonucleotide, as specified in Tables 75-77. ISIS 387916 was included in the assay as a benchmark oligonucleotide. The qRT-PCR assay for molecular beacon for the A allele was conducted with the annealing temperature at 56.5° C. The qRT-PCR assay for molecular beacon for the C allele was conducted with the annealing temperature at 62.0° C. Primer probe set RTS2617 was used to measure the total HTT mRNA reduction. The results of the assay are presented in Tables 77-79 as percent inhibition over the PBS control. The results demonstrate that the SNP-specific ISIS oligonucleotides specifically target the C allele of rs7685686 compared to the A allele (Table 80).







TABLE 77







Dose-dependent antisense inhibition of the A allele


of rs7685686 in GM04022 fibroblasts















0.06
0.19
0.56
1.67
5.00
15.00
IC50


ISIS No
μM
μM
μM
μM
μM
μM
(μM)

















387916
33
40
53
90
99
98
0.56


435879
0
0
50
29
38
47
10.8


460209
14
4
54
73
81
95
0.53


476333
2
44
41
77
91
86
0.64
















TABLE 78







Dose-dependent antisense inhibition of the C allele of


rs7685686 in GM04022 fibroblasts


















1.67
5.00
15.00
IC50


ISIS No
0.06 μM
0.19 μM
0.56 μM
μM
μM
μM
(μM)

















