ANTI-SENSE OLIGOMERS FOR THE TREATMENT OF CONDITIONS AND DISEASES BASED ON RNA DECAY MEDIATED BY NON

专利摘要:
alternative splicing events in genes can lead to non-productive mrna transcripts, which in turn can lead to aberrant protein expression, and therapeutic agents that can target alternative splicing events in genes can modulate the level of expression of functional proteins in patients and / or inhibit aberrant protein expression. such therapeutic agents can be used to treat a condition or disease caused by protein deficiency.
公开号:BR112020007881A2
申请号:R112020007881-6
申请日:2018-10-23
公开日:2020-12-22
发明作者:Isabel Aznarez；Enxuan Jing；Jacob Kach；Aditya Venkatesh；Juergen Scharner；Baruch Ticho；Gene Liau
申请人:Stoke Therapeutics, Inc.；
IPC主号:

专利说明:

[001] [001] This order claims the benefit of US Provisional Order No. 62 / 575,924, filed on October 23, 2017, and US Provisional Order No. 62 / 667,200, filed on May 4, 2018, each of which is incorporated into this specification. by reference in its entirety. FUNDAMENTALS
[002] [002] Alternative gene splicing events can lead to non-productive mRNA transcripts which, in turn, can lead to aberrant protein expression, and therapeutic agents that can target alternative gene splicing events can modulate the level of expression of functional proteins in patients and / or inhibit the expression of aberrant protein. These therapeutic agents can be used to treat a condition or disease caused by protein deficiency. SUMMARY
[003] [003] This specification describes, in certain modalities, a method of modulating the expression of a target protein by a cell that has an mRNA that comprises a nonsense mediated RNA decay inducer (NMD exon) and encodes the target protein, the method comprising the contact of a therapeutic agent with the cell, whereby the therapeutic agent modulates the splicing of the NMD exon of the mRNA thereby modulating the level of processed mRNA that encodes the target protein, and modulating the expression of the target protein in the cell, where the target protein is selected from the group consisting of: AKT3 proteins,
[004] [004] This specification describes, in certain modalities, a method of treating a disease or condition in an individual in need by modulating the expression of a target protein in an individual's cell, comprising: contact of the individual's cell with a therapeutic agent that modulates the splicing of a nonsense-mediated decay-inducing mRNA exon (NMD exon) by an mRNA in the cell, where the mRNA comprises the NMD exon and encodes the target protein thereby modulating the level of processed mRNA encoding the target protein, and modulating the expression of the target protein in the individual's cell, where the target protein is selected from the group consisting of: AKT3, CACNA1A, CBS, CD46, CFH, CHD2 proteins, CLN3, COL11A2, COL4A3, COL4A4, COL4A4, CR1, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERN1, GALE, GUCY2F, GUCY2F, HEXA, HEXA, MAPK3, MBD5, MBD5, MBD5, MUTO, MYD, NF2, NIPBL, NR1H4, NSD1, NSD1, NSD1, NSD1, OPA1, OPA1, PCCA, PKP2, PPARA, PRPF3, PRPF3, SCN2A, SC N8A, SCN8A, SCN9A, SEMA3C, SEMA3D, SIRT3, STK11, STK11, SYNGAP1, TOPORS and VCAN.
[005] [005] In some embodiments, the therapeutic agent: (a) binds to a targeted portion of the mRNA that encodes the target protein; (b) modulates the binding of a factor involved in splicing the NMD exon; or (c) a combination of (a) and (b).
[006] [006] In some embodiments, the therapeutic agent interferes with the binding of the factor involved in splicing the NMD exon to a region of the target portion. In some embodiments, the target portion is proximal to the NMD exon. In some embodiments, the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the 5 'end of the NMD exon. In some embodiments, the target portion is at least about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides , about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 20 nucleotides, about 10 nucleotides , about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the 5 'end of the NMD exon. In some modalities, the target portion is, at most, about
[007] [007] In some embodiments, the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides , about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site selected from the group consisting of: GRCh38 / hg38 : chr1 243564388; GRCh38 / hg38: chr19 13236618; GRCh38 / hg38: chr21 43060012; GRCh38 / hg38: chr1 207775610; GRCh38 / hg38: chr1 196675450; GRCh38 / hg38: chr15 92998149; GRCh38 / hg38: chr16 28479765; GRCh38 / hg38: chr6 33183698; GRCh38 / hg38: chr2 227296487;
[008] [008] In some embodiments, the target portion is about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564388; GRCh38 / hg38: chr19 13236618; GRCh38 / hg38: chr21 43060012; GRCh38 / hg38: chr1 207775610; GRCh38 / hg38: chr1 196675450; GRCh38 / hg38: chr15 92998149; GRCh38 / hg38: chr16 28479765; GRCh38 / hg38: chr6 33183698; GRCh38 / hg38: chr2 227296487; GRCh38 / hg38: chr2 227144833; GRCh38 / hg38: chr2 227015360; GRCh38 / hg38: chr1 207637688; GRCh38 / hg38: chr19 47835403; GRCh38 / hg38: chr1 59904516; GRCh38 / hg38: chr1 26442335; GRCh38 / hg38: chr1 28230252; GRCh38 / hg38: chr2 88582824; GRCh38 / hg38: chr17 64102804; GRCh38 / hg38: chr1 23798484; GRCh38 / hg38: chrX 109383446; GRCh38 / hg38: chrX 109439175; GRCh38 / hg38: chr15 72362466; GRCh38 / hg38: chr15 72345776; GRCh38 / hg38: chr16 30115645; GRCh38 / hg38: chr2 148460219; GRCh38 / hg38: chr2 148490695; GRCh38 / hg38: chr2 148505761; GRCh38 / hg38: chr6 49436597; GRCh38 / hg38: chr19 50230825; GRCh38 / hg38: chr6 75867431; GRCh38 / hg38: chr17 31249955; GRCh38 / hg38: chr22 29628658; GRCh38 / hg38: chr5 37048127; GRCh38 / hg38: chr12 100499841; GRCh38 / hg38: chr5 177169394; GRCh38 / hg38: chr5 177200761; GRCh38 / hg38: chr5 177247924; GRCh38 / hg38: chr5 177275947; GRCh38 / hg38: chr3 193628509; GRCh38 / hg38: chr3 193603500; GRCh38 / hg38: chr13 100305751; GRCh38 / hg38: chr12 32894778; GRCh38 / hg38: chr22 46203575; GRCh38 / hg38: chr1 150327557; GRCh38 / hg38: chr1 150330401; GRCh38 / hg38: chr2 165327155; GRCh38 / hg38: chr12 51688758; GRCh38 / hg38: chr12 51780202; GRCh38 / hg38: chr2 166304329; GRCh38 / hg38: chr7 80794957; GRCh38 / hg38: chr7 85059541; GRCh38 / hg38: chr11 226081; GRCh38 / hg38: chr19 1216268; GRCh38 / hg38: chr19 1221621; GRCh38 / hg38: chr6
[009] [009] In some embodiments, the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides , about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream from the genomic site selected from the group consisting of: GRCh38 / hg38 : chr1 243564285; GRCh38 / hg38: chr19 13236449; GRCh38 / hg38: chr21 43059730; GRCh38 / hg38: chr1 207775745; GRCh38 / hg38: chr1 196675529; GRCh38 / hg38: chr15 92998261; GRCh38 / hg38: chr16 28479644; GRCh38 / hg38: chr6 33183634; GRCh38 / hg38: chr2 227296526; GRCh38 / hg38: chr2 227144653; GRCh38 / hg38: chr2 227015283; GRCh38 / hg38: chr1 207637848; GRCh38 / hg38: chr19 47835579; GRCh38 / hg38: chr1 59904366; GRCh38 / hg38: chr1 26442372; GRCh38 / hg38: chr1 28230131; GRCh38 / hg38: chr2 88582755; GRCh38 / hg38: chr17 64102673; GRCh38 / hg38: chr1 23798311; GRCh38 / hg38: chrX 109383365; GRCh38 / hg38: chrX 109439038; GRCh38 / hg38: chr15 72362376; GRCh38 / hg38: chr15 72345677; GRCh38 / hg38: chr16 30115595; GRCh38 / hg38: chr2 148460304; GRCh38 / hg38: chr2 148490787; GRCh38 / hg38: chr2 148505830; GRCh38 / hg38: chr6 49436522; GRCh38 / hg38: chr19 50230999; GRCh38 / hg38: chr6 75867523; GRCh38 / hg38: chr17 31250125; GRCh38 / hg38: chr22 29628773; GRCh38 / hg38: chr5 37048354; GRCh38 / hg38: chr12 100500024; GRCh38 / hg38: chr5 177169559; GRCh38 / hg38: chr5 177200783; GRCh38 / hg38: chr5 177248079; GRCh38 / hg38: chr5
[010] [010] In some embodiments, the target portion is about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream from the genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564285; GRCh38 / hg38: chr19 13236449; GRCh38 / hg38: chr21 43059730; GRCh38 / hg38: chr1 207775745; GRCh38 / hg38: chr1 196675529; GRCh38 / hg38: chr15 92998261; GRCh38 / hg38: chr16 28479644; GRCh38 / hg38: chr6 33183634; GRCh38 / hg38: chr2 227296526; GRCh38 / hg38: chr2 227144653; GRCh38 / hg38: chr2 227015283; GRCh38 / hg38: chr1 207637848; GRCh38 / hg38: chr19 47835579; GRCh38 / hg38: chr1 59904366; GRCh38 / hg38: chr1 26442372; GRCh38 / hg38: chr1 28230131; GRCh38 / hg38: chr2 88582755; GRCh38 / hg38: chr17 64102673; GRCh38 / hg38: chr1 23798311; GRCh38 / hg38: chrX 109383365; GRCh38 / hg38: chrX 109439038; GRCh38 / hg38: chr15 72362376; GRCh38 / hg38: chr15
[011] [011] In some embodiments, the target portion is located in an intronic region between two canonical exonic regions of the mRNA encoding the target protein, and in which the intronic region contains the NMD exon. In some embodiments, the target portion overlaps at least partially with the NMD exon. In some embodiments, the target portion overlaps at least partially with an intron upstream or downstream of the NMD exon. In some embodiments, the target portion comprises NMD-intron 5 'exon junction or NMD-intron 3' exon junction. In some embodiments, the target portion is within the NMD exon. In some embodiments, the target portion comprises about 5, 6, 7, 8, 9, 10, 11, 12,
[012] [012] In some embodiments, the mRNA encoding the target protein comprises a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a sequence selected from the group consisting of IDS. SEQ. Nos: 135-191. In some embodiments, the mRNA encoding the target protein is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a sequence selected from the group consisting of IDS. SEQ. Nos: 1-5, 12, 19-21, 25, 26, 28, 30, 33, 35, 38, 40, 41, 44, 45, 51, 53, 55-57 and 192-211. In some embodiments, the target portion of the mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous nucleic acids in a sequence selected from the group consisting of IDS. SEQ. Nos: 135-191. In some embodiments, the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to at least 8 contiguous nucleic acids from a sequence selected from the group consisting of IDS. SEQ. Nos: 135-191.
[013] [013] In some embodiments, the target portion of the mRNA is within the nonsense mediated RNA decay inducer selected from the group consisting of: GRCh38 / hg38: chr1 243564285 243564388; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261;
[014] [014] In some embodiments, the target portion of the mRNA is upstream or downstream of the exon-inducing RNA mediated by nonsense selected from the group consisting of: GRCh38 / hg38: chr1 243564285 243564388; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261; GRCh38 / hg38: chr16 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698; GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chr1 207637688 207637848; GRCh38 / hg38: chr19 47835403 47835579; GRCh38 / hg38: chr1 59904366 59904516; GRCh38 / hg38: chr1 26442335 26442372; GRCh38 / hg38: chr1 28230131 28230252; GRCh38 / hg38: chr2 88582755 88582824; GRCh38 / hg38: chr17 64102673 64102804; GRCh38 / hg38: chr1 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chr15 72362376 72362466; GRCh38 / hg38: chr15 72345677 72345776; GRCh38 / hg38: chr16 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chr19 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chr17 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chr12 100499841 100500024;
[015] [015] In some embodiments, the target portion of the mRNA comprises an exon-intron junction of the exon selected from the group consisting of: GRCh38 / hg38: chr1 243564285 243564388; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261; GRCh38 / hg38: chr16 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698; GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chr1 207637688 207637848; GRCh38 / hg38: chr19 47835403 47835579; GRCh38 / hg38: chr1 59904366 59904516; GRCh38 / hg38: chr1 26442335 26442372; GRCh38 / hg38: chr1 28230131 28230252; GRCh38 / hg38: chr2 88582755 88582824;
[016] [016] In some embodiments, the target protein produced is either a full-length protein or a wild-type protein.
[017] [017] In some embodiments, the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA that encodes the target protein.
[018] [018] In some embodiments, the disease or condition is induced by a loss-of-function mutation in the target protein.
[019] [019] In some embodiments, the disease or condition is associated with haploinsufficiency of a gene that encodes the target protein, and in which the individual has a first allele that encodes a functional target protein, and a second allele by which the protein Target is not produced or is produced at a reduced level or a second allele that encodes a non-functional target protein or a partially functional target protein. In some modalities, the disease or condition is selected from the group consisting of: Sotos Syndrome 1; Beckwith-Wiedemann syndrome; Migraine, hemiplegic relative, 1; Episodic ataxia, type 2; Epileptic encephalopathy, arising in childhood; Wagner's syndrome 1; Optical atrophy type 1; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Neurofibromatosis type 1; Early childhood epileptic encephalopathy, 11; Convulsions, benign family children, 3; Cognitive deficit with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Convulsions, benign family children, 5; Via (SNC); 16p11.2 deletion syndrome; Mental retardation, autosomal dominant 1; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Deafness, autosomal dominant 13; Retinal dystrophy of rod cones-2; Deafness, autosomal dominant 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Deafness, autosomal dominant 22; Neurofibromatosis type 2; Mental retardation, autosomal dominant 5; Epilepsy, generalized, with febrile seizures plus, type 7; and Febrile seizures, family, 3B.
[020] [020] In some modalities, the disease or condition is associated with an autosomal recessive mutation of a gene that encodes the target protein, in which the individual has a first coding allele, whereby: (i) the target protein is not produced or is produced at a reduced level, compared to a wild type allele; or (ii) the target protein produced is non-functional or partially functional, compared to a wild-type allele, and a second allele, whereby: (iii) the target protein is produced at a reduced level, compared to an allele wild-type and the target protein produced is at least partially functional, compared to a wild-type allele; or (iv) the target protein produced is partially functional, compared to a wild type allele. In some modalities, the disease or condition is selected from the group consisting of: Alport's syndrome; Ceroid, neuronal lipofuscinosis, 3; Galactose epimerase deficiency; Homocystinuria, types responsive and unresponsive to B6; Methyl malonic aciduria; Propionic acidosis; Retinitis pigmentosa 59; Tay-Sachs disease;
[021] [021] In some embodiments, the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA that encodes the target protein and increases the expression of the target protein in the cell. In some embodiments, the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA that encodes the target protein. In some embodiments, the exclusion of the NMD exon from the processed mRNA encoding the target protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times , about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 up to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times at least about 1.5 times, at least about 2 times, eg it at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to exclusion of the NMD exon of the processed mRNA that encodes the target protein in a control cell. In some embodiments, the therapeutic agent decreases the level of the processed mRNA that encodes the target protein in the cell. In some embodiments, the level of the processed mRNA that encodes the target protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to a processed mRNA level that encodes the target protein in a control cell.
[022] [022] In some embodiments, the therapeutic agent decreases the expression of the target protein in the cell. In some embodiments, a level of the target protein produced in the cell brought into contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times,
[023] [023] In some embodiments, the disease or condition is induced by a gain-of-function mutation in the target protein. In some embodiments, the individual has an allele by which the target protein is produced at an increased level or an allele that encodes a mutant target protein that exhibits increased activity in the cell.
[024] [024] In some embodiments, the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA encoding the target protein and decreases the expression of the target protein in the cell. In some embodiments, the target protein comprises SCN8A. In some embodiments, the disease or condition comprises a disease of the central nervous system. In some embodiments, the disease or condition comprises epilepsy. In some modalities, the disease or condition comprises
[025] [025] In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and the antisense oligomer comprises a phosphorodiamidate morpholino, a blocked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2 ' -Fluorine or a 2'-O-methoxyethyl portion.
[026] [026] In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a portion of modified sugar. In some embodiments, each serving of sugar is a serving of modified sugar.
[027] [027] In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 25 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[028] [028] In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% complementary to the target portion of the mRNA.
[029] [029] In some modalities, the method also comprises the assessment of the level of mRNA or the level of expression of the target protein.
[030] [030] In some modalities, the individual is a human. In some embodiments, the individual is a non-human animal. In some embodiments, the individual is a fetus, an embryo or a child. In some embodiments, the cells are ex vivo. In some modalities, the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal or intravenous injection to the individual. In some embodiments, the method further comprises administering a second therapeutic agent to the individual.
[031] [031] In some embodiments, the second therapeutic agent is a small molecule. In some embodiments, the second therapeutic agent is an antisense oligomer. In some embodiments, the second therapeutic agent corrects intron retention.
[032] [032] In some modalities, the disease or condition is selected from the group consisting of: 16p11.2 deletion syndrome; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Ceroid, neuronal lipofuscinosis, 3; Cognitive deficit with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Convulsions, benign family children, 5; Retinal dystrophy of cones
[033] [033] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS
[034] [034] The unprecedented characteristics of the disclosure are presented with particularity in the attached claims. A better understanding of the characteristics and advantages of the present disclosure will be obtained by reference to the following detailed description which presents illustrative modalities, in which the principles of the disclosure are used and in the accompanying drawings, in which:
[035] [035] FIG. 1 depicts a schematic representation of a target mRNA containing a nonsense-mediated decay-inducing mRNA exon (NMD exon mRNA) and the therapeutic agent-mediated exclusion of nonsense-mediated mRNA decay to increase expression of nonsense full-length target protein or functional RNA. FIG. 1A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, a pre-mRNA transcript from a target gene is spliced to generate mRNA, and that mRNA is exported to the cytoplasm and translated into the target protein. For this target gene, some fraction of the mRNA contains a nonsense-mediated decay-inducing mRNA exon (NMD exon mRNA) that is degraded in the cytoplasm thus leading to the absence of production of the target protein.
[036] [036] FIG. 1B shows an example of the same cell divided into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, for example, an antisense oligomer (ASO), promotes the exclusion of nonsense-mediated decay-inducing mRNA exon and results in an increase in mRNA, which, in turn, translates into levels higher target protein. FIG. 1C is a schematic representation of therapeutic ASO-mediated exclusion of a nonsense-mediated decay-inducing mRNA exon, which transforms a non-productive mRNA into a productive mRNA and increases the expression of the full-length target protein by the productive mRNA.
