WO2011097643A1 - Selective reduction of allelic variants - Google Patents
Selective reduction of allelic variants Download PDFInfo
- Publication number
- WO2011097643A1 WO2011097643A1 PCT/US2011/024103 US2011024103W WO2011097643A1 WO 2011097643 A1 WO2011097643 A1 WO 2011097643A1 US 2011024103 W US2011024103 W US 2011024103W WO 2011097643 A1 WO2011097643 A1 WO 2011097643A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- wing
- modified
- compound
- modified oligonucleotide
- sugar
- Prior art date
Links
- 0 CC*C(C(C(*)OC12COCCP)C1OC)(C2(C)P)P Chemical compound CC*C(C(C(*)OC12COCCP)C1OC)(C2(C)P)P 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/22—Anxiolytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/12—Ophthalmic agents for cataracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/11—Antisense
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/334—Modified C
- C12N2310/3341—5-Methylcytosine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/341—Gapmers, i.e. of the type ===---===
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/34—Allele or polymorphism specific uses
Definitions
- 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 diseases.
- SNP single nucleotide polymorphism
- HD Huntington's Disease
- CAG polymorphic trinucleotide
- HIT polymorphic trinucleotide
- 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 ⁇ 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.
- 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.
- 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.
- Figure 1 provides the mRNA and genomic HTT sequence showing SNP positions.
- SNPs may be associated with a mutant allele, the expression of which causes disease.
- the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease.
- the expressed gene product of a mutant allele results in gain of function causing disease.
- 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.
- the antisense compound is an antisense oligonucleotide
- 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.
- 2'-0-methoxyethyl refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring.
- a 2'-0-methoxyethyl modified sugar is a modified sugar.
- 2'-0-methoxyethyl nucleotide means a nucleotide comprising a 2'-0-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.
- 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.
- administering 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.
- Allelic pair 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
- 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.
- Motant 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.
- 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 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.
- 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(CH 3 )-0-2 ⁇
- “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'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0-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.
- 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.
- a dose may be administered in one, two, or more boluses, tablets, or injections.
- 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.
- 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.
- 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.
- 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 DNArRNA 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.
- at least one of said sugar-modified high-affinity nucleosides confers a ATm 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.
- At least one of the high-affinity sugar modifications confers about 2 or more, 3 or more, or 4 or more degrees per modification.
- 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 tricycloDNA nucleosides.
- 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.
- complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
- “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.
- Microtif 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).
- 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 monomelic 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).
- RNA ribonucleic acids
- DNA deoxyribonucleic acids
- siRNA small interfering ribonucleic acids
- miRNA microRNAs
- 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.
- 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.
- Oligomer means a polymer of linked monomelic 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.
- 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.
- 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.
- 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.
- SNP single nucleotide polymorphism
- Side effects means physiological responses attributable to a treatment other than the desired effects.
- side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise.
- increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
- 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 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.
- 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 occuring nucleobases, sugar moieties, and internucleoside linkages.
- an unmodified nucleotide is an RNA nucleotide (i.e. ⁇ -D-ribonucleosides) or a DNA nucleotide (i.e. ⁇ -D-deoxyribonucleoside).
- 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.
- the allelic variant contains a single nucleotide polymorphism (SNP).
- the SNP is a differentiating polymorphism.
- a SNP is associated with a mutant allele.
- a SNP is in linkage disequilibrium with another polymorphism that is associated with or is causative of disease.
- a mutant allele is associated with disease.
- mRNA and protein expression of a mutant allele is associated with disease.
- the expressed gene product of a mutant allele results in
- genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt- Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009 ); GFAP gene encoding glial fibrillary acidic protein involved in
- Alexander disease J. Neurosci. 2006, 26:111623; alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia- 1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in
- Pelizaeus-Merzbacher disease NeuroMol Med. 2007, 4: 73
- DYT1 gene encoding torsinA protein involved in Torsion dystonia
- alpha-B crystalline gene encoding alpha-B crystalline protein involved in protein aggregation diseases, including cardiomyopathy (Cell 2007, 130: 427); alphal -antitrypsin gene encoding alphal -antitrypsin protein involved in chronic obstructive pulmonary disease (COPD), liver disease and hepatocellular carcinoma (New Engl J Med.
- COPD chronic obstructive pulmonary disease
- Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J.
- PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum.
- alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5: 167); GNAS gene encoding G proteins involved in congenital visual defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci.
- AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab.
- CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RPl gene encoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTP
- 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.
- the antisense compound is an antisense oligonucleotide.
- the antisense compound is not a ribozyme, a double stranded siRNA, or an shRNA.
- the antisense oligonucleotide may have one or more modified sugar(s), nucleobase(s), or internucleoside linkage(s).
- the antisense oligonucleotide is complementary to the SNP site.
- 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.
- the antisense oligonucleotide is 100% complementary to the SNP site.
- 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.
- 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.
- 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.
- selective reduction of mRNA and protein expression of an allelic variant of a gene containing a SNP occurs in a cell or tissue.
- the cell or tissue is in an animal.
- the animal is a human.
- described herein are compounds comprising a modified antisense oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a single nucleotide
- the modified oligonucleotide 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 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, or positions 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the modified oligonucleotide, as counted from the 5' terminus of the gap, aligns with the single nucleotide polymorphism.
- the single nucleotide polymorphism site is on a mutant allele that is associated with a disease. In certain embodiments, the single nucleotide polymorphism site contains a differentiating polymorphism.
- the modified antisense oligonucleotide consists of 12 to 20 linked nucleosides. In certain embodiments, modified antisense oligonucleotide consists of 15 to 20 linked nucleosides. In certain embodiments, the modified antisense oligonucleotide consists of 15 to 19 linked nucleosides.
- position 8, 9, or 10 of the modified oligonucleotide as counted from the 5' terminus of the modified oligonucleotide, or positions 4, 5, or 6 of the modified
- oligonucleotide as counted from the 5' terminus of the gap, aligns with the single nucleotide polymorphism.
- the gap region is 7-11 nucleosides in length
- the 5' wing region is 1- 6 nucleobases in length
- the 3' wing region is 1-6 nucleobases in length.
- 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.
- the wing-gap-wing motif is any one of the group consisting of 2-9-6, 4-9-5, and 4-11-4.
- At least one intemucleoside linkage is a modified intemucleoside linkage.
- each intemucleoside linkage is a phosphorothioate intemucleoside linkage.
- At least one nucleoside comprises a modified nucleobase.
- the modified nucleobase is a 5'-methylcytosine.
- At least one nucleoside of at least one of the wing regions comprises a modified sugar or sugar surrogate.
- each of the nucleosides of each wing region comprises a modified sugar or sugar surrogate.
- the sugar or sugar surrogate is a 2'-0-methoxyethyl modified sugar.
- 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.
- the non-bicyclic 2 '-modified nucleoside is a 2'-0-methoxyethyl nucleoside.
- the 4' to 2' bicyclic nucleoside is 4'-CH(CH3)-0-2' bicyclic nucleoside.
- the modified antisense oligonucleotide consisting of 17 linked nucleosides and wherein position 9 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism.
- the wing-gap-wing motif is 2-9-6.
- described herein are compounds comprising a modified
- oligonucleotide consisting of 18 linked nucleosides and 90% complementary to a differentiating polymorphism
- the modified oligonucleotide comprises a wing-gap- wing motif, wherein position 9 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein the wing-gap- wing motif is 4-9-5.
- described herein are compounds comprising a modified
- oligonucleotide consisting of 19 linked nucleosides and 90% complementary to a differentiating polymorphism
- the modified oligonucleotide comprises a wing-gap- wing motif, wherein position 10 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein the wing-gap- wing motif is 4-11- 4.
- described herein are compounds comprising a modified
- oligonucleotide consisting of 15 to 19 linked nucleosides and fully complementary to a
- the modified oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; and at least one high-affinity sugar modification.
- the modified oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; and at least one high-affinity sugar modification.
- the modified oligonucleotide comprises a wing-gap-wing motif, wherein position 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligon
- oligonucleotide is 100% complementary to the single nucleotide polymorphism site.
- At least one of the wing regions comprises a Wgh-affinity sugar modification.
- the high-affinity sugar modification is a bicyclic sugar.
- the bicyclic sugar comprises a 4'-CH(CH3)-0-2' bridge.
- at least one of positions 2, 3, 6, 9, 10, 11, 13, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide comprises the at least one high-affinity sugar modification.
- oligonucleotide as counted from the 5' terminus of the modified oligonucleotide, comprises the at least one Wgh-afftnity sugar modification.
