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WO2016027168A2 - Oligonucléotides modulateurs d'épissage et leurs procédés d'utilisation - Google Patents

Oligonucléotides modulateurs d'épissage et leurs procédés d'utilisation Download PDF

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Publication number
WO2016027168A2
WO2016027168A2 PCT/IB2015/001917 IB2015001917W WO2016027168A2 WO 2016027168 A2 WO2016027168 A2 WO 2016027168A2 IB 2015001917 W IB2015001917 W IB 2015001917W WO 2016027168 A2 WO2016027168 A2 WO 2016027168A2
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scn8a
smo
sequence
mrna
exon
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PCT/IB2015/001917
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WO2016027168A3 (fr
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Melanie TALLENT
Nicole LYKENS
Gordon LUTZ
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Lifesplice Pharma Llc
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Priority to EP15833669.3A priority Critical patent/EP3183347A4/fr
Priority to GB1707988.0A priority patent/GB2547586A/en
Priority to CA2958524A priority patent/CA2958524A1/fr
Priority to AU2015304945A priority patent/AU2015304945B2/en
Priority to US15/504,952 priority patent/US11198874B2/en
Priority to PCT/IB2015/001917 priority patent/WO2016027168A2/fr
Publication of WO2016027168A2 publication Critical patent/WO2016027168A2/fr
Publication of WO2016027168A3 publication Critical patent/WO2016027168A3/fr
Priority to US17/549,098 priority patent/US20220098596A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
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    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • 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
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    • 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
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present invention in general relates in general to therapeutic compositions, and in particular to a sequence designed to modulate the splicing of a SCN8A pre-mRNA.
  • SMEI is a relatively rare but catastrophic form of childhood epilepsy characterized by the development of seizures in previously healthy infants that advance to include multiple seizure types such as myoclonus, partial seizures, febrile induced, and the absence episodes by age 2. Progressive developmental and behavioral impairments manifest along with the recurrent and varied seizure episodes that are typically unresponsive to currently available antiepileptic drugs (Dravet et. al., 2005). Additionally, motor abnormalities occur in 20-60% SMEI children (Dravet et. al., 2005). Greater availability of genetic testing and advances in mutational screening now allow for better detection and earlier diagnosis of this severe childhood epilepsy, making early intervention and cure a possibility. Thus, there is a significant and urgent need for the development of novel therapeutic approaches in these patients.
  • the SCNIA gene encodes the a subunit for a voltage-gated sodium (VGS or Nav) channel (Navl .1), one of a family of 10 paralogous pore-forming alpha subunits (SCN) expressed in the human central nervous system (CNS) ), peripheral nerves, and other areas of the body such as the heart.
  • VGS or Nav voltage-gated sodium
  • SCN paralogous pore-forming alpha subunits
  • peripheral nerves and other areas of the body such as the heart.
  • SCN8A is a VGS channel subunit which functionally opposes the currents produced by SCNIA containing channels.
  • SCN8A- containing (Navl.6) channels are highly expressed in excitatory neurons (including hippocampal and purkinje neurons), and function to drive excitatory neuron repetitive firing (Chen et. al., 2008; Raman et. al, 1997).
  • SCNIA-containing sodium channels are expressed in GABAergic inhibitory neurons, particularly in hippocampal (Yu et. al., 2006) and purkinje interneurons (Raman et. al, 1997).
  • SCNIA R168H mutant mice a GEFS+ model, sodium channel activity in interneurons is impaired, leading to decreased GABAergic inhibition, and the overall effect of the mutation is hyperexcitability and increased seizure susceptibility (Martin et.
  • SCNIA knockout (KO) SMEI mice exhibit significantly reduced firing and sodium current density in cortical and hippocampal interneurons, with no change in excitatory pyramidal neurons (Ogiwara et. al., 2007; Yu et. al., 2006), suggesting a common lack of inhibitory balance as the cause of SMEI and GEFS+.
  • reducing SCN8A function can "rescue" pro-seizure phenotypes in both SCNIA R168H and SCNIA knockout mice (Hawkins et. al., 2011; Martin et. al., 2007; Meisler et. al., 2010).
  • SCN8A partial loss-of- function mutations alone cause ataxia and neuromuscular degeneration, but increased kainate- and flurothyl-induced seizure thresholds in mice (Martin et. al., 2007).
  • SCNIA knockouts or SCNIA R168H mutant mice crossing either SCNIA knockouts or SCNIA R168H mutant mice with an SCN8A partial loss-of-function mutant mouse, normalized flurothyl-induced seizure thresholds and extended lifespan in both lines (Hawkins et. al., 2011; Martin et. al., 2007; Meisler et. al., 2010).
  • SCN8A levels to diminish SCN8A-mediated excitation therapeutically rebalances inhibitory deficits caused by loss-of-function SCNIA mutations.
  • VGS channel a subunits undergo several alternative pre-mRNA splicing events, some of these splicing events regulate the inhibitory and excitatory balance of sodium currents in the CNS.
  • SCN8A pre-mRNA undergoes mutually exclusive alternative splicing at both exon 5 and exon 18 during development to form 5N (neonatal), or 5A (adult) and 18N (neonatal), or 18A (adult) isoforms, respectively.
  • Inclusion of the 18N exon introduces a premature stop codon into the transcript to yield a nonfunctional truncated SCN8A 18N isoform (Plummer et.
  • SNP single nucleotide polymorphism
  • SCN1A is a member of a family of voltage gated Na+ (VGS) channel a subunits, and is expressed largely in inhibitory GABAergic interneurons of the central nervous system (CNS).
  • VGS voltage gated Na+
  • SCN8 channels conversely, are expressed on excitatory neurons, and thus these two VGS channel subunits reciprocally regulate network excitation. Accordingly, partial loss of SCN8A function can "rescue" pro-febrile seizure phenotypes in both SCN1A R168H mutant mice and SCN1A knockout mice (Hawkins et. al., 2011; Martin et. al., 2007; Meisler et. al., 2010).
  • certain embodiments of the invention provide a splice modulating oligonucleotide (SMO), comprising a sequence designed to modulate the splicing of a SCN8A pre-mRNA, wherein the SMO sequence specifically binds to a sequence in the SCN8A pre-mRNA.
  • SMO splice modulating oligonucleotide
  • compositions comprising an SMO described herein.
  • Certain embodiments of the invention provide a pharmaceutical composition comprising an SMO described herein and a pharmaceutically acceptable carrier.
  • Certain embodiments of the invention provide a method of modulating splicing of an SCN8A pre-mRNA comprising contacting a cell with an effective amount of an SMO or a composition described herein.
  • Certain embodiments of the invention provide a method of treating or preventing a disease, disorder or condition in subject (e.g., a mammal, e.g., a human), comprising administering an SMO or composition as described herein to the subject.
  • Certain embodiments of the invention provide an SMO or a composition as described herein for the prophylactic or therapeutic treatment of a disease, disorder or condition in a subject.
  • Certain embodiments of the invention provide the use of an SMO or a composition as described herein to prepare a medicament for treating disease, disorder or condition in a subject.
  • Certain embodiments of the invention provide an SMO or a composition as described herein for use in medical therapy.
  • Certain embodiments of the invention provide an SMO or a composition as described herein for use in treating a disease, disorder or condition. BRIEF DESCRIPTION OF THE DRAWINGS
  • LSP-GRl is shown on the left as a dark grey bar
  • LSP-GR3 is shown on the right as a light grey bar.
  • the bar representing the cortex is shown on the left as a light grey bar and the bar representing the hippocampus is shown on the right as a dark grey bar.
  • FIGS. 1D-F ICV injection of LSP-GRl (GR1) protected neonatal mice from KA- induced seizures, and prevented status epilepticus (SE)-induced increase in AMPA-R (a)EPSCs.
  • FIG. IE ICV injection of LSP-GRl (GR1) protected neonatal mice from KA- induced seizures, and prevented status epilepticus (SE)-induced increase in AMPA-R (a)EPSCs.
  • FIG. 2 C Whole-cell patch- clamp recordings of aEPSCs from CA1 pyramidal neurons in P12 mice. SE induction at P10, followed 2 hr later by ICV injection of saline, produced a large increase in aEPSC amplitude compared to naive (no SE) mice (p ⁇ 0.001).
  • FIGS. 2A-D Comparison of top candidate SCN8A exon 18A skipping SMOs.
  • FIG. 2A Ten SMOs were tested in vivo for ability to direct SCN8A exon 18A skipping via paradigms involving 1, 2, or 3 bilateral ICV injections at doses of 2 or 4 ⁇ g per ventricle in neonatal pups between the ages of P3-7.
  • 18A-2 SEQ ID: 1324
  • 18A-3 SEQ ID: 1327
  • 18A-4 SEQ ID: 1317
  • 18A-5 SEQ ID: 1306)
  • 18A-8 SEQ ID: 1307
  • 18A-9 SEQ ID: 1422
  • 18A-10 SEQ ID: 1541
  • FIG. 2B A single submaximal dose (2 ⁇ g bilateral - 4 ⁇ g total) was given by ICV injection in P3-5 neonatal mouse pups for each candidate compound to examine small differences in splicing efficiency for the most potent of the compounds during initial screeening, relative to saline (negative control, dotted line at 1.0) and compared to LSP-GRl (positive control) .
  • FIGS. 2E-F Comparison of candidate SCN8A exon 5A skipping SMOs.
  • FIGS. 3A-K SCN8A E5A Splicing SMOs.
  • FIG. 3A Human SCN8A target sequences for E5A splicing: 7nt of the Intron 5' to Exon 5A + entire 92 nt of Exon 5A + 5nt ofIntron 5.
  • FIG. 3B SCN8A E5A 24 mer SMO sequences.
  • FIG. 3C SCN8A E5A 23 mer SMO sequences.
  • FIG. 3 D SCN8A E5A 22 mer SMO sequences.
  • FIG. 3E SCN8A E5A 21 mer SMO sequences.
  • FIG. 3F SCN8A E A 20 mer SMO sequences.
  • FIG. 3G Human SCN8A target sequences for E5A splicing: 7nt of the Intron 5' to Exon 5A + entire 92 nt of Exon 5A + 5nt ofIntron 5.
  • FIG. 3B SCN8A E5A 24
  • FIG. 3H SCN8A E5A 18 mer SMO sequences.
  • FIG. 31 SCN8A E5A 17 mer SMO sequences.
  • FIG. 3J SCN8A E5A 16 mer SMO sequences.
  • FIG. 3K SCN8A E5A 15 mer SMO sequences.
  • FIGS. 4A-D SCN8A E18A Splicing SMOs.
  • FIG. 4A Human SCN8A target sequences internal to Intron 18 near the 5' splice site, and corresponding preferred SCN8AN SMO sequences for skipping Exon 18 A.