[037] [037] FIG. 2 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CD46 gene. The identification of the NMD-inducing exon in the CD46 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CD46 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 207770363 207783291, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[038] [038] FIG. 3 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the COL11A2 gene. The identification of the NMD-inducing exon in the COL11A2 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the COL11A2 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr6 33181172 33184144, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[039] [039] FIG. 4 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CR1 gene. The identification of the NMD-inducing exon in the CR1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CR1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 207630622 207639396, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[040] [040] FIG. 5 depicts the identification of an exon mRNA-inducing exon (NMD) mediated by exemplary nonsense in the CRX gene. The identification of the NMD-inducing exon in the CRX gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CRX gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr19 47834545 47836242, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[041] [041] FIG. 6 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the DNAJC8 gene. The identification of the NMD-inducing exon in the DNAJC8 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the DNAJC8 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 28229025 28232920, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[042] [042] FIG. 7 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MYH14 gene. The identification of the NMD-inducing exon in the MYH14 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MYH14 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr19 50230625 50231929, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[043] [043] FIG. 8 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SEMA3C gene. The identification of the NMD-inducing exon in the SEMA3C gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SEMA3C gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr7 80789529 80798091, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[044] [044] FIG. 9 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the VCAN gene. The identification of the NMD-inducing exon in the VCAN gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the VCAN gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 83542270 83545536, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[045] [045] FIG. 10 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the OPAI gene. The identification of the NMD-inducing exon in the OPAI gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the OPAI gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr3 193626204 193631611, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[046] [046] FIG. 11 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the COL4A3 gene. The identification of the NMD-inducing exon in the COL4A3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the COL4A3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 227295318 227297673, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[047] [047] FIG. 12 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the DHDDS gene. The identification of the NMD-inducing exon in the DHDDS gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the DHDDS gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 26438286 26442730, shown in the middle panel. Bioinformatics analysis identified an exon-like sequence (bottom panel, sequence highlighted in capital letters)
[048] [048] FIG. 13 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CFH gene. The identification of the NMD-inducing exon in the CFH gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CFH gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 196673964 196675988, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[049] [049] FIG. 14 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the AKT3 gene. The identification of the NMD-inducing exon in the AKT3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the AKT3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 243563849 243572925, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[050] [050] FIG. 15 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the TOPORS gene. The identification of the NMD-inducing exon in the TOPORS gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the TOPORS gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr9 32550970 32552433, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[051] [051] FIG. 16 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the PRPF3 gene. The identification of the NMD-inducing exon in the PRPF3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the PRPF3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 150325883 150328319, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[052] [052] FIG. 17 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the PRPF3 gene. The identification of the NMD-inducing exon in the PRPF3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the PRPF3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 150328468 150332683, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[053] [053] FIG. 18 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NIPBL gene. The identification of the NMD-inducing exon in the NIPBL gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NIPBL gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 37046201 37048501, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[054] [054] FIG. 19 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CBS gene. The identification of the NMD-inducing exon in the CBS gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CBS gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr21 43059305 43060440, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[055] [055] FIG. 20 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the PKP2 gene. The identification of the NMD-inducing exon in the PKP2 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the PKP2 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr12 32879034 32896508, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[056] [056] FIG. 21 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the COL4A4 gene. The identification of the NMD-inducing exon in the COL4A4 gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the COL4A4 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 227144560 227147412, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[057] [057] FIG. 22 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the COL4A4 gene. The identification of the NMD-inducing exon in the COL4A4 gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the COL4A4 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 227012299 227022047, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[058] [058] FIG. 23 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CYP2J2 gene. The identification of the NMD-inducing exon in the CYP2J2 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CYP2J2 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 59901104 59904870, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[059] [059] FIG. 24 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the PPARA gene. The identification of the NMD-inducing exon in the PPARA gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the PPARA gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr22 46198592 46215172, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[060] [060] FIG. 25 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SEMA3D gene. The identification of the NMD-inducing exon in the SEMA3D gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SEMA3D gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr7 85055860 85065423, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[061] [061] FIG. 26 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the ERN1 gene. The identification of the NMD-inducing exon in the ERN1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the ERN1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr17 64098242 64129975, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[062] [062] FIG. 27 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the GUCY2F gene. The identification of the NMD-inducing exon in the GUCY2F gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the GUCY2F gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chrX 109382213 109385183, shown in the middle panel. Bioinformatics analysis identified an exon-like sequence (bottom panel, sequence highlighted in capital letters)
[063] [063] FIG. 28 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the GUCY2F gene. The identification of the NMD-inducing exon in the GUCY2F gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the GUCY2F gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chrx 109430397 109441350, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[064] [064] FIG. 29 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SCN2A gene. The identification of the NMD-inducing exon in the SCN2A gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SCN2A gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 165326986 165331329, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[065] [065] FIG. 30 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SCN8A gene. The identification of the NMD-inducing exon in the SCN8A gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SCN8A gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr12 51687221 51689004, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[066] [066] FIG. 31 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SCN8A gene. The identification of the NMD-inducing exon in the SCN8A gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the SCN8A gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr12 51774364 51786541, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[067] [067] FIG. 32 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SCN9A gene. The identification of the NMD inducing exon in the SCN9A gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the SCN9A gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 166304123 166305791, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[068] [068] FIG. 33 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CLN3 gene. The identification of the NMD-inducing exon in the CLN3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CLN3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr16 28477879 28482104, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[069] [069] FIG. 34 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MAPK3 gene. The identification of the exon-inducing exon
[070] [070] FIG. 35 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NF1 gene. The identification of the NMD-inducing exon in the NF1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NF1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr17 31249120 31252937, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[071] [071] FIG. 36 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MBD5 gene. The identification of the NMD-inducing exon in the MBD5 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MBD5 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 148502511 148510059, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[072] [072] FIG. 37 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MBD5 gene. The identification of the NMD-inducing exon in the MBD5 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MBD5 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 148458873 148462581, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[073] [073] FIG. 38 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MBD5 gene. The identification of the NMD-inducing exon in the MBD5 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MBD5 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr2 148490596 148502435, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[074] [074] FIG. 39 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NF2 gene. The identification of the NMD-inducing exon in the NF2 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NF2 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr22 29604114 29636750, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[075] [075] FIG. 40 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MYO6 gene. The identification of the NMD-inducing exon in the MYO6 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MYO6 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr6 75867107 75870646, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[076] [076] FIG. 41 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the SYNGAP1 gene. The identification of the NMD-inducing exon in the SYNGAP1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SYNGAP1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr6 33447935 33451759, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[077] [077] FIG. 42 depicts the identification of a mRNA decay inducing exon (NMD) mediated by exemplary nonsense in the SIRT3 gene. The identification of the NMD-inducing exon in the SIRT3 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the SIRT3 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr11 224241 230451, shown in the middle panel. Bioinformatics analysis identified an exon-like sequence (bottom panel, sequence highlighted in capital letters)
[078] [078] FIG. 43 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CACNA1A gene. The identification of the NMD-inducing exon in the CACNA1A gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphic representation of the CACNA1A gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr19 13235732 13241520, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[079] [079] FIG. 44 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the CHD2 gene. The identification of the NMD-inducing exon in the CHD2 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the CHD2 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr15 92997404 92998498, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[080] [080] FIG. 45 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NSD1 gene. The identification of the NMD-inducing exon in the NSD1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NSD1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 177136032 177191883, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[081] [081] FIG. 46 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NSD1 gene. The identification of the NMD-inducing exon in the NSD1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NSD1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 177192021 177204119, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[082] [082] FIG. 47 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NSD1 gene. The identification of the NMD-inducing exon in the NSD1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NSD1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 177246798 177248180, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[083] [083] FIG. 48 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NSD1 gene. The identification of the NMD-inducing exon in the NSD1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the NSD1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr5 177273786 177280564, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[084] [084] FIG. 49 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the EIF2AK3 gene. The identification of the exon-inducing exon
[085] [085] FIG. 50 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the GALE gene. The identification of the NMD-inducing exon in the GALE gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the GALE gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr1 23798232 23798614, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[086] [086] FIG. 51 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the HEXA gene. The identification of the NMD-inducing exon in the HEXA gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the HEXA gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr15 72356652 72375719, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[087] [087] FIG. 52 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the HEXA gene. The identification of the NMD-inducing exon in the HEXA gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the HEXA gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr15 72345552 72346234, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[088] [088] FIG. 53 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the NR1H4 gene. The identification of the NMD-inducing exon in the NR1H4 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The upper panel shows a graphical representation of the NR1H4 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr12 100493403 100505574, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[089] [089] FIG. 54 depicts the identification of an exon mRNA-inducing exon (NMD) mediated by exemplary nonsense in the STK11 gene. The identification of the NMD-inducing exon in the STK11 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the STK11 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr19 1207204 1218416, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[090] [090] FIG. 55 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the STK11 gene. The identification of the NMD-inducing exon in the STK11 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the STK11 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr19 1221341 1221948, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[091] [091] FIG. 56 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the PCCA gene. The identification of the NMD-inducing exon in the PCCA gene with the use of RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the PCCA gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr13 100302999 100307191, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[092] [092] FIG. 57 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the MUT gene. The identification of the NMD-inducing exon in the MUT gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the MUT gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr6 49435625 49440205, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[093] [093] FIG. 58 depicts the identification of an exon-inducing mRNA (NMD) mediated by exemplary nonsense in the OPA1 gene. The identification of the NMD-inducing exon in the OPA1 gene using RNA sequencing is shown, visualized in the UCSC genome browser. The top panel shows a graphical representation of the OPA1 gene at scale. Peaks corresponding to the RNA sequencing readings were identified at the intron GRCh38 / hg38: chr3 193593374 193614710, shown in the middle panel. The bioinformatics analysis identified an exon-type sequence (bottom panel, sequence highlighted in capital letters) flanked by splice sites 3 ’and 5’. The inclusion of this exon leads to the introduction of a premature termination codon making the transcript an NMD target.
[094] [094] FIG. 59 depicts confirmation of NMD-inducing exon by treatment with puromycin or cycloheximide in various cell lines. RT-PCR analysis using total RNA of cells treated with water, treated with DMSO, treated with puromycin or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 8x (GRCh38 / hg38: chr1 243564285 243564388) of the gene AKT3.
[095] [095] FIG. 60 depicts an exemplary ASO Walk around the 8x exon region (GRCh38 / hg38: chr1 243564285 243564388) from AKT3. The graphic representation of an ASO Walk performed for around the exon region 8x (GRCh38 / hg38: chr1 243564285 243564388) of AKT3 that aims at sequences upstream of the splice site 3 ', through the splice site 3', exon 8x, through the 5 'splice site, and downstream of the 5' splice site, is shown. ASOs were designed to cover these regions by changing 5 nucleotides at once.
[096] [096] FIG. 61 depicts the ASO Walk of the 8x exon region (GRCh38 / hg38: chr1 243564285 243564388) of AKT3 evaluated by Taqman-qPCR by reverse transcription. A graph of the rate of change of the AKT3 productive mRNA product in relation to the Simulated is plotted.
[097] [097] FIG. 62 depicts confirmation of NMD-inducing exon through treatment with cycloheximide in several cell lines. RT-PCR analysis using total RNA from cells treated with DMSO or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 14x (GRCh38 / hg38: chr13 100305751 100305834) of the PCCA gene.
[098] [098] FIG. 63 depicts an exemplary ASO Walk around the 14x exon region (GRCh38 / hg38: chr13 100305751 100305834) of PCCA. The graphic representation of an ASO Walk performed around the region of the exon 14x (GRCh38 / hg38: chr13 100305751 100305834) of PCCA that aims at sequences upstream of the splice site 3 ', through the splice site 3', exon 14x, through the 5 'splice site, and downstream of the 5' splice site, is shown. ASOs were designed to cover these regions by changing 5 nucleotides at once.
[099] [099] FIG. 64 depicts ASO Walk from the 14x exon region (GRCh38 / hg38: chr13 100305751 100305834) of PCCA evaluated by reverse transcription Taqman -qPCR and RT-PCR. A graph of the rate of change of the productive PCR mRNA product in relation to the Simulated (gray) and percentage of change in inclusion of the NMD exon (black) is plotted.
[100] [100] FIG. 65 depicts confirmation of NMD-inducing exon through treatment with puromycin or cycloheximide in various cell lines, as well as confirmation of NMD-inducing exon in brain and retinal samples. RT-PCR analysis using total RNA from cells treated with water, treated with DMSO, treated with puromycin or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 7x (GRCh38 / hg38: chr3 193628509 193628616) of the gene OPA1.
[101] [101] FIG. 66 depicts an exemplary ASO Walk around the 7x exon region (GRCh38 / hg38: chr3 193628509 193628616) by OPA1. The graphic representation of an ASO Walk performed for around the exon 7x region (GRCh38 / hg38: chr3 193628509 193628616) of OPA1 that aims at sequences upstream of the splice site 3 ', through the splice site 3', exon 7x, through the 5 'splice site, and downstream of the 5' splice site, is shown. ASOs were designed to cover these regions by changing 5 nucleotides at once or 3 nucleotides through the splice regions site.
[102] [102] FIGS. 67 and 68 depict ASO Walk from the 7x exon region (GRCh38 / hg38: chr3 193628509 193628616) from OPA1 evaluated by Taqman RT-qPCR. Graphs of the rate of change of the productive OPA1 mRNA product in relation to the Simulated are plotted.
[103] [103] FIG. 69 depicts confirmation of NMD-inducing exon through treatment with cycloheximide in ReNCell VM and existence of NMD-inducing exon mRNA (NF1) in both human and monkey cortex. RT-PCR analysis using total RNA of cells treated with DMSO or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 31x (GRCh38 / hg38: chr17 31249955 31250125) of the NF1 gene.
[104] [104] FIG. 70 depicts an exemplary ASO Walk around the 31x exon region (GRCh38 / hg38: chr17 31249955 31250125) of NF1. The graphical representation of an ASO Walk performed for around the exon 31x region (GRCh38 / hg38: chr17 31249955 31250125) of NF1 that aims at sequences upstream of the splice site 3 ', through the splice site 3', exon 31x, through the 5 'splice site, and downstream of the 5' splice site, is shown. ASOs were designed to cover these regions by changing 5 nucleotides at once.
[105] [105] FIG. 71 depicts ASO Walk from the 31x exon region (GRCh38 / hg38: chr17 31249955 31250125) of NF1 evaluated by RT-PCR (upper) and RT-Taqman-qPCR (lower). Results of RT-PCR that indicate a decrease in exon 31x and a graph of the ratio of change of the NF1 productive mRNA product to the Simulated are shown.
[106] [106] FIG. 72 depicts confirmation of NMD-inducing exon by treatment with puromycin or cycloheximide in various cell lines. RT-PCR analysis using total RNA from cells treated with water, treated with DMSO, treated with puromycin or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 18x (GRCh38 / hg38: chr6 33448789 33448868) of the gene SYNGAP1.
[107] [107] FIG. 73 depicts an exemplary ASO Walk around the 18x exon region (GRCh38 / hg38: chr6 33448789 33448868) from SYNGAP1. The graphic representation of an ASO Walk made for around the 18x exon region (GRCh38 / hg38: chr6
[108] [108] FIG. 74 depicts ASO Walk from the 18x exon region (GRCh38 / hg38: chr6 33448789 33448868) of SYNGAP1 evaluated by RT-PCR (upper) and Taqman-qPCR (lower). Graphs of the% inclusion of exon 18x and the rate of change of the SYNGAP1 productive mRNA product in relation to the Simulated are plotted (top and bottom, respectively).
[109] [109] FIG. 75 depicts confirmation of NMD-inducing exon by treatment with cycloheximide. RT-PCR analysis using total RNA from cells treated with DMSO or treated with cycloheximide confirmed the presence of a band that corresponds to the NMD-inducing exon 30x (GRCh38 / hg38: chr15 92998149 92998261) of the CHD2 gene. Also shown is the RT-PCR analysis that demonstrates the presence of mRNA containing exon NMD 30x exon in mouse cortex, non-human and human primate samples.
[110] [110] FIG. 76 depicts an exemplary ASO Walk around the 30x exon region (GRCh38 / hg38: chr15 92998149 92998261) by CHD2. The graphic representation of an ASO Walk performed for around the exon region 30x (GRCh38 / hg38: chr15 92998149 92998261) of CHD2 that aims at sequences upstream of the splice site 3 ', through the splice site 3', exon 30x, through the 5 'splice site, and downstream of the 5' splice site, is shown. ASOs were designed to cover these regions by changing 5 nucleotides at once.
[111] [111] FIG. 77 depicts ASO Walk from the 30x exon region
[112] [112] FIG. 78 depicts changes induced by different ASOs in the non-productive exon levels of CHD2 (exon 30x (GRCh38 / hg38: chr15 92998149 92998261)) and productive CHD2 mRNA. DETAILED DESCRIPTION
[113] [113] Alternative splicing events in the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GHALE , GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRXF2, , SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, ERF, SEMA3, NF2, SEM3 , GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 can lead to non-productive mRNA transcripts which, in turn, can lead to aberrant protein expression, and therapeutic agents that can target alternative splicing events in the gene ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRINA, GRINA, GRINA KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NI PBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTANS, TEK, TS3, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2,
[114] [114] One of the alternative splicing events that can lead to non-productive mRNA transcripts is the inclusion of an extra exon in the mRNA transcript that can induce nonsense-mediated mRNA decay. The present disclosure provides compositions and methods for modulating alternative splicing of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOLL FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PFB, PRP RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAF, MY6, MY6, MY6, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 to increase the production of mature protein-encoding mRNA and thus ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CDK, CD55, protein , CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP, MECP, MECP , NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAF, MY6, MY6, MY6, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or translated functional SYNGAP1.
[115] [115] Intervening sequences or introns are removed by a large, highly dynamic RNA-protein complex called a spliceosome, which orchestrates complex interactions between primary transcripts, small nuclear RNAs (snRNAs) and a large number of proteins. Spliceosomes are assembled ad hoc on each intron in an orderly manner, starting with recognition of the 5 'splice site (5's) by U1 snRNA or the splice site 3' (3's) via U2, which involves the connection of the auxiliary factor U2 (U2AF) to the region 3'ss to facilitate the connection of U2 to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65 kD subunit encoded by U2AF2 (U2AF65), which binds to the polypyrimidine tract (PPT), and a 35 kD subunit encoded by U2AF1 (U2AF35), which interacts with highly conserved AG dinucleotides in 3's and stabilizes the U2AF65 binding. In addition to the BPS / PPT and 3’ss / 5’ss units, precise splicing requires sequences or auxiliary structures that activate or repress splice site recognition, known as intronic or exonic splice enhancers or silencers. These elements allow genuine splice sites to be recognized among a large excess of cryptic or pseudo-sites in the genome of higher eukaryotes, which have the same sequences, but a much larger number of authentic sites by an order of magnitude. Although they often have a regulatory function, the exact mechanisms of their activation or repression are poorly understood.