- each of nucleoside positions 2, 3, 13, and 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, comprise the at least one high-affinity sugar modification.
- the Wgh-affinity sugar modification is a bicyclic sugar.
- the bicyclic sugar comprises a 4'-CH(CH3)-0-2' bridge.
- the wing-gap-wing motif is any of the group consisting of 3-9-3, 4- 9-4, and 5-9-5.
- described herein are compounds comprising a modified
- oligonucleotide consisting of 15, 17, or 19 linked nucleosides and fully complementary to a differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap- wing motif, wherein position 6, 8, 10, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; and at least one high-affinity sugar modification.
- At least one of positions 2, 3, 6, 9, 10, 11, 13, or 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, comprises the at least one high-affinity sugar modification.
- the high-affinity sugar modification is a bicyclic sugar.
- the bicyclic sugar comprises a 4'-CH(CH3)-0-2' bridge.
- the wing-gap- wing motif is any of the group consisting of 3-9-3, 4- 9-4, and 5-95.
- described herein are compounds comprising a modified
- the modified oligonucleotide consisting of 15 linked nucleosides and 90% complementary to a differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap-wing motif, wherein position 8 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, aligns with the differentiating polymorphism; and at least one high-affinity sugar modification.
- the modified oligonucleotide is 100% complementary to the differentiating polymorphism.
- each of nucleoside positions 2, 3, 13, and 14 of the modified oligonucleotide, as counted from the 5' terminus of the modified oligonucleotide, comprise the at least one high-affinity sugar modification.
- the high-affinity sugar modification is a bicyclic sugar.
- the bicyclic sugar comprises a 4'-CH(CH3)-0-2' bridge.
- the wing-gap- wing motif is 3-9-3.
- described herein are methods of selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism in a cell, tissue, or animal, comprising administering to the cell, tissue, or animal a compound comprising a modified oligonucleotide complementary to a differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap-wing motif and 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 differentiating polymorphism.
- the modified oligonucleotide is 90% complementary to the single differentiating polymorphism. In certain embodiments, the modified oligonucleotide is 95% complementary to the single nucleotide polymorphism site. In certain embodiments, the modified oligonucleotide is 100% complementary to the single nucleotide polymorphism site.
- the single nucleotide polymorphism site is from 12 to 30 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is from 15 to 25 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is from 17 to 22 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is 17 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is 18 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is 19 nucleobases in length. In certain embodiments, the single nucleotide polymorphism site is 20 nucleobases in length.
- the allelic variant is associated with disease. In certain embodiments, the allelic variant is associated with disease.
- the disease is Huntington's Disease.
- the modified oligonucleotide is a single-stranded oligonucleotide.
- at least one intemucleoside linkage is a modified intemucleoside linkage.
- each intemucleoside linkage is a phosphorothioate intemucleoside linkage.
- At least one nucleoside comprises a modified nucleobase.
- the at least one modified nucleobase is a 5'-methylcytosine.
- At least one nucleoside comprises a modified sugar.
- the modified sugar is a Wgh-affinity sugar modification.
- the high-affinity sugar is a bicyclic sugar.
- each bicyclic sugar comprises a 4'- CH(CH 3 )-0-2' bridge.
- nucleoside positions 2, 3, 13, and 14 of the modified oligonucleotide, counting from the 5' terminus of the modified oligonucleotide comprises a nucleoside having a bicyclic sugar wherein the bicyclic sugar comprises a 4'-CH(CH 3 )-0-2' bridge.
- each of nucleoside positions 2, 3, 13, and 14 of the modified oligonucleotide, counting from the 5' terminus of the modified oligonucleotide, comprises a bicyclic sugar wherein the bicyclic sugar comprises a 4'-CH(CH 3 )-0-2' bridge.
- the at least one modified sugar comprises a 2'-0-methoxyethyl.
- each nucleoside positioned in a wing segment of the modified oligonucleotide comprises a 2'-0-methoxyethyl modification.
- the wing-gap- wing motif is any of the group consisting of 2-9-6, 3-
- the modified oligonucleotide is not a ribozyme, a double stranded siRNA, or an shRNA.
- the single nucleotide polymorphism site is on a mutant allele that is associated with disease. In certain embodiments, the single nucleotide polymorphism site contains a differentiating polymorphism.
- the modified antisense oligonucleotide consists of 12 to 20 linked nucleosides. In certain embodiments, the modified antisense oligonucleotide consists of 15 to 19 linked nucleosides.
- the gap region is 7 to 11 nucleosides in length
- the 5' wing region is 1 to 6 nucleobases in length
- 3' wing region is 1 to 6 nucleobases in length.
- at least one nucleoside of at least one of the wing regions comprises a modified sugar or sugar surrogate.
- each of the nucleosides of each wing region comprises a modified sugar or sugar surrogate.
- the sugar or sugar surrogate is a 2'-0- methoxyethyl modified sugar.
- 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.
- the non-bicyclic 2'-modified nucleoside is a 2'-0-methoxyethyl nucleoside.
- 4' to 2' bicyclic nucleoside is a 4'-CH(CH3)-0-2' bicyclic nucleoside.
- described herein are methods of selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism in a cell, tissue, or animal, comprising administering to the cell, tissue, or animal a compound comprising a modified oligonucleotide complementary to a differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap-wing motif and 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 differentiating polymorphism.
- described herein are methods of selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism in a cell, tissue, or animal, comprising administering to the cell, tissue, or animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and complementary to a differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap- wing motif and 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 differentiating polymorphism; and wherein the allelic variant is a mutant allele.
- the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt- Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome,
- any disease
- described herein are methods of treating Huntington's Disease, comprising selectively reducing expression of an allelic variant of a gene containing a single nucleotide polymorphism in a cell, tissue, or animal, comprising administering to the cell, tissue, or animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and complementary to differentiating polymorphism, wherein the modified oligonucleotide comprises a wing-gap-wing motif and 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 differentiating polymorphism; and wherein the allelic variant is associated with Huntington's Disease.
- position 8, 9, or 10 of the modified oligonucleotide as counted fromt eh 5' terminus of the modified oligonucleotide, or positions 4, 5, or 6 of the modified oligonucleotide, as counted from the 5' terminus of the gap, aligns with the single nucleotide polymorphism.
- SNPs Single Nucleotide Polymorphisms
- Single-nucleotide polymorphisms 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).
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
- an antisense compound is an antisense oligonucleotide. In certain embodiments, the antisense compound is not a ribozyme, a double stranded siRNA, or an shRNA.
- 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.
- 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.
- 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.
- 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.
- the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleosides.
- antisense oligonucleotides targeted to a nucleic acid may be shortened or truncated.
- 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.
- 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.
- the additional subunit may be located at the 5' or 3' end of the antisense compound.
- 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.
- 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.
- an antisense compound such as an antisense oligonucleotide
- introduce mismatch bases without eliminating activity.
- Gautschi et al 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.
- 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 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 R A:DNA duplex.
- Antisense compounds having a gapmer motif are considered chimeric antisense compounds.
- a gapmer an internal region having a plurality of nucleotides that supports R aseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region.
- the gap segment 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.
- 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.
- 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.
- 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.
- the SNP anneals to a nucleobase at position 4, 5, 6, or 7 counting from the 5' terminus of the gap segment.
- the SNP anneals to a nucleobase at position 4 or 5 beginning from the 5' terminus of the gap segment.
- the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
- the bicyclic moiety may be a cEt having the formula 4'-CH(CH 3 )-0-2.'
- 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.
- 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 mtervening 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.
- X and Z are the same, in other
- 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.
- 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.
- 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.
- antisense compounds targeted to a nucleic acid possess a 2-9-6 gapmer motif or a 6-9-2 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 3-9-3 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 3-9-5 gapmer motif or 5-9-3 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 4-9-5 gapmer motif or 5-9-4 gapmer motif.
- 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.
- antisense compounds targeted to a nucleic acid possess a 5-9-5 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 5-8-6 gapmer motif or a 6-8-5 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 6-7-6 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 6-8-5 gapmer motif or a 5-8-6 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 3-9-4 gapmer motif or a 4-9-3 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 5-7-5 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 4-7-4 gapmer motif.
- antisense compounds targeted to a nucleic acid possess a 5-10-5 gapmer motif.
- an antisense compound targeted to a nucleic acid has a gap- widened motif.
- 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.
- antisense compounds are referred to as mixed wing antisense compounds (see WO 2008/049085).
- 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.
- 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.
- 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.
- the modifications and combination of modifications at the 3' wing and at the 5' wing may be the same or they may be different.