  • FIG. 4B Human SCN8A target sequences at the 5' splice site, and corresponding preferred SCN8AN SMO sequences for skipping Exon 18A. The entire target sequence covers 5' splice site, and is 100% conserved between mouse and human. It is noted that the 5' splice site cannot be targeted while being specific for SCN8A because of too much identity with SCN1A.
  • FIG. 4C Human SCN8A target sequences internal to Intron 18 near the 5' splice site, and corresponding preferred SCN8AN SMO sequences for skipping Exon 18 A.
  • FIG. 4B Human SCN8A target sequences at the 5' splice site, and corresponding preferred SCN8AN SMO
  • the present invention has utility as a medical treatment of seizure disorders, neurological disorders, and cancers; as well as novel compositions for the detection of susceptibility thereto.
  • SCNIA loss-of-function mutations are the major cause of Dravet spectrum pediatric epilepsies, including generalized epilepsy with febrile seizure plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome (Claes et. al., 2001).
  • the major therapeutic indication for modulating the splicing of SCN8A is to correct the excitatory/inhibitory imbalance in the brain caused by loss-of-function mutations in SCNIA.
  • SCNIA and SCN8A can to be thought of as opposing aspects that must balance exactly for normal brain function. If the amount of normal SCNIA function is reduced due to a mutation, then the present invention serves to reduce SCN8A function, to rebalance the scale.
  • An inventive process to control SCN8A function is by controlling the mRNA splicing to code for an alpha subunit protein that either doesn't allow the resulting VGS channel to function as a sodium channel or exhibits reduced sodium channel kinetics. Based on SCNIA knock out mouse studies, reducing SCN8 mediated excitation is a logical strategy for rebalancing the reduced inhibitory input caused by SCNIA mutations.
  • SCN8A subunits are naturally alternatively spliced at two specific sites of interest. Exon 18 is alternatively spliced to form 18N (neonatal) and 18A (adult) iso forms. Inclusion of the 18N exon yields a truncated nonfunctional SCN8A-18N (Plummer et. al., 1997).
  • SMOs are designed to overcome several barriers to successful drug development. In contrast to classic antisense compounds and siRNAs, SMOs do not recruit degradation enzymes (RNAseH, dicer) and therefore do not cause off-target degradation of transcripts. SMOs bind to their targets with exceptional potency, specificity, and negligible off-target effects (Eckstein 2007)
  • our proposed SMOs will be designed for complete selectivity in targeting SCN8A isoform expression without affecting any other highly related VGS channel subunits.
  • regulation of SCN8A exon 18A splicing is differentially controlled in non-neuronal cells, thus SMOs can be designed specifically to modulate splicing in the CNS such that release from the CNS during normal metabolism is unlikely to have on-target effects outside of the CNS (Zubovic et. al., 2012), and vice versa.
  • the SCN8A gene is nearly 100% conserved between mouse and human surrounding the SMO target sites, such that SMOs validated in the mouse model will be directly applicable to the clinic.
  • SCN8A The strategy of specifically reducing function only of the Na+ channel subunit that counterbalances SCN1A input (SCN8A) should be more effective with fewer adverse effects than non-selective VGS channel blockers. Further, by changing alternative splicing, an SMO directed against exon 5A will specifically reduce excitatory channel properties, rather than simply decreasing overall Navl.6 channel function.
  • the modulation of SCN8A pre-mRNA splicing may also be used to treat a variety of diseases and disorders. Specifically, the SMOs described herein, which target SCN8A pre- mRNA, may also be used to treat certain neurological disorders and cancer as described below.
  • the present invention encompasses a class of compounds known as splice modulating oligonucleotides (SMOs) that modulate pre-mRNA splicing, thereby affecting expression and functionality of a specific protein in a cell; where the pre-mRNA is SCN8A.and the protein is Navl.6
  • SMOs splice modulating oligonucleotides
  • An SMO specifically binds to a complementary sequence on a pre-mRNA at an exon or intron splice suppressor or splice enhancer site, or at an intron- exon splice site, or at a variety of sites on the pre-mRNA containing various other motifs that are predicted to affect splicing.
  • an SMO specifically binds to a splice enhancer site, or an intron-exon splice site, the adjacent exon is excluded from the resulting mRNA. Additionally, an SMO may specifically bind to a splice suppressor site or an intron- exon site and the adjacent exon is included in the resulting mRNA. Finally, an SMO may specifically bind to a splice enhancer site or an intron-exon splice site and shift the reading frame of the pre-mRNA so that the resulting protein is truncated. In some cases, the resulting protein is a limited-function, or non-functional protein relative to the native protein.
  • an exonic or intronic splice enhancer or suppressor motif may be found anywhere within the exon and the flanking introns.
  • an SMO may either fully or partially overlap a predicted exonic or intronic splice enhancer or suppressor site in proximity to an intron-exon boundary and/or be complementary to the predicted 3' or 5' splice sites.
  • the present invention is directed, in specific embodiments to oligonucleotides referred to herein as splice modulating oligonucleotides (SMOs), suitable for use in modulating splicing of a target transcript pre-mRNA.
  • SCN8A pre-mRNA splicing is modulated to correct the excitatory/inhibitory imbalance in the brain caused by loss-of- function mutations in SCN1A.
  • SCN8A pre-mRNA splicing is modulated to treat any disease or disorder to which reducing or increasing input from SCN8A containing voltage gated sodium channels is therapeutic.
  • SCN8A pre-mRNA splicing is also modulated as a tool for studying SCN8A both in vitro and in vivo.
  • SMOs are operative as therapeutics, gene therapy, genotyping a subject, and as part of a business method in which any of the aforementioned tasks are accomplished in exchange for financial remuneration.
  • certain embodiments of the invention provide an SMO based on the consensus sequence of sodium channel, voltage-gated, type VIII (Navl.6), alpha subunit (SCN8A) (OMIM: 600702; Genbank AB027567.1), including upstream and downstream nucleotides (see, e.g., FIGS. 3A-K. and 4A-D).
  • the present invention also includes a pharmaceutical composition including an SMO suitable for modulating splicing of a target pre-mRNA both in vitro and in vivo (e.g., SCN8A pre-mRNA).
  • SMOs are used according to the methods of the invention to modulate splicing of SCN8A pre-mRNA.
  • these SMOs are used according to the methods of the invention to modulate splicing of SCN8A pre-mRNA to exclude exon 5 A or exon 18A or a combination thereof.
  • FIGS. 3A-K and 4A-D depict exemplary SMOs useful for modulating splicing of SCN8A pre-mRNA (e.g., to exclude exon 5A or exon 18A).
  • certain embodiments of the invention provide a splice modulating oligonucleotide (SMO) that specifically binds to a SCN8A pre-mRNA (i.e., a pre-mRNA that undergoes splicing to form an mRNA encoding a SCN8A protein).
  • SMO splice modulating oligonucleotide
  • the inventive SMO specifically binds a complementary sequence of the SCN8A pre-mRNA.
  • the SMO includes a sequence designed to modulate the splicing of an SCN8A pre-mRNA.
  • the SMO includes a sequence that specifically binds to an exon, an intron, a 5' untranslated region (UTR), a 3' UTR, a splice junction, an exon:exon splice junction, an exonic splicing silencer (ESS), an exonic splicing enhancer (ESE), an intronic splicing silencer (ISS), ,an intronic splicing enhancer (ISE), or a combination of any of the aforementioned in the SCN8A pre-mRNA.
  • UTR 5' untranslated region
  • ESE exonic splicing enhancer
  • ISS intronic splicing silencer
  • ISE intronic splicing enhancer
  • the SMO includes a sequence that specifically binds to exon 5A, exon 5N, exon 18 A, exon 18N, intron 4, intron 5, intron 4A, intron 4N, intron A, intron 5N, intron 17, intron 18, intron 17A, intron 17N, intron 18A , intron 18N or a combination of any of the aforementioned of the SCN8 A pre-mRNA (see, e.g., Example 1 and FIGS. 3A-K. and 4A-D).
  • hybridizing refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art.
  • the term refers to hybridization of an SMO with a substantially complementary sequence contained within a complementary sequence of a target complementary sequence of the SCN8A pre-mRNA molecule, to the substantial exclusion of hybridization of the SMO with a pre-mRNA that has a non-complementary sequence.
  • Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art. It is appreciated that these conditions are largely dictated by cellular conditions for in vivo applications.
  • the term "complementary” or “complementarity” refers to a degree of antiparallel relationship between a strand of SMO and a pre-mRNA molecule In some instances, the complementarity between an inventive SMO and a pre- mRNA is between 80 and 99.9%., while in other instance, the complementarity to a pre- mRNA by an inventive SMO is 100%.
  • the SMO of the invention may be defined generally as a nucleotide sequence (or oligonucleotide) a portion of which is capable of hybridizing with the target nucleic acid to exact an antisense activity on the target nucleic acid.
  • the inventive SMO may be defined functionally as a nucleotide sequence (or oligonucleotide) a portion of which is complementary to and capable of hybridizing with the target nucleic acid sequence to exact a splice modulation in the target RNA of at least 10% for a given subject as measured by target RNA levels.
  • the target nucleic acid an SCN8A pre-mRNA.
  • splice modulation refers to molecular manipulation of pre-mRNA splicing to direct the final composition of the mRNA transcript. It is appreciated that complementarity to the target pre-mRNA alone is not sufficient to produce an inventive SMO.
  • the location of SMO binding ie blocking splicing motifs in the pre-mRNA, and thermodynamics of binding at that site, as well as secondary structure of the pre-mRNA are among the factors that determine whether splice modulation occurs and the magnitude thereof
  • Tm 81.5°C.+16.6 Log [Na+]+0.41(% G+C)-0.63(% formamide)-600/#bp in duplex
  • the stringency of the ex vivo hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the SMO with a target therefor, the hybridization is usually carried out at salt and temperature conditions that are 20-25 °C below the calculated Tm of the hybrid. Wash conditions should be as stringent as possible for the degree of identity of the probe for the target. In general, wash conditions are selected to be approximately 12-20°C below the Tm of the hybrid.
  • a moderate stringency hybridization is defined as hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 2xSSC and 0.5% SDS at 55°C for 15 minutes.
  • a high stringency hybridization is defined as hybridization in 6xSSC, 5x.Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in l.times.SSC and 0.5% SDS at 65°C for 15 minutes.
  • a very high stringency hybridization is defined as hybridization in 6xSSC, 5x.Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in O.lxSSC and 0.5% SDS at 65°C for 15 minutes.
  • Examples of additional conditions under which a nucleotide sequence (or oligonucleotide or SMO sequence) is capable of hybridizing with the target RNA include 400 raM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C hybridization for 12-16 hours; followed by washing) and hybridization at 70°C in IX SSC or 50°C in IX SSC, 50% formamide followed by washing at 70°C in 0.3X SSC or hybridization at 70°C in 4X SSC or 50°C in 4X SSC, 50% formamide followed by washing at 67°C in IX SSC.