[116] [116] The decision to splice or not can typically be modeled as a stochastic process, rather than a deterministic process, so that even more defined splicing signals can sometimes splice incorrectly. However, under normal conditions, pre-mRNA splicing proceeds at surprisingly high fidelity. This is attributed, in part, to the activity of adjacent exonic and intronic auxiliary cis-acting splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) of exonic or intronic splicing based on their ability to stimulate or inhibit splicing, respectively. Although there is now evidence that some auxiliary cis-acting elements may act by influencing the kinetics of spliceosome assembly, for example, in the arrangement of the complex between U1 snRNP and the 5's, it seems very likely that many elements will work in harmony with proteins trans-acting RNA binding agents (RBPs). For example, the family of RBPs rich in serine and arginine (SR proteins) is a family of conserved proteins that play a crucial role in defining exons. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of ESSs in the neighborhood. The repressive effects of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter the recruitment of central splicing factors to adjacent splice sites. In addition to their roles in the regulation of splicing, it is suggested that silencing elements have a role in the repression of pseudoexons, sets of intronic trap splice sites with the typical spacing of an exon, but without a functional open reading frame. ESEs and ESSs, in cooperation with their cognate trans-acting RBPs, represent important components in a set of splicing controls that specify how, where and when mRNAs are assembled by their precursors.
[117] [117] The sequences that mark exon-intron boundaries are degenerate signals of varying potencies that can occur at high frequency within human genes. In multi-exon genes, different pairs of splice sites can be linked together in many different combinations, creating a diverse array of transcripts from a single gene. This is commonly called alternative pre-mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported by the nucleus and translated into functional polypeptides, different mRNA isoforms from a single gene can vary markedly in their translation efficiency. Those mRNA isoforms with premature termination codons (PTCs) of at least 50 bp upstream of an exon junction complex are likely to be targeted for degradation by the nonsense-mediated mRNA (NMD) decay pathway. Mutations in traditional (BPS / PPT / 3's / 5's) and auxiliary splicing motifs can cause aberrant splicing, for example, exon-skipping or inclusion of cryptic (or pseudo-) exon or splice site activation, and contribute significantly to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.
[118] [118] Considering that exon-intron limits can occur in any of the three positions of a codon, it is clear that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons that are evenly divisible by 3 can be skipped or included in the mRNA without any change in the reading frame. Splicing events that do not have compatible phases will induce a frame shift. Unless reversed by downstream events, frame changes can certainly lead to one or more PTCs, probably resulting in subsequent NMD degradation. NMD is a translation-coupled mechanism that eliminates mRNAs that contain PTCs. NMD can act as a surveillance route that exists in all eukaryotes. NMD can reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. The translation of these aberrant mRNAs could, in some cases,
[119] [119] An NMD-inducing exon (NIE) is an exon or a pseudo-exon that is a region within an intron and can activate the NMD pathway if included in a mature RNA transcript. In constitutive splicing events, the intron that contains an NIE is usually spliced, but the intron or a portion of it (for example, NIE) can be retained during alternative or aberrant splicing events. Mature mRNA transcripts that contain this NIE may be non-productive due to changes in the frame that induce the NMD pathway. The inclusion of an NIE in mature RNA transcripts may impair gene expression. MRNA transcripts that contain an NIE can be termed "NIE-containing mRNA" or "NMD exon mRNA" in the present disclosure.
[120] [120] Cryptic splice sites (or splice pseudo-sites) have the same splicing recognition sequences as genuine splice sites, but are not used in splicing reactions. They outnumber genuine splice sites in the human genome by an order of magnitude and are normally repressed by molecular mechanisms hitherto poorly understood. Cryptic 5 'splice sites have the NNN / GUNNNN or NNN / GCNNNN consensus, where N is any nucleotide and / is the exon-intron boundary. Cryptic 3 'splice sites have the NAG / N consensus. Their activation is positively influenced by surrounding nucleotides that make them more similar to the optimal consensus of authentic splice sites, specifically MAG / GURAGU and YAG / G, respectively, where M is C or A, R is G or A, and Y is C or U.
[121] [121] Splice sites and their regulatory sequences can be readily identified by those skilled in the art using appropriate publicly available algorithms, listed, for example, in Kralovicova, J. and Vorechovsky, I. (2007) “Global Control of Aberrant Splice Site Activation by Auxiliary Splicing Sequences: Evidence for a Gradient in Exon and Intron Definition ”. Nucleic Acids Res., 35, 6,399-6,413, (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095810/pdf/gk m680.pdf).
[122] [122] Cryptic splice sites or regulatory splicing sequences can compete for RNA-binding proteins, for example, U2AF, with an NIE splice site. In some embodiments, an agent can bind to a cryptic splice site or splicing regulatory sequence to prevent binding of RNA-binding proteins and, thus, favoring the binding of RNA-binding proteins to NIE splice sites.
[123] [123] In some embodiments, the cryptic splice site may not comprise the 5 'or 3' splice site of the NIE. In some embodiments, the cryptic splice site can be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides or at least 200 nucleotides upstream of the 5 'NIE splice site. In some embodiments, the cryptic splice site may be at least 10 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides downstream of the 3 'NIE splice site.
[124] [124] In some embodiments, the methods of the present disclosure explore the presence of NIE in the pre-mRNA transcribed by the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 genes, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, CR1, CR3, COL, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. Splicing of the identified NIE pre-mRNA species from ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRFF3, PRFI RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, SEM, MY3, MY3, MY4, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 to produce mature functional ABCR4 mRNA, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRN7 , COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN,
[125] [125] In some embodiments, the diseases or conditions that can be treated or improved with the use of the method or composition disclosed in this specification are not directly associated with the target protein (gene) that the therapeutic agent targets. In some embodiments, a therapeutic agent provided in this specification may target a protein (gene) that is not directly associated with a disease or condition, but modulation of target protein (gene) expression can treat or ameliorate the disease or condition. For example, targeting genes such as CD46, CFH, CR1, DNAJC8, EIF2AK3, ERN1, GUCY2F, GUCY2F, SEMA3C, SEMA3D, SIRT3 or AKT3 by a therapeutic agent provided in this specification can treat or improve eye diseases or conditions. In some modalities, targeting the CD46, CFH, CR1, DNAJC8, EIF2AK3, ERN1, GUCY2F, GUCY2F, SEMA3C, SEMA3D, SIRT3 or AKT3 genes may be indicated for the Via (eye). In some modalities, targeting a gene such as SCN8A can treat or ameliorate diseases of the central nervous system, for example, epilepsy, for example, Dravet's Syndrome. In some embodiments, these target genes, such as SCN8A, are indicated for Via (central nervous system) or Via (central nervous system, epilepsy).
[126] [126] In various embodiments, the present disclosure provides a therapeutic agent that can target ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A4, COL4A4 mRNA transcripts , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC2 , PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CRY, COLD, MYC3, , MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 to modulate the level of splicing or protein expression. The therapeutic agent can be a small molecule,
[127] [127] In some embodiments, the ASO targets a sequence about 4 to about 300 nucleotides upstream (or 5 ') from the 5' end of the NIE. In some embodiments, ASO targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides or about 250 to about 300 nucleotides upstream (or 5 ') from the 5' end of the NIE region. In some embodiments, the ASO may target a sequence of more than 300 nucleotides upstream of the 5 'end of the NIE. In some embodiments, the ASO targets a sequence of about 4 to about 300 nucleotides downstream (or 3 ') from the 3' end of the NIE. In some embodiments, ASO targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides or about 250 to about 300 nucleotides downstream of the 3 'end of the NIE. In some embodiments, the ASO targets a sequence of more than 300 nucleotides downstream of the 3 'end of the NIE.
[128] [128] In some embodiments, the pre-mRNA transcript containing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS , ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PKP2, , PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, MY2, CR1, MY, , SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity for any of the IDS. SEQ. Nos 1-59 or 192-211. In some embodiments, the NIE pre-mRNA transcript ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAO FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRFF3, PRFI RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, SEM, MY3, MY3, MY4, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100 % string identity for any of the IDS. SEQ. Nos: 60-191.
[129] [129] In some embodiments, the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, pre-mRNA transcript containing NIE (or NMD exon mRNA)
[130] [130] In some embodiments, the ASO targets exon 8x of a pre-mRNA containing NIE of ABCB4 comprising exon 8 of NIE, exon 9x of a pre-mRNA containing NIE of ASS1 comprising exon 9 of NIE , exon 16x of a pre-mRNA containing NIE of ATP8B1 comprising exon 16 of NIE, exon 1x of a pre-mRNA containing NIE of BAG3 comprising exon 1 of NIE, exon 31x of a pre-mRNA containing NAC of CACNA1A comprising exon 31 of NIE, exon 36x of a pre-mRNA containing NIE of CACNA1A comprising exon NIE 36, exon 37x of a pre-mRNA containing NIE of CACNA1A comprising exon NIE 37, exon 3x of a pre-mRNA that contains CIE NIE that comprises NIE exon 3, 12x exon of a CBS NIE pre-mRNA that comprises NIE exon 12, 1x exon of a pre-mRNA that contains CD55 NIE that comprises exon 1 of NIE, exon 16x of a pre-mRNA containing NIE of CDKL5 comprising exon 16 of NIE, exon 3x of pre-mRNA containing NIE of CFH comprising exon 3 of NIE, exon 30x of a pre-mRNA containing CHD2 NIE comprising exon 30 of NIE, exon 4x of the pre-mRNA containing NIE of CHRNA7 comprising exon 4 of NIE, exon 1x of pre-mRNA containing NIE of CISD2 comprising exon 1 of NIE, exon 15x of the pre-mRNA that contains NIE of CLN3 that comprises exon 15 of NIE, exon 11x of a pre-mRNA that contains NIE of COL4A3 that comprises exon 11 of NIE, exon 41x of a pre- mRNA containing COL4A3 NIE comprising exon 41 of NIE, exon 22x from a pre-mRNA containing NIE from COL4A4 comprising exon 22 from NIE, exon 44x from a pre-mRNA containing COL4A4 which comprises exon 44 of NIE, exon 20x of pre-mRNA containing NIE of DEPDC5 comprising exon 20 of NIE, exon 2x of a pre-mRNA containing NIE of DHDDS comprising exon 2 of NIE, exon 3x of a pre-mRNA containing ELOVL4 NIE which comprises NIE exon 3, 5x exon of a FAH NIE pre-mRNA which comprises NIE exon 5, 4x of the pre-mRNA which contains FXN NIE which comprises exon 4 of NIE, exon 4x of a pre-mRNA containing NIE of GALE comprising exon 4 of NIE, exon 3x of a pre-mRNA containing NIE of GBE1 comprising exon 3 of NIE, exon 11x of pre -mRNA containing GRIN2A NIE comprising exon 11 of NIE, exon 1x of pre-mRNA containing NIE of GRN comprising exon 1 of NIE, exon 2x of a pre-mRNA containing NIE of HEXA comprising exon 2 of NIE, exon 2x of a pre-mRNA containing NIE of KANSL1 comprising exon 2 of NIE, exon 1x of a pre-mRNA containing NIE of KCNQ2 comprising exon 1 of NIE, exon 50x of a pre- mRNA containing KMT2D NIE comprising exon 50 of NIE, exon 8x of pre-mRNA containing NIE of MAPK3 comprising exon 8 of NIE, exon 13x of pre-mRNA containing NIE of MBD5 comprising exon 13 of NIE, 2x exon of a pre-mRNA containing NIE of MECP2 comprising exon 2 of NIE, exon 11x of pre-mRNA containing NIE of MUT comprising exon 11 of NIE, exon 31x of pre-mRNA containing NIE of NF1 that comprises exon 31 of NIE, exon 7x of a pre-mRNA containing NIE of NIPBL comprising exon 7 of NIE, exon 38x of a pre-mRNA containing NIE of NIPBL comprising exon 38 of NIE, exon 11x of a pre-mRNA containing NSD1 NIE comprising exon 11 of NIE, exon 6x of a pre-mRNA containing NIE of OPA1 comprising exon 6 of NIE, exon 28x of a pre-mRNA containing OIE1 which comprises exon 28 of NIE, exon 1x of pre-mRNA that contains NIE of OPTN that comprises exon 1 of NIE, exon 1x of pre-mRNA that contains PCIE NIE that comprises exon
[131] [131] In some embodiments, ASO targets a sequence of about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5 ') from the 5' end of ABCB4 exon 8x, 9x exon of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, is exx of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x is DExDC5x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of G RN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NF1, exon 7x NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, exon 1x of PCCA, exon 5x of PCCB, exon 6x of PCCB, exon 4x of PKP2,
[132] [132] In some embodiments, ASO targets a sequence at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream (or 5 ') from the 5' end of ABCB4 exon 8x, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CD55, exx 16x of exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x exon 2x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exx on 1x GRN, 2x exon HEXA, 2x exon KANSL1, 1x exon KCNQ2, exon 50x KMT2D, exon 8x MAPK3, exon 13x MBD5, exon 2x MECP2, exon 11x MUT, exon 31x NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, exon 1x of PCCA, exon 5x of PCCB, exon 6x of PCCB, exon 4x of PKP2, exon 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3A, exon 7x of SCN3A, exon 4x of SCN8A, isxon of exon 20x of SCN8A, exon 6x of SCN9A, exon 24x of SHANK3, exon 3x of SLC25A13, exon 6x of SLC25A13, exon 9x of SLC25A13, exon 11x of SLC25A13, exon 13x of SLC25A13, exon 1x of SLC25A13 exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN. In some modalities, the ASO targets a sequence at most about 1,500 nucleotides, about
[133] [133] In some embodiments, the ASO targets a sequence of about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3 ') of the 3' end of ABCB4 exon 8x, 9x exon of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, is exx of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x is DExDC5x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GR N, 2x exon of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NF1, exon 7x NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, exon 1x of PCCA, exon 5x of PCCB, exon 6x of PCCB, exon 4x of PKP2, exon 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3A, exon 7x of SCN3A, exon 4x of SCN8A, exon 6x of SCN8, exon 6x of SCN8 SCN8A, exon 6x of SCN9A, exon 24x of SHANK3, exon 3x of SLC25A13, exon 6x of SLC25A13, exon 9x of SLC25A13, exon 11x of SLC25A13, exon 13x of SLC25A13, exon 1x of SLC6A1, isxon of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN.
[134] [134] In some embodiments, ASO targets a sequence at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream (or 3 ') from the 3' end of the ABCB4 exon 8x, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CD55, exx 16x of exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x exon 2x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exo n 1x GRN, exon 2x HEXA, exon 2x KANSL1, exon 1x KCNQ2, exon 50x KMT2D, exon 8x MAPK3, exon 13x MBD5, exon 2x MECP2, exon 11x MUT, exon 31x NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, exon 1x of PCCA, exon 5x of PCCB, exon 6x of PCCB, exon 4x of PKP2, exon 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3A, exon 7x of SCN3A, exon 4x of SCN8A, isxon of exon 20x from SCN8A, exon 6x from SCN9A, exon 24x from SHANK3, exon 3x from SLC25A13,
[135] [135] In some embodiments, the ASO has a sequence complementary to the targeted portion of the NMD exon mRNA according to any of the IDS. SEQ. Nos: 60-191.
[136] [136] In some embodiments, the ASO targets a sequence upstream of the 5 'end of an NIE. For example, ASOs that target a sequence upstream of the 5 'end of an NIE (for example, exon 8x of ABCB4, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A , exon 37x from CACNA1A, exon 3x from CBS, exon
[137] [137] In some embodiments, ASOs target a sequence that contains an exon-intron boundary (or junction). For example, ASOs that target a sequence that contains an exon-intron boundary may comprise a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complementary to at least 8 contiguous nucleic acids of any of the IDS. SEQ.
