Splice modulating oligonucleotides and methods of use thereof

Abstract

A splice modulating oligonucleotide (SMO), is provided having 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. Certain embodiments of the invention provide methods of using the SMOs described herein, including methods of treating or preventing epilepsy or a Dravet Spectrum disorder in subject (e.g., a mammal, e.g., a human), including the administration of an SMO or composition described herein to the subject. A method of using the SMOs is described herein to treat spinal cord injury, cancer, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, autism, hemiplegic migraine, multiple sclerosis, CNS infections, Parkinson's and Huntington's disease, or other neurological diseases or disorders in which excitotoxicity or hyperexcitability contributes to the pathology.

What is claimed is: 
     
         1 . A method of modulating splicing of an SCN8A pre-mRNA comprising:
 contacting a plurality of cells with splice modulating oligonucleotide (SMO) that specifically binds a complementary sequence of a pre-mRNA that undergoes splicing to form an mRNA encoding the voltage gated sodium channel subunit SCN8A   , wherein the SMO sequence directs exclusion of exon 5A or exon 18A in the SCN8A pre-mRNA;   in the plurality of cells expressing SCN8A pre-mRNA.   
     
     
         2 . The method of  claim 1  wherein the plurality of cells are in vitro. 
     
     
         3 . The method of  claim 1  wherein the plurality of cells are in vivo. 
     
     
         4 . The method of  claim 1  wherein the SMO specifically binds to a complementary sequence on a pre-mRNA in at least one of the group consisting of an intron-exon splice site, an exonic splice enhancer (ESE) site, and an intronic splice enhancer (ISE) site, in the plurality of cells to produce at least a 5 percent decrease in exon 18A inclusion in an SCN8A RNA compared to baseline untreated cells and alters expression of SCN8A or one or more isoforms thereof. 
     
     
         5 . The method of  claim 1  wherein the SMO specifically binds to a complementary sequence on a pre-mRNA in at least one of the group consisting of an intron-exon splice site, an exonic splice enhancer (ESE) site, and an intronic splice enhancer (ISE) site, in the plurality of cells to produce at least a 5 percent decrease in exon 5A inclusion in an SCN8A RNA compared to baseline untreated cells and alters expression of SCN8A or one or more isoforms thereof. 
     
     
         6 . The method of  claim 1  wherein the plurality of cells are in vivo and the SMO sequence is administered into a subject and into contact with the plurality of cells through a route of oral, rectal, intracerebroventricular, intracranial, intratumoral, intrathecal, intracisternal, epidural, intravaginal, parenteral, intravenous, intramuscular, subcutaneous, local, intraperitoneal, transdermal, or by inhalation or as a buccal or nasal spray. 
     
     
         7 . The method of  claim 6  wherein the subject has a disorder with symptoms, the symptoms being reduced by reduced excitation functionality of an SCN8A protein encoded by the SCN8A RNA. 
     
     
         8 . The method of any one of  claims 1  to  5  wherein the splice modulating oligonucleotide (SMO) sequence is completely selective towards the SCN8A pre-mRNA relative to highly related voltage gated sodium channel subunits. 
     
     
         9 . The method of any one of  claims 1  to  5  wherein the plurality of cells are human cells. 
     
     
         10 . The method of any one of  claims 1  to  5  wherein the plurality of cells are mouse cells. 
     
     
         11 . The method of any one of  claims 1  to  5  wherein the plurality of cells are rat cells. 
     
     
         12 . The method of any one of  claims 1  to  5  wherein the plurality of cells are non-human primate cells. 
     
     
         13 . A composition for performing the method of  claim 1  comprising:
 a splice modulating oligonucleotide (SMO) sequence consisting of 15 to 24 nucleotides that are complementary to an exonic or intronic sequence within intron 4, exon 5A, exon 5N, intron 5A, or intron 5N, intron 17, exon 18A, exon 18N, intron 18A, or intron 18N an SCN8A pre-mRNA and an optional one or two additional nucleotides; and 
 a carrier for delivery of the SMO sequence to a plurality of cells. 
 
     
     
         14 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos.: 26, 33, or 40. 
     
     
         15 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos.: 1306, 1307, 1324, 1327, 1422, or 1541. 
     
     
         16 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos.:
 4-39, 86-120, 169-202, 253-285, 338-369, 424-454, 511-540, 599-627, 688-715, or 778-804. 
 
     
     
         17 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos: 295-1309, 1352-1356, 1410-1424, 1469-1483, 1529-1543, 1590-1604, 1652-1666, 1715-1729, 1779-1793, 1844-1858, 1861-1869, 1889-1896, 1917-1923, 1945-1950, 1973-1977, 2001-2004, 2029-2031, 2057-2058, or 2085. 
     
     
         18 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos: 4-60, 86-142, 169-225, 253-309, 338-394, 424-480, 511-567, 599-655, 688-744, or 778-834. 
     
     
         19 . The composition of  claim 11  wherein the SMO sequence comprises one of SEQ ID. Nos: 860-964, 1261-1275, 1295-1309, 1317-1332, 1352-1356, 1374-1390, 1410-1424, 1432-1449, 1469-1483, 1491-1509, 1529-1543, 1551-1570, 1590-1604, 1612-1632, 1652-1666, 1674-1695, 1715-1729, 1737-1759, 1779-1793, 1801-1824, 1844-1858 1861-1869, 1889-1896, 1917-1923, 1945-1950, 1973-1977, 2001-2004, 2029-2031, 2057-2058, or 2085. 
     
     
         20 . The composition of  claims 13  to  19  wherein at least one nucleotide in said SMO contains a non-naturally occurring modification comprising at least one of a chemical composition of phosphorothioate 2′-O-methyl, phosphorothioate 2′-MOE, locked nucleic acid (LNA) including a constrained ethyl nucleic acid (cEt), peptide nucleic acid (PNA), phosphorodiamidate morpholino, cholesterol modified or any combination thereof. 
     
     
         21 . The composition of any one of  claims 13  to  19  wherein at least one of the 15 to 24 nucleotides is a phosphorothioate 2′-O-methyl modified nucleotide.