  • the hybridization temperature for hybrids less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, as determined according to the following equations.
  • Tm melting temperature
  • Tm (°C ) 2 (number of A + T bases) + 4 (number of G + C bases).
  • the SMO includes a sequence designed to modulate the splicing of an SCN8A pre-mRNA (e.g., to exclude exon 5A or exon 18A), wherein the SMO has at least about 60% (e.g., about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) complementarity to an SCN8A pre-mRNA, and wherein the SMO sequence is 14 to 26 nucleotides long (e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides long).
  • the SMO includes a sequence designed to bind with complementarity to an SCN8A pre-mRNA and modulate the splicing of exon 5A/5N in the SCN A pre-mRNA.
  • the SMO includes a sequence designed to bind with complementarity to an SCN8A pre-mRNA and exclude exon 5A from a resulting SCN8A niRNA.
  • the SMO includes a sequence designed to bind with complementarity to an SCN8A pre-mRNA and include exon 5N in a resulting SCN8A mRNA.
  • the SMO includes a sequence that specifically binds to a 3' or 5' splice site of SCN8A exon 5 A. In certain inventive embodiments, the SMO includes a sequence that specifically binds to an exon 5 A exonic splice enhancer (ESE) sequence within an SCN8A pre-mRNA. In certain inventive embodiments, the SMO includes a sequence that specifically binds to an exon 5A intronic splice enhancer (ISE) sequence within an SCN8A pre-mRNA. In certain inventive embodiments, the SMO includes a sequence that specifically binds to an exon 5N intronic splice silencer (ISS) sequence within an SCN8A pre-mRNA.
  • ESE exonic splice enhancer
  • ISE exonic splice enhancer
  • ISS exon 5N intronic splice silencer
  • the SMO includes a sequence that specifically binds to an exon 5N exonic splice silencer (ESS) sequence within an SCN8A pre-mRNA.
  • the SMO includes a sequence that specifically binds to exon 5 A of the SCN8 A pre-mRNA (e.g., binds to a complementary sequence in exon 5A (either partially or wholly within exon 5A)).
  • the SMO includes a sequence designed to modulate the splicing of exon 18A/18N in the SCN8A pre-mRNA.
  • the SMO includes a sequence designed to bind with complementarity to an SCN8A pre- mRNA and exclude exon 18A from the resulting SCN8A mRNA.
  • the SMO includes a sequence designed to bind with complementarity to an SCN8A pre-mRNA and include exon 18N in a resulting SCN8A mRNA.
  • the nucleic acid includes a sequence that specifically binds to a 3 ' or 5' splice site of SCN8A exon 18 A.
  • the nucleic acid includes a sequence that specifically binds to an exon 18 A exonic splice enhancer (ESE) sequence within an SCN8A pre-mRNA.
  • the nucleic acid includes a sequence that specifically binds to an exon 18A intronic splice enhancer (ISE) sequence within an SCN8A pre-mRNA.
  • the SMO includes a sequence that specifically binds to an exon 18N intronic splice silencer (ISS) sequence within an SCN8A pre-mRNA.
  • the SMO includes a sequence that specifically binds to an exon 18N exonic splice silencer (ESS) sequence within an SCN8A pre-mRNA.
  • the SMO includes a sequence that specifically binds to exon 18A of the SCN8A pre-mRNA (e.g., binds to a complementary sequence in exon 18A (either partially or wholly within exon 18A)).
  • the SMO includes a sequence that has at least about 60% complementarity with a SCN8A pre-mRNA sequence.
  • the sequence has at least about 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% complementarity with a SCN8A pre-mRNA sequence.
  • the SMO includes a sequence that has at least about 60% complementarity with SEQ ID NO:l, 858, 965, 1252, or 1859. In certain inventive embodiments, the sequence has at least about 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% complementarity with SEQ ID NO: 1, 858, 965, 1252, or 1859.
  • the SMO includes a sequence that has at least about 60% sequence identity with SEQ ID NOs:2, 859, 966, 1253, or 1860.
  • the sequence has at least about 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with SEQ ID NOs: 2, 859, 966, 1253, or 1860.
  • the SMO sequence is about 14 to about 26 nucleotides long (e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides long). In certain inventive embodiments, the SMO is about 15 to about 24 nucleotides long.
  • the SMO is about 14 to about 26 nucleotides and includes between about 6 and 24 contiguous nucleotides (i.e., about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides) of any one of SEQ ID NOs: 3-857. In certain inventive embodiments, the SMO includes between about 10 to about 24 contiguous nucleotides of any one of SEQ ID NOs: 3-857. In certain inventive embodiments, the SMO includes about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides of any one of SEQ ID NOs: 3-857.
  • the SMO is about 14 to about 26 nucleotides and includes between about 6 and 24 contiguous nucleotides (i.e., about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides) of any one of SEQ ID NOs:860-964, 967-1251, 1254-1858 and 1861-2140.
  • the SMO includes between about 10 to 24 contiguous nucleotides of any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861-2140.
  • the SMO includes about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 contiguous nucleotides of any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861-2140.
  • the SMO includes a sequence that has at least 60% sequence identity with any one of SEQ ID NOs: 3-857.
  • the sequence has at least 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with any one of SEQ ID NOs. 3-857.
  • the sequence is selected from any one of SEQ ID NOs: 3-857.
  • the SMO is a sequence that has at least 60% sequence identity with any one of SEQ ID NOs: 3-857.
  • the sequence has at least 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with any one of SEQ ID NOs: 3-857.
  • the sequence is selected from any one of SEQ ID NOs: 3-857.
  • the SMO includes a sequence that has at least 60% sequence identity with any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861- 2140.
  • the sequence has at least 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with any one of SEQ ID NOs:860-964, 967-1251, 1254-1858 and 1861-2140.
  • the sequence is selected from any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861-2140.
  • the SMO has a sequence that has at least 60% sequence identity with any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861- 2140.
  • the sequence has at least 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with any one of SEQ ID NOs:860-964, 967-1251, 1254-1858 and 1861-2140.
  • the sequence is selected from any one of SEQ ID NOs: 860-964, 967-1251, 1254-1858 and 1861-2140.
  • sequence is selected from any one of SEQ ID NOs: 860-964. [0059] In certain inventive embodiments, the sequence is selected from any one of SEQ ID NOs: 967-1251.
  • the sequence is selected from any one of SEQ ID NOs: 1254-1858. In certain inventive embodiments, the sequence is SEQ ID NO: 1324.
  • sequence is selected from any one of SEQ ID NOs: 1861-2140.
  • compositions including an SMO described herein are provided.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition includes a pharmaceutically acceptable carrier.
  • the route of SMO administration is oral, rectal, intraventricular, intracranial, intratumoral, intrathecal, intracisternal, epidural, intravaginal, parenteral, intravenous, intramuscular, subcutaneous, local, intraperitoneal, transdermal, by inhalation or as a buccal or nasal spray.
  • the exact amount of SMO required will vary from subject to subject, depending on the age, weight and general condition of the subject, the severity of the disease that is being treated, the mode of administration, and the like. An appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • the SMO can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include an effective amount of the selected SMO in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents.
  • pharmaceutically acceptable is meant a material that is not biologically, or otherwise undesirable, which can be administered to a subject along with the selected SMO without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert customary excipient such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents
  • compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the SMO in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above- mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example
  • compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.
  • Ophthalmic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
  • An oligonucleotide of the invention i.e. the SMO
  • SMO can be synthesized using any procedure known in the art, including chemical synthesis, enzymatic ligation, organic synthesis, and biological synthesis.
  • an RNA molecule e.g., an SMO
  • an SMO is prepared chemically.
  • Methods of synthesizing RNA and DNA molecules are known in the art, in particular, the chemical synthesis methods as described in Verma and Eckstein (1998) Annul Rev. Biochem. 67:99-134.
  • RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
  • the RNA may be used with no or a minimum of purification to avoid losses due to sample processing.
  • the oligonucleotides of the present invention are modified to improve stability in serum or growth medium for cell cultures, or otherwise to enhance stability during delivery to subjects and/or cell cultures.
  • the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they include only purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine by 2'-deoxythymidine, or cytosine by 5'-methylcytosine, can be tolerated without affecting the efficiency of oligonucleotide reagent-induced modulation of splice site selection.
  • the absence of a 2' hydroxyl may significantly enhance the nuclease resistance of the oligonucleotides in tissue culture medium.
  • the oligonucleotides may contain at least one modified nucleotide analogue at any position within the sequence, including the entirety of the SMO sequence.
  • the nucleotide analogues may be located at positions where the target-specific activity, e.g., the splice modulating activity is not substantially effected, e.g., in a region at the 5'-end and/or the 3'-end of the oligonucleotide molecule, or a combination of such sites to increase stability against enzymatic degradation while preserving functionality compared to a base SMO containing only nucleotides naturally occurring in the host.
  • the ends may be stabilized by incorporating modified nucleotide analogues.
  • nucleotide analogues operative herein include sugar- and/or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphodiester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphorothioate group.
  • the 2' OH-group is replaced by a group of CH 3 , H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or ON, where R is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl and halo is F, CI, Br or I.
  • the 2' OH-group is replaced by O-CH 3 also known as 2'0-methyl modification
  • Other specific nucleotide analogues include nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to phosphorothioate derivatives and acridine substituted nucleotides, , 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-
  • Oligonucleotides of the invention also may be modified with chemical moieties (e.g., cholesterol) that improve the in vivo pharmacological properties of the oligonucleotides.
  • chemical moieties e.g., cholesterol
  • oligonucleotides of the invention as few as one and as many as all nucleotides of the oligonucleotide can be modified.
  • a 20-mer oligonucleotide (e.g., oligoribonucleotide) of the invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modified nucleotides.
  • the modified oligonucleotides (e.g., oligoribonucleotides) of the invention will contain as few modified nucleotides as are necessary to achieve a desired level of in vivo stability and/or bio- accessibility while maintaining cost effectiveness.
  • SMOs of the invention include oligonucleotides synthesized to include any combination of modified bases disclosed herein in order to optimize function.
  • an SMO of the invention includes at least two different modified bases.
  • an SMO of the invention may include alternating 2' O-methyl substitutions and LNA bases or constrained ethyl nucleic acid (cEt) bases.
  • An oligonucleotide of the invention can be an a-anomeric nucleic acid molecule.
  • An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual a-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641).
  • the oligonucleotide can also include a 2'-0-methylribonucleotide (Inoue et al, 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • the oligonucleotides of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acid molecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.
  • PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs.
  • the oligonucleotides of the invention can also be formulated as morpholino oligonucleotides.
  • an operative SMO has an oligonucleotide modification that includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the linkage is preferably a methylene ( ⁇ CH2 ⁇ ) n group (such as an ethyl or methoxymethyl group) bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226, the entire contents of which are incorporated by reference herein.