[138] [138] In some embodiments, ASO targets exon 8x of a pre-mRNA containing NIE of ABCB4 comprising exon 8 of NIE, exon 9x of a pre-mRNA containing NIE of ASS1 comprising exon 9 of NIE , exon 16x of a pre-mRNA containing NIE of ATP8B1 comprising exon 16 of NIE, exon 1x of a pre-mRNA containing NIE of BAG3 comprising exon 1 of NIE, exon 31x of a pre-mRNA containing NAC of CACNA1A comprising exon 31 of NIE, exon 36x of a pre-mRNA containing NIE of CACNA1A comprising exon NIE 36, exon 37x of a pre-mRNA containing NIE of CACNA1A comprising exon NIE 37, exon 3x of a pre-mRNA that contains CIE NIE that comprises NIE exon 3, 12x exon of a CBS NIE pre-mRNA that comprises NIE exon 12, 1x exon of a pre-mRNA that contains CD55 NIE that comprises exon 1 of NIE, exon 16x of a pre-mRNA containing NIE of CDKL5 comprising exon 16 of NIE, exon 3x of pre-mRNA containing NIE of CFH comprising exon 3 of NIE, exon 30x of a pre-mRNA containing CHD2 NIE comprising exon 30 of NIE, exon 4x of the pre-mRNA containing NIE of CHRNA7 comprising exon 4 of NIE, exon 1x of pre-mRNA containing NIE of CISD2 comprising exon 1 of NIE, exon 15x of the pre-mRNA that contains NIE of CLN3 that comprises exon 15 of NIE, exon 11x of a pre-mRNA that contains NIE of COL4A3 that comprises exon 11 of NIE, exon 41x of a pre- mRNA containing COL4A3 NIE comprising exon 41 of NIE, exon 22x of a pre-mRNA containing NIE of COL4A4 comprising exon 22 of NIE, exon 44x of a pre-
[139] [139] In some embodiments, the target portion of the pre-mRNA that contains NIE of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PK1 PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A, CR1, MYC, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some embodiments, hybridization of an ASO to the target portion of the NIE pre-mRNA results in exon-skipping of at least one NIE within intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and subsequently increases the production of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A protein, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMTD, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN,
[140] [140] In some embodiments, the methods and compositions of the present disclosure are used to increase the expression of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4 , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC2 , PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CRY, COLD, MYC3, , MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 by exon-skipping induction of a pre-mRNA pseudo-exon containing ATBP4, N1, AT1, ABC1 BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSD MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRP F3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A,
[141] [141] In some embodiments, the methods described in this specification are used to increase the production of a functional protein or RNA from ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, NIPBL, NSD1, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, CRU, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. As used in this specification, the term “functional” refers to the amount of activity or function of a protein or RNA of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 , COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH,
[142] [142] In some embodiments, the method is a method of increasing the expression of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PK1 PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A,
[143] [143] In some embodiments, the method is a method of increasing the expression of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1,
[144] [144] In some embodiments, the method is a method of increasing the expression of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PK1 PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A,
[145] [145] In some embodiments, the method is a method of increasing the expression of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PK1 PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A, CR1, MYC, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4,
[146] [146] In related modalities, the method is a method of using an ASO to increase the expression of a functional protein or RNA. In some embodiments, an ASO can be used to increase protein expression ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 , FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PRF31 , RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAF3, MY6, MY2 , SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 in cells of an individual that have a pre-mRNA containing NIE that encodes the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CACNA1A, CBS CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, KMT2D, MAPK3, KMT2D, MAPK3 MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFO X2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYHD, SEMA3, SEM, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1, in which the individual has a disability, for example, Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceroid, neuronal lipofuscinosis, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive intrahepatic family 1; Type II citrullinaemia; Citrullinaemia, Type 1; Cognitive deficit with or without cerebral ataxia; Cornélia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, arising in childhood; Early childhood epileptic encephalopathy, 11; Early childhood epileptic encephalopathy, 12; Early childhood epileptic encephalopathy, 13; Early childhood epileptic encephalopathy, 2; Episodic ataxia, type 2; Familial focal epilepsy; Febrile seizures, familial, 3B; Friedreich's ataxia; Friedreich's ataxia with retained reflexes; Galactose epimerase deficiency; Glaucoma 3, primary congenital, E; Glycogen storage disease IV; GRN-related frontotemporal dementia; Homocystinuria, types responsive and unresponsive to B6; HSAN2D, autosomal recessive; Congenital insensitivity to pain; Kabuki syndrome; Koolen-De Vries syndrome; Mental retardation, autosomal dominant 1; Methyl malonic aciduria; Migraine, Myoclonic-atonic epilepsy; family hemiplegic, 1; Neurofibromatosis type 1; Opioid addiction; Optical atrophy type 1; Phelan-McDermid syndrome; Propionic acidosis; Primary open-angle glaucoma; Propionic acidosis; Retinitis pigmentosa 11; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Retinitis pigmentosa 59; Rett syndrome; Convulsions, benign family children, 3; Convulsions, benign family children, 5; Smith-Magenis syndrome; Sotos Syndrome 1; Beckwith-Wiedemann syndrome; Stargardt's disease 3; Tay-Sachs disease; Tuberous sclerosis; Tyrosinemia, type I; Wagner's syndrome 1; West syndrome; Wolfram 2 syndrome / NAFLD; 15q13.3 microdeletion; 16p11.2 deletion syndrome; Deafness, autosomal dominant 13; Retinal dystrophy of rod cones-2; Deafness, autosomal dominant 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Deafness, autosomal dominant 22; Neurofibromatosis type 2; NASH; or Autosomal dominant mental retardation 5, in the amount or function of an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 protein , FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PR3
[147] [147] In some embodiments, the pre-mRNA transcript that contains NIE that encodes the protein that causes the disease or condition is targeted by the ASOs described in that specification. In some embodiments, a pre-mRNA transcript that contains NIE that encodes a protein that does not cause the disease is targeted by ASOs. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway can be improved by targeting a pre-mRNA that contains NIE that encodes a second protein, thereby increasing the production of the second protein . In some embodiments, the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which causes the disease or condition).
[148] [148] In some embodiments, the individual has: (a) a first mutant allele whereby: (i) the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, NIPBL, NSD1, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, CRU, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11,
[149] [149] In some embodiments, the level of mRNA encoding an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 , FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PR3
[150] [150] In some embodiments, an individual treated using the methods of the present disclosure expresses an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4 protein , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC2 , PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CRY, COLD, MYC3, , MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 partially functional by an allele, in which the protein ABCB4, ASS1, ATP8B1, BAG, CACNA1, BAG3, CACNA1, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MB5, MB5 NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2 , SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, SEMO3, N3, SEM3, , ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or partially functional SYNGAP1 can be caused by a frame change mutation, a nonsense mutation, a missense mutation or a partial gene deletion.
[151] [151] As used in this specification, a “pre-
[152] [152] In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of pre-mRNAs that contain NIE transcribed by the gene encoding the target protein in a cell. In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of pre-mRNAs that contain NIE transcribed by the gene encoding the target protein in a cell, where the population of pre-mRNAs that contain NIE comprises two or more pseudo-exons included. In some embodiments, an antisense oligomer targeting the most abundant pseudoexon in the population of pre-mRNAs that contain NIE encoding the target protein induces exon-skipping of one or two or more pseudoexons in the population, including the pseudoexon to which the oligomer antisense is targeted or binds. In some embodiments, the target region is in a pseudo-exon which is the most abundant pseudo-exon in a pre-mRNA that contains NIE encoding the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7 protein , CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, NUT, NSF1, NIP1, , OPTN, PCCA, PCCB, PKP2,
[153] [153] The degree of exon inclusion can be expressed as a percentage of exon inclusion, for example, the percentage of transcripts in which a certain pseudoexon is included. Briefly, the percentage of exon inclusion can be calculated as the percentage of the amount of RNA transcripts with the inclusion of exon, over the sum of the average of the amount of RNA transcripts with exon inclusion plus the average of the amount of RNA transcripts excluding exon.
[154] [154] In some embodiments, an included pseudoexon is an exon that is identified as an included pseudoexon based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50%, inclusion. In embodiments, an included pseudoexon is an exon that is identified as an included pseudoexon based on a determination of about 5% to about 100%, about 5% to about 95%, about 5% to about 90 %, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65 %, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40 %, about 5%
[155] [155] In some embodiments, cell contact with an ASO that is complementary to a targeted portion of an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7 pre-mRNA transcript , CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, NUT, NSF1, NIP1, , OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOC, TSC2, U3, TS3 , CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 results in an increase in the amount of protein ABCB4, ASS1, BAP3, AT1, BAP3, ATP8 CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCN2, KMC2 MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI 1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH
[156] [156] In some embodiments, cell contact with an ASO that is complementary to a targeted portion of an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7 pre-mRNA transcript , CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, NUT, NSF1, NIP1, , OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOC, TSC2, U3, TS3 , CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 results in an increase in the amount of mRNA that encodes the ABCB4 protein, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, GRN, HEX KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1 , PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, CR6O, MY6, CRYO , NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1, including the mature mRNA encoding the target protein. In some embodiments, the amount of mRNA encoding an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7 protein,
[157] [157] The NIE can be any length. In some embodiments, the NIE comprises a complete sequence of an intron, when it can then be called intron retention. In some embodiments, the NIE can be a portion of the intron. In some embodiments, the NIE can be a portion of the 5 'end of an intron, including a 5'ss string.
[158] [158] The inclusion of a pseudo-exon can lead to a change in frame and the introduction of a premature termination codon (PIC) in the mature mRNA transcript, making the transcript a target for NMD. The mauro mRNA transcript that contains NIE can be transcribed from non-productive mRNA that does not lead to protein expression. The PIC can be present in any position downstream of an NIE. In some modalities, the PIC can be present in any exon downstream of an NIE. In some modalities, the PIC may be present within the NIE. For example, the inclusion of exon 8x of ABCB4, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 3x of CBS, exon 3x of CBS , 1x exon of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4 , exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH,
[159] [159] In various embodiments of the present disclosure, compositions and methods comprising a therapeutic agent are provided to modulate the level of protein expression ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIP1, NIP1, NIP1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TS3, U3, U3 CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. In some embodiments, compositions and methods to modulate the alternative splicing of ABCM4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3 are provided in this specification. COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPACA, PCTB, PCO, PCTB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, CR1, CRC, AKC3, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. In some embodiments, compositions and methods for inducing exon-skipping in the pre-mRNA splicing of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 are provided in this specification. COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1,
[160] [160] A therapeutic agent disclosed in this specification may be an NIE-repressing agent. A therapeutic agent can comprise a polynucleic acid polymer. According to one aspect of the present disclosure, in that specification a method of treating or preventing a condition or disease associated with a protein deficiency ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2 is provided , CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, NIP, MIP, NUT , OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1,
[161] [161] When reference is made to reducing the inclusion of NIE in the mature mRNA, the reduction may be complete, for example, 100%, or it may be partial. The reduction can be clinically significant. The reduction / correction can be in relation to the level of inclusion of NIE in the individual without treatment or in relation to the amount of inclusion of NIE in a population of similar individuals. The reduction / correction can be at least 10% less inclusion of NIE in relation to the average individual or to the individual before treatment. The reduction can be at least 20% less inclusion of NIE in relation to an average individual or to the individual before treatment. The reduction can be at least 40% less inclusion of NIE in relation to an average individual or to the individual before treatment. The reduction can be at least 50% less inclusion of NIE in relation to an average individual or to the individual before treatment. The reduction can be at least 60% less inclusion of NIE in relation to an average individual or to the individual before treatment. The reduction can be at least 80% less inclusion of NIE in relation to an average individual or to the individual before treatment. The reduction may be at least 90% less inclusion of NIE in relation to an average individual or to the individual before treatment.
[162] [162] When reference is made to increased levels of protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH , FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF1 , RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, SEM, MYF3, MYH , EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or active SYNGAP1, the increase can be clinically significant. The increase may be in relation to the protein level ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1,
[163] [163] In embodiments in which the NIE-repressing agent comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length. The polynucleic acid polymer can be about 19 nucleotides in length.
[164] [164] The polynucleic acid polymer sequence can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% complementary to a target sequence of an mRNA transcript, for example, a partially processed mRNA transcript. The sequence of the polynucleic acid polymer can be 100% complementary to a target sequence of a pre-mRNA transcript.
[165] [165] The polynucleic acid polymer sequence can have 4 or fewer pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence can have 3 or fewer pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence can have 2 or less pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence can have 1 or less pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence may have no pairing errors for a target sequence of the pre-mRNA transcript.
[166] [166] The polynucleic acid polymer can hybridize specifically to a target sequence of the pre-mRNA transcript. For example, the polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence complementarity for a sequence-
[167] [167] The polynucleic acid polymer comprising a sequence of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 99.5% of sequence identity for a selected sequence from the group consisting of IDS. SEQ. Nos: 60-
[168] [168] When reference is made to a polynucleic acid polymer sequence, those skilled in the art will understand that one or more substitutions can be tolerated, optionally two substitutions can be tolerated in the sequence, so that it maintains the ability to hybridize to target sequence; or, when the substitution is in a target sequence, the ability to be recognized as the target sequence. References to string identity can be determined by aligning BLAST strings using standard / standardized parameters. For example, the string can have 99% identity and still work according to the present disclosure. In other modalities, the sequence can have 98% identity and still work according to the present disclosure. In another modality, the sequence can have 95% identity and still work according to the present disclosure. In another modality, the sequence can have 90% identity and still work according to the present disclosure.
[169] [169] A composition is provided in this specification that comprises an antisense oligomer that induces exon-skipping by binding to a target portion of a pre-mRNA that contains ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55 NIE , CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MACHUT, MBK2, , NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, TSA2, , VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. As used in this specification, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer such as a polynucleotide, which comprises nucleobases that hybridize to a target nucleic acid sequence (e.g., a pre-mRNA containing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, NIE FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRFF3, PRFI RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, SEM, MY3, MY3, MY4, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1) by Watson-Crick base pairing or oscillation base pairing (GU). The ASO can have the exact sequence complementary to the target sequence or almost complementarity (for example, enough complementarity to bind to the target sequence and increase splicing at a splice site). ASOs are designed to bind (hybridize) to a target nucleic acid (for example, a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the desired (target) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (for a few different sites than a target nucleic acid). The design of an ASO can take into account the occurrence of the nucleic acid sequence of the target portion of the pre-mRNA transcript or a sequence of nucleic acids sufficiently similar at other locations in the genome or pre-mRNA or cell transcriptome, so that the The likelihood that the ASO will link to other sites and cause “off-target” effects is limited. Any antisense oligomers known in the art, for example, in PCT Application No. PCT / US2014 / 054151, published as WO 2015/035091, entitled “Reducing Nonsense-Mediated mRNA Decay”, incorporated by reference in this specification, can be used to the practice of the methods described in this specification.
[170] [170] In some embodiments, ASOs "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA that contains NIE. Typically this hybridization occurs at a Tm substantially greater than 37 ° C, preferably at least 50 ° C and, typically, between 60 ° C to approximately
[171] [171] Oligomers, for example, oligonucleotides, are "complementary" to each other when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide if hybridization can occur between one strand of the first and the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) is quantifiable in terms of the proportion (for example, the percentage) of bases on opposite strands that are supposed to form hydrogen bonds between them, according to base pairing rules generally accepted. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence complementarity for a target region within the target nucleic acid sequence they target. For example, an ASO in which 18 out of 20 nucleobases of the oligomeric compound are complementary to a target region and therefore would hybridize specifically would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases would be grouped together or spaced with complementary nucleobases and need not be contiguous with each other or with complementary nucleobases. The percentage of complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment research tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol. , 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[172] [172] An ASO does not need to hybridize for all nucleobases in a target sequence and the nucleobases to which it hybridizes can be contiguous or non-contiguous. ASOs can hybridize over one or more segments of a pre-mRNA transcript, so that intervening or adjacent segments are not involved in the hybridization event (for example, a loop structure or hairpin structure can be formed). In certain embodiments, an ASO hybridizes to non-contiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases to which the ASO does not hybridize.
[173] [173] The ASOs described in this specification include nucleobases that are complementary to the nucleobases present in a targeted portion of a pre-mRNA that contains NIE. The term "ASO" encompasses oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridization to a complementary nucleobase in a target mRNA, but does not comprise a portion of sugar, for example, a peptide nucleic acid (PNA). ASOs can comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or which have a modified framework. In some embodiments, all ASO nucleotides are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described in this specification will be evident to those skilled in the art and can be found, for example, in US Patent No. 8,258,109 B2, US Patent No. 5,656,612 , US Patent Publication No. 2012/0190728 and Dias and Stein, Mol. Cancer Ther. 2002, 347-355, incorporated in this specification by reference in its entirety.
[174] [174] One or more nucleobases from an ASO can be any naturally occurring, unmodified nucleobase, for example, adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase from so that it is capable of hydrogen bonding to a nucleobase present in a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine.
[175] [175] The ASOs described in this specification also comprise a framework structure that connects the components of an oligomer. The terms "frame structure" and "oligomer bonds" can be used interchangeably and refer to the connection between ASO monomers. In naturally occurring oligonucleotides, the framework comprises a 3'-5 'phosphodiester bond that connects sugar portions of the oligomer. The framework structure or oligomer bonds of the ASOs described in this specification may include (without limitation) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, phosphoramidate and the like. See, for example, LaPlanche et al., Nucleic Acids Res. 14: 9,081 (1986); Stec et al, J. Am. Chem. Soc. 106: 6,077 (1984), Stein et al., Nucleic Acids Res. 16: 3,209 (1988), Zon et al., Anti-Cancer Drug Design 6: 539 (1991); Zon et al, “Oligonucleotides and Analogues: A Practical Approach,” pages 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al, U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90: 543 (1990). In some embodiments, the ASO backbone does not contain phosphorus, but instead contains peptide bonds, for example, in a peptide nucleic acid (PNA), or linkage groups that include carbamate, amides and linear hydrocarbon groups and cyclical. In some embodiments, the modification of the framework is a phosphotioate bond. In some embodiments, the modification of the framework is a phosphoramidate bond.
[176] [176] In modalities, the stereochemistry in each of the phosphorus internucleotide bonds in the ASO framework is random. In some modalities, the stereochemistry in each of the phosphorus internucleotide bonds in the ASO framework is controlled and is not random. For example, U.S. Patent Application Publication No. 2014/0194610, “Methods for the Synthesis of Functionalized Nucleic Acids”,
[177] [177] In some modalities, ASO has a non-random mix of Rp and Sp configurations in its phosphorus internucleotide bonds. For example, it has been suggested that a mixture of Rp and Sp is needed in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan et al., 2014, “Synthesis, Biophysical Properties and Biologic Activity of Second Generation Antisense Oligonucleotides Containing Chiral
[178] [178] In some embodiments, an ASO used in disclosure methods including, without limitation, any of the ASOs presented in this specification, comprises a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous nucleic acids from any of the IDS. SEQ. Nos: 60-191, comprises about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp , at least about 80% Sp, at least about 85% Sp, at least about 90% Sp or at least about 95% Sp, with the rest Rp or about 100% Sp. modalities, an ASO used in disclosure methods including, without limitation, any of the ASOs presented in this specification, comprises a string with at least about 80%, 85%, 90%, 95%, 97% or 100% identity of sequence for a region comprising at least 8 contiguous nucleic acids of any of the IDS. SEQ. Nos: 60-191, comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp , about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about from 90% to about 100% Sp or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30 % to about 70% Sp, about 40% to about 60% Sp or about 45% to about 55% Sp, with the rest Rp.
[179] [179] Any of the ASOs described in this specification may contain a sugar portion comprising ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar or sugar analog portion, including a morpholino ring. Non-limiting examples of modified sugar moieties include 2 'substitutions such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl , 2'F; N3 ’-> P5’ phosphoramidate, 2’dimethylaminooxyethoxy, 2’dimethylaminoethoxyethoxy, 2’-guanidinium, 2’-O-guanidinium ethyl, modified carbamate sugars and modified bicyclic sugars. In some embodiments, the sugar portion modification is selected from 2’-O-Me, 2’F and 2’MOE. In some embodiments, modifying the sugar portion is an extra bridge, for example, to a blocked nucleic acid (LNA). In some embodiments, the sugar analog contains a morpholino ring, for example, morpholino phosphorodiamidate (PMO). In some embodiments, the sugar portion comprises a ribofuranosyl or 2'-deoxyribofuranosyl modification. In some embodiments, the sugar portion comprises 2'-O-methyloxyethyl 2'4'-forced modifications (cMOE). In some embodiments, the sugar portion comprises cEt 2'-O 2 ', 4'-forced ethyl BNA modifications. In some embodiments, the sugar portion comprises tricyclo-DNA (tcDNA) modifications. In some embodiments, the sugar portion comprises ethylene-nucleic acid (ENA) modifications. In some embodiments, the sugar portion comprises MCE modifications. Modifications are known in the art and described in the literature, for example, by Jarver et al, 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications”, Nucleic Acid Therapeutics 24 (1): 37-47, incorporated by reference for this purpose in this specification.