- 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.
- 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.
- an antisense compound comprises at least one wing comprising at least one MOE substituted nucleoside and at least one high affinity modification.
- the at least one MOE substituted nucleoside and the at least one high affinity are in the 3' wing.
- the at least one MOE substituted nucleoside and the at least one high affinity are in the 5' wing.
- an antisense compound comprises 1, 2 or 3 high affinity modifications in the 5' and/or 3' wings.
- 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,
- 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.
- a target region is a structurally defined region of the target nucleic acid.
- 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.
- 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.
- the desired effect is a reduction in mRNA target nucleic acid levels of a particular allelic variant.
- 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.
- 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.
- 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).
- 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 H7T 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.
- 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.
- the antisense compounds provided herein are specifically hybridizable with the nucleic acid of a particular allelic variant.
- 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.
- 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).
- 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.
- 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.
- the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
- 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).
- 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.
- "fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid.
- 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.
- 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.
- 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.
- non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound.
- the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound.
- two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-contiguous.
- a non- complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
- 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.
- 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.
- 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.
- the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment.
- the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment.
- the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment.
- 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.
- 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.
- an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability.
- 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.
- 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.
- a portion of the antisense compound is compared to an equal length portion of the target nucleic acid.
- 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.
- a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid.
- 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.
- 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 intemucleoside linkages of the oligonucleotide.
- 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.
- RNA and DNA The naturally occuring intemucleoside linkage of RNA and DNA is a 3' to 5'
- Antisense compounds having one or more modified, i.e. non-naturally occurring, intemucleoside linkages are often selected over antisense compounds having naturally occurring intemucleoside 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 intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom.
- Representative phosphorus containing intemucleoside 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.
- antisense compounds comprise one or more modified
- each intemucleoside linkage of an antisense compound is a phosphorothioate intemucleoside linkage.
- 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.
- nucleosides comprise a chemically modified ribofuranose ring moieties.
- substitutent groups including 5' and 2' substituent groups
- BNA bicyclic nucleic acids
- Examples of chemically modified sugars include 2 -F- 5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 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
- 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'-0(CH2)20CH3 substituent groups.
- 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.
- antisense compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4' to 2' bicyclic nucleoside.
- 4' to 2' bicyclic nucleosides include but are not limited to one of the formulae: 4'-(CH 2 )-0-2' (LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 ) 2 -0-2' (ENA); 4 ⁇ - ⁇ ( ⁇ 3 )-0-2' and 4'-CH(CH 2 0CH 3 )-0-2* (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH.3)(CH 3 )-0-2' (and analogs thereof see published International Application
- bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L- ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
- x 0, 1, or 2;
- n 1, 2, 3, or 4;
- the bridge of a bicyclic sugar moiety is , -[C(R a )(R b )] n -,
- the bridge is 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(R)-2' and 4'-CH 2 - N(R)-0-2'- wherein each R is, independently, H, a protecting group or C ! -C 12 alkyl.
- bicyclic nucleosides are further defined by isomeric configuration.
- a nucleoside comprising a 4'-2' methylene-oxy bridge may be in the a-L
- bicyclic nucleosides include, but are not limited to, (A) a-L- Methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') BNA , (C) Ethyleneoxy (4'-(CH 2 )2-0-2') BNA , (D) Aminooxy (4'-CH 2 -0-N(R)-2') BNA, (E) Oxyamino (4'-CH 2 -N(R)-0- 2') BNA, and (F) Methyl(methyleneoxy) (4'-CH(CH 3 )-0-2') BNA, (G) methylene-thio (4'-CH 2 -S- 2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH 2 -CH(CH 3 )- 2') BNA
- Bx is a heterocyclic base moiety
- Rc is C1-C12 alkyl or an amino protecting group
- T a 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.
- bicyclic nucleoside having Formula II having Formula II:
- Bx is a heterocyclic base moiety
- T a 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;
- Z a is C ! -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted Ci-Ce alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
- Bx is a heterocyclic base moiety
- T a and T b 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;
- bicyclic nucleoside having Formula IV having Formula IV:
- Bx is a heterocyclic base moiety
- T a and T b 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 Ci-C 6 alkyl, substituted C!-C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl;
- each q a , q b , q c and q d is, independently, H, halogen, Q-Q alkyl, substituted C!-C 6 alkyl, C 2 - C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl, Q-C 6 alkoxyl, substituted C ! -C 6 alkoxyl, acyl, substituted acyl, C C 6 aminoalkyl or substituted C C 6 aminoalkyl;
- bicyclic nucleoside having Formula V having Formula V:
- Bx is a heterocyclic base moiety
- T a and T 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;
- qg and q h are each, independently, H, halogen, C ! -C 12 alkyl or substituted C ⁇ -C ⁇ 2 alkyl.
- BNA methyleneoxy (4'-CH 2 -0-2') 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.
- bicyclic nucleoside having Formula VI having Formula VI:
- Bx is a heterocyclic base moiety
- 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.
- nucleosides refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties.
- sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
- 2 '-modified sugar means a furanosyl sugar modified at the 2' position.
- 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.
- 2'- substituent groups can also be selected from: C ⁇ - Ci2 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , 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.
- modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000).
- 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.
- 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, CJ. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA) or those compounds having Formula X:
- T 3 and T 4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T 3 and T 4 is an
- internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T 3 and T 4 is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
- qi, 3 ⁇ 42, 3, 3 ⁇ 44, q 5 > 3 ⁇ 46 and q 7 are each independently, H, Ci-C 6 alkyl, substituted Q-Q alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl; and
- Ri and R 2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJA, SJi, N 3 , J 2 and CN, wherein X is O, S or NJi and each J l5 J 2 and J3 is, independently, H or -Ce alkyl.
- the modified THP nucleosides of Formula X are provided wherein qm, qn, p, qr, qs, qt and q lake are each H. In certain embodiments, at least one of q m , q n , q p , q r , q s , qt and q u is other than H. In certain embodiments, at least one of q m , q n , q p , q r , q s , qt and q u is methyl. In certain embodiments, THP nucleosides of Formula X are provided wherein one of Ri and R 2 is F. In certain embodiments, Ri is fluoro and R 2 is H; Ri is methoxy and R 2 is H, and R is methoxyethoxy and R 2 is H.
- 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'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.
- 2'-OMe or “2'-OCH 3 " or “2'-0-methyl” each refers to a nucleoside comprising a sugar comprising an -OCH 3 group at the 2' position of the sugar ring.
- 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 (R A) and/or deoxyribonucleosides (DNA).
- Such ring systems can undergo various additional substitutions to enhance activity.
- nucleobase moieties 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.
- antisense compounds comprise one or more nucleotides having modified sugar moieties.
- the modified sugar moiety is 2'-MOE.
- the 2'-MOE modified nucleotides are arranged in a gapmer motif.
- the modified sugar moiety is a cEt.
- the cEt modified nucleotides are arranged throughout the wings of a gapmer motif.
- 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.
- 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
- 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-CH 3 ) 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
- 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 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
- antisense compounds comprise one or more modified nucleobases.
- gap-widened antisense oligonucleotides comprise one or more modified nucleobases.
- the modified nucleobase is 5-methylcytosine.
- each cytosine is a 5-methylcytosine.
- 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.
- pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
- PBS is a diluent suitable for use in compositions to be delivered parenterally.
- employed in the methods described herein is a pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent.
- the pharmaceutically acceptable diluent is PBS.
- the antisense compound is an antisense oligonucleotide.
- 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.
- 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 January 16, 2003.
- 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.
- Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
- 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, CA).
- Antisense oligonucleotides are mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) 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, CA). Antisense oligonucleotide is mixed with
- LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) 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.
- 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
- 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, CA) according to the manufacturer's recommended protocols.
- 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, CA and used according to manufacturer's instructions.
- 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, CA) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
- RNA 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.
- 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, CA). 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, CA). 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, OR). 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, CA).
- 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 (ELIS A), 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, MI), 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.
- 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.
- 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.
- 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.
- 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
- the compounds and compositions as described herein are administered parenterally.
- 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.
- 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
- intraparenchymal administration intrathecal administration, or intracerebroventricular
- parenteral administration is by injection.
- the injection may be delivered with a syringe or a pump.
- the injection is a bolus injection.
- the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.
- methods of specifically localizing a pharmaceutical agent decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50.
- EC50 median effective concentration
- the pharmaceutical agent in an antisense compound as further described herein.
- the targeted tissue is brain tissue.
- the targeted tissue is striatal tissue.
- decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.
- 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.