387916
41
42
46
86
95
92
0.54


435879
0
0
75
60
68
81
2.9


460209
35
48
76
84
88
92
0.19


476333
22
60
75
84
90
93
0.15
















TABLE 79







Dose-dependent antisense inhibition of total HTT


mRNA in GM04022 fibroblasts


















5.00
15.00


ISIS No
0.06 μM
0.19 μM
0.56 μM
1.67 μM
μM
μM
















387916
32
59
49
89
98
99


435879
0
0
42
25
41
62


460209
26
27
54
75
84
96


476333
25
51
58
82
92
90
















TABLE 80







IC50 ratio (A/C) in GM04022 fibroblasts










ISIS No
Ratio







387916
1.0



435879
4.2



460209
2.8



476333
4.3










Example 19
Allele-Specific Inhibition Measured in Cortical Neurons from BacHD and YAC18 Mice
In order to identify potential SNPs for screening of human allele-specific ISIS oligonucleotides, the HTT mRNA of YAC 18 and BACHD mice were sequenced by the Goldengate 96SNP assay. It was determined that the BAC and YAC mice carried different alleles at several key SNP positions (Table 72) and could therefore be used as a screening tool for allele-specific knockdown. Each of the SNP positions chosen for targeting in the mouse strains were also compared to human HD chromosomes. For each target, approximately 50% of the human FID population is heterozygous for the target expressed in the BACHD mice, but not the YAC18 mice.
In order to verify the allele-specificity of the ISIS oligonucleotides (described in Examples 2, 9, 17 and 18), the antisense oligonucleotides, ISIS 460207, targeting SNP rs362331; ISIS 460209, targeting SNP rs7685686; ISIS 435879, targeting SNP rs7685686; ISIS 476333, targeting SNP rs7685686; ISIS 460210, targeting SNP rs2298969; ISIS 435874, targeting SNP rs4690072; ISIS 460208, targeting SNP rs4690072; ISIS 435331, targeting SNP rs2024115; and ISIS 435871, targeting SNP rs363088, were tested for their effect on HTT protein levels in BACHD and YAC18 cortical neurons. ISIS 387916, which has no human or murine SNP target site, was used as the benchmark. ISIS 387916 is cross-reactive with murine HTT mRNA (GENBANK Accession No. NM—010414.1, designated herein as SEQ ID NO: 286) at target start site 5763 with one mismatch. It was expected that treatment with the allele-specific antisense oligonucleotides would cause significant inhibition of HTT mRNA in the BACHD neurons and not in the YAC18 neurons. It was also expected that treatment with ISIS 387916 would cause inhibition of HTT mRNA in both sets of neurons.
YAC18 cultures were prepared from E16.5 pregnant female YAC18 (line 60, +/+) mice who had been bred with YAC18 (line 60, +/+) males. All progeny are thus homozygous YAC18 (line 60), facilitating pooled cortical cultures. BACHD E16.5 embryos were isolated from pregnant BACHD (+/−) mice who had been bred with pregnant BACHD (+/−) male mice, necessitating single pup cultures and genotyping. Single cortices were isolated, using caution to prevent cross-contamination of samples. Each dissociated cortex was used to seed 5 wells of a 6-well plate. After genotyping, only BACHD (+/−) cultures were used for ASO treatment. The antisense oligonucleotides were added to the supplementary media fed to the neurons on division 2. Cells were harvested after 8 days with into 1 mL of media using a cell scraper. Cells were centrifuged at 2,500 rpm for 5 min at 4° C. and the pellets were resuspended in a buffer of 50 mM Tris, pH=8.0, 150 mM NaCl, 1% Igepal, 40 mM (β-glycerophosphate, 10 mM NaF, 1× Roche complete protease inhibitor, 1 mM Sodium Orthovanadate and 800 μM PMSF. The lysates were centrifuged after 15 mM incubation and protein concentration was measured with the DC assay (BioRad).
Protein lysates were run on low-bis gels to separate huntingtin alleles (resolving gel—2001:Acrylamide:BIS (10% acrylamide, 0.5% BIS, 375 mMTris pH 8.8; stacking gel—4% Acrylamide-BIS(29:1), 156 mM Tris pi-16.8; Running buffer—25 mM Tris, 190 mM Glycine, 0.1% SDS+10 μM beta-mercaptoethanol added fresh). After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Hybond-C Extra; GE Healthcare Bio-Sciences) at 90V for 40′ to allow samples to penetrate the stacking gel and then at 190V for 2.5 h to resolve proteins.
Primary antibodies specific for human HTT and murine calnexin protein were used at 1:10,000 dilutions. HRP-conjugated anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch Laboratories) was used for visualizing proteins using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Protein bands were quantified using ImageJ software and normalized to calnexin levels. Tables 81-91 provide the percentage inhibition relative to the untreated control sample. The percentage inhibition of human HTT protein levels in BACHD and YAC18 neurons are presented.