  • the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 6:958- 976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the invention also includes molecular beacon nucleic acid molecules having at least one region which is complementary to a nucleic acid molecule of the invention, such that the molecular beacon is useful for quantitating the presence of the nucleic acid molecule of the invention in a sample.
  • a "molecular beacon" nucleic acid is a nucleic acid molecule including a pair of complementary regions and having a fluorophore and a fluorescent quencher associated therewith. The fluorophore and quencher are associated with different portions of the nucleic acid in such an orientation that when the complementary regions are annealed with one another, fluorescence of the fluorophore is quenched by the quencher.
  • the SMO includes at least one nucleotide that contains a non-naturally occurring modification including at least one of a chemical composition of phosphorothioate 2'-0-methyl, phosphorothioate 2'-MOE, locked nucleic acid (LNA) peptide nucleic acid (PNA), phosphorodiamidate morpholino, or any combination thereof.
  • a chemical composition of phosphorothioate 2'-0-methyl, phosphorothioate 2'-MOE locked nucleic acid (LNA) peptide nucleic acid (PNA), phosphorodiamidate morpholino, or any combination thereof.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • the SMO includes at least one 2'-0-methyl nucleotide. In certain inventive embodiments, the SMO includes at least two 2 -O-methyl nucleotides. In certain inventive embodiments, the SMO includes at least three 2'-0-methyl nucleotides. In certain inventive embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the SMO nucleotides are 2'-0-methyl modified.
  • the SMO includes at least one nucleotide with a phosphorothioate linkage. In certain inventive embodiments, the SMO includes at least two nucleotides with phosphorothioate linkages. In certain inventive embodiments, the SMO includes at least three nucleotides with phosphorothioate linkages. In certain inventive embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the SMO nucleotides include phosphorothioate linkages.
  • the SMO includes at least one phosphorothioate 2'- O-methyl modified nucleotide. In certain inventive embodiments, the SMO includes at least two phosphorothioate 2'-0-methyl modified nucleotides. In certain inventive embodiments, the SMO includes at least three phosphorothioate 2'-0-methyl modified nucleotides. In certain inventive embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the SMO nucleotides are phosphorothioate 2'-0-methyl modified.
  • modifications include a bicyclic sugar moiety similar to the LNA has also been described (see U.S. Pat. No. 6,043,060) where the bridge is a single methylene group which connect the 3'-hydroxyl group to the 4' carbon atom of the sugar ring thereby forming a 3'-C,4'-C-oxymethylene linkage.
  • oligonucleotide modifications include cyclohexene nucleic acids (CeNA), in which the furanose ring of a DNA or RNA molecule is replaced with a cyclohexenyl ring to increase stability of the resulting complexes with RNA and DNA complements (Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602).
  • other bicyclic and tricyclic nucleoside analogs are included in the SMO.
  • the target RNA e.g., pre-mRNA, e.g., SCN8A pre-mRNA
  • oligonucleotides of the invention is highly sequence specific.
  • oligonucleotides containing nucleotide sequences perfectly complementary, having 100% complementarity to a portion of the target RNA are exposed to target RNA for blocking of sequence elements within the target RNA.
  • 100% sequence complementarity between the oligonucleotide and the target RNA is not required to practice the present invention.
  • the invention may tolerate sequence variations that might be expected due to genetic mutation, wobble base pairing, strain polymorphism, or evolutionary divergence.
  • oligonucleotide sequences with insertions, deletions, and single point mutations relative to the target sequence may also be effective for SMO - mediated splice modulation.
  • oligonucleotide sequences with nucleotide analog substitutions or insertions can be effective for splice modulation.
  • sequence identity e.g., 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, and any and all whole or partial increments there between the oligonucleotide and the target RNA, e.g., target pre-mRNA, is preferred.
  • oligonucleotide (SMO) sequences may be more tolerant to mismatches than other oligonucleotide sequences.
  • SMO oligonucleotide
  • One of ordinary skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, between an SMO and a target nucleic acid, such as by determining melting temperature (Tm) and evaluating the effect of chemical modifications on the Tm and hybridization stringency. Tm can be calculated by techniques that are familiar to one of ordinary skill in the art. Techniques and calculations as described in Freier et al.
  • sequence identity in the context of two nucleic acid sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned by sequence comparison algorithms or by visual inspection. For example, sequence identity may be used to reference a specified percentage of residues that are the same across the entirety of the two sequences when aligned.
  • the term "substantial identity" of polynucleotide sequences means that a polynucleotide includes a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%; at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%; at least 90%, 91%, 92%, 93%, or 94%; or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • Sequence identity including determination of sequence complementarity or homology for nucleic acid sequences, may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity i.e., a local alignment.
  • a preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment).
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment).
  • a preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
  • sequence identity for two sequences is based on the greatest number of consecutive identical nucleotides between the two sequences (without inserting gaps). For example, the percent sequence identity between Sequence A and B below would be 87.5% (Sequence B is 14/16 identical to Sequence A), whereas the percent sequence identity between Sequence A and C would be 25% (Sequence C is 4/16 identical to Sequence A).
  • nucleic acids, oligonucleotides, SMOs, or a portion thereof may have a defined percent identity to a SEQ ID NO, or a another LifeSplice compound.
  • a sequence is identical to the SMO sequence disclosed herein if it has the same nucleobase pairing ability. This identity may be over the entire length of the nucleotide sequence, or in a portion of the nucleotide sequence e.g., nucleobases 1-20 of a 300-mer may be compared to a 20-mer to determine percent identity of the nucleic acid to the SEQ ID NO described herein.
  • Percent identity is calculated according to the number of nucleotide bases that have identical base pairing corresponding to the SEQ ID NO or SMO compound to which it is being compared.
  • the non-identical bases may be adjacent to each other, dispersed throughout the nucleotide sequence, or both. For example, a 18-mer having the same sequence as nucleobases 3-20 of a 24-mer SMO is 75% identical to the 24-mer SMO. Alternatively, a 24-mer containing six nucleobases not identical to another 24-mer is also 75% identical to the 24-mer. Similarly a 15-mer having the same sequence as nucleobases 1-15 of a 100-mer is 15% identical to the 100-mer. Such calculations are well within the ability of those skilled in the art.
  • nucleic acid sequence need not have an identical sequence to those described herein to function similarly to the SMO compound described herein.
  • Shortened versions of SMO compounds taught herein, or non- identical versions of the SMO compounds taught herein, are also provided.
  • Non-identical versions can include at least one base replaced with a different base with different pairing activity (e.g., G can be replaced by C, A, or T), wobble base pairing, or sequences are those wherein each base does not have the same pairing activity (e.g. by the nucleic acid sequence being shorter or having at least one abasic site) as the SMOs disclosed herein.
  • the oligonucleotide may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) a portion of which is capable of hybridizing with the target RNA (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 5O 0 C or 70°C hybridization for 12-16 hours; followed by washing).
  • target RNA e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 5O 0 C or 70°C hybridization for 12-16 hours; followed by washing.
  • Additional preferred hybridization conditions include hybridization at 70°C in IX SSC or 50°C in IX SSC, 50% formamide followed by washing at 70°C in 0.3X SSC or hybridization at 70°C in 4X SSC or 50°C in 4X SSC, 50% formamide followed by washing at 67°C in IX SSC.
  • the present invention provides compositions and methods for modulating SCN8A pre-mRNA splicing using an SMO of the invention (e.g., to abrogate disease-causing mutations in a protein, such as SCN1A).
  • an SMO may modulate pre-mRNA splicing by removing an exon (e.g., exon 5A or exon 18A) or including an exon (e.g., exon 5N or exon 18N) in order to alter protein isoform expression (e.g., to enhance expression of isoforms with reduced excitatory function).
  • an SMO as described herein may modulate SCN8A pre-mRNA by excluding exon 5 A in the resulting SCN8A mRNA.
  • SMOs may be used to modify SCN8A channel properties, i.e., to reduce sodium currents.
  • an SMO described herein may modulate SCN8A pre-mRNA by excluding exon 18A in the resulting SCN8A mRNA.
  • SMOs may be used to generate a truncated SCN8A protein that is non-functional as a sodium channel, or that is not even translated into a SCN8 protein.
  • certain inventive embodiments of the invention provide a method of modulating splicing of an SCN8A pre-mRNA, either in vitro or in vivo including contacting a cell with an effective amount of an SMO or composition described herein.
  • the SMO specifically binds to a SCN8A pre-mRNA sequence (e.g., at an intron/exon splice site, ESE and/or ISE), thereby excluding exon 5 A or exon 18A from a resulting SCN8A mRNA.
  • Certain inventive embodiments of the invention provide a method of modulating splicing of an SCN8A pre-mRNA including contacting a cell with an effective amount of an SMO that specifically binds to a complementary sequence on the pre-mRNA at a intron-exon splice site, ESE and/or ISE, wherein when the SMO specifically binds to the complementary sequence, exon-18A or exon-5A is excluded from the resulting mRNA, and wherein the resulting mRNA encodes an SCN8A protein.
  • Certain inventive embodiments of the invention provide a method of modulating splicing of an SCN8A pre-mRNA including contacting a cell with an effective amount of an SMO that specifically binds to a complementary sequence on the pre-mRNA at a intron-exon splice site, ESE and/or ISE, wherein when the SMO specifically binds to the complementary sequence, exon-18N or exon-5N is included in the resulting mRNA, and wherein the resulting mRNA encodes an SCN8A protein.
  • Certain inventive embodiments of the invention provide a method of reducing neuronal excitability including contacting a cell with an effective amount of an SMO or composition described herein.
  • SCN8A loss-of function mutation or knockout results in increased seizure threshold to chemoconvulsant induced seizures (Martin et. al., 2007), thus the SMOs that modulate SCN8A isoform expression (e.g., decrease E5A or E18A; FIGS. 3A-K. and 4A-D) are viable therapeutics for other types of refractory pediatric and adult epilepsies; some that have dysfunctional SCNIA and others that do not.
  • SMOs have the potential be treat various diseases or disorders in which CNS hyperexcitability and/or excitotoxicity have been implicated as having a significant contribution to disease pathology through dysfunction of SCNIA or SCN8A. Additionally, there are hundreds of SCNIA and SCN8A mutations attributed to a variety of epilepsy syndromes aside from the Dravet spectrum epilepsies (Oliva et. al., 2012).
  • Epileptic encephalopathy is characterized by onset of variable types of seizures in infancy including generalized tonic-clonic, atypical absence, partial, apneic attack, febrile convulsion, and loss of tone and consciousness, which are refractory to typical anti-seizure drugs (Ohba et.
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection as infrequently as every 1-6 months or by continuous infusion via pump (ie Omaya Reservoir) directly into the hippocampus.
  • Dosing for peripheral indications ie SUDEP from cardiac arrythmia
  • SMOs in the present invention that mediate splice modulation of SCN8A channel alpha subunits to reduce functional channel expression (E18A) or modulate channel properties (E5A) are strong therapeutic candidates.