- a mutant allele 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.
- 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.
- the antisense oligonucleotides have a sequence that is 12-30 nucleobases in lenth. 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
- 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.
- 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, nuceobases in length. In certain embodiments, the antisense oligonucleotides have a sequence that is 16-19 nucleobases in lenth. In certain
- 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.
- oligonucleotides of various lengths 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.
- 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 oligonuceotide and/or positions 1-9 counting from the 5' end of the gap.
- 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 oligonuceotide and/or positions 1-9 counting from the 5' end of the gap.
- 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
- 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 oligonuceotide and/or positions 4-6 counting from the 5' end of the gap.
- 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.
- the effect of the length of the gap, 5' wing, and 3' wing was tested.
- wing-gap- wing combinations were shown to be beneficial for a SNP targeting antisense oligonucleotide.
- 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
- 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-
- each nucleoside of each wing of the modified antisense oligonucleotide has a 2'-MOE modification.
- each nucleoside of each wing of the modified antisense oligonucleotide has a high affinity modification.
- 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.
- the antisense oligonucleotide has one or more 2'-MOE modifications in the wings and/or one or more high affinity modifications in the wings.
- the high affinity modification is a cEt modification.
- the antisense oligonucleotide has a high affinity modification at positions 2, 3, 13, and 14 of the antisense oligonucleotide (counting from the 5' terminus).
- the antisense oligonuceotide has one, two, three, or four high affinity modifications in at least one of the wings.
- the antisense oligonuceotide has one, two, three, or four high affinity modifications in each of the 5' and 3' wings independently.
- 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 oligonuceotide 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 oligonuceotide has a high affinity
- the antisense oligonuceotide 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 oligonuceotide has alternating 2'-MOE and high affinity modification in at least one of the 5' and 3 'wings.
- the compound comprises an antisense oligonucleotide
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 1-9 begining 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.
- the single nucleotide polymorphism site contains a
- the single nucleotide polymorphism site is on a mutant allele.
- the mutant allele is associated with disease.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 1-9 begining 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.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 1-9 begining 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.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 4-7 begining 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.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 4-6 begining 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.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 4-6 begining 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.
- 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.
- 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 begining from the 5' terminus of the antisense oligonuceotide or positions 4-6 begining 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.
- 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.
- 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 oligonuceotide or positions 4-6 begining 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.
- 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.
- 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 oligonuceotide or positions 1-9 begining 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.
- 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.
- 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 oligonuceotide or positions 4-6 begining 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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)-0-2' bridge.
- 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.
- the compound comprises a modified antisense oligonucleotide consisting of 15 to 19 linked nucleosides and fully complementary to a single nucleotide
- 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(CH 3 )-0-2' bridge.
- 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
- 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(CH 3 )-0-2' bridge.
- 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.
- 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'-0-methoxyethyl sugar.
- 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.
- 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'-0-methoxyethyl sugar.
- 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.
- 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(CH 3 )-0-2' bridge.
- 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.
- the compound comprises a modified antisense oligonucleotide consisting of 17 to 19 linked nucleosides and fully complementary to a single nucleotide
- modified antisense oligonucleotide comprises a wing-gap-wing motif, wherein position 5, 6, or 7 of the gap segment aligns with the single nucleotide
- 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(CH 3 )-0-2' bridge.
- the wing-gap-wing motif is any one of the group consisting of 2-9-6, 3-9-3, 3-
- 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(CH 3 )-0-2' bridge.
- 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-
- 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.
- 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.
- 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).
- 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).
- 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).
- the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions described herein.
- the individual has an allelic variant associated with a disease or disorder.
- the pharmaceutical compositions provided herein preferentially target a SNP.
- the SNP is a differentiating polymorphism.
- the gene and associated disease are any of the following: APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371 : 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt- Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009 ); GFAP gene encoding glial fibrillary acidic protein involved in Alexander disease (J. Neurosci. 2006, 26:111623); alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest.
- SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia-1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in Pelizaeus- Merzbacher disease (NeuroMol Med.
- PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci.
- CaSR gene encoding the calcium sensing receptor protein involved in primary hypercalciuria (Kidney Int. 2007, 71 : 1155); alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord.
- AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab.
- CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RPl gene encoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTP
- the disease is a neurodegenerative disorder.
- the neurodegenerative disorder is Huntington's Disease.
- the targeted SNP is one or more of: rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rsl l731237, rs362296, rsl0015979, rs7659144, rs363096, rs362273, rsl6843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125,
- 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.
- 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.
- administering 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.
- the mutant nucleic acid is huntingtin nucleic acid
- the mRNA is huntingtin mRNA
- the protein is huntingtin protein.
- 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 any of Huntington's Disease, Alzheimer's Disease, Crutzfeldt- Jakob Disease, Fatal Familial Insomnia, Huntington's Disease, Alexander Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS), Dentato-Rubral and Pallido-Luysian Atrophy, Spinocerebellar Ataxia 1, Pelizaeus-Merzbacher Disease, Torsion Dystonia, Cardiomyopathy, Chrome Obstructive Pulmonary Disease (COPD), liver disease and hepatocellular carcinoma, SLE, Hypercholesterolemia, breast tumors, Asthma, Type 1 Diabetes, Rheumatoid Arthritis, Graves Disease, Spinal and Bulbar Muscular Atrophy, Kennedy's Disease, progressive childhood posterior subcapsular cataracts, Cholesterol Gallstone Disease, Arthrosclerosis, cardiovascular disease, primary hypertension
- one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents.
- 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.
- 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.
- 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.
- one or more pharmaceutical compositions of the present invention are coadministered with another pharmaceutical agent to treat an undesired effect of that other
- 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.
- 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.
- Example 1 Single nucleotide polymorphisms (SNPs) in the huntingtin (HTT) gene sequence
- HTT genomic sequence designated herein as SEQ ID NO: 1 (NT_006081.18 truncated from nucleotides 1566000 to 1768000) was aligned with the HTTmRNA, designated herein as SEQ ID NO: 2 (NM_0021 11.6), using the EMBL-EBI sequence database (ClustalW2,
- '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.
- SNPs Single Nuclear Polymorphisms
- Example 2 Design of antisense oligonucleotides targeting huntingtin gene SNPs and inhibition of Hrr 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 H7 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
- HTT rnRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
- Results are presented as percent inhibition of H7 mRNA, relative to untreated control cells.
- ISIS 387916 (TCTCTATTGCACATTCCAAG, 5-10-5 MOE (SEQ ID NO: 6)
- 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.
- oligonucleotides are further described in Table 3.
- the percent inhibition of H7TmRNA 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 H pre-mRNA, which is SEQ ID NO: 1.
- 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 H7 mRNA levels were measured by quantitative real-time PCR. Human HTT primer probe set RTS2617 was used to measure mRNA levels. H7 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. IC 50 values are also provided in Tables 5, 6, and 7.
- Example 4 Dose-dependent antisense inhibition of human huntingtin mRNA levels in Corieil 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 H7TmRNA 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 H T mRNA relative to untreated control cells. 1 ⁇ 2 0 values are also provided in Tables 8, 9, and 10.
- 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.
- antisense oligonucleotides were designed targeting SNP RS Nos. rs2857936, rsl2506200, rs762855, and rsl006798 (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.
- 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.
- each nucleotide in the 5' wing segment and each nucleotide in the 3' wing segment has a 2'-MOE modification.
- All cytosine nucleobases throughout each gapmer are 5- methylcytosines.