[180] [180] In some embodiments, each ASO monomer is modified in the same way, for example, each ASO framework bond comprises a phosphorothioate bond or each ribose sugar portion comprises a 2'-O-methyl modification. These modifications that are present in each of the monomeric components of an ASO are called "uniform modifications". In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate bonds and sugar moieties that comprise morpholino (morpholino) rings. Combinations of different modifications for an ASO are called "mixed modifications" or "mixed chemicals".
[181] [181] In some modalities, the ASO comprises one or more modifications to the framework. In some embodiments, the ASO comprises one or more modifications of the sugar portion. In some embodiments, the ASO comprises one or more modifications to the framework and one or more modifications to the sugar portion. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate framework. In some embodiments, ASO comprises a morpholino phosphorodiamidate (PMO). In some embodiments, ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (for example, a nucleobase, sugar portion, framework) described in this specification can be modified to obtain desired ASO properties or activities or to reduce unwanted ASO properties or activities. For example, an ASO or one or more components of any ASO can be modified to increase the binding affinity for a target sequence in a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (ie RNase H); increase the uptake of ASO in a cell and / or in the nucleus of a cell; change the pharmacokinetics or pharmacodynamics of ASO; and / or modulate the ASO half-life.
[182] [182] In some embodiments, ASOs are composed of 2'-O- (2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs composed of these nucleotides are especially well suited to the methods revealed in this specification; it was demonstrated that oligomers that have these modifications have significantly increased resistance to degradation by nuclease and increased bioavailability, which makes them suitable, for example, for oral release in some modalities described in this specification. See, for example, Geary et al, J. Pharmacol. Exp. Ther. 2001; 296 (3): 890-7; Geary et al, J. Pharmacol. Exp. Ther. 2001; 296 (3): 898-904.
[183] [183] ASO synthesis methods will be known to those skilled in the art. Alternatively or in addition, ASOs can be obtained from a commercial source.
[184] [184] Unless otherwise specified, the left end of the single stranded nucleic acid sequences (eg, pre-mRNA transcript, oligonucleotide, ASO etc.) is the 5 'end and the left direction of acid sequences single or double stranded nuclei is called the 5 'direction. Similarly, the right end or direction of a nucleic acid sequence (single or double stranded) is the 3 'end or direction. Generally, a region or sequence that is 5 'to a reference point in a nucleic acid is referred to as "upstream" and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream ”. Generally, the 5 'direction or end of an mRNA is where the initiation or start codon is located, while the 3' end or direction is where the termination codon is located. In some respects, nucleotides that are upstream of a reference point in a nucleic acid can be designated by a negative number, while nucleotides that are downstream of a reference point can be designated by a positive number. For example, a reference point (for example, an exon-exon junction in mRNA) can be designated as the “zero” site and a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one”, for example, “-1”, while a nucleotide that is directly adjacent and downstream from the reference point is designated “plus one”, for example, “+1”.
[185] [185] In some embodiments, ASOs are complementary to (and bind to) a targeted portion of a pre-mRNA that contains ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, NIE, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF, NF, NF OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, AK3, TS3, TS3, TS3, TS3, TS3 COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 which is downstream (in the 3 'direction) of the 5' splice site. '(or 3' end of the NIE) of the exon included in a pre-mRNA containing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4 , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, TOLCA1, TSLC2 UBE3A, VCAN,
[186] [186] In some embodiments, ASOs are complementary to (and bind to) a target portion of a pre-mRNA that contains ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, NIE, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF, NF, NF OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, AK3, TS3, TS3, TS3, TS3, TS3 COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 that is upstream (in the 5 'direction) of the splice site '(or 3' end) of the exon included in a pre-mRNA containing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5 , DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPLC, TS3 VCAN, AKT3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2,
[187] [187] In some embodiments, ASOs are complementary to a target region of a pre-mRNA that contains ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, CR1, CR3, COL, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 that is upstream (in the 5 'direction) from the splice site 3' (or 5 'end) ) of the exon included in a pre-mRNA containing NIE of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 , FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1 , OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, CD, TS3, TS3, TS3, TS3 , COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 (for example, in the direction designated by negative numbers). In some embodiments, ASOs are complementary to a targeted portion of the pre-mRNA that contains ABCIE4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, NIE DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA,
[188] [188] In some embodiments, ASOs are complementary to a target region of a pre-mRNA that contains ABCIE4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 NIE, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, CR1, CR3, COL, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 which is downstream (in the 3 'direction) of the splice site 3' (end 5 ') of the exon included in a pre-mRNA containing NIE of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAO FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OP A1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC3, TSC3 COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 (for example, in the direction designated by positive numbers). In some embodiments, ASOs are complementary to a targeted portion of the pre-mRNA that contains ABCIE4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, NIE DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC, PCP PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13,
[189] [189] In some embodiments, the target portion of the pre-mRNA that contains NIE of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PK1 PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A, VCAN, AKT3, CD46, COL11A, CR1, MYC, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1 is within the +100 region in relation to the 5 'splice site (3' end) of the included exon up to -100 with respect to to the 3 'splice site (5' end) of the included exon. In some embodiments, the target portion of the pre-mRNA containing NIE of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 , FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PRF31 , RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS,
[190] [190] ASOs can be of any length suitable for specific binding and increased effective splicing. In some embodiments, ASOs consist of 8 to 50 nucleobases. For example, the ASO can have 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 40, 45 or 50 nucleobases in length. In some embodiments, ASOs consist of more than 50 nucleobases. In some embodiments, the ASO is 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases,
[191] [191] In some embodiments, two or more ASOs are used with different chemicals, but complementary to the same target portion of the pre-mRNA that contains NIE. In some embodiments, two or more ASOs are used that are complementary to different target portions of the NIE-containing pre-mRNA.
[192] [192] In some embodiments, the antisense oligonucleotides of the disclosure are chemically linked to one or more moieties or conjugates, for example, a targeting moiety or other conjugate that increases the oligonucleotide activity or cellular uptake. These portions include,
[193] [193] In some embodiments, the nucleic acid to be targeted by an ASO is a pre-mRNA that contains ABCIE4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 NIE , COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2,
[194] [194] Pharmaceutical compositions or formulations comprising the agent, for example, antisense oligonucleotide, of the described compositions and for use in any of the described methods, can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the literature published. In embodiments, a pharmaceutical composition or formulation for the treatment of an individual comprises an effective amount of any antisense oligomer as described in that specification, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof. The pharmaceutical formulation comprising an antisense oligomer can further comprise a pharmaceutically acceptable excipient, diluent or carrier.
[195] [195] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without unnecessary toxicity, irritation, allergic response, etc. and are compatible with a reasonable risk / benefit ratio (see, for example, S.M.
[196] [196] In some embodiments, the compositions are formulated in any of several possible dosage forms, such as, without limitation, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. In some embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may also contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. In embodiments, a pharmaceutical formulation or composition of the present disclosure includes, without limitation, a solution, emulsion, microemulsion, foam or formulation containing liposomes (for example, cationic or non-cationic liposomes).
[197] [197] The pharmaceutical composition or formulation described in this specification may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients, as appropriate and well known to those skilled in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, for example, liposomes that comprise one or more specialized lipids. These specialized lipids result in liposomes with an increased circulation half-life. In embodiments, a sterically stabilized liposome comprises one or more glycolipids, or is derivatized with one or more hydrophilic polymers, for example, a portion of polyethylene glycol (PEG). In some embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in pharmaceutical products, formulations and emulsions is well known in the art. In embodiments, the present disclosure employs a penetration promoter to effect the efficient release of the antisense oligonucleotide, for example, to assist diffusion across cell membranes and / or to increase the permeability of a lipophilic drug. In some embodiments, penetration promoters are a surfactant, fatty acid, bile salt, chelating agent or non-chelating non-surfactant.
[198] [198] In some embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. Combined therapies
[199] [199] In some embodiments, the ASOs disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the (one or more) additional therapeutic agents may comprise a small molecule. For example, the (one or more) additional therapeutic agents may comprise a small molecule described in WO 2016128343 A1, WO 2017053982 A1, WO 2016196386 A1, WO 201428459 A1, WO 201524876 A2, WO 2013119916 A2 and WO 2014209841 A2, which are incorporated by reference in that specification in its entirety. In some embodiments, the (one or more) additional therapeutic agents comprise an ASO that can be used to correct intron retention. Treatment of Individuals
[200] [200] Any of the compositions provided in this specification can be administered to an individual. The term "person" can be used interchangeably with "individual" or "patient". An individual can be a mammal, for example, a human or animal such as, for example, a non-human primate, a rodent, a rabbit, a mouse, a mouse, a horse, a monkey, a goat, a cat, a dog , a cow, a pig or a sheep. In modalities, the individual is a human. In modalities, the individual is a fetus, an embryo or a child. In other embodiments, the individual may be another eukaryotic organism, for example, a plant. In some embodiments, the compositions provided in that specification are administered to an ex vivo cell.
[201] [201] In some embodiments, the compositions provided in this specification are administered to an individual as a method of treating a disease or disorder. In some modalities, the individual has a genetic disease, for example, any of the diseases described in this specification. In some modalities, the individual is at risk of having a disease, for example, any of the diseases described in this specification. In some embodiments, the individual is at increased risk of having a disease or disorder caused by insufficient protein or insufficient protein activity. If an individual is "at an increased risk" of having a disease or disorder caused by insufficient protein or insufficient protein activity, the method involves preventive or prophylactic treatment. For example, an individual may be at an increased risk of having this disease or disorder because of the family's history of the disease. Typically, individuals at an increased risk of having this disease or disorder benefit from prophylactic treatment (for example, by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in the womb, for example, by administering the ASO composition to the fetus directly or indirectly (for example, through the mother).
[202] [202] Pathways suitable for administering ASOs of the present disclosure may vary depending on the type of cell to which ASO release is desired. Multiple tissues and organs are affected by Dravet's syndrome, with the brain being the tissue most significantly affected. The ASOs of the present disclosure can be administered to patients parenterally, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection.
[203] [203] In modalities, the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the antisense oligonucleotide in question through the blood-brain barrier by any method known in the art. For example, the release of agents by administering an adenovirus vector to motor neurons in muscle tissue is described in US Patent No. 6,632,427, “Adenoviral-Vector-Mediated Gene Transfer into Medullary Motor Neurons”, incorporated in this specification by reference . The release of vectors directly to the brain, for example, to the striatum, to the thalamus, to the hippocampus or to the substantia nigra, is described, for example, in US Patent No. 6,756,523, “Adenovirus Vectors for the Transfer of Foreign Genes into Cells of the Central Nervous System Particularly in Brain ”, incorporated in this specification by reference.
[204] [204] In some embodiments, antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to a substance known in the art to promote penetration or transport across the blood-brain barrier, for example, an antibody to the transferrin receptor. In modalities, the antisense oligonucleotide is linked to a viral vector, for example, to make the antisense compound more effective or to increase transport across the blood-brain barrier. In modalities, the osmotic rupture of the blood-brain barrier is aided by sugar infusion, for example, meso erythritol, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, dulcitol, myoinositol, L (-) fructose, D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+)
[205] [205] In some embodiments, an ASO of the development is coupled with a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), a noradrenaline reuptake inhibitor (NRI), a reuptake inhibitor norepinephrine-dopamine (NDRI) and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI), using methods described, for example, in US Patent No.
[206] [206] In some embodiments, individuals treated using the methods and compositions are assessed for improvement in condition using any methods known and described in the art.
[207] [207] Also within the scope of this disclosure are methods for identifying or determining ASOs that induce exon-skipping of a pre-mRNA containing ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, NIE CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, NBL, MECP2, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TSE, TS3, TS3, CD46, COL11A2, CR1, CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. For example, a method may comprise the identification or determination of ASOs that induce pseudoexon skipping of a pre-mRNA that contains ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2 NIE , CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, NIPBL, NSD1, NIPBL, NSD1, , PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UCE3, COL, , CRX, DNAJC8, MYH14, MYO6, NF2, SEMA3C, SEMA3D, EIF2AK3, ERN1, GUCY2F, SIRT3, NR1H4, STK11, PPARA, CYP2J2 or SYNGAP1. ASOs that hybridize specifically to different nucleotides within the target region of the pre-mRNA can be evaluated to identify or determine ASOs that increase the rate and / or extent of splicing of the target intron. In some modalities,
[208] [208] An evaluation round, referred to as a "walk" ASO, can be performed using ASOs that have been designed to hybridize to a pre-mRNA target region. For example, ASOs used in the ASO Walk can be covered every 5 nucleotides from approximately 100 nucleotides upstream from the included exon 3 'splice site (for example, an exon sequence portion located upstream from the target exon / included) up to approximately 100 nucleotides from a 3 'splice site downstream of the target exon / included and / or from approximately 100 nucleotides upstream from the included exon 5' splice site to approximately 100 nucleotides from a target exon splice 5 'downstream of the target / included exon (for example, a portion of the exon sequence located downstream of the target / included exon). For example, a first ASO of 15 nucleotides in length can be designed to hybridize specifically to nucleotides +6 to +20 with respect to the splice site 3 'of the target / included exon. A second ASO can be designed to hybridize specifically to nucleotides +11 to +25 with respect to the splice site 3 'of the target / included exon. ASOs are designed to span the target region of the pre-mRNA. In modalities, ASOs can be covered more closely, for example, every 1, 2, 3 or 4 nucleotides. In addition, ASOs can be covered from 100 nucleotides to a 5 'splice site downstream, up to 100 nucleotides upstream of the 3' splice site. In some modalities, ASOs can be covered from around
[209] [209] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, a sequence that is not expected to hybridize to the target region) are released, for example, by transfection, into a cell line relevant to disease that expresses the target pre-mRNA (for example, a pre-mRNA that contains NIE described in that specification). The exon-skipping effects of each of the ASOs can be assessed by any method known in the art, for example, by (RT) -PCR with reverse transcriptase using primers that span the splice junction, as described in Example 4 A reduction or absence of a longer RT-PCR product produced using primers that span the region containing the included exon (for example, including NIE flanking exons) in cells treated with ASO, when compared to cells treated with
[210] [210] A second round of evaluation, called a "micro-walk" ASO, can be performed using ASOs that have been designed to hybridize to a pre-mRNA target region. The ASOs used in the ASO micro-walk are covered every 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA which, when hybridized to an ASO, results in exon-skipping (or increased NIE splicing).
[211] [211] Regions defined by ASOs that promote splicing of the target intron are explored in more detail by means of a micro-walk ASO, which involves ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18 -25 nt.
[212] [212] As described for the ASO Walk above, the ASO micro-walk is performed by releasing one or more ASOs, or a control ASO (an ASO with a scrambled sequence, a sequence that is not expected to hybridize to the region- target), for example, by transfection, into a disease-relevant cell line that expresses the target pre-mRNA. The splicing-inducing effects of each of the ASOs can be evaluated by any method known in the art, for example, by (RT) -PCR with reverse transcriptase using primers that transpose the NIE, as described in this specification (see , for example, Example 4). A reduction or absence of a longer RT-PCR product produced using primers that transpose the NIE in cells treated with ASO, when compared to cells treated with ASO control, indicates that exon-skipping (or splicing of the target intron) containing an NIE) has been increased. In some embodiments, the efficiency of exon-skipping (or the splicing efficiency for the intron splice that contains the NIE), the ratio of pre-mRNA spliced to unspliced, the rate of splicing, or the extent of splicing can be increased using the ASOs described in this specification. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO has achieved the desired effect (for example, increased production of functional protein). Any method known in the art for assessing and / or quantifying protein production, for example, Western blotting, flow cytometry, immunofluorescence microscopy and ELISA, can be used.
[213] [213] ASOs that, when hybridized to a region of a pre-mRNA, result in exon-skipping (or increased splicing of the intron that contains an NIE) and increased protein production, can be tested in vivo using animal models, for example, models in transgenic mice in which the full-length human gene was knocked-in or in disease models in humanized mice. Appropriate pathways for administering ASOs may vary depending on the disease and / or the types of cells to which the release of ASOs is desired. ASOs can be administered, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection. After administration, the cells, tissues and / or organs of the animals in the model can be evaluated to determine the effect of treatment with ASO, for example, by evaluating the splicing (efficiency, rate, extent) and protein production by methods known in the art and described in that specification. Animal models can also be any phenotypic or behavioral indication of the disease or severity of the disease.
[214] [214] Also within the scope of the present disclosure is a method for identifying or validating an NMD-inducing exon in the presence of an NMD inhibitor, for example, cycloheximide. An exemplary method is provided in Example 2. SPECIFIC MODALITIES
[215] [215] Mode A1. A method of treating Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceroid, neuronal lipofuscinosis, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive intrahepatic family 1; Type II citrullinaemia; Citrullinaemia, Type 1; Cognitive deficit with or without cerebral ataxia; Cornélia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy,
[216] [216] Mode A2. The A1 mode method, where the target protein is ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAHV , FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF1 , RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN.
[217] [217] A3 mode. A method of increasing protein expression ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF3, RAI SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN by a cell that has an mRNA that contains a nonsense mediated RNA decay inducing exon (mDNA of the NMR exon code and NMR code) protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2 HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCA, SCA, SCN SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN, the method comprising the cell contact an agent that binds to a targeted portion of the NMD exon mRNA encoding the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A4, COL4A4 , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC2 , PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN, for which it is an exon excluded from the NMD exon mRNA encoding the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAO GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRP2, RPF, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE 3A or VCAN thereby increasing the level of mRNA encoding the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4,
[218] [218] A4 format. The method of any of modalities A1 to A3, in which the nonsense mediated RNA decay inducing exon is amended by the NMD exon mRNA encoding the target protein or functional RNA.
[219] [219] A5 mode. The method of any of the modalities A1 to A4, in which the target protein does not comprise a sequence of amino acids encoded by the exons inducing RNA decay mediated by nonsense.
[220] [220] Mode A6. The method of any of modalities A1 to A5, wherein the target protein is a full-length target protein.
[221] [221] Mode A7. The method of any of the modalities A1 to A6, in which the agent is an antisense oligomer (ASO) complementary to the target portion of the NMD exon mRNA.
[222] [222] Mode A8. The method of any of the modalities A1 to A7, in which the mRNA is pre-mRNA.
[223] [223] Mode A9. The method of any of the modalities A1 to A8, in which the contact comprises the contact of the therapeutic agent with the mRNA, in which the mRNA is in a cell nucleus.
[224] [224] Mode A10. The method of any of modalities A1 to A9, wherein the target protein or functional RNA corrects a deficiency in the target protein or functional RNA in the individual.