  • SMO dosing for spinal cord injury can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months, or as otherwise necessary.
  • Voltage-gated sodium channels are also expressed in non-excitable cells such as macrophages and neoplastic cells.
  • a functional splice variant containing E18A of SCN8A, is required for podosome and invadopodia formation in macrophages.
  • SCN8A is as the alpha subunit of NaV1.6. Absence of functional NaV1.6 through a naturally occurring mutation (med) in mouse peritoneal macrophages inhibited podosome formation (Carrithers et. al, 2009).
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months, continuous ICV infusion via pump (ie Omaya Reservoir), or bolus delivery (ie Omaya Reservoir) directly into the tumor vasculature.
  • Dosing for peripheral indications can be achieved through monthly subcutaneous injections.
  • ALS Amyotrophic Lateral Sclerosis
  • SMOs recited herein that reduce SCN8A 5A and 18A isoforms also are expected to provide a potent anti-inflammatory response in the CNS (see Section 10 below), and therefore are expected to provide therapeutic benefit to ALS patients via a dual mechanism.
  • SMO dosing for ALS can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months, or as otherwise necessary for efficacy and patient compliance.
  • AD Alzheimer 's disease
  • Reduced SCNIA the alpha subunit of Navl .1 expression in inhibitory interneurons and parvalbumin cells are found both in mouse models of AD and AD patients (Verret et. al., 2012).
  • restoring normal levels of SCNIA in the brain of AD mice reduced epileptiform discharges, memory deficits, and increased survival.
  • neuronal network excitatory imbalance produces debilitating brain pathology.
  • An innovative SMO-based therapeutic approach to rebalance the net inhibitory plus excitatory synaptic drive from reduced SCNIA expression in AD is to reduce the counterbalancing SCN8A synaptic drive using optimal SMOs that reduce either SCN8A E18A (FIGS. 4A-D) or the SCN8A E5A (FIG. 3A-K) isoform expression, reducing overall synaptic input from the SCN8A-containing VGS channels.
  • SMOs SCN8A E18A
  • FIG. 3A-K SCN8A E5A
  • this strategy may feasibly be accomplished via reducing SCN8A E5A- or E18A-containing isoforms of SCN8A.
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months or continuous infusion via pump (ie Omaya Reservoir) directly into the lateral ventricles, or as otherwise necessary for efficacy and patient compliance.
  • VGS voltage-gated sodium
  • SCN8A NaV1.6
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months or continuous infusion via pump (ie Omaya Reservoir) directly into the lateral ventricles, or as otherwise necessary for efficacy and patient compliance.
  • Autism has been linked to de novo SCN1A mutations (O'Roak et. al., 2011; O'Roak et. al., 2012).
  • patients with Dravet spectrum epilepsies may also exhibit austistic behaviors due to SCN1A mutations (Han et. al., 2012), thus rebalancing the excitatory and inhibitory inputs in the brain can be accomplished through reducing SCN8A E18A or E5A expression which could provide therapeutic benefit to autistic patients.
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months or continuous infusion via pump (ie Omaya Reservoir) directly into the lateral ventricles, or as otherwise necessary for efficacy and patient compliance.
  • FHM Familial Hemiplegic Migraine
  • SCN1A- containing VGS channel function Gargus and Tournay 2007; Silberstein and Dodick 2013
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months or continuous infusion via pump (ie Omaya Reservoir) directly into the lateral ventricles, or as otherwise necessary for efficacy and patient compliance.
  • SCN8A-containing VGS channels in demyelinated axons activates a Na+-Ca2+ exchanger that imports Ca2+ into the axon, leading to axonal injury and eventually axonal degeneration (Waxman 2006).
  • SCN8A is upregulated in microglia of MS patients and in animal models of MS (Black and Waxman 2012).
  • SMOs reducing SCN8A function with SMOs ⁇ see, e.g., FIG. 3A-K. and 4A- D), would both reduce microglial activation and axonal injury/degeneration; providing therapeutic benefit to MS patients via two distinct mechanisms.
  • SMO dosing for CNS manifestations can be accomplished by direct bolus intrathecal injection at a frequency of every 1-6 months or continuous infusion via pump (ie Omaya Reservoir) directly into the lateral ventricles, or as otherwise necessary for efficacy and patient compliance.
  • Peripheral neuropathic pain (including post-herpetic neuralgia and diabetic neuropathy) : There is indirect evidence of increased persistent Na + currents at nodes of Ranvier due to changes in expression of Na v l.6 in diabetic neuropathy (Morris et. al., 2012). Development of neuropathic pain depends on axonal hyperexcitability due to increased nodal Na + currents, which is potentiated by lack of glycemic control, and this cascade is suggested to be responsible for neuropathic pain/paresthesia in diabetic neuropathy (Misawa et. al., 2009).
  • PPN Post-herpetic neuralgia
  • DRG dormant varicella zoster virus
  • Varicella zoster virus infection is associated with a significant increase in Nav 1 6 mRNA, which significantly increased Na+ current amplitude (Kennedy et. al., 2013).
  • Carpal tunnel In carpal tunnel syndrome, persistent Na+ current becomes altered across the carpal tunnel region leading to injury, inflammation, and ectopic impulse generation (Kuwabara et. al., 2006).
  • Nav 1 6 (and SCN8A) is highly expressed in the peripheral nodes of Ranvier (Morris et. al., 2012).
  • Sodium channel blockers such as
  • Mexiletine have been sown to be useful, thus SMO treatment to alter splicing of SCN8A, specifically to reduce expression of 18A or 5 A containing isoforms individually or in combination may produce long term relief of symptoms or prevent need for surgery. Dosing for peripheral indications can be achieved through monthly local subcutaneous,
  • SMO dosing may also be accomplished by epidural injection at the affected spinal level at a frequency of every 1-6 months, or as otherwise necessary for efficacy and patient compliance.
  • Cardiovascular disease or disorder e.g., hypertension, congestive heart failure, ischemia/reperfusion, arrhythmias
  • Arrhythmia and Ischemia and reperfusion injury It is thought that ventricular and atrial expression of Nav 1.6, in part, allows for a slow persistent Na+ current based Nav channel leak leading to arrhythmia or contributing to ischemia and reperfusion injury (Morris et. al., 2012).
  • current sodium channel blocking strategies to ameliorate cardiac ischemic and reperfusion damage, including block of the Na+ H+ exchanger have so far been therapeutically ineffective (Weiss et. al., 2010) necessitating novel therapeutic approaches.
  • SMO dosing for cardiac indications can be achieved through monthly subcutaneous injections, or as otherwise necessary for efficacy and patient compliance.
  • SCN8A expression is upregulated in activated microglia, and blocking SCN8A activity with nonselective Na+ channel blockers prevents microglia activation (Black and Waxman 2012).
  • many neurological diseases/disorders with a neuroinflammatory component including but not limited to CNS infections, stroke, ALS, Alzheimer's disease, Parkinson's disease, Huntington's disease (Fernandes et. al., 2014), and aging and age-related disorders (Norden and Godbout 2013) may be treatable using the highly selective SCN8A SMOs (FIGS. 3A-K. and 4A-D) of the present invention.
  • a SCN8A pre-mRNA may be an alternatively spliced, aberrantly spliced, overexpressed or unwanted pre-mRNA (e.g., a SCN8A pre-mRNA including exon 5A or exon 18 A) that encodes a protein that results in, causes, produces, or pre-disposes a subject to a disease or disorder.
  • splicing of a SCN8A pre-mRNA is not a cause of a disease or disorder, but modulation of the splicing of the SCN8A pre-mRNA reduces at least one symptom of the disease or disorder.
  • the invention provides a method of preventing in a subject, a disease, disorder, or condition associated with SCN8A pre-mRNA splicing, the method including administering to the subject an SMO or composition described, or vector, or transgene encoding same.
  • certain inventive embodiments of the invention provide a method of treating or preventing a disease, disorder or condition in subject (e.g., a mammal, e.g., a human), including administering an SMO or composition described herein to the subject.
  • subject e.g., a mammal, e.g., a human
  • the disease, disorder or condition is a neurological disease, disorder or condition.
  • the neurological disease, disorder or condition is epilepsy (e.g., a Dravet spectrum epilepsy), a disease or disorder associated with CNS hyperexcitability and/or excitotoxicity, a spinal cord injury, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), traumatic brain injury (TBI), autism, hemiplegic migraine, multiple sclerosis or a neuroinflammatory associated disease or disorder.
  • the neuroinflammatory associated disease or disorder is a CNS infection, stroke, ALS, AD, Parkinson's disease, Huntington's disease, aging or aging related disorders.
  • the disease, disorder or condition is pain mediated by SCN8A regulation.
  • the pain mediated disease, disorder or condition is peripheral neuropathic pain or carpal tunnel syndrome.
  • the disease, disorder or condition is cardiovascular mediated by SCN8A regulation.
  • the cardiovascular mediated disease, disorder or condition is hypertension, congestive heart failure, ischemia reperfusion, or arrhythmia.
  • the disease, disorder or condition is cancer mediated by SCN8A regulation.
  • the cancer is brain cancer mediated by SCN8A regulation.
  • Certain inventive embodiments of the invention provide a method of treating or preventing epilepsy or a Dravet Spectrum disorder in subject (e.g., a mammal, e.g., a human), including administering an SMO or composition described herein to the subject.
  • subject e.g., a mammal, e.g., a human
  • the Dravet Spectrum disorder is caused by a SCN1A mutation.
  • the Dravet Spectrum disorder is febrile seizures, generalized epilepsy with febrile seizure plus (GEFS+) or Dravet syndrome (severe myoclonic epilepsy of infancy or SMEI).
  • the administration reduces SCN8A excitatory function.
  • the SMO specifically binds to a SCN8A pre- mRNA sequence, wherein when the SMO specifically binds to the SCN8A pre-mRNA sequence, exon 5A is excluded in the resulting SCN8A mRNA, and wherein the resulting mRNA encodes a SCN8A protein.
  • the SMO specifically binds to a SCN8A pre- mRNA sequence, wherein when the SMO specifically binds to the SCN8A pre-mRNA sequence, exon 18A is excluded in the resulting SCN8A mRNA, and wherein the resulting mRNA encodes a SCN8A protein.
  • the SCN8A protein has reduced excitatory function.
  • Certain inventive embodiments of the invention provide an SMO as described herein for the prophylactic or therapeutic treatment of a disease or disorder in a subject mediated by SCN8A regulation.
  • Certain inventive embodiments of the invention provide the use of an SMO as described herein to prepare a medicament for treating a disease or disorder in a subject mediated by SCN8A regulation.
  • Certain inventive embodiments of the invention provide an SMO as described herein for use in medical therapy.
  • Certain inventive embodiments of the invention provide an SMO as described herein for use in treating a disease or disorder mediated by SCN8 A regulation.