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Molecular Biology (AREA)
- Diabetes (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Physical Education & Sports Medicine (AREA)
- Obesity (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Immunology (AREA)
- Pulmonology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Urology & Nephrology (AREA)
- Ophthalmology & Optometry (AREA)
Abstract
Description
Claims
Priority Applications (15)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11740542.3A EP2534248B1 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
DK11740542.3T DK2534248T3 (en) | 2010-02-08 | 2011-02-08 | SELECTIVE REDUCTION OF ALLELVARIANS |
CA2789005A CA2789005A1 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
AU2011213562A AU2011213562B2 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
US13/577,616 US8957040B2 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
EP19164928.4A EP3561060A1 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
JP2012552932A JP6006120B2 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
EP17206749.8A EP3321361B1 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
IL221272A IL221272A (en) | 2010-02-08 | 2012-08-02 | Selective reduction of allelic variants |
US14/581,235 US20150329859A1 (en) | 2010-02-08 | 2014-12-23 | Selective reduction of allelic variants |
AU2016234914A AU2016234914B2 (en) | 2010-02-08 | 2016-09-28 | Selective reduction of allelic variants |
IL254191A IL254191B (en) | 2010-02-08 | 2017-08-28 | Selective reduction of allelic variants |
US15/961,567 US20190002877A1 (en) | 2010-02-08 | 2018-04-24 | Selective reduction of allelic variants |
AU2018260866A AU2018260866A1 (en) | 2010-02-08 | 2018-11-07 | Selective reduction of allelic variants |
US17/577,832 US20220403386A1 (en) | 2010-02-08 | 2022-01-18 | Selective Reduction of Allelic Variants |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US30246910P | 2010-02-08 | 2010-02-08 | |
US61/302,469 | 2010-02-08 | ||
US37163510P | 2010-08-06 | 2010-08-06 | |
US61/371,635 | 2010-08-06 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/577,616 A-371-Of-International US8957040B2 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
US14/581,235 Continuation US20150329859A1 (en) | 2010-02-08 | 2014-12-23 | Selective reduction of allelic variants |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011097643A1 true WO2011097643A1 (en) | 2011-08-11 |
Family
ID=44355846
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/024103 WO2011097643A1 (en) | 2010-02-08 | 2011-02-08 | Selective reduction of allelic variants |
Country Status (9)
Country | Link |
---|---|
US (4) | US8957040B2 (en) |
EP (3) | EP3561060A1 (en) |
JP (4) | JP6006120B2 (en) |
AU (3) | AU2011213562B2 (en) |
CA (1) | CA2789005A1 (en) |
DK (1) | DK2534248T3 (en) |
ES (1) | ES2733708T3 (en) |
IL (2) | IL221272A (en) |
WO (1) | WO2011097643A1 (en) |
Cited By (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012109395A1 (en) * | 2011-02-08 | 2012-08-16 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
WO2013159108A2 (en) | 2012-04-20 | 2013-10-24 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
WO2013138353A3 (en) * | 2012-03-12 | 2013-11-14 | Santaris Pharma A/S | Compositions and methods for modulation of atxn3 expression |
WO2014059356A2 (en) | 2012-10-12 | 2014-04-17 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP2742056A1 (en) * | 2011-08-11 | 2014-06-18 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
WO2014121287A2 (en) | 2013-02-04 | 2014-08-07 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
US9605019B2 (en) | 2011-07-19 | 2017-03-28 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
US9617547B2 (en) | 2012-07-13 | 2017-04-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant |
US9695211B2 (en) | 2008-12-02 | 2017-07-04 | Wave Life Sciences Japan, Inc. | Method for the synthesis of phosphorus atom modified nucleic acids |
US9695418B2 (en) | 2012-10-11 | 2017-07-04 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleosides and uses thereof |
US9982257B2 (en) | 2012-07-13 | 2018-05-29 | Wave Life Sciences Ltd. | Chiral control |
US10144933B2 (en) | 2014-01-15 | 2018-12-04 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator |
US10149905B2 (en) | 2014-01-15 | 2018-12-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having antitumor effect and antitumor agent |
US10160969B2 (en) | 2014-01-16 | 2018-12-25 | Wave Life Sciences Ltd. | Chiral design |
US10167309B2 (en) | 2012-07-13 | 2019-01-01 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
US10307434B2 (en) | 2009-07-06 | 2019-06-04 | Wave Life Sciences Ltd. | Nucleic acid prodrugs and methods of use thereof |
US10322173B2 (en) | 2014-01-15 | 2019-06-18 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent |
US10428019B2 (en) | 2010-09-24 | 2019-10-01 | Wave Life Sciences Ltd. | Chiral auxiliaries |
US10479995B2 (en) | 2015-07-22 | 2019-11-19 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
EP3452596A4 (en) * | 2016-05-04 | 2020-03-18 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
WO2021084495A1 (en) | 2019-11-01 | 2021-05-06 | Novartis Ag | The use of a splicing modulator for a treatment slowing progression of huntington's disease |
US11293025B2 (en) | 2015-09-25 | 2022-04-05 | Ionis Pharmaceuticals, Inc. | Compositions and methods for modulating Ataxin 3 expression |
US11382918B2 (en) | 2017-06-28 | 2022-07-12 | Ptc Therapeutics, Inc. | Methods for treating Huntington's Disease |
US11395822B2 (en) | 2017-06-28 | 2022-07-26 | Ptc Therapeutics, Inc. | Methods for treating Huntington's disease |
EP4035659A1 (en) | 2016-11-29 | 2022-08-03 | PureTech LYT, Inc. | Exosomes for delivery of therapeutic agents |
US11407753B2 (en) | 2017-06-05 | 2022-08-09 | Ptc Therapeutics, Inc. | Compounds for treating Huntington's disease |
US11434488B2 (en) | 2018-05-09 | 2022-09-06 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing ATXN3 expression |
WO2022189363A1 (en) * | 2021-03-08 | 2022-09-15 | Les Laboratoires Servier | Antisense oligonucleotides for inhibiting alpha-synuclein expression |
WO2022208170A1 (en) | 2021-03-29 | 2022-10-06 | Novartis Ag | The use of the splicing modulator brataplam for slowing progression of huntington's disease |
US11583548B2 (en) | 2016-11-10 | 2023-02-21 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing ATXN3 expression |
US11638706B2 (en) | 2015-12-10 | 2023-05-02 | Ptc Therapeutics, Inc. | Methods for treating Huntington's disease |
US11780839B2 (en) | 2018-03-27 | 2023-10-10 | Ptc Therapeutics, Inc. | Compounds for treating Huntington's disease |
US11820985B2 (en) | 2019-03-26 | 2023-11-21 | University Of Massachusetts | Modified oligonucleotides with increased stability |
US11827882B2 (en) | 2018-08-10 | 2023-11-28 | University Of Massachusetts | Modified oligonucleotides targeting SNPs |
EP4010476A4 (en) * | 2019-08-09 | 2023-12-27 | University Of Massachusetts | Chemically modified oligonucleotides targeting snps |
US11858941B2 (en) | 2018-06-27 | 2024-01-02 | Ptc Therapeutics, Inc. | Heterocyclic and heteroaryl compounds for treating Huntington's disease |
US11896669B2 (en) | 2016-01-31 | 2024-02-13 | University Of Massachusetts | Branched oligonucleotides |
US12049627B2 (en) | 2017-06-23 | 2024-07-30 | University Of Massachusetts | Two-tailed self-delivering siRNA |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013148283A1 (en) * | 2012-03-30 | 2013-10-03 | Isis Pharmaceuticals, Inc. | Compositions and methods for modulating tau expression for reducing seizure and modifying a neurodegenerative syndrome |
WO2014078749A1 (en) * | 2012-11-15 | 2014-05-22 | The Regents Of The University Of California | Splice modulating oligonucleotides that inhibit cancer |
JP6629716B2 (en) * | 2013-03-14 | 2020-01-15 | アイオーニス ファーマシューティカルズ, インコーポレーテッドIonis Pharmaceuticals,Inc. | Compositions and methods for modulating tau expression |