[225] [225] Mode A11. The method of any of modalities A1 to A10, in which the cells are in or are from an individual with a condition caused by a deficient quantity or activity of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MF2 NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTANS, TE3, TS3
[226] [226] Mode A12. The method of any of modalities A1 to A11, in which the deficient amount of the target protein is caused by haploinsufficiency of the target protein, in which the individual has a first allele that encodes a functional target protein, and a second allele by which the target protein is not produced or a second allele that encodes a non-functional target protein, and where the antisense oligomer binds to a targeted portion of the NMD exon mRNA transcribed by the first allele.
[227] [227] Mode A13. The method of any of modalities A1 to A11, in which the individual has a condition caused by a disorder that results from a deficiency in the quantity or function of the target protein, in which the individual has: (a) a first mutant allele by which: (i) the target protein is produced at a reduced level, compared to production by a wild type allele, (ii) the target protein is produced in a form that has reduced function, compared to a protein of the type equivalent wild type, or (iii) the target protein is not produced, and (b) a second mutant allele whereby: (i) the target protein is produced at a reduced level, compared to production by a wild type allele , (ii) the target protein is produced in a form that has reduced function, compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and where when the individual has a first mutant allele (a) (iii), the second mutant allele is (b) (i) or (b) (ii) and where, when the individual has a second mutant allele (b) (iii), the first mutant allele is (a) (i) or (a) (ii), and the NMD exon mRNA is transcribed by the first mutant allele which is (a ) (i) or (a) (ii), and / or the second allele which is (b) (i) or (b) (ii).
[228] [228] Mode A14. The A13 method, in which the target protein is produced in a form that has reduced function, compared to the equivalent wild-type protein.
[229] [229] Mode A15. The A13 modality method, in which the target protein is produced in a form that is fully functional, compared to the equivalent wild-type protein.
[230] [230] A16 mode. The method of any of modalities A1 to A15, in which the target portion of the NMD exon mRNA is within the nonsense mediated RNA decay inducing exon.
[231] [231] Mode A17. The method of any of the modalities A1 to A15, in which the target portion of the NMD exon mRNA is upstream or downstream of the nonsense mediated RNA decay inducing exon.
[232] [232] Mode A18. The method of any of modalities A1 to A17, in which the NMD exon mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for anyone of the IDS. SEQ. Nos: 60-134.
[233] [233] Mode A19. The method of any of modalities A1 to A17, in which the NMD exon mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for IDS. SEQ. Nos: 1-59.
[234] [234] A20 mode. The method of any of the modalities A1 to A17, in which the target portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: IDS. SEQ. Nos: 60-134.
[235] [235] A21 mode. The method of any of modalities A1 to A20, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97% or 100%
[236] [236] Mode A22. The method of any of the modalities A1 to A15, in which the target portion of the NMD exon mRNA is within the ABCB4 nonsense 8x mediated RNA decay indon, ASS1 exon 9x, ATP8B1 exon 16x, exon 16x exon BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, is exon of 4x CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOLL FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of KMT2D, exon 8x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPA1 OPTN, 1x PCCA exon, 5x PCCB exon, 6x exon of PCCB, exon 4x of PKP2, exon 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3A, exon 6x of SCN3A, exon 7x of SCN of SCN8A, exon 6x of SCN8A, exon 20x of SCN8A, exon 6x of SCN9A, exon 24x of SHANK3, exon 3x of SLC25A13, exon 6x of SLC25A13, exon 9x of SLC25A13, exon 11x of SLC25A13, isxonx of SLC6A1, exon 12x of SPTAN1, exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN.
[237] [237] Mode A23. The method of any of modalities A1 to A15, in which the target portion of the NMD exon mRNA is upstream or downstream of the ABCB4 nonsense 8x-mediated RNA decay exon, ASS1 9x exon, ATP8B1 exon 9x , 1x exon of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, 1x exon of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD , exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, ELV 2x , exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D , exon 8x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1 , 1x exon of OPTN, 1x exon of PCCA, 5x exon of PCCB, exon 6x of PCCB, exon 4x of PKP2, exon 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3, exon 6x of SCN3 from SCN3A, exon 4x from SCN8A, exon 6x from SCN8A, exon 20x from SCN8A, exon 6x from SCN9A, exon 24x from SHANK3, exon 3x from SLC25A13, exon 6x from SLC25A13, exon 9x from SLC25A13, exon 11x13 of SLC25A13, exon 1x of SLC6A1, exon 12x of SPTAN1, exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN.
[238] [238] Mode A24. The A1 to A15 method, wherein the target portion of the NMD exon mRNA comprises an exon-exon 8x exon of ABCB4, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of
[239] [239] Mode A25. The method of any of modalities A1 to A24, wherein the target protein produced is full-length protein or wild-type protein.
[240] [240] A26 mode. The method of any of modalities A1 to A25, in which the total amount of mRNA encoding the target protein or functional RNA produced in the cell placed in contact with the antisense oligomer is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times , about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least ce about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to the total amount of mRNA encoding the target protein or functional RNA produced in a control cell.
[241] [241] A27 mode. The method of any of modalities A1 to A25, in which the total amount of mRNA encoding the target protein or functional RNA produced in the cell placed in contact with the antisense oligomer is increased by about 20% to about 300% , about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100% , about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150% , about 50% to about 200%, about 50% to about
[242] [242] A28 mode. The method of any of modalities A1 to A25, in which the total amount of target protein produced by the cell placed in contact with the antisense oligomer is increased by about 1.1 to about 10 times, about 1.5 up to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 up to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times , about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about d and 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to the total amount of target protein produced by the control cell.
[243] [243] Mode A29. The method of any of modalities A1 to A25, in which the total amount of target protein produced by the cell placed in contact with the antisense oligomer is increased by about 20% to about 300%, about 50% to about about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 10%, at least about 20%, at least about 50%, at least about 100 %, at least about 150%, at least about 200%, at least about 250% or at least about 300%, compared to the total amount of target protein produced by the control cell.
[244] [244] A30 mode. The method of any of modalities A1 to 29, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[245] [245] A31 mode. The method of any of the modalities A1 to A30, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a phosphorodiamidate morpholino, a blocked nucleic acid, a peptide nucleic acid, a 2 ' -O-methyl, a 2'-Fluorine or a 2'-O-methoxyethyl moiety.
[246] [246] A32 mode. The method of any of modalities A1 to A31, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar portion.
[247] [247] Mode A33. The A32 method, in which each portion of sugar is a portion of modified sugar.
[248] [248] Mode A34. The method of any of the modalities A1 to A33, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 30 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 15 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[249] [249] A35 mode. The method of any of modalities A1 to A34, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% complementary to the targeted portion of the NMD exon mRNA encoding the protein.
[250] [250] A36 mode. The method of any of the modalities A1 to A35, in which the method still comprises the evaluation of mRNA or protein expression ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, NIPBL, NSD1, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCE
[251] [251] A37 mode. The method of any of the modalities A1 to A36, in which Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceroid, neuronal lipofuscinosis, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive intrahepatic family 1; Type II citrullinaemia; Citrullinaemia, Type 1; Cognitive deficit with or without cerebral ataxia; Cornélia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, arising in childhood; Early childhood epileptic encephalopathy, 11; Early childhood epileptic encephalopathy, 12; Early childhood epileptic encephalopathy, 13; Early childhood epileptic encephalopathy, 2; Episodic ataxia, type 2; Familial focal epilepsy; Febrile seizures, familial, 3B; Friedreich's ataxia; Ataxia of
[252] [252] Mode A38. The method of any of the modalities A1 to A37, in which the individual is a human.
[253] [253] Mode A39. The method of any of modalities A1 to A38, in which the individual is a non-human animal.
[254] [254] A40 mode. The method of any of modalities A1 to A39, in which the individual is a fetus, an embryo or a child.
[255] [255] A41 mode. The method of any of modalities A1 to A40, in which the cells are ex vivo.
[256] [256] A42 mode. The method of any of modalities A1 to A41, in which the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection of the individual.
[257] [257] Mode A43. The method of any of modalities A1 to A42, wherein the method further comprises administering a second therapeutic agent to the individual.
[258] [258] Mode A44. The method of modality A43, in which the second therapeutic agent is a small molecule.
[259] [259] Mode A45. The method of modality A43, in which the second therapeutic agent is an ASO.
[260] [260] A46 mode. The method of any of the modalities A43 to A45, in which the second therapeutic agent corrects intron retention.
[261] [261] Mode A47. An antisense oligomer as used in a method of any of A1 to A46 modalities.
[262] [262] A48 mode. An antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 60-134.
[263] [263] A49 mode. A pharmaceutical composition comprising the antisense oligomer of the A47 or A48 modality and an excipient.
[264] [264] A50 mode. A method of treating an individual in need, which comprises administering the pharmaceutical composition of modality A49 to the individual, in which administration is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.
[265] [265] A51 mode. A composition comprising a therapeutic agent for use in a method of increasing the expression of a target protein or functional RNA by cells to treat Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceroid, neuronal lipofuscinosis, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive intrahepatic family 1; Type II citrullinaemia; Citrullinaemia, Type 1; Cognitive deficit with or without cerebral ataxia; Cornélia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, arising in childhood; Early childhood epileptic encephalopathy, 11;
[266] [266] A52 mode. A composition comprising a therapeutic agent for use in a method of treating a condition associated with the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC, PCP PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN in an individual in need of step expression, method understanding protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRINA, GRE , KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN2A, SCN2A, SCN2A, SCN2A, SCN2A, SCN2A,
[267] [267] Mode A53. The composition of modality A52, in which the condition is a disease or disorder.
[268] [268] A54 mode. The composition of modality A53, in which the disease or disorder is Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary; Arrhythmogenic right ventricular dysplasia 9; Autism spectrum disorder; Cardiomyopathy, dilated, 1HH; Myopathy, myofibrillar 6; Ceroid, neuronal lipofuscinosis, 3; Cholestasis, intrahepatic, of pregnancy, 3; Cholestasis, progressive intrahepatic family 1; Type II citrullinaemia; Citrullinaemia, Type 1; Cognitive deficit with or without cerebral ataxia; Cornélia de Lange; Early-onset epileptic encephalopathy; Epilepsy-aphasia spectrum; Epilepsy, generalized, with febrile seizures plus, type 7; Epileptic encephalopathy, arising in childhood; Early childhood epileptic encephalopathy, 11; Early childhood epileptic encephalopathy, 12; Early childhood epileptic encephalopathy, 13; Early childhood epileptic encephalopathy, 2; Episodic ataxia, type 2; Familial focal epilepsy; Febrile seizures, familial, 3B; Friedreich's ataxia; Friedreich's ataxia with retained reflexes; Galactose epimerase deficiency; Glaucoma 3, primary congenital, E; Glycogen storage disease IV; GRN-related frontotemporal dementia; Homocystinuria, types responsive and unresponsive to B6; HSAN2D, autosomal recessive; Congenital insensitivity to pain; Kabuki syndrome; Koolen-De Vries syndrome; Mental retardation, autosomal dominant 1; Methyl malonic aciduria; Migraine, Myoclonic-atonic epilepsy; family hemiplegic, 1; Neurofibromatosis type 1; Opioid addiction; Optical atrophy type 1; Via (eye); Route (central nervous system, epilepsy); Phelan-McDermid syndrome; Propionic acidosis; Primary open-angle glaucoma; Propionic acidosis; Retinitis pigmentosa 11; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Retinitis pigmentosa 59; Rett syndrome; Convulsions, benign family children, 3; Convulsions, benign family children, 5; Smith-Magenis syndrome; Sotos Syndrome 1; Beckwith-Wiedemann syndrome; Stargardt's disease 3; Tay-Sachs disease; Tuberous sclerosis; Tyrosinemia, type I; Wagner's syndrome 1; West syndrome; Wolfram 2 syndrome / NAFLD; 15q13.3 microdeletion; or 16p11.2 deletion syndrome.
[269] [269] A55 mode. The composition of any of the modalities A52 to 54, wherein the protein and mRNA of the NMD exon of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4 , DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC2 , PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN are encoded by the ABC, AB1, CNA3 gene, ,
[270] [270] A56 mode. The composition of any of the modalities A51 to A55, in which the exons inducing RNA decay mediated by nonsense is spliced by the NMD exon mRNA encoding the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5 protein, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MF2 NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTANS, TE3, TS3
[271] [271] A57 mode. The composition of any of the modalities A51 to A56, wherein the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 , FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PRF31 , RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN do not comprise an amino acid sequence encoded by the RNA exon decay-inducing exon.
[272] [272] A58 mode. The composition of any of the modalities A51 to A57, wherein the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2,
[273] [273] A59 mode. The composition of any of the modalities A51 to A58, wherein the therapeutic agent is an antisense oligomer (ASO) complementary to the target portion of the NMD exon mRNA.
[274] [274] A60 mode. The composition of any of the modalities A51 to A59, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer targets a portion of the NMD exon mRNA that is within the decay inducing exon RNA mediated by nonsense.
[275] [275] A61 mode. The composition of any of the modalities A51 to A59, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer targets a portion of the NMD exon mRNA that is upstream or downstream of the exon nonsense mediated RNA decay inducer.
[276] [276] A62 mode. The composition of any of the modalities A51 to A61, in which the target protein is ABCB4, ASS1,
[277] [277] A63 mode. The A62 modality composition, in which the NMD exon mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for any of the IDS. SEQ. Nos: 60-134.
[278] [278] A64 mode. The composition of modality A62, in which the NMD exon mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for SEQ ID NO: 1-59.
[279] [279] A65 mode. The A62 modality composition, wherein the target portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous nucleic acids of SEQ ID NO: 60-134.
[280] [280] A66 mode. The composition of any of the modalities A62 to A65, in which the target portion of the NMD exon mRNA is within the ABCB4 nonsense 8x mediated RNA decay inductor, ASS1 exon 9x, ATP8B1 exon 16x, 1x exon 8P exon BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, is exon of 4x CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon
[281] [281] A67 mode. The composition of any of the modalities A62 to A65, in which the target portion of the NMD exon mRNA is upstream or downstream of the ABCB4 nonsense 8x mediated RNA decay exon, ASS1 9x exon, ATP8B1 exon 16x , 1x exon of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, 1x exon of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD , exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, ELV 2x , exon 5x of FAH, exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of
[282] [282] A68 mode. The composition of any of the modalities A62 to A65, wherein the target portion of the NMD exon mRNA comprises an exon-intron junction of exon 8x of ABCB4, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 4x of CHRNA7, exon of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon 5x of FAH of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of MAPK3, exon 13x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of
[283] [283] A69 mode. The composition of any of the modalities A62 to A68, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95% , 97% or 100% complementary to a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 60-134.
[284] [284] A70 mode. The composition of any of the modalities A51 to A69, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA or a mature wild-type mRNA.
[285] [285] A71 mode. The composition of any of the modalities A51 to A70, wherein the target protein produced is full-length protein or wild-type protein.
[286] [286] Mode A72. The composition of any of the modalities A51 to A71, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[287] [287] A73 mode. The composition of any of the modalities A51 to A72, in which the therapeutic agent is an antisense oligomer (ASO) and in which said antisense oligomer is an antisense oligonucleotide.
[288] [288] A74 mode. The composition of any of the modalities A51 to A73, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a phosphorodiamidate morpholino, a blocked nucleic acid, a peptide nucleic acid, a 2 '-O-methyl, a 2'-fluorine or a 2'-O-methoxyethyl moiety.
[289] [289] A75 mode. The composition of any of the modalities A51 to A74, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar portion.
[290] [290] A76 mode. The composition of modality A75, in which each portion of sugar is a portion of modified sugar.
[291] [291] A77 mode. The composition of any of the modalities A51 to A76, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases , 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases , 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases , 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases , 12 to
[292] [292] A78 mode. A pharmaceutical composition comprising the therapeutic agent of any of the compositions of modalities A51 to A77, and an excipient.
[293] [293] Mode A79. A method of treating an individual in need, which comprises administering the pharmaceutical composition of modality A78 to the individual, in which administration is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.
[294] [294] A80 mode. The method of any of the modalities A51 to A79, wherein the method further comprises administering a second therapeutic agent to the individual.
[295] [295] A81 mode. The A80 method, in which the second therapeutic agent is a small molecule.
[296] [296] A82 mode. The method of modality A80, in which the second therapeutic agent is an ASO.
[297] [297] A83 mode. The method of any of the modalities A80 to A82, in which the second therapeutic agent corrects intron retention.
[298] [298] Mode A84. A pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3 mRNA transcript COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or RNA
[299] [299] A85 mode. The pharmaceutical composition of modality A84, wherein the mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOLL FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PFB, PRP RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN is an NMD exon NMD mRNA transcript of ABCB4, CBA1, ASS1, BPA CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1,
[300] [300] A86 mode. The pharmaceutical composition of modality A84 or A85, in which the target portion of the NMD exon mRNA is within the ABCB4 nonsense mediated RNA decay inducing exon, ASS1 exon 9x, ATP8B1 exon 16x, BAG3 exon 16x, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7 exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOVL4, is 3x of ELOVL4, and exon 4x of FXN, exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, 1x exon of OPTN, 1x PCCA exon, 5x PCCB exon, 6 exon x from PCCB, exon 4x from PKP2, exon 23x from PLCB1, exon 3x from PRPF3, exon 9x from PRPF31, exon 1x from RAI1, exon 5x from RBFOX2, exon 13x from SCN2A, exon 6x from SCN3A, exon 7 from SCN3A, exon 7 4x of SCN8A, exon 6x of SCN8A, exon 20x of SCN8A, exon 6x of SCN9A, exon 24x of SHANK3, exon 3x of SLC25A13, exon 6x of SLC25A13, exon 9x of SLC25A13, exon 11x of SLC25A13, isxonx 1x of SLC6A1, exon 12x of SPTAN1, exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of
[301] [301] A87 mode. The pharmaceutical composition of the A84 or A85 modality, in which the target portion of the NMD exon mRNA is upstream or downstream of the ABCB4 nonsense 8x mediated RNA decay indon, ASS1 exon 9x, ATP8B1 exon 16x, exon 16 1x BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, is exon 30x of CHD2, is 4x CHRNA7, 1x exon of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOx 5x FAH, exon 4x FXN, exon 4x GALE, exon 3x GBE1, exon 11x GRIN2A, exon 1x GRN, exon 2x HEXA, exon 2x KANSL1, exon 1x KCNQ2, exon 50x KMT2D, exon 8x MAPK3, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, éon 1x of OPTN, exon 1x of PCCA, exon 5 x from PCCB, exon 6x from PCCB, exon 4x from PKP2, exon 23x from PLCB1, exon 3x from PRPF3, exon 9x from PRPF31, exon 1x from RAI1, exon 5x from RBFOX2, exon 13x from SCN2A, exon is 6x from SCN3 7x SCN3A, exon 4x SCN8A, exon 6x SCN8A, exon 20x SCN8A, exon 6x SCN9A, exon 24x SHANK3, exon 3x SLC25A13, exon 6x SLC25A13, exon 9x SLC25A13, isxonx 13x of SLC25A13, exon 1x of SLC6A1, exon 12x of SPTAN1, exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN.