TABLE 81







HTT SNPs in BACHD and YAC18 mice and


correlation with human HTT SNPs












Allele
Allele
Allele present
% of human



present in
present in
in human patients
patients



YAC18
BACHD
with high
heterozgous at


SNP
Mice
Mice
CAG repeats
the SNP position














rs2024115
G
A
A
48


rs2298969
G
A
A
52


rs362331
C
T
T
49


rs363088
G
T
T
38


rs4690072
T
A
A
49


rs7685686
G
A
A
49
















TABLE 82







Effect of antisense inhibition by ISIS


387916 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
69
81



BACHD
84
90

















TABLE 83







Effect of antisense inhibition by ISIS 435331,


targeting rs2024115 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
0
0



BACHD
39
43

















TABLE 84







Effect of antisense inhibition by ISIS 460210,


targeting rs2298969 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
31
51



BACHD
79
89

















TABLE 85







Effect of antisense inhibition by ISIS 460207,


targeting rs362331 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
0
0



BACHD
29
44

















TABLE 86







Effect of antisense inhibition by ISIS 435871,


targeting rs363088 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
0
0



BACHD
51
68

















TABLE 87







Effect of antisense inhibition by ISIS 435874,


targeting rs4690072 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
9
5



BACHD
30
44

















TABLE 88







Effect of antisense inhibition by ISIS 460208,


targeting rs4690072 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
1
8



BACHD
54
68

















TABLE 89







Effect of antisense inhibition by ISIS 460209,


targeting rs7685686 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
12
32



BACHD
72
83

















TABLE 90







Effect of antisense inhibition by ISIS 435879,


targeting rs7685686 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
0
7



BACHD
36
58

















TABLE 91







Effect of antisense inhibition by ISIS 476333,


targeting rs7685686 in BACHD and YAC18 neurons










500 nM
1500 nM















YAC18
46
61



BACHD
89
91

## Claims

1. A method of selectively reducing the expression of a mutant huntingtin allele in a cell, tissue, or animal comprising administering to the cell, tissue, or animal a compound comprising a modified, single-stranded antisense oligonucleotide complementary to the mutant huntingtin allele at a position comprising a single nucleotide polymorphism, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a wing-gap-wing motif with a 5′ wing region positioned at the 5′ end of a deoxynucleoside gap, and a 3′ wing region positioned at the 3′ end of the deoxynucleoside gap, wherein position 8, 9, or 10 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the single nucleotide polymorphism, wherein the expression of the mutant huntingtin allele is selectively reduced, and wherein the single nucleotide polymorphism is rs362306, rs362331, rs2298969, rs7685686, rs4690072, rs2024115, or rs363088.

2. The method of claim 1, wherein at least one nucleoside comprises a modified sugar.

3. The method of claim 2, wherein the modified sugar is a high-affinity sugar modification.

4. The method of claim 3, wherein the high-affinity sugar is a bicyclic sugar.

5. The method of claim 4, wherein each bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.

6. The method of claim 3, wherein the at least one modified sugar comprises a 2′-O-methoxyethyl.

7. The method of claim 1, wherein the wing-gap-wing motif is any of the group consisting of 2-9-6, 3-9-3, 3-9-4, 3-9-5, 4-7-4, 4-9-4, 4-9-5, 4-10-5, 4-11-4, 4-11-5, 5-7-5, 5-8-6, 5-9-3, 5-9-5, 5-10-4, 5-10-5, 6-7-6, 6-8-5, and 6-9-2.

8. The method of claim 7, wherein each nucleoside positioned in the wing segment of the modified oligonucleotide comprises a 2′-O-methoxyethyl modification.

9. The method of claim 7, wherein at least one of the wing regions comprises a 4′ to 2′ bicyclic nucleoside and at least one of the remaining wing nucleosides is a non-bicyclic 2′-modified nucleoside.

10. The method of claim 9, wherein the non-bicyclic 2′-modified nucleoside is a 2′-O-methoxyethyl nucleoside.

11. The method of claim 9, wherein the 4′ to 2′ bicyclic nucleoside is a 4′-CH(CH3)—O-2′ bicyclic nucleoside.

12. The method of claim 1, wherein at least one nucleoside comprises a modified nucleobase.

13. The method of claim 12, wherein the at least one modified nucleobase is a 5′-methylcytosine.

14. The method of claim 1, wherein the modified oligonucleotide is 95% complementary to the mutant huntingtin allele.

15. The method of claim 1, wherein the modified oligonucleotide is 100% complementary to the mutant huntingtin allele.

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

17. The method of claim 16, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

18. A method of treating, ameliorating, or slowing the onset or progression of Huntington's Disease, comprising administering to an animal a compound comprising a modified, single-stranded antisense oligonucleotide complementary to a mutant huntingtin allele at a position on the allele comprising a single nucleotide polymorphism site, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a wing-gap-wing motif with a 5′ wing region positioned at the 5′ end of a deoxynucleoside gap, and a 3′ wing region positioned at the 3′ end of the deoxynucleoside gap, wherein position 8, 9, or 10 of the modified oligonucleotide, as counted from the 5′ terminus of the modified oligonucleotide, aligns with the single nucleotide polymorphism, wherein the compound administered to the animal treats, ameliorates or slows the onset or progression Huntington's Disease by selectively reducing the mutant huntingtin allele, and wherein the single nucleotide polymorphism site is rs362306, rs362331, rs2298969, rs7685686, rs4690072, rs2024115, or rs363088.

19. The method of claim 1, wherein the single nucleotide polymorphism is rs2298969, rs7685686, rs4690072, rs2024115, or rs363088.

20. The method of claim 18, wherein the single nucleotide polymorphism is rs2298969, rs7685686, rs4690072, rs2024115, or rs363088.