TWI657819B (en) | 2013-07-19 | 2019-05-01 | 美商Ionis製藥公司 | Compositions for modulating tau expression |
GB201410693D0 (en) | 2014-06-16 | 2014-07-30 | Univ Southampton | Splicing modulation |
JP6867945B2 (en) | 2014-10-03 | 2021-05-12 | コールド スプリング ハーバー ラボラトリー | Targeted enhancement of nuclear gene output |
SG11201802870RA (en) | 2015-10-09 | 2018-05-30 | Univ Southampton | Modulation of gene expression and screening for deregulated protein expression |
US10370667B2 (en) | 2015-11-18 | 2019-08-06 | Rosalind Franklin University Of Medicine And Science | Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease |
US9840710B2 (en) | 2015-11-18 | 2017-12-12 | Rosalind Franklin University Of Medicine And Science | Antisense compounds targeting leucine-rich repeat kinase 2 (LRRK2) for the treatment of parkinsons disease |
AU2016370653A1 (en) | 2015-12-14 | 2018-06-21 | Cold Spring Harbor Laboratory | Antisense oligomers for treatment of Autosomal Dominant Mental Retardation-5 and Dravet Syndrome |
US11096956B2 (en) | 2015-12-14 | 2021-08-24 | Stoke Therapeutics, Inc. | Antisense oligomers and uses thereof |
JOP20190065A1 (en) | 2016-09-29 | 2019-03-28 | Ionis Pharmaceuticals Inc | Compounds and methods for reducing tau expression |
EP4303321A3 (en) | 2017-08-25 | 2024-07-24 | Stoke Therapeutics, Inc. | Antisense oligomers for treatment of conditions and diseases |
MX2020011695A (en) | 2018-05-04 | 2021-02-26 | Stoke Therapeutics Inc | Methods and compositions for treatment of cholesteryl ester storage disease. |
WO2021178374A2 (en) * | 2020-03-05 | 2021-09-10 | Synerk Inc. | Compounds and methods for reducing apoe expression |
AU2021270720A1 (en) | 2020-05-11 | 2022-12-08 | Stoke Therapeutics, Inc. | OPA1 antisense oligomers for treatment of conditions and diseases |
Citations (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2699508A (en) | 1951-12-21 | 1955-01-11 | Selectronics Inc | Method of mounting and construction of mounting for low frequency piezoelectric crystals |
US2699808A (en) | 1944-10-06 | 1955-01-18 | Mark W Lowe | Apparatus for peeling tomatoes |
WO1994014226A1 (en) | 1992-12-14 | 1994-06-23 | Honeywell Inc. | Motor system with individually controlled redundant windings |
US5656408A (en) | 1996-04-29 | 1997-08-12 | Xerox Corporation | Coated carrier particles |
WO1999014226A2 (en) | 1997-09-12 | 1999-03-25 | Exiqon A/S | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues |
US6670461B1 (en) | 1997-09-12 | 2003-12-30 | Exiqon A/S | Oligonucleotide analogues |
US20040171570A1 (en) | 2002-11-05 | 2004-09-02 | Charles Allerson | Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
WO2004106356A1 (en) | 2003-05-27 | 2004-12-09 | Syddansk Universitet | Functionalized nucleotide derivatives |
WO2005021570A1 (en) | 2003-08-28 | 2005-03-10 | Gene Design, Inc. | Novel artificial nucleic acids of n-o bond crosslinkage type |
US20060063730A1 (en) * | 2004-09-17 | 2006-03-23 | Monia Brett P | Enhanced antisense oligonucleotides |
WO2007134181A2 (en) | 2006-05-11 | 2007-11-22 | Isis Pharmaceuticals, Inc. | 5'-modified bicyclic nucleic acid analogs |
US20080039618A1 (en) | 2002-11-05 | 2008-02-14 | Charles Allerson | Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
US7399845B2 (en) | 2006-01-27 | 2008-07-15 | Isis Pharmaceuticals, Inc. | 6-modified bicyclic nucleic acid analogs |
WO2008101157A1 (en) | 2007-02-15 | 2008-08-21 | Isis Pharmaceuticals, Inc. | 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom |
WO2008143774A2 (en) | 2007-05-01 | 2008-11-27 | University Of Massachusetts | Methods and compositions for locating snp heterozygosity for allele specific diagnosis and therapy |
WO2008147930A2 (en) | 2007-05-23 | 2008-12-04 | Medtronic, Inc. | Methods and kits for linking polymorphic sequences to expanded repeat mutations |
WO2009135322A1 (en) | 2008-05-09 | 2009-11-12 | The Universtity Of British Columbia | Methods and compositions for the treatment of huntington's disease |
US20090318536A1 (en) * | 2006-11-27 | 2009-12-24 | Iss Pharmaceuticals, Inc. | Methods for treating hypercholesterolemia |
US8623108B2 (en) | 2009-01-14 | 2014-01-07 | Boehler Edelstahl Gmbh & Co Kg | Wear-resistant material |
US9778708B1 (en) | 2016-07-18 | 2017-10-03 | Lenovo Enterprise Solutions (Singapore) Pte. Ltd. | Dual sided latching retainer for computer modules |
US9984408B1 (en) | 2012-05-30 | 2018-05-29 | Amazon Technologies, Inc. | Method, medium, and system for live video cooperative shopping |
Family Cites Families (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6582908B2 (en) | 1990-12-06 | 2003-06-24 | Affymetrix, Inc. | Oligonucleotides |
US5801154A (en) | 1993-10-18 | 1998-09-01 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of multidrug resistance-associated protein |
US6770748B2 (en) | 1997-03-07 | 2004-08-03 | Takeshi Imanishi | Bicyclonucleoside and oligonucleotide analogue |
JP3756313B2 (en) | 1997-03-07 | 2006-03-15 | 武 今西 | Novel bicyclonucleosides and oligonucleotide analogues |
US20030228597A1 (en) | 1998-04-13 | 2003-12-11 | Cowsert Lex M. | Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation |
CA2363077A1 (en) | 1999-03-01 | 2000-09-08 | Variagenics, Inc. | Methods for targeting rna molecules |
US6753422B2 (en) | 1999-03-01 | 2004-06-22 | O'brien Thomas G. | Odc allelic analysis method for assessing carcinogenic susceptibility |
AU776362B2 (en) | 1999-05-04 | 2004-09-09 | Roche Innovation Center Copenhagen A/S | L-ribo-LNA analogues |
US6525191B1 (en) | 1999-05-11 | 2003-02-25 | Kanda S. Ramasamy | Conformationally constrained L-nucleosides |
US20020187931A1 (en) | 2000-04-13 | 2002-12-12 | Michael Hayden | Modulating cell survival by modulating huntingtin function |
JP2005504020A (en) | 2001-07-03 | 2005-02-10 | アイシス・ファーマシューティカルス・インコーポレーテッド | Nuclease resistant chimeric oligonucleotide |
AU2002326589B2 (en) | 2001-08-07 | 2008-06-05 | University Of Delaware | Compositions and methods for the prevention and treatment of Huntington's disease |
US20050096284A1 (en) * | 2002-02-20 | 2005-05-05 | Sirna Therapeutics, Inc. | RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA) |
US8090542B2 (en) | 2002-11-14 | 2012-01-03 | Dharmacon Inc. | Functional and hyperfunctional siRNA |
ES2485848T3 (en) * | 2003-09-12 | 2014-08-14 | University Of Massachusetts | RNA interference for the treatment of disorders related to function gain |
CA2538252C (en) | 2003-09-18 | 2014-02-25 | Isis Pharmaceuticals, Inc. | 4'-thionucleosides and oligomeric compounds |
WO2005121372A2 (en) * | 2004-06-03 | 2005-12-22 | Isis Pharmaceuticals, Inc. | Double strand compositions comprising differentially modified strands for use in gene modulation |
EP2322657B1 (en) | 2005-06-28 | 2014-11-05 | Medtronic, Inc. | Methods and sequences to preferentially suppress expression of mutated huntingtin gene. |
WO2007089611A2 (en) | 2006-01-26 | 2007-08-09 | Isis Pharmaceuticals Inc. | Compositions and their uses directed to huntingtin |
CN103554205A (en) | 2006-05-05 | 2014-02-05 | Isis制药公司 | Compounds and methods for modulating expression of gccr |
CA2662704A1 (en) * | 2006-07-07 | 2008-01-10 | University Of Massachusetts | Rna silencing compositions and methods for the treatment of huntington's disease |
ES2377327T5 (en) | 2006-10-18 | 2020-04-28 | Ionis Pharmaceuticals Inc | Antisense compounds |
TW200838551A (en) * | 2006-11-27 | 2008-10-01 | Isis Pharmaceuticals Inc | Methods for treating hypercholesterolemia |
AU2008260277C1 (en) | 2007-05-30 | 2014-04-17 | Isis Pharmaceuticals, Inc. | N-substituted-aminomethylene bridged bicyclic nucleic acid analogs |
WO2008154401A2 (en) | 2007-06-08 | 2008-12-18 | Isis Pharmaceuticals, Inc. | Carbocyclic bicyclic nucleic acid analogs |