  • oligonucleotides of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to SCN8A pre-mRNA.
  • the SMO enhances exclusion of exon 5A or enhances inclusion of exon 5N during splicing of a SCN8A pre-mRNA.
  • the SMO enhances exclusion of exon 5N or enhances inclusion of exon 5A during splicing of a SCN8A pre-mRNA.
  • the SMO enhances exclusion of exon 18A or enhances inclusion of exon 18A during splicing of a SCN8A pre-mRNA. In still other embodiments, the SMO enhances exclusion of exon 18N or enhances inclusion of exonl8A during splicing of a SCN8A pre-mRNA.
  • the hybridization can be by conventional Watson-Crick base pairing by nucleotide complementarity and/or wobble pairing of U-G nucleic acids to form a stable duplex. Wobble base pairing can also be accomplished with Inosine (I-A, I-U, I-C, I-G), where I is inosine. Hybridization can also occur, for example, in the case of an oligonucleotide which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • oligonucleotides may be delivered using, e.g., methods involving liposome-mediated uptake, lipid conjugates, polylysine-mediated uptake, nanoparticle-mediated uptake, and receptor-mediated endocytosis, as well as additional non-endocytic modes of delivery, such as microinjection, permeabilization (e.g., streptolysin-0 permeabilization, anionic peptide permeabilization), electroporation, and various non-invasive non-endocytic methods of delivery that are known in the art (refer to Dokka and Rojanasakul, Advanced Drug Delivery Reviews 44, 35-49, incorporated in its entirety herein by reference). Methods of delivery may also include the following.
  • Cationic Lipids Naked nucleic acids (e.g., DNA/RNA) can be introduced into cells in vivo by complexing the nucleic acid with cationic lipids or encapsulating the nucleic acid in cationic liposomes.
  • naked nucleic acids e.g., DNA/RNA
  • cationic lipids e.g., DNA/RNA
  • Suitable cationic lipid formulations include N-[-l-(2,3- dioleoyloxy)propyl]N,N,N-triethylammonium chloride (DOTMA) and a 1 : 1 molar ratio of l,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. et al. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human Gene Therapy 4:781-788).
  • DOTMA N-[-l-(2,3- dioleoyloxy)propyl]N,N,N-triethylammonium chloride
  • DMRIE dioleoyl phosphatidylethanolamine
  • DOPE dioleoyl phosphatidylethanolamine
  • Naked nucleic acids can also be introduced into cells in vivo by complexing the nucleic acid to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the nucleic acid-ligand complex to the receptor facilitates uptake of the nucleic acid by receptor-mediated endocytosis.
  • a cation such as polylysine
  • a nucleic acid-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
  • Carrier mediated SMO delivery may also involve the use of lipid-based compounds which are not liposomes.
  • lipofectins and cytofectins are lipid-based positive ions that bind to negatively charged nucleic acids and form a complex that can ferry the nucleic acid across a cell membrane.
  • Another method of * carrier mediated transfer involves receptor-based endocytosis.
  • a ligand (specific to a cell surface receptor) is made to form a complex with a nucleic acid or SMO of interest and then delivered to the bodyTarget cells that have the cell surface receptor will specifically bind the ligand and transport the ligand-DNA complex into the cell.
  • Oligonucleotides may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the RNA using methods known in the art for introducing nucleic acid (e.g., DNA) into cells in vivo.
  • nucleic acid e.g., DNA
  • the oligonucleotides of the invention can be delivered to a subject by any art- recognized method.
  • peripheral blood injection of the oligonucleotides of the invention can be used to deliver the reagents via diffusive and/or active means.
  • the oligonucleotides of the invention can be modified to promote crossing of the blood-brain- barrier (BBB) to achieve delivery of said reagents to neuronal cells of the central nervous system (CNS).
  • BBB blood-brain- barrier
  • CNS central nervous system
  • the oligonucleotides of the invention can be delivered by transdermal methods (e.g., via incorporation of the oligonucleotide reagent(s) of the invention into, e.g., emulsions, with such oligonucleotides optionally packaged into liposomes).
  • transdermal and emulsion/liposome-mediated methods of delivery are described for delivery of antisense oligonucleotides in the art, e.g., in U.S. Pat. No. 6,965,025, the contents of which are incorporated in their entirety by reference herein.
  • the oligonucleotides of the invention may also be delivered via an implantable device (e.g., an infusion pump or other such implantable device). Design of such a device is an art- recognized process.
  • an implantable device e.g., an infusion pump or other such implantable device.
  • the SMO is delivered parenterally, for example by intravenous or subcutaneous injections.
  • an SMO is delivered directly into the cerebral spinal fluid (CSF) of a subject.
  • Delivery of an SMO into the CSF of a subject may be accomplished by any means known in the art, including, but not limited to, epidural injection or intrathecal injection or intrathecal injection using an infusion pump, or direct brain delivery with a pump or other device.
  • SMOs are conjugated to a peptide to facilitate delivery of the SMO across the blood brain barrier (BBB) following parenteral administration to a subject.
  • BBB blood brain barrier
  • the SMO may be either directly conjugated to the peptide or indirectly conjugated to the peptide via a linker molecule such as a poly amino acid linker, or by electrostatic interaction.
  • Peptides useful in delivering SMOs across the BBB include, but are not limited to, peptides derived from the rabies virus glycoprotein (RVG) that specifically bind to the nicotinic acetylcholine receptor (AchR) present on neurons and the vascular endothelium of the BBB thereby allowing transvascular delivery, probably by receptor-mediated transcytosis (Kumar et al., 2007, Nature 448.39-43, encompassed by reference in its entirety); Kunitz domain- derived peptides called angiopeps (Demeule et al., 2008, J. Neurochem. 106: 1534-1544; Demeule et al, 2008, J. Pharmacol. Exp. Ther.
  • RVG rabies virus glycoprotein
  • AchR nicotinic acetylcholine receptor
  • Recombinant methods known in the art can also be used to achieve oligonucleotide reagent-induced modulation of splicing in a target nucleic acid.
  • vectors containing oligonucleotides can be employed to express, e.g., an antisense oligonucleotide to modulate splicing of an exon of a targeted pre-mRNA.
  • RNA expression may be assayed by use of a reporter or drug resistance gene whose protein product is easily assayed.
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucuronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopal
  • multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • quantitation of the amount of gene expression allows one to determine a degree of modulation which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.
  • oligonucleotides may result in modulation in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells).
  • Quantitation of gene expression in a cell may show similar amounts of modulation at the level of accumulation of target mRNA or translation of target protein.
  • the efficiency of modulation may be determined by assessing the amount of gene product in the cell; pre- mRNA or mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the oligonucleotide reagent, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.
  • An SMO of the invention may be administered to a subject in a pharmaceutical composition.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Pharmaceutical compositions can be prepared as described below.
  • exon 5N-containing SCN8A protein production is enhanced in a treated cell, cell extract, organism or patient, with an enhancement of exon 5N-containing SCN8A protein levels of at least about 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 7-, 10-, 20-, 100-fold and higher values being exemplary.
  • exon 18N-containing SCN8A protein production is enhanced in a treated cell, cell extract, organism or patient, with an enhancement of exon 18N-containing SCN8A protein levels of at least about 1.1-, 1.2-, 1.5-, 2-, 3-, 4-, 5-, 7-, 10-, 20-, 100-fold and higher values being exemplary.
  • Enhancement of gene expression refers to the presence (or observable increase) in the level of protein and/or mRNA product from a target RNA. Specificity refers to the ability to act on the target RNA without manifest effects on other genes of the cell.
  • RNA solution hybridization nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell analysis
  • the oligonucleotide i.e. the SMO
  • Higher doses e.g., at least 5, 10, 100, 500 or 1000 copies per cell
  • lower doses may also be useful for specific applications.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, parenteral, intranasal, buccal, or another route of administration.
  • Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.
  • a "unit dose" is discrete amount of the pharmaceutical composition including a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may include between 0.1% and 100% (w/w) active ingredient.
  • a pharmaceutical composition of the invention may further include one or more additional pharmaceutically active agents.
  • Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, intratumoral, and kidney dialytic infusion techniques.
  • Formulations of a pharmaceutical composition suitable for parenteral administration include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline.
  • Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further include one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • the pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may include, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Formulations suitable for nasal administration may, for example, include from about as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further include one or more of the additional ingredients described herein.
  • a pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance including an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may include a powder or an aerosolized or atomized solution or suspension including the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may fiirther include one or more of the additional ingredients described herein.
  • additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials.
  • compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.
  • the therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions including a splice modifying oligonucleotide of the invention to practice the methods of the invention.
  • the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
  • the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • Kits for practicing the methods of the invention are further provided.
  • kit is intended any manufacture (e.g., a package or a container) including at least one reagent, e.g., at least one SMO for specifically enhancing inclusion of exon 5N in SCN8A protein (i.e., for enhancing the exclusion of exon 5A), for the treatment of a disease, disorder or condition, e.g., a Dravet Spectrum Epilepsy.
  • the kit includes at least one SMO for specifically enhancing the inclusion of exon 18N in SCN8A protein (i.e., for enhancing the exclusion of exon 18 A), for the treatment of a disease, disorder or condition, e.g., a Dravet Spectrum Epilepsy.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and including instructional material for its use.
  • Positive, negative, and/or comparator controls may be included in the kits to validate the activity and correct usage of reagents employed in accordance with the invention.
  • Controls may include samples, such as tissue sections, cells fixed on glass slides, etc., known to be either positive or negative for the presence of the biomarker of interest.
  • the design and use of controls is standard and well within the routine capabilities of those of ordinary skill in the art.
  • Standard techniques are used for nucleic acid and peptide synthesis.
  • the techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2001 , Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.
  • 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 change in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
  • Antisense compound means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • antisense compounds include single-stranded and double-stranded compounds, such as, SMOs, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
  • Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy/steric block based mechanisms, including, without limitation uniform modified oligonucleotides. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.
  • a "disease” is a state of health of subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
  • a "disorder" in an subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject 's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject 's state of health.
  • the subject is an animal. In more preferred embodiments, the subject is a mammal. In most preferred embodiments, the subject is a human.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, or the frequency with which such a symptom is experienced by a subject, or both, is reduced.
  • an effective amount refers to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • exonic regulatory elements refers to sequences present on pre-mR A that enhance or suppress splicing of an exon.
  • An exonic regulatory element that enhances splicing of an exon is an exonic splicing enhancer (ESE).
  • An exonic regulatory element that suppresses splicing of an exon is an exonic splicing suppressor (ESS).
  • An intronic regulatory element that enhances splicing of an exon is an intronic splicing enhancer (ISE).
  • ISS intronic splicing suppressor
  • "Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition and/or compound of the invention in a kit.
  • the instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container which contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.
  • nucleic acid is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfbne linkages, and combinations of such linkages.
  • the term also includes other modified nucleic acids as described herein.
  • nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).
  • nucleic acid typically refers to large polynucleotides.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • nucleic acid may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences.
  • gene e.g., genomic DNA, and even synthetic DNA sequences.
  • sequences that include any of the known base analogs of DNA and RNA.
  • RNA essential RNA
  • mRNA is any RNA that specifies the order of amino acids in a protein. It is produced by transcription of DNA by RNA polymerase. In eukaryotes, the initial RNA product (primary transcript, including introns and exons) undergoes processing to yield a functional mRNA (i.e. , a mature mRNA), which is then transported to the cytoplasm for translation.
  • Precursor mRNA or “pre-mRNA” includes the primary transcript and RNA processing intermediates; the spliceosome assembles on a pre-mRNA and carries out RNA splicing.
  • fragment or “portion” is meant a full length or less than full length of the nucleotide sequence.
  • a "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions.
  • splice variant and splice isoform may be used interchangeably to denote different mRNAs which are a product of which may or may not encode the same protein, but are a result of differential splicing from the same initial pre-mRNA sequence.
  • SCN8A exon 18A inclusion generates the SCN8A 18A mRNA transcript variant
  • SCN8A exon 18N inclusion generates the SCN8A 18N mRNA transcript variant.
  • SCN8A exon 5A inclusion generates the SCN8A 5A mRNA transcript variant
  • SCN8A exon 5N inclusion generates the SCN8A 5N mRNA transcript variant.
  • nucleotide sequence variants of the invention will have in at least one embodiment 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.
  • isolated and/or purified refer to in vitro isolation of a nucleic acid, e.g., a DNA or RNA molecule from its natural cellular environment, and from association with other components of the cell or test solution (e.g. RNA pool), such as nucleic acid or polypeptide, so that it can be sequenced, replicated, and/or expressed.
  • a nucleic acid e.g., a DNA or RNA molecule from its natural cellular environment, and from association with other components of the cell or test solution (e.g. RNA pool), such as nucleic acid or polypeptide, so that it can be sequenced, replicated, and/or expressed.
  • the RNA or DNA is “isolated” in that it is free from at least one contaminating nucleic acid with which it is normally associated in the natural source of the RNA or DNA and is preferably substantially free of any other mammalian RNA or DNA.
  • the phrase "free from at least one contaminating source nucleic acid with which it is normally associated" includes the case where the nucleic acid is reintroduced into the source or natural cell but is in a different chromosomal location or is otherwise flanked by nucleic acid sequences not normally found in the source cell, e.g. , in a vector or plasmid.
  • Nucleic acid molecules having base substitutions ⁇ i.e., variants are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the nucleic acid molecule.
  • nucleotide molecule As used herein, the term “derived” or “directed to” with respect to a nucleotide molecule means that the molecule has complementary sequence identity to a particular molecule of interest.
  • the direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the "coding strand”; sequences on the DNA strand which are located 5' to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3' to a reference point on the DNA are referred to as "downstream sequences.”
  • variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may results form, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
  • Polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term “protein” typically refers to large polypeptides.
  • peptide typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the ammo-terminus; the right-hand end of a polypeptide sequence is the carboxyl-terminus.
  • a "polynucleotide” means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid.
  • A refers to adenosine
  • C refers to cytidine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 60 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U” replaces "T.”
  • recombinant DNA as used herein is defined as DNA produced by joining pieces of DNA from different sources.
  • recombinant polypeptide as used herein is defined as a polypeptide produced by using recombinant DNA methods.
  • telomere binding molecule such as an SMO, which recognizes and binds to another molecule or feature (i.e., the target pre-mRNA), but does not substantially recognize or bind other molecules or features in a sample (i.e.., other non-target pre-mRNAs).
  • Two nucleic acids substantially recognize or bind to each other when at least about 50%, preferably at least about 60% and more preferably at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T, A:U and G.C nucleotide pairs).
  • two nucleic acids substantially recognize or bind to each other when at least about 90%-100% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T, A:U and G:C nucleotide pairs).
  • the molecule may be an antibody. Chemical modification of the nucleic acid in part determines hybridization energy and thus how many base pairs are required for specific binding of the SMO nucleic acid sequence and a target nucleic acid sequences. Such calculations are well within the ability of those skilled in the art.
  • splice defect of a protein is meant a defective protein resulting from a defect in the splicing of an RNA encoding a protein.
  • treatment refers to reversing, alleviating, delaying the onset of, inhibiting the progress of, and/or preventing a disease or disorder, or one or more symptoms thereof, to which the term is applied in a subject.
  • treatment may be applied after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered prior to symptoms (e.g., in light of a history of symptoms and/or one or more other susceptibility factors), or after symptoms have resolved, for example to prevent or delay their reoccurrence.
  • novel SMOs were designed to specifically and potently skip selected alternatively spliced exons in SCN8A and the efficacy of these SMOs was subsequently validated in mouse models of epilepsy.
  • AMPA-Rs are the major excitatory neurotransmitter receptors in the CNS.
  • the we 11- validated mechanism for reducing network hyperexcitability and excitotoxicity is that reducing GluA- flip exon expression produce AMPA-Rs with much lower sensitivity to glutamate, greatly increased desensitization, and reduced Ca 2+ permeability.
  • GR1 greatly reduced AMP A excitatory post-synaptic currents (aEPSCs) (FIG. IF).
  • aEPSCs excitatory post-synaptic currents
  • an SMO directed against exon 5A specifically reduces excitatory channel properties, rather than simply downregulating overall expression.
  • splice regulation of 18A is known to be differentially controlled in non-neuronal cells, thus SMO that escapes from the CNS in active form during normal metabolism is unlikely to affect splicing, or have on-target effects outside of the CNS (Zubovic et. al., 2012).
  • SMOs do not recruit degradation enzymes (RNAseH, dicer) and therefore do not cause off-target degradation of transcripts.
  • SMOs bind to their targets with exceptional potency, specificity, and negligible off-target effects (Eckstein 2007). Two SMOs are showing great promise in clinical trials for treating Duchene muscular dystrophy and Spinal muscular atrophy (Disterer et. al., 2014; Porensky and Burghes 2013)
  • SMOs described herein can be used to treat, e.g., Dravet spectrum epilepsies refractory to current therapies.
  • SMOs are designed for complete selectivity in targeting SCN8A isoform expression without affecting any other highly related VGS channel subunits.
  • the SCN8A gene is nearly 100% conserved between mouse and human surrounding the SMO target sites, such that SMOs validated in the mouse model is directly applicable to humans. It has been clearly documented that SMOs are widely distributed and biologically active throughout the CNS after direct delivery to CSF without the necessity of a carrier (Smith et.
  • SMOs alone do not cross the blood-brain barrier when taken orally or parenterally.
  • SMOs are administered intrathecally, intracerebroventricularly (ICV), or potentially intranasally (via aerosolized nose spray).
  • Intrathecal osmotic pumps are currently used in over 500,000 patients to treat chronic pain and spasticity, and are well- tolerated.
  • SMOs delivered via spinal intrathecal injections have been shown to reach the brain in rodents and non-human primates (Hua et. al., 2010; Kordasiewicz et. al., 2012; Smith et. al., 2006; Williams et.
  • SMOs Splice Modulating Oligonucleotides
  • SMOs Splice modulating oligonucleotides
  • SMOs are designed and validated that specifically and potently modulate SCN8A pre-mRNA splicing to decrease expression of the 18A and 5 A isoforms and determine the dose-response profile of the top 2 SMOs (one each for 18A and 5A skipping) in normal mice.
  • Candidate SMOs are developed that target splicing of both human and mouse SCN8A pre-mRNA to reduce expression of the 18A and 5 A isoforms.
  • a proven set of molecular engineering tools are used to identify ranked panels of SMOs that decrease the expression of the SCN8 exon 18A and 5 A isoforms. The process is refined iteratively to select the most potent SMO candidates for further testing.
  • SMOs are developed to facilitate specific skipping of exons 5 A and 18 A, resulting in significantly reduced excitatory function of SCN8 channels.
  • 2'OMe steric block oligomers modulate pre-mRNA splicing through high affinity binding to complementary sequences containing specific splicing elements, resulting in potent and efficient skipping of the targeted exon (Aartsma-Rus et. al., 2005; Aartsma-Rus et. al., 2006; Buvoli et. a , 2007; Wheeler et. al., 2007) (see, FIG. 1C).
  • Pre-mRNA splicing is controlled by the spliceosome, a large ribonucleoprotein (RNP) complex with many auxiliary proteins and small non-coding RNAs. These factors bind to specific splice enhancer and suppressor sequences (motifs) on pre- mRNAs near intron-exon boundaries and coordinate the splicing of pre-mRNA to mRNA. Exons 18A/18N and exons 5A/5N are mutually exclusive cassette exons.
  • mice C57/BL6 mice are used as they are the background strain of the SCN1A mutant GEFS+ mice to be tested below. While complete SCN8A KO causes a severe phenotype in mice including motor system degeneration and early lethality (Martin et. al., 2007; Meisler et. al., 2004) and haplosinsufficient SCN8A mice exhibit spike wave discharges characteristic of absence seizures (Papale et. al., 2009), similar mutations have been found in humans with only mild impact on cognition (Trudeau et. al., 2006).
  • SCN8A haploinsufficiency is adequate to modify the Dravet's phenotype of SCN1A mutant mice, without causing an adverse phenotype (Hawkins et. al., 2011; Martin et. al., 2007; Meisler et. al., 2010), however adverse effects may limit SMO dosing in WT mice. All mice are monitored daily for gross signs of toxicity including weight loss, paralysis, and tremor. For all studies described herein, groups are weight, sex, and litter-matched to reduce phenotypic variability.
  • Step 1 Identification of conservation between human and mouse SCN8A sequences. Alignments of the highly conserved SCN1-11A gene sequences have been performed to ensure specificity of SMO sites targeting SCN8A splicing, and complete conservation between mouse and human. Thus, SMOs developed and tested in mice can be translated directly to human use.
  • Step 2 Identification of ESE/ESS/ISE motifs surrounding the 3 ' and 5 ' splice sites of alternatively spliced exons in SCN8A pre-mRNA. Splice modulation sites for SCN8A exons 5 A and 18A have completely conserved regulatory motifs between mouse and human. ESE motifs were defined using ESE Finder (Cartegni et. al., 2003) RESCUE-ESE (Dravet et. al., 2011; Fairbrother et. al., 2002) and PESX (Zhang and Chasin 2004). ESS elements were predicted by PESX, and the two hexamer data set analysis by FAS-ESS (Wang et. al, 2004) tool. Finally, ISE motifs are predicted using the ACESCAN2 application (Yeo et. al., 2005; Yeo et. al., 2007).
  • Step 3 RNA Structure and Thermodynamics of SCN8A target sequences.