[302] [302] A88 mode. The pharmaceutical composition of modality A84 or A85, wherein the target portion of the NMD exon mRNA comprises an exon-exon 8x exon of ABCB4, exon 9x of ASS1, exon 16x of ATP8B1, exon 1x of BAG3, exon 31x of CACNA1A, exon 36x of CACNA1A, exon 37x of CACNA1A, exon 3x of CBS, exon 12x of CBS, exon 1x of CD55, exon 16x of CDKL5, exon 3x of CFH, exon 30x of CHD2, exon 4x of CHRNA7, exon 1x of CISD2, exon 15x of CLN3, exon 11x of COL4A3, exon 41x of COL4A3, exon 22x of COL4A4, exon 44x of COL4A4, exon 20x of DEPDC5, exon 2x of DHDDS, exon 3x of ELOVL4, exon 5x of FAH, exon exon 4x of GALE, exon 3x of GBE1, exon 11x of GRIN2A, exon 1x of GRN, exon 2x of HEXA, exon 2x of KANSL1, exon 1x of KCNQ2, exon 50x of KMT2D, exon 8x of MAPK3, exon 13x of MBD5, exon 13x of MBD5, exon 2x of MECP2, exon 11x of MUT, exon 31x of NF1, exon 7x of NIPBL, exon 38x of NIPBL, exon 11x of NSD1, exon 6x of OPA1, exon 28x of OPA1, exon 1x of OPTN, exon 1x of PCCA, exon 5x of PCCB, exon 6x of PCCB, exon 4x of PKP2, exo n 23x of PLCB1, exon 3x of PRPF3, exon 9x of PRPF31, exon 1x of RAI1, exon 5x of RBFOX2, exon 13x of SCN2A, exon 6x of SCN3A, exon 7x of SCN3A, exon 4x of SCN8A, exon exon 20x of SCN8A, exon 6x of SCN9A, exon 24x of SHANK3, exon 3x of SLC25A13, exon 6x of SLC25A13, exon 9x of SLC25A13, exon 11x of SLC25A13, exon 13x of SLC25A13, exon 1x of SLC25A13 exon 10x of TEK, exon 15x of TEK, exon 1x of TOPORS, exon 11x of TSC2, exon 30x of TSC2, exon 1x of UBE3A or exon 7x of VCAN.
[303] [303] A89 mode. The pharmaceutical composition of any of the modalities A84 to A88, wherein the NMD exon mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A,
[304] [304] A90 mode. The pharmaceutical composition of modality A84 or A88, wherein the NMD exon mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5 , DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP , PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN comprise a sequence with at least about 90%, 85% 85%, 85% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity for any of the IDS. SEQ. Nos: 60-134.
[305] [305] A91 mode. The pharmaceutical composition of modality A84, wherein the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[306] [306] A92 mode. The pharmaceutical composition of modality A84, in which the antisense oligomer is an antisense oligonucleotide.
[307] [307] A93 mode. The pharmaceutical composition of modality A84, wherein the antisense oligomer comprises a morpholine phosphorodiamidate, a blocked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluorine or a 2'-O- methoxyethyl.
[308] [308] A94 mode. The pharmaceutical composition of modality A84, in which the antisense oligomer comprises at least a portion of modified sugar.
[309] [309] A95 mode. The pharmaceutical composition of modality A84, in which the antisense oligomer comprises 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[310] [310] A96 mode. The pharmaceutical composition of modality A84 or A85, in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or is 100% complementary to a targeted portion of the NMD exon mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOLL , FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF1 , RBFOX2, SCN2A,
[311] [311] A97 mode. The pharmaceutical composition of modality A84 or A85, wherein the target portion of the NMD exon mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPACA, PCTB, PCO, PCTB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN is within a selected IDC sequence. SEQ. Nos: 60-134.
[312] [312] A98 mode. The pharmaceutical composition of modality A84, in which the antisense oligomer comprises a sequence of nucleotides that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% sequence identity for a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 60-
[313] [313] A99 mode. The pharmaceutical composition of modality A84, in which the antisense oligomer comprises a sequence of nucleotides that is identical to a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 60-134.
[314] [314] A100 mode. The pharmaceutical composition of any of the modalities A84 to A99, in which the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection or intravenous injection.
[315] [315] A101 mode. The method of any of the modalities A84 to A100, wherein the method further comprises administering a second therapeutic agent to the individual.
[316] [316] Mode A102. The A101 method, in which the second therapeutic agent is a small molecule.
[317] [317] A103 mode. The method of modality A101, in which the second therapeutic agent is an ASO.
[318] [318] Mode A104. The method of any of the modalities A101 to A103, in which the second therapeutic agent corrects intron retention.
[319] [319] A105 mode. An induction method of processing a deficient mRNA transcript of ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, FAOVL4 , FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF1 , RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN to facilitate the removal of an RNA decay inducing exon mediated by a nonsense mRNA to produce a mRNA ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRINA, GRINA, GRINA KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN2, SCN2 SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN fully pro ceased encoding a functional form of an ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3 protein,
[320] [320] A106 mode. A method of treating an individual who has a condition caused by a deficient amount or activity of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCC, PCP PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN, which understands the administration of an individual to an individual a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 60-134.
[321] [321] A107 mode. A method of treating Alport's Syndrome; Amyotrophic lateral sclerosis (ALS); Angelman syndrome; Aphasia, progressive primary;
[322] [322] A108 mode. A method of increasing the expression of the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF3, RAI SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN by a cell that has an mRNA that contains a nonsense mediated RNA decay inducing exon (mDNA of the NMR exon code and NMR code) protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2 HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF31, RAI1, RBFOX2, SCA, SCA, SCN SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN, the method comprising the contact of the cell with an agent that modulates the splicing of the NMD exon mRNA encoding the ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5 protein , DHDDS, ELOVL4, FAH, FXN, GALE, GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP , PRPF3, PRPF31, RAI1, RBFOX2, SCN2A, SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN, by which the decon-inducing exon is deemed to be a decon- exon NMD mRNA encoding the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4, FAH, FX GBE1, GRIN2A, GRN, HEXA, KANSL1, KCNQ2, KMT2D, MAPK3, MBD5, MECP2, MUT, NF1, NIPBL, NSD1, OPA1, OPTN, PCCA, PCCB, PKP2, PLCB1, PRPF3, PRPF3, RAI SCN3A, SCN8A, SCN9A, SHANK3, SLC25A13, SLC6A1, SPTAN1, TEK, TOPORS, TSC2, UBE3A or VCAN thereby increasing the level of mRNA encoding the protein ABCB4, ASS1, ATP8B1, BAG3, CACNA1A, CBS, CD55, CDKL5, CFH, CHD2, CHRNA7, CISD2, CLN3, COL4A3, COL4A4, DEPDC5, DHDDS, ELOVL4 FAH, FXN, GALE, GBE1,
[323] [323] A109 mode. The A107 or A108 method, wherein the agent: (a) binds to a targeted portion of the NMD exon mRNA that encodes the target protein or functional RNA; (b) binds to one or more components of a spliceosome; or (c) a combination of (a) and (b). ADDITIONAL SPECIFIC MODALITIES
[324] [324] Modality 1. A method of modulating the expression of a target protein by a cell that has an mRNA that comprises a nonsense mediated RNA decay inducing exon (NMD exon) and encodes the target protein, the method comprising the contact of a therapeutic agent with the cell, whereby the therapeutic agent modulates the splicing of the NMD exon of the mRNA, thereby modulating the level of processed mRNA that encodes the target protein, and modulating the expression of the target protein in the cell , in which the target protein is selected from the group consisting of: AKT3 proteins,
[325] [325] Modality 2. A method of treating a disease or condition in an individual in need by modulating the expression of a target protein in an individual's cell, comprising: contact of the individual's cell with a therapeutic agent that modulates the splicing of a nonsense-mediated decay-inducing mRNA exon (NMD exon) by an mRNA in the cell, where the mRNA comprises NMD exon and encodes the target protein, thereby modulating the level of processed mRNA that encodes the protein- target, and modulating the expression of the target protein in the individual's cell, where the target protein is selected from the group consisting of: AKT3, CACNA1A, CBS, CD46, CFH, CHD2, CLN3, COL11A2, COL4A3, COL4A4 proteins, COL4A4, CR1, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERN1, GALE, GUCY2F, GUCY2F, HEXA, HEXA, MAPK3, MBD5, MBD5, MBD5, MUT, MYH14, MYO6, NF1, NSF2, NF2, NF2, NF2 NSD1, NSD1, NSD1, OPA1, OPA1, PCCA, PKP2, PPARA, PRPF3, PRPF3, SCN2A, SCN8A, SCN8A, SCN9A, SEMA3C, SEMA3D, SIRT3, STK11, S TK11, SYNGAP1, TOPORS and VCAN.
[326] [326] Mode 3. The method of mode 1 or 2, in which the therapeutic agent: (a) binds to a targeted portion of the mRNA that encodes the target protein; (b) modulates a bond of a factor involved in splicing the NMD exon; or (c) the combination of (a) and (b).
[327] [327] Mode 4. The method of mode 3, in which the therapeutic agent interferes with a binding of the factor involved in splicing the NMD exon to a region of the target portion.
[328] [328] Mode 5. The method of mode 3 or 4, where the target portion is proximal to the NMD exon.
[329] [329] Modality 6. The method of any of modalities 3 to 5, in which the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream 5 'end of the NMD exon.
[330] [330] Modality 7. The method of any of modalities 3 to 6, in which the target portion is at least about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides , about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the 5 'end of the NMD exon.
[331] [331] Modality 8. The method of any of modalities 3 to 5, in which the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of 3 'end of the NMD exon.
[332] [332] Mode 9. The method of any of modes 3 to 5 or 8, in which the target portion is at least about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide downstream of the 3 'end of the NMD exon.
[333] [333] Mode 10. The method of any one of modalities 3 to 5, in which the target portion is, at most, about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564388; GRCh38 / hg38: chr19 13236618; GRCh38 / hg38: chr21 43060012; GRCh38 / hg38: chr1 207775610; GRCh38 / hg38: chr1 196675450; GRCh38 / hg38: chr15 92998149; GRCh38 / hg38: chr16 28479765; GRCh38 / hg38: chr6 33183698; GRCh38 / hg38: chr2 227296487; GRCh38 / hg38: chr2 227144833; GRCh38 / hg38: chr2 227015360; GRCh38 / hg38: chr1 207637688; GRCh38 / hg38: chr19 47835403; GRCh38 / hg38: chr1 59904516; GRCh38 / hg38: chr1 26442335; GRCh38 / hg38: chr1 28230252; GRCh38 / hg38: chr2 88582824; GRCh38 / hg38: chr17 64102804; GRCh38 / hg38: chr1 23798484; GRCh38 / hg38: chrX 109383446; GRCh38 / hg38: chrX 109439175; GRCh38 / hg38: chr15 72362466; GRCh38 / hg38: chr15 72345776; GRCh38 / hg38: chr16 30115645; GRCh38 / hg38: chr2 148460219; GRCh38 / hg38: chr2 148490695; GRCh38 / hg38: chr2 148505761; GRCh38 / hg38: chr6 49436597; GRCh38 / hg38: chr19 50230825; GRCh38 / hg38: chr6 75867431; GRCh38 / hg38: chr17 31249955; GRCh38 / hg38: chr22 29628658; GRCh38 / hg38: chr5 37048127; GRCh38 / hg38: chr12 100499841; GRCh38 / hg38: chr5 177169394; GRCh38 / hg38: chr5 177200761; GRCh38 / hg38: chr5 177247924; GRCh38 / hg38: chr5 177275947; GRCh38 / hg38: chr3 193628509; GRCh38 / hg38: chr3 193603500; GRCh38 / hg38: chr13 100305751; GRCh38 / hg38: chr12 32894778; GRCh38 / hg38: chr22 46203575; GRCh38 / hg38: chr1 150327557; GRCh38 / hg38: chr1 150330401; GRCh38 / hg38: chr2 165327155; GRCh38 / hg38: chr12 51688758; GRCh38 / hg38: chr12 51780202; GRCh38 / hg38: chr2
[334] [334] Mode 11. The method of any of modes 3 to 5 or 10, in which the target portion is about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564388; GRCh38 / hg38: chr19 13236618; GRCh38 / hg38: chr21 43060012; GRCh38 / hg38: chr1 207775610; GRCh38 / hg38: chr1 196675450; GRCh38 / hg38: chr15 92998149; GRCh38 / hg38: chr16 28479765; GRCh38 / hg38: chr6 33183698; GRCh38 / hg38: chr2 227296487; GRCh38 / hg38: chr2 227144833; GRCh38 / hg38: chr2 227015360; GRCh38 / hg38: chr1 207637688; GRCh38 / hg38: chr19 47835403; GRCh38 / hg38: chr1 59904516; GRCh38 / hg38: chr1 26442335; GRCh38 / hg38: chr1 28230252; GRCh38 / hg38: chr2 88582824; GRCh38 / hg38: chr17 64102804; GRCh38 / hg38: chr1 23798484; GRCh38 / hg38: chrX 109383446; GRCh38 / hg38: chrX 109439175; GRCh38 / hg38: chr15 72362466; GRCh38 / hg38: chr15 72345776; GRCh38 / hg38: chr16 30115645; GRCh38 / hg38: chr2 148460219; GRCh38 / hg38: chr2 148490695; GRCh38 / hg38: chr2 148505761; GRCh38 / hg38: chr6 49436597; GRCh38 / hg38: chr19 50230825; GRCh38 / hg38: chr6 75867431; GRCh38 / hg38: chr17
[335] [335] Modality 12. The method of any of modalities 3 to 5, in which the target portion is at most about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564285; GRCh38 / hg38: chr19 13236449; GRCh38 / hg38: chr21 43059730; GRCh38 / hg38: chr1 207775745; GRCh38 / hg38: chr1 196675529; GRCh38 / hg38: chr15 92998261; GRCh38 / hg38: chr16 28479644; GRCh38 / hg38: chr6 33183634; GRCh38 / hg38: chr2 227296526; GRCh38 / hg38: chr2 227144653; GRCh38 / hg38: chr2 227015283; GRCh38 / hg38: chr1 207637848; GRCh38 / hg38:
[336] [336] Mode 13. The method of any of modes 3 to 5 or 12, in which the target portion is about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the genomic site selected from the group consisting of: GRCh38 / hg38: chr1 243564285; GRCh38 / hg38: chr19 13236449; GRCh38 / hg38: chr21 43059730; GRCh38 / hg38: chr1 207775745; GRCh38 / hg38: chr1 196675529; GRCh38 / hg38: chr15 92998261; GRCh38 / hg38: chr16 28479644; GRCh38 / hg38: chr6 33183634; GRCh38 / hg38: chr2 227296526; GRCh38 / hg38: chr2 227144653; GRCh38 / hg38: chr2 227015283; GRCh38 / hg38: chr1 207637848; GRCh38 / hg38: chr19 47835579; GRCh38 / hg38: chr1 59904366; GRCh38 / hg38: chr1 26442372; GRCh38 / hg38: chr1 28230131; GRCh38 / hg38: chr2 88582755; GRCh38 / hg38: chr17 64102673; GRCh38 / hg38: chr1 23798311; GRCh38 / hg38: chrX 109383365; GRCh38 / hg38: chrX 109439038; GRCh38 / hg38: chr15 72362376; GRCh38 / hg38: chr15 72345677; GRCh38 / hg38: chr16 30115595; GRCh38 / hg38: chr2 148460304; GRCh38 / hg38: chr2 148490787; GRCh38 / hg38: chr2 148505830; GRCh38 / hg38: chr6 49436522; GRCh38 / hg38: chr19 50230999; GRCh38 / hg38: chr6 75867523; GRCh38 / hg38: chr17 31250125; GRCh38 / hg38: chr22 29628773; GRCh38 / hg38: chr5 37048354; GRCh38 / hg38: chr12 100500024; GRCh38 / hg38: chr5 177169559; GRCh38 / hg38: chr5 177200783; GRCh38 / hg38: chr5 177248079; GRCh38 / hg38: chr5 177276101; GRCh38 / hg38: chr3 193628616; GRCh38 / hg38: chr3 193603557; GRCh38 / hg38: chr13 100305834; GRCh38 / hg38: chr12 32894516; GRCh38 / hg38: chr22 46203752; GRCh38 / hg38: chr1 150327652; GRCh38 / hg38: chr1 150330498; GRCh38 / hg38: chr2 165327202; GRCh38 / hg38: chr12 51688849; GRCh38 / hg38: chr12 51780271; GRCh38 / hg38: chr2 166304238; GRCh38 / hg38: chr7 80794854; GRCh38 / hg38: chr7 85059498; GRCh38 / hg38: chr11 225673; GRCh38 / hg38: chr19 1216398; GRCh38 / hg38: chr19 1221846; GRCh38 / hg38: chr6
[337] [337] Modality 14. The method of any of modalities 3 to 13, in which the target portion is located in an intronic region between two canonical exonic regions of the mRNA encoding the target protein, and in which the intronic region contains the NMD exon.
[338] [338] Mode 15. The method of any of modes 3 to 14, in which the target portion overlaps at least partially with the NMD exon.
[339] [339] Mode 16. The method of any of modes 3 to 15, in which the target portion overlaps at least partially with an intron upstream or downstream of the NMD exon.
[340] [340] Mode 17. The method of any of modes 3 to 16, wherein the target portion comprises NMD exon junction - 5 'intron or NMD exon 3' junction.
[341] [341] Mode 18. The method of any of modes 3 to 16, in which the target portion is within the NMD exon.
[342] [342] Mode 19. The method of any of modes 1 to 18, wherein the target portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive NMD exon nucleotides.
[343] [343] Mode 20. The method of any of modalities 1 to 19, in which the mRNA encoding the target protein comprises a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a selected sequence from the group consisting of IDS. IN
[344] [344] Mode 21. The method of any of modalities 1 through 20, in which the mRNA encoding the target protein is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a selected sequence from the group consisting of IDS. SEQ. Nos: 1-5, 12, 19-21, 25, 26, 28, 30, 33, 35, 38, 40, 41, 44, 45, 51, 53, 55-57 and 192-211.