ATE462787T1 (en) | 2007-06-18 | 2010-04-15 | Commissariat Energie Atomique | REVERSIBLE SIRNA-SILENCING OF A MUTATED AND ENDOGENOUS HUNTINGTON WILD-TYPE AND ITS APPLICATION TO THE TREATMENT OF HUNTINGTON'S DISEASE |
ES2376507T5 (en) | 2007-07-05 | 2015-08-31 | Isis Pharmaceuticals, Inc. | 6-disubstituted bicyclic nucleic acid analogs |
US8987435B2 (en) | 2008-10-24 | 2015-03-24 | Isis Pharmaceuticals, Inc. | Oligomeric compounds and methods |
AU2011213563B2 (en) * | 2010-02-08 | 2015-12-24 | Ionis Pharmaceuticals, Inc. | Selective reduction of allelic variants |
-
2011
- 2011-02-08 JP JP2012552932A patent/JP6006120B2/en active Active
- 2011-02-08 EP EP19164928.4A patent/EP3561060A1/en active Pending
- 2011-02-08 WO PCT/US2011/024103 patent/WO2011097643A1/en active Application Filing
- 2011-02-08 EP EP17206749.8A patent/EP3321361B1/en not_active Revoked
- 2011-02-08 US US13/577,616 patent/US8957040B2/en active Active
- 2011-02-08 ES ES17206749T patent/ES2733708T3/en active Active
- 2011-02-08 EP EP11740542.3A patent/EP2534248B1/en active Active
- 2011-02-08 CA CA2789005A patent/CA2789005A1/en not_active Abandoned
- 2011-02-08 AU AU2011213562A patent/AU2011213562B2/en active Active
- 2011-02-08 DK DK11740542.3T patent/DK2534248T3/en active
-
2012
- 2012-08-02 IL IL221272A patent/IL221272A/en active IP Right Grant
-
2014
- 2014-12-23 US US14/581,235 patent/US20150329859A1/en not_active Abandoned
-
2016
- 2016-04-11 JP JP2016078592A patent/JP6316334B2/en active Active
- 2016-09-28 AU AU2016234914A patent/AU2016234914B2/en active Active
-
2017
- 2017-08-28 IL IL254191A patent/IL254191B/en unknown
-
2018
- 2018-03-23 JP JP2018055560A patent/JP6652984B2/en active Active
- 2018-04-24 US US15/961,567 patent/US20190002877A1/en not_active Abandoned
- 2018-11-07 AU AU2018260866A patent/AU2018260866A1/en not_active Abandoned
-
2020
- 2020-01-24 JP JP2020009595A patent/JP2020089382A/en active Pending
-
2022
- 2022-01-18 US US17/577,832 patent/US20220403386A1/en active Pending
Patent Citations (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2699808A (en) | 1944-10-06 | 1955-01-18 | Mark W Lowe | Apparatus for peeling tomatoes |
US2699508A (en) | 1951-12-21 | 1955-01-11 | Selectronics Inc | Method of mounting and construction of mounting for low frequency piezoelectric crystals |
WO1994014226A1 (en) | 1992-12-14 | 1994-06-23 | Honeywell Inc. | Motor system with individually controlled redundant windings |
US5656408A (en) | 1996-04-29 | 1997-08-12 | Xerox Corporation | Coated carrier particles |
WO1999014226A2 (en) | 1997-09-12 | 1999-03-25 | Exiqon A/S | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues |
US6670461B1 (en) | 1997-09-12 | 2003-12-30 | Exiqon A/S | Oligonucleotide analogues |
US20080039618A1 (en) | 2002-11-05 | 2008-02-14 | Charles Allerson | Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
US20040171570A1 (en) | 2002-11-05 | 2004-09-02 | Charles Allerson | Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
WO2004106356A1 (en) | 2003-05-27 | 2004-12-09 | Syddansk Universitet | Functionalized nucleotide derivatives |
WO2005021570A1 (en) | 2003-08-28 | 2005-03-10 | Gene Design, Inc. | Novel artificial nucleic acids of n-o bond crosslinkage type |
US20060063730A1 (en) * | 2004-09-17 | 2006-03-23 | Monia Brett P | Enhanced antisense oligonucleotides |
US7399845B2 (en) | 2006-01-27 | 2008-07-15 | Isis Pharmaceuticals, Inc. | 6-modified bicyclic nucleic acid analogs |
WO2007134181A2 (en) | 2006-05-11 | 2007-11-22 | Isis Pharmaceuticals, Inc. | 5'-modified bicyclic nucleic acid analogs |
US20070287831A1 (en) | 2006-05-11 | 2007-12-13 | Isis Pharmaceuticals, Inc | 5'-modified bicyclic nucleic acid analogs |
US20090318536A1 (en) * | 2006-11-27 | 2009-12-24 | Iss Pharmaceuticals, Inc. | Methods for treating hypercholesterolemia |
WO2008101157A1 (en) | 2007-02-15 | 2008-08-21 | Isis Pharmaceuticals, Inc. | 5'-substituted-2'-f modified nucleosides and oligomeric compounds prepared therefrom |
WO2008143774A2 (en) | 2007-05-01 | 2008-11-27 | University Of Massachusetts | Methods and compositions for locating snp heterozygosity for allele specific diagnosis and therapy |
WO2008147930A2 (en) | 2007-05-23 | 2008-12-04 | Medtronic, Inc. | Methods and kits for linking polymorphic sequences to expanded repeat mutations |
WO2009135322A1 (en) | 2008-05-09 | 2009-11-12 | The Universtity Of British Columbia | Methods and compositions for the treatment of huntington's disease |
US8623108B2 (en) | 2009-01-14 | 2014-01-07 | Boehler Edelstahl Gmbh & Co Kg | Wear-resistant material |
US9984408B1 (en) | 2012-05-30 | 2018-05-29 | Amazon Technologies, Inc. | Method, medium, and system for live video cooperative shopping |
US9778708B1 (en) | 2016-07-18 | 2017-10-03 | Lenovo Enterprise Solutions (Singapore) Pte. Ltd. | Dual sided latching retainer for computer modules |
Non-Patent Citations (24)
Title |
---|
"Huntington's Disease Collaborative Research Group", CELL, vol. 72, no. 6, 1993, pages 971 - 83 |
ADV EXP MED BIOL., vol. 613, 2008, pages 203 |
ADV. APPL. MATH., vol. 2, 1981, pages 482 489 |
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 410 |
BRAIN RES., vol. 877, 2000, pages 379 |
CELL, vol. 130, 2007, pages 427 |
DRAGATSIS ET AL., NAT. GENET, vol. 26, 2000, pages 300 - 306 |
FREIER ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 4429 - 4443 |
GENE, vol. 234, 1999, pages 177 |
GENE, vol. 371, 2006, pages 68 |
GENOME RES., vol. 10, 2000, pages 895 |
J. CLIN. INVEST., vol. 111, 2003, pages 145 |
J. NEUROSCI., vol. 26, 2006, pages 111623 |
LOMBARDI ET AL., J EXP NEUROL, vol. 217, 2009, pages 312 - 319 |
NASIR ET AL., CELL, vol. 81, no. 5, 1995, pages 811 - 23 |
NAT. MED., vol. 3, 1997, pages 1009 |
NEUROMOL MED., vol. 4, 2007, pages 73 |
NEW ENGL J MED., vol. 346, 2002, pages 45 |
PROTEIN SCI., vol. 12, 2003, pages 953 |
SCIENCE, vol. 281, 1998, pages 1851 |
See also references of EP2534248A4 |
TRENDS BIOTECHNOL., vol. 18, 2000, pages 77 |
TRENDS MOL. MED., vol. 7, 2001, pages 479 |
ZHANG; MADDEN, GENOME RES., vol. 7, 1997, pages 649 656 |
Cited By (63)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10329318B2 (en) | 2008-12-02 | 2019-06-25 | Wave Life Sciences Ltd. | Method for the synthesis of phosphorus atom modified nucleic acids |
US9695211B2 (en) | 2008-12-02 | 2017-07-04 | Wave Life Sciences Japan, Inc. | Method for the synthesis of phosphorus atom modified nucleic acids |
US10307434B2 (en) | 2009-07-06 | 2019-06-04 | Wave Life Sciences Ltd. | Nucleic acid prodrugs and methods of use thereof |
US10428019B2 (en) | 2010-09-24 | 2019-10-01 | Wave Life Sciences Ltd. | Chiral auxiliaries |
EP2673361B1 (en) | 2011-02-08 | 2016-04-13 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
WO2012109395A1 (en) * | 2011-02-08 | 2012-08-16 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
EP3467109A1 (en) * | 2011-02-08 | 2019-04-10 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
US10017764B2 (en) | 2011-02-08 | 2018-07-10 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
EP3067421A1 (en) * | 2011-02-08 | 2016-09-14 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
US10280192B2 (en) | 2011-07-19 | 2019-05-07 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
US9605019B2 (en) | 2011-07-19 | 2017-03-28 | Wave Life Sciences Ltd. | Methods for the synthesis of functionalized nucleic acids |
EP3205725A1 (en) * | 2011-08-11 | 2017-08-16 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP2742056A1 (en) * | 2011-08-11 | 2014-06-18 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP2742056B1 (en) | 2011-08-11 | 2017-01-11 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
US11732261B2 (en) | 2011-08-11 | 2023-08-22 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP2742056A4 (en) * | 2011-08-11 | 2015-03-25 | Isis Pharmaceuticals Inc | Selective antisense compounds and uses thereof |
EP3556859A1 (en) * | 2011-08-11 | 2019-10-23 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