  • the RNA Structure program (Mathews et. al., 2004) predicts secondary structure of target sequences and thermodynamic properties of all potential SMOs targeting SCN8A. Additionally, sequence motifs and structures known or predicted to cause immune stimulation or other toxicities, are screened for, and avoided.
  • Step 4 BLAST analysis of potential off-target hybridization. All candidate SMOs are screened using BLASTN analysis for potential hybridization to off-target sites in the human/mouse genomes. SMOs with greater than 85% off-target hybridization to any other known gene product are not considered.
  • Step 5 Prioritization of SMOs based on combined properties. Thermodynamic properties between SMOs and their target, and self-se If binding energies of SMOs, splice site strength, and splicing motifs are combined to establish top candidate SMOs for empirical evaluation of splicing specificity and efficiency. These parameters used to predict top candidate SMOs are all contained in the above referenced oligonucleotide and RNA structure predictive software.
  • In vivo splicing efficacy of top candidate SMOs are tested in neonatal pups. Splicing efficacy of the top ranked SMOs determined above are validated using well-established in vivo screening protocol in neonatal mice by ICV delivery, and measuring transcript levels with real-time PCR. This testing determines the most effective SMOs (one each targeting SCN8A 5A and 18A exons). Dose-response and dose-timing profile of lead SMOs in reducing SCN8A 5 A and 18A expression, respectively, are performed in normal mice and examined at P15, and P42 (6 weeks of age). Dose-response measures both mRNA expression by QPCR and protein expression by Western blot.
  • SMO potency increases the therapeutic index. Specificity of SMOs that pass the initial screen for potency are confirmed against other highly conserved SCN subunits using QPCR (as done for GR1; FIG. 1A). For all in vivo studies, treated and control animals are litter-matched to reduce variability. FVBs are the preferred strain for SMO screening because of their large litter size, and good maternal care. FVB neonatal mice are given free-hand bilateral injections of SMO on post-natal (P)l, P3, and P5 into the lateral ventricles and brain tissues are harvested at P10 as previously described (Williams et. al., 2009).
  • Cortex and hippocampus are rapidly dissected; RNA isolated, converted cDNA using Multiscribe with random hexamer primers.
  • SMOs splice modulating oligonucleotides
  • the threshold to flurothyl-induced seizures in normal mice after optimized dosing of the SMOs is determined, as SCN8A loss-of-function mutations increase seizure thresholds to flurothyl (Martin et. al., 2007). Also, the efficacy of SMOs is determined (skipping SCN8A 5A andl8A exons) at extending lifespan and reducing spontaneous seizures in a mouse model of GEFS+ (SCN1A R1648H).
  • SMO treatment The effect of SMO treatment on seizure threshold in normal mice is determined. Based on the dose-response data determined above, three SMO doses (25, 50, 75% splicing) are selected for testing in P15 and 5-6 week old mice to examine seizure threshold responses to flurothyl induced seizures. SMO potency and efficacy determines dosing for further experimentation. [0231] The two top SMOs (18A and 5 A) are assessed for efficacy in reducing the number of spontaneous seizures in SCNIATM 1111 mice (Martin et. al., 2010), as a correlative measure to survival.
  • SMOs splice modulating oligonucleotides
  • SMOs are a class of synthetic RNA based compounds that bind directly to a complementary sequence on pre-mRNA and function by sterically blocking or weakening interactions between elements of the splice machinery and the pre-mRNA.
  • the 18A and 18N exons are mutually exclusive cassette exons such that when one exon is excluded the other exon is included.
  • directing splicing to exclude (skip) the SCN8A exon 18A results in inclusion of exon 18N (truncated isoform) and thereby effectively reduces expression of the full length functional 18A isoform.
  • the 5A/5N exons are also mutually exclusive cassette exons, and directing splicing to skip SCN8A exon 5A result in inclusion of exon 5N (decreased gain isoform) and to reduce expression of the undesirable increased gain 5A isoform.
  • mice with the SCN8A med + mutation exhibit resistance to flurothyl induced seizure by 5-6 weeks of age (Martin et. al., 2007).
  • the SMO-mediated reduction of SCN8A 18A or 5 A isoform expression modulates SCN8A- mediated sodium current in a similar manner to the SCN8A "med" mutation.
  • the optimal injection frequency as determined above to maintain effect from P 15 to 6 weeks in WT mice is used for testing a range of SMO doses in increasing flurothyl seizure threshold in P15 and 5-6 week old WT mice, as physiological validation of our SMO strategy.
  • SCN8A 18A or 5 A isoforms can increase lifespan and ameliorate seizure susceptibility in SCNIA R1648H knock-in mice. Similar to SCNIA KO mice, homozygous R1648H (SCN1A RH RH ) mice exhibit weight loss, spontaneous seizures, and susceptibility to febrile seizures starting at P14-16 and lethality by P16-26 (Martin et. al., 2010).
  • heterozygous SCN1A + mutant mice show a less severe phenotype than SCN1A + - knockout mice with only -15% exhibiting spontaneous seizures in adulthood, but do have increased susceptibility to flurothyl and hyperthermia induced seizures by 5-6 weeks of age (Martin et. al., 2010).
  • SCN1A R1648H mutant mice are raised in-house on a C57BL/6 background with care, husbandry, and genotyping performed as described previously (Martin et. al, 2010).
  • Flurothyl seizures are performed as previously described, and outcome measures include latency to initial myoclonic jerk (MJ) and generalized tonic-clonic seizure (GTCS) (Martin et. al., 2007).
  • SMO efficacy in reducing in spontaneous seizures in SCN1A m/RH mice Starting at P15, SCN1A ⁇ *TM* 1 mice are evaluated for 4hrs daily on 3 consecutive days with number of observed behavioral_seizures recorded. Efficacy of the two top SMOs (18A and 5 A SMOs) are determined by reduction in number of spontaneous seizures in the SCNIA 1 TM 111 mice (Martin et. al., 2010) as compared to saline littermate controls (Table 4).
  • SCN1A RH/RH mice Efficacy of treatment with the two top SMOs (18A and 5 A) is evaluated by survival in SCN1A RH/RH mice.
  • SCN1A mice exhibit weight-loss starting at ⁇ P15 corresponding to the onset of spontaneous seizures, and die at -P18.5 without treatment (Martin et. al., 2010).
  • the SCNl A mice treated with 18A or 5 A SMO are also assessed daily for righting reflex, body weight, and survival compared to litter matched saline controls (Table 4).
  • SCN8 channels Reduction of sodium current through SCN8 channels, either by reducing full length functional channels (18A SMO) or by altering channel kinetics to a lower gain (5 A SMO), reduces seizure frequency and increases survival in mutant SCN1A RH/RH mice.
  • the SCNIATM mouse model was chosen in this application, rather than SCNl A mouse model, due to lack of success in transferring the highly fragile SCNl A knockout breeders from their home colony.
  • SCN1A RH/+ mice are a model of GEFS+, a less severe Dravet spectrum epilepsy, homozygous SCNl A 8 TM 111 mice present a severe, Dravet-like phenotype.
  • Antzelevitch C. et.al, "Electrophysiologic basis for the antiarrhythmic actions of ranolazine", Heart Rhythm., 8 (8), 1281-1290 (2011).
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  • Dravet, C. et.al. "Severe myoclonic epilepsy in infancy: Dravet syndrome", Adv.Neurol, 95 71-102 (2005).
  • Eckstein, F. "The versatility of oligonucleotides as potential therapeutics", Expert.Opin.Biol.Ther., 7 (7), 1021-1034 (2007).
  • Fletcher E. V. et.al., "Alternative splicing modulates inactivation of type 1 voltage- gated sodium channels by toggling an amino acid in the first S3-S4 linker", J.Biol.Chem., 286 (42), 36700-36708 (2011).
  • Garry E. M. et.al., "Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs", Pain, 118 (1-2), 97-111 (2005).
  • Gazina E. V. et.al., "Differential expression of exon 5 splice variants of sodium channel alpha subunit mRNAs in the developing mouse brain", Neuroscience, 166 (1), 195- 200 (2010).
  • Hua Y. et.al., "Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model", Genes Dev., 24 (15), 1634-1644 (2010).
  • Kennedy P. G. et.al., "Varicella-zoster viruses associated with post-herpetic neuralgia induce sodium current density increases in the ND7-23 Nav-1.8 neuroblastoma cell line", PLoS.ONE., 8 (1), e51570-(2013).
  • Meisler, M. H. et.al. “Sodium channel gene family: epilepsy mutations, gene interactions and modifier effects", J.Physiol, 588 (Pt 11), 1841-1848 (2010).
  • Meisler, M. H. et.al. “Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions", Genetica, 122 (1), 37-45 (2004).
  • Singh R. et.al., "Generalized epilepsy with febrile seizures plus: a common childhood-onset genetic epilepsy syndrome", Ann.Neurol, 45 (1), 75-81 (1999).
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Abstract

Cette invention concerne un oligonucléotide modulateur d'épissage (SMO) ayant une séquence conçue pour moduler l'épissage du pré-ARNm de SCN8A, la séquence SMO se liant spécifiquement à une séquence dans le pré-ARNm de SCN8A. Certains modes de réalisation selon l'invention concernent des procédés d'utilisation des SMO ci-décrits, dont des méthodes destinées à traiter ou à prévenir l'épilepsie ou un trouble du type spectre de Dravet chez un sujet (p. ex., un mammifère, p. ex., un sujet humain), comprenant l'administration d'un SMO ou d'une composition selon l'invention au sujet. Une méthode d'utilisation des SMO destinée à traiter une lésion de la moelle épinière, le cancer, la sclérose latérale amyotrophique, la maladie d'Alzheimer, la lésion cérébrale traumatique, l'autisme, la migraine hémiplégique, la sclérose en plaques, les infections du SNC, la maladie de Parkinson et la maladie d'Huntington, ou autres maladies neurologiques ou troubles dans lesquels l'excitotoxicité ou l'hyperexcitabilité contribue à la pathologie, est en outre décrite.
PCT/IB2015/001917 2014-08-20 2015-10-17 Oligonucléotides modulateurs d'épissage et leurs procédés d'utilisation WO2016027168A2 (fr)

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GB1707988.0A GB2547586A (en) 2014-08-20 2015-10-17 Splice modulating oligonucleotides and methods of use thereof(In the PCT request)
CA2958524A CA2958524A1 (fr) 2014-08-20 2015-10-17 Oligonucleotides modulateurs d'epissage et leurs procedes d'utilisation
AU2015304945A AU2015304945B2 (en) 2014-08-20 2015-10-17 Splice modulating oligonucleotides and methods of use thereof
US15/504,952 US11198874B2 (en) 2014-08-20 2015-10-17 SCN8A splice modulating oligonucleotides and methods of use thereof
PCT/IB2015/001917 WO2016027168A2 (fr) 2014-08-20 2015-10-17 Oligonucléotides modulateurs d'épissage et leurs procédés d'utilisation
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