WO2013138353A3 (en) * | 2012-03-12 | 2013-11-14 | Santaris Pharma A/S | Compositions and methods for modulation of atxn3 expression |
WO2013159108A2 (en) | 2012-04-20 | 2013-10-24 | Isis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
EP3336189A1 (en) * | 2012-04-20 | 2018-06-20 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
US9914922B2 (en) | 2012-04-20 | 2018-03-13 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
EP2839006A4 (en) * | 2012-04-20 | 2015-12-16 | Isis Pharmaceuticals Inc | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
US11566245B2 (en) | 2012-04-20 | 2023-01-31 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleotides and uses thereof |
US9617547B2 (en) | 2012-07-13 | 2017-04-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant |
US9982257B2 (en) | 2012-07-13 | 2018-05-29 | Wave Life Sciences Ltd. | Chiral control |
US10590413B2 (en) | 2012-07-13 | 2020-03-17 | Wave Life Sciences Ltd. | Chiral control |
US10167309B2 (en) | 2012-07-13 | 2019-01-01 | Wave Life Sciences Ltd. | Asymmetric auxiliary group |
US9695418B2 (en) | 2012-10-11 | 2017-07-04 | Ionis Pharmaceuticals, Inc. | Oligomeric compounds comprising bicyclic nucleosides and uses thereof |
EP3459549A1 (en) * | 2012-10-12 | 2019-03-27 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
WO2014059356A2 (en) | 2012-10-12 | 2014-04-17 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP4086347A3 (en) * | 2012-10-12 | 2023-01-11 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
WO2014059356A3 (en) * | 2012-10-12 | 2015-07-16 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
US11236335B2 (en) | 2012-10-12 | 2022-02-01 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP2906256A4 (en) * | 2012-10-12 | 2016-08-10 | Ionis Pharmaceuticals Inc | Selective antisense compounds and uses thereof |
WO2014121287A2 (en) | 2013-02-04 | 2014-08-07 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
US10260069B2 (en) | 2013-02-04 | 2019-04-16 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
WO2014121287A3 (en) * | 2013-02-04 | 2014-10-23 | Isis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
EP3778618A1 (en) * | 2013-02-04 | 2021-02-17 | Ionis Pharmaceuticals, Inc. | Selective antisense compounds and uses thereof |
US10144933B2 (en) | 2014-01-15 | 2018-12-04 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having immunity induction activity, and immunity induction activator |
US10322173B2 (en) | 2014-01-15 | 2019-06-18 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent |
US10149905B2 (en) | 2014-01-15 | 2018-12-11 | Shin Nippon Biomedical Laboratories, Ltd. | Chiral nucleic acid adjuvant having antitumor effect and antitumor agent |
US10160969B2 (en) | 2014-01-16 | 2018-12-25 | Wave Life Sciences Ltd. | Chiral design |
US10479995B2 (en) | 2015-07-22 | 2019-11-19 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US11293025B2 (en) | 2015-09-25 | 2022-04-05 | Ionis Pharmaceuticals, Inc. | Compositions and methods for modulating Ataxin 3 expression |
US11638706B2 (en) | 2015-12-10 | 2023-05-02 | Ptc Therapeutics, Inc. | Methods for treating Huntington's disease |
US11896669B2 (en) | 2016-01-31 | 2024-02-13 | University Of Massachusetts | Branched oligonucleotides |
US10724035B2 (en) | 2016-05-04 | 2020-07-28 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
EP3452596A4 (en) * | 2016-05-04 | 2020-03-18 | Wave Life Sciences Ltd. | Oligonucleotide compositions and methods thereof |
US11583548B2 (en) | 2016-11-10 | 2023-02-21 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing ATXN3 expression |
EP4035659A1 (en) | 2016-11-29 | 2022-08-03 | PureTech LYT, Inc. | Exosomes for delivery of therapeutic agents |
US11407753B2 (en) | 2017-06-05 | 2022-08-09 | Ptc Therapeutics, Inc. | Compounds for treating Huntington's disease |
US12049627B2 (en) | 2017-06-23 | 2024-07-30 | University Of Massachusetts | Two-tailed self-delivering siRNA |
US11395822B2 (en) | 2017-06-28 | 2022-07-26 | Ptc Therapeutics, Inc. | Methods for treating Huntington's disease |
US11382918B2 (en) | 2017-06-28 | 2022-07-12 | Ptc Therapeutics, Inc. | Methods for treating Huntington's Disease |
US11780839B2 (en) | 2018-03-27 | 2023-10-10 | Ptc Therapeutics, Inc. | Compounds for treating Huntington's disease |
US11434488B2 (en) | 2018-05-09 | 2022-09-06 | Ionis Pharmaceuticals, Inc. | Compounds and methods for reducing ATXN3 expression |
US11858941B2 (en) | 2018-06-27 | 2024-01-02 | Ptc Therapeutics, Inc. | Heterocyclic and heteroaryl compounds for treating Huntington's disease |
US11827882B2 (en) | 2018-08-10 | 2023-11-28 | University Of Massachusetts | Modified oligonucleotides targeting SNPs |
US11820985B2 (en) | 2019-03-26 | 2023-11-21 | University Of Massachusetts | Modified oligonucleotides with increased stability |
EP4010476A4 (en) * | 2019-08-09 | 2023-12-27 | University Of Massachusetts | Chemically modified oligonucleotides targeting snps |
WO2021084495A1 (en) | 2019-11-01 | 2021-05-06 | Novartis Ag | The use of a splicing modulator for a treatment slowing progression of huntington's disease |
WO2022189363A1 (en) * | 2021-03-08 | 2022-09-15 | Les Laboratoires Servier | Antisense oligonucleotides for inhibiting alpha-synuclein expression |
WO2022208170A1 (en) | 2021-03-29 | 2022-10-06 | Novartis Ag | The use of the splicing modulator brataplam for slowing progression of huntington's disease |
Also Published As
Publication number | Publication date |
---|---|
AU2016234914B2 (en) | 2018-08-09 |
AU2011213562A1 (en) | 2012-08-23 |
JP6006120B2 (en) | 2016-10-12 |
IL254191B (en) | 2021-07-29 |
US20150329859A1 (en) | 2015-11-19 |
JP2016171800A (en) | 2016-09-29 |
US20220403386A1 (en) | 2022-12-22 |
EP3321361B1 (en) | 2019-03-27 |
IL221272A (en) | 2017-09-28 |
JP2018117630A (en) | 2018-08-02 |
EP2534248B1 (en) | 2018-08-29 |
US20190002877A1 (en) | 2019-01-03 |
US20130046007A1 (en) | 2013-02-21 |
AU2018260866A1 (en) | 2018-11-22 |
JP6652984B2 (en) | 2020-02-26 |
AU2016234914A1 (en) | 2016-10-20 |
CA2789005A1 (en) | 2011-08-11 |
JP2020089382A (en) | 2020-06-11 |
DK2534248T3 (en) | 2018-11-19 |
EP3321361A1 (en) | 2018-05-16 |
JP2013518604A (en) | 2013-05-23 |
JP6316334B2 (en) | 2018-04-25 |
EP2534248A1 (en) | 2012-12-19 |
IL221272A0 (en) | 2012-10-31 |
ES2733708T3 (en) | 2019-12-02 |
US8957040B2 (en) | 2015-02-17 |
EP2534248A4 (en) | 2014-09-17 |
IL254191A0 (en) | 2017-10-31 |
EP3561060A1 (en) | 2019-10-30 |
AU2011213562B2 (en) | 2016-07-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220403386A1 (en) | Selective Reduction of Allelic Variants | |
US20200377946A1 (en) | Selective Reduction of Allelic Variants | |
AU2013331434B2 (en) | Compositions for modulating C90RF72 expression | |
AU2015231130B2 (en) | Compositions for modulating Ataxin 2 expression | |
WO2014062686A1 (en) | Methods for modulating c9orf72 expression | |
WO2016167780A1 (en) | Compositions for modulating expression of c9orf72 antisense transcript | |
WO2016112132A1 (en) | Compositions for modulating expression of c9orf72 antisense transcript | |
EP2480667A1 (en) | Modulation of ttc39 expression to increase hdl | |
EP3265564A1 (en) | Methods for modulating mecp2 expression |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11740542 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011213562 Country of ref document: AU Ref document number: 2011740542 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 221272 Country of ref document: IL |
|
ENP | Entry into the national phase |
Ref document number: 2789005 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012552932 Country of ref document: JP |
|
ENP | Entry into the national phase |
Ref document number: 2011213562 Country of ref document: AU Date of ref document: 20110208 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13577616 Country of ref document: US |