[345] [345] Mode 22. The method of any one of modalities 3 to 21, in which the target portion of the mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% identity sequence for a region comprising at least 8 contiguous nucleic acids from a sequence selected from the group consisting of IDS. SEQ. Nos: 135-
[346] [346] Mode 23. The method of any of modes 1 to 22, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to at least 8 contiguous nucleic acids from a sequence selected from the group consisting of IDS. SEQ. Nos: 135-191.
[347] [347] Modality 24. The method of any of modalities 3 to 23, in which the target portion of the mRNA is within the exon inducing RNA mediated by nonsense selected from the group consisting of: GRCh38 / hg38: chr1 243564285 243564388 ; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261;
[348] [348] Modality 25. The method of any of modalities 3 to 23, in which the target portion of the mRNA is upstream or downstream of the exon inducing RNA mediated by nonsense selected from the group consisting of: GRCh38 / hg38 : chr1 243564285 243564388; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261; GRCh38 / hg38: chr16 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698; GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chr1 207637688 207637848; GRCh38 / hg38: chr19 47835403 47835579; GRCh38 / hg38: chr1 59904366 59904516; GRCh38 / hg38: chr1 26442335 26442372; GRCh38 / hg38: chr1 28230131 28230252; GRCh38 / hg38: chr2 88582755 88582824; GRCh38 / hg38: chr17 64102673 64102804; GRCh38 / hg38: chr1 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chr15 72362376 72362466; GRCh38 / hg38: chr15 72345677 72345776; GRCh38 / hg38: chr16 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chr19 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chr17 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354;
[349] [349] Modality 26. The method of any of modalities 3 to 23, wherein the target portion of the mRNA comprises an exon-intron junction of the exon selected from the group consisting of: GRCh38 / hg38: chr1 243564285 243564388; GRCh38 / hg38: chr19 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chr1 207775610 207775745; GRCh38 / hg38: chr1 196675450 196675529; GRCh38 / hg38: chr15 92998149 92998261; GRCh38 / hg38: chr16 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698; GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chr1 207637688 207637848; GRCh38 / hg38: chr19 47835403 47835579; GRCh38 / hg38: chr1 59904366 59904516; GRCh38 / hg38: chr1 26442335 26442372; GRCh38 / hg38: chr1 28230131 28230252;
[350] [350] Modality 27. The method of any of modalities 1 to 26, wherein the target protein produced is a full-length protein or a wild-type protein.
[351] [351] Mode 28. The method of any of modes 1 to 27, in which the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA encoding the target protein.
[352] [352] Mode 29. The method of mode 28, in which exclusion of the NMD exon from the processed mRNA that encodes the target protein in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 up to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times, at least about 1.5 times, eg it at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times compared to excluding the NMD exon from the processed mRNA encoding the target protein in a control cell.
[353] [353] Mode 30. The method of mode 28 or 29, in which the therapeutic agent increases the level of the processed mRNA that encodes the target protein in the cell.
[354] [354] Mode 31. The method of any of modes 28 to 30, in which the level of processed mRNA that encodes the target protein produced in the cell brought into contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 up to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times , about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to a level of the processed mRNA that encodes the target protein in a control cell.
[355] [355] Mode 32. The method of any of the modes 28 to 31, in which the therapeutic agent increases the expression of the target protein in the cell.
[356] [356] Mode 33. The method of any of modes 28 to 32, in which a level of the target protein produced in the cell brought into contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times , about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least c about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times , compared to a level of the target protein produced in a control cell.
[357] [357] Mode 34. The method of any of modes 2 to 33, in which the disease or condition is induced by a loss-of-function mutation in the target protein.
[358] [358] Modality 35. The method of modality 34, in which the disease or condition is associated with haploinsufficiency of a gene that encodes the target protein, and in which the individual has a first allele that encodes a functional target protein, and a second allele by which the target protein is not produced or is produced at a reduced level, or a second allele that encodes a non-functional target protein or a partially functional target protein.
[359] [359] Modality 36. The method of any of modalities 2 to 35, in which the disease or condition is selected from the group consisting of: Sotos Syndrome 1; Beckwith-Wiedemann syndrome; Migraine, family hemiplegic, 1; Episodic ataxia, type 2; Epileptic encephalopathy, arising in childhood; Wagner's syndrome 1; Optical atrophy type 1; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Neurofibromatosis type 1; Early childhood epileptic encephalopathy, 11; Convulsions, benign family children, 3; Cognitive deficit with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Convulsions, benign family children, 5; Via (SNC); 16p11.2 deletion syndrome; Mental retardation, autosomal dominant 1; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Deafness, autosomal dominant 13; Retinal dystrophy of rod cones-2; Deafness, autosomal dominant 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Deafness, autosomal dominant 22; Neurofibromatosis type 2; Mental retardation, autosomal dominant 5; Epilepsy, generalized, with febrile seizures plus, type 7; and Febrile seizures, family, 3B.
[360] [360] Modality 37. The method of any of modalities 2 to 36, in which the disease or condition is associated with an autosomal recessive mutation of a gene encoding the target protein, in which the individual has a first coding allele, whereby: (i) the target protein is not produced or is produced at a reduced level, compared to a wild-type allele; or (ii) the target protein produced is non-functional or partially functional, compared to a wild-type allele, and a second allele whereby: (iii) the target protein is produced at a reduced level, compared to an allele of the wild type and the target protein produced is at least partially functional, compared to a wild type allele; or (iv) the target protein produced is partially functional, compared to a wild type allele.
[361] [361] Mode 38. The method of mode 37, in which the disease or condition is selected from the group consisting of: Alport syndrome; Ceroid, neuronal lipofuscinosis, 3; Galactose epimerase deficiency; Homocystinuria, types responsive and unresponsive to B6; Methyl malonic aciduria; Propionic acidosis; Retinitis pigmentosa 59; Tay-Sachs disease; Congenital insensitivity to pain; and HSAN2D, autosomal recessive.
[362] [362] Mode 39. The method of any of modes 34 to 39, in which the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA that encodes the target protein and increases the expression of the target protein in the cell.
[363] [363] Mode 40. The method of any of modalities 1 to 27, in which the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA encoding the target protein.
[364] [364] Mode 41. The method of mode 40, in which exclusion of the NMD exon from the processed mRNA that encodes the target protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 up to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times, at least about 1.5 times, eg it at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times compared to excluding the NMD exon from the processed mRNA encoding the target protein in a control cell.
[365] [365] Mode 42. The method of mode 40 or 41, in which the therapeutic agent decreases the level of the processed mRNA that encodes the target protein in the cell.
[366] [366] Mode 43. The method of any of modes 40 to 42, in which the level of processed mRNA that encodes the target protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1 , 5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to a level of the processed mRNA that encodes the target protein in a control cell.
[367] [367] Mode 44. The method of any of modes 40 to 43, in which the therapeutic agent decreases the expression of the target protein in the cell.
[368] [368] Mode 45. The method of any of modes 40 to 44, in which a level of the target protein produced in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times , about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least c about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times , compared to a level of the target protein produced in a control cell.
[369] [369] Mode 46. The method of either mode 2 to 27 or 40 to 45, in which the disease or condition is induced by a gain-of-function mutation in the target protein.
[370] [370] Mode 47. The method of mode 46, in which the individual has an allele by which the target protein is produced at an increased level or an allele that encodes a mutant target protein that exhibits increased activity in the cell.
[371] [371] Mode 48. The method of mode 46 or 47, in which the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA encoding the target protein and decreases the expression of the target protein in the cell.
[372] [372] Mode 49. The method of mode 40, wherein the target protein comprises SCN8A.
[373] [373] Mode 50. The method of mode 49, wherein the disease or condition comprises a disease of the central nervous system.
[374] [374] Mode 51. The method of mode 50, in which the disease or condition comprises epilepsy.
[375] [375] Mode 52. The method of mode 51, in which the disease or condition comprises Dravet's Syndrome.
[376] [376] Mode 53. The method of any of modes 1 to 52, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[377] [377] Modality 54. The method of any of modalities 1 to 53, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluorine or a 2'-O-methoxyethyl moiety.
[378] [378] Mode 55. The method of any of modes 1 to 54, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar portion.
[379] [379] Mode 56. The method of mode 55, in which each portion of sugar is a portion of modified sugar.
[380] [380] Modality 57. The method of any of modalities 1 to 56, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to
[381] [381] Mode 58. The method of any of modalities 3 to 57, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% complementary to the target portion of the mRNA.
[382] [382] Modality 59. The method of any of the modalities 1 to 58, wherein the method still comprises the evaluation of the level of mRNA or the level of expression of the target protein.
[383] [383] Mode 60. The method of any one of modalities 1 to 59, in which the individual is a human.
[384] [384] Mode 61. The method of any of modes 1 to 59, in which the individual is a non-human animal.
[385] [385] Mode 62. The method of any of modes 2 to 60, in which the individual is a fetus, embryo or child.
[386] [386] Mode 63. The method of any of modalities 1 to 62, in which the cells are ex vivo.
[387] [387] Mode 64. The method of any of modes 2 to 62, in which the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal or intravenous injection to the individual.
[388] [388] Mode 65. The method of any of modes 2 to 62 or 64, wherein the method still comprises the administration of a second therapeutic agent to the individual.
[389] [389] Mode 66. The method of any of modes 1 to 65, in which the second therapeutic agent is a small molecule.
[390] [390] Mode 67. The method of any of modalities 1 to 65, in which the second therapeutic agent is an antisense oligomer.
[391] [391] Mode 68. The method of any of the modalities 1 to 67, in which the second therapeutic agent corrects intron retention.
[392] [392] Mode 69. The method of any of modes 2 to 68, in which the disease or condition is selected from the group consisting of: 16p11.2 deletion syndrome; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Ceroid, neuronal lipofuscinosis, 3; Cognitive deficit with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Convulsions, benign family children, 5; Retinal dystrophy of cones-rods-2; Cornélia de Lange; Deafness, autosomal dominant 13; Deafness, autosomal dominant 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Epilepsy, generalized, with febrile seizures plus, type 7; Febrile seizures, familial, 3B; Congenital insensitivity to pain; HSAN2D, autosomal recessive; Epileptic encephalopathy, arising in childhood; Early childhood epileptic encephalopathy, 11; Convulsions, benign family children, 3;
[393] [393] The present disclosure will be more specifically illustrated by the following Examples. However, it must be understood that the present disclosure is not limited by these examples in any way. Example 1: Identification of NMD-inducing exon inclusion events in RNAseq transcripts using Next Generation Sequencing
[394] [394] Shotgun sequencing of the entire transcriptome is performed using new generation sequencing to reveal a snapshot of transcripts produced by the genes described in this specification to identify NIE inclusion events. For this purpose, polyA + RNA from human cell nuclear and cytoplasmic fractions is isolated and cDNA libraries are constructed using the “Illumina’s TruSeq Stranded mRNA Library Prep Kit”. The libraries were sequenced pair-end resulting in readings of 100 nucleotides that are mapped to the human genome (February 2009, assembly GRCh37 / hg19). FIGS. 2-58 depict the identification of different exons inducing mRNA (NMD) decay mediated by exemplary nonsense in various genes.
[395] [395] Exemplary intron genes and sequences are summarized in Table 1 and Table 2 (IDS. SEQ. Nos. Indicate the corresponding nucleotide sequences represented by the Gen. ID. Nos.). The sequence for each intron is summarized in Table 3 and Table 4.
[396] [396] RT-PCR analysis using cytoplasmic RNA from human cells treated with DMSO or treated with puromycin or cycloheximide and exon primers can confirm the presence of a band that corresponds to an NMD-inducing exon. The identity of the product is confirmed by sequencing. The densitometry analysis of the bands is performed to calculate the percentage of inclusion of the NMD exon of total transcript. The treatment of cells with cycloheximide or puromycin to inhibit NMD may lead to an increase in the product that corresponds to the NMD-inducing exon in the cytoplasmic fraction. FIGS. 59, 62, 65, 69, 72 and 75 depict confirmation of exemplary NIE exons in various gene transcripts using treatment with cycloheximide or puromycin, respectively.
[397] [397] An ASO Walk is performed for the NMD exon region that targets sequences immediately upstream of the 3 'splice site, through the 3' splice site, the NMD exon, through the 5 'splice site, and downstream splice site using 2Os-MOE ASOs, PS framework. ASOs are designed to cover these regions by changing 5 nucleotides at once. FIGS. 60, 63, 66, 70, 73 and 76 depict ASO Walk for several exemplary NIE exon regions, respectively. Example 4: ASO Walk of NMD exon region evaluated by RT-PCR
[398] [398] ASO Walk sequences can be evaluated, for example, by RT-PCR. PAGE can be used to display SYBR-Safe stained RT-PCR products simulated (Simulated), treated with control SMN ASO (SMN) or treated with a 2ʹ-MOE ASO that targets NMD exon regions as described in that specification at a concentration of 20 μM in human cells by gymnotic uptake. Products that correspond to the inclusion of the NMD exon and of full length are quantified and the percentage of inclusion of the NMD exon is plotted. Full-length products can be standardized for internal RPL32 control and the rate of change compared to the Simulated can be plotted. FIGS. 71 and 78 depict the RT-PCR evaluation of several exemplary ASO Walks throughout exemplary NIE exon regions, respectively. Example 5: ASO Walk of NMD exon region evaluated by RT-qPCR.
[399] [399] Amplification results by RT-qPCR-Verde-SYBR normalized to RPL32 can be obtained using the same ASO capture experiment that can be evaluated by RT-PCR-SYBR-Safe, and can be plotted as a reason for change in Simulated to confirm the results of RT-PCR-SYBR-Safe. FIGS. 61, 64, 67, 68, 71, 74, 77 and 78 depict the evaluation using RT-qPCR of several exemplary ASO Walks throughout exemplary NIE exon regions, respectively. Example 6: Dose-dependent effect of selected ASOs in cells treated with CXH.
[400] [400] PAGE can be used to display SYBR-Safe stained RT-PCR products treated in a simulated manner (Simulated, RNAiMAX alone) or treated with 2ʹ-MOE ASOs that target NMD exons at concentrations of 30 nM, 80 nM and 200 nM in mouse or human cells by transfection of RNAiMAX. Products that correspond to the inclusion of NMD exon and of full length are quantified and the percentage of inclusion of NMD exon can be plotted. Full-length products can also be standardized for internal HPRT control and the rate of change from the Simulated can be plotted. Example 7: Intravitreal injection (IVT) of selected ASOs
[401] [401] PAGEs of SY-Safe stained RT-PCR products from mice injected with PBS (1 μl) (-) or ASOs or Cep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac. ., 2015) injected with ASO of 2ʹ-MOE (1 μl) (+) at a concentration of 10 mM. Products that correspond to the inclusion of the NMD exon and of full length (are quantified and the percentage of inclusion of the NMD exon can be plotted. Products of full length can be normalized for GAPDH internal control and ratio of change of injected with ASO in relation to those injected with PBS can be plotted Example 8: Intracerebroventricular injection (ICV) of selected ASOs.
[402] [402] PAGEs of RT-PCR products stained with SYBR-Safe from uninjected brain mice (- without ASO control) or 300 μg of Cep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac., 2015), injected with ASO 2ʹ-MOE. Products that correspond to the inclusion of NMD exon and of full length can be quantified and the percentage of inclusion of NMD exon can be plotted. Taqman-PCR can be performed using two different probes that span the NMD exon junctions and the products can be normalized for internal control of GAPDH and ratio of change in brains injected with ASO in relation to brains injected with Cep290 can be plotted.
[403] [403] Although preferred modalities of the present disclosure have been shown and described in this specification, it will be obvious to those skilled in the art that these modalities are provided as an example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from disclosure. It should be understood that several alternatives to the disclosure modalities described in this specification can be used in the disclosure practice. The following claims are intended to define the scope of the disclosure and that the methods and structures within the scope of those claims and their equivalents are covered by it.

权利要求:
Claims (69)
[1]
1. Method of modulating the expression of a target protein by a cell that has an mRNA that comprises an RNA-mediated decay-inducing exon (NMD exon) and encodes the target protein, the method characterized by understanding the contact of a therapeutic agent in the cell, by which the therapeutic agent modulates the splicing of the NMD exon of the mRNA, thus modulating the level of processed mRNA that encodes the target protein and modulates the expression of the target protein in the cell, where the target protein is selected from the group consisting of in: AKT3, CACNA1A, CBS, CD46, CFH, CHD2, CLN3, COL11A2, COL4A3, COL4A4, COL4A4, CRl, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERNl, GALE, GUCY2F, GUCYA, HEXA MBD5, MBD5, MUT, MYH14, MY06, NF1, NF2, NIPBL, NR1H4, NSD 1, NSD1, NSD 1, NSD1, OPA1, OPA1, PCCA, PKP2, PPARA, PRPF3, PRPF3, SCN2A, SCN8A, SCNA SCN9A, SEMA3C, SEMA3D, SIRT3, STK11, STK11, SYNGAP1, TOPORS and VCAN.
[2]
2. Method to treat a disease or condition in an individual in need of it, modulating the expression of a target protein in a subject's cell, characterized by understanding: the contact of the subject's cell with a therapeutic agent that modulates the splicing of a non-sense exon mediated mRNA decay inducer (NMD exon) of a mRNA in the cell, where the mRNA comprises the NMD exon and encodes the target protein, thereby modulating the level of processed mRNA encoding the target protein and modulating expression of the target protein in the subject's cell, where the target protein is selected from the group consisting of: AKT3, CACNA1A, CBS, CD46, CFH, CHD2, CLN3, COL1 1A2, COL4A3, COL4A4, COL4A4, CRl, CRX, CYP2J2, DHDDS, DNAJC8, EIF2AK3, ERNl, GALE, GUCY2F,
GUCY2F, HEXA, HEXA, MAPK3, MBD5, MBD5, MBD5, MUT, MYH14, MY06, N06, NF1, NF2, NIPBL, NR1H4, NSD1, NSD1, NSD1, NSD1, OPA1, OPA1, PCCA, PKP2, PRARA , PRPF3, SCN2A, SCN8A, SCN8A, SCN9A, SEMA3C, SEMA3D, SIRT3, STK11, STK11, SYNGAP1, TOPORS and VCAN.
[3]
Method according to claim 1 or 2, characterized in that the therapeutic agent (a) binds to a target portion of the mRNA encoding the target protein; (b) modulates the binding of a factor involved in the NMD exon bond; or (c) a combination of (a) and (b).
[4]
4. Method according to claim 3, characterized by the fact that the therapeutic agent interferes with the binding of the factor involved in the splicing of the NMD exon to a region of the target portion.
[5]
5. Method according to claim 3, characterized by the fact that the target portion is proximal to the NMD exon.
[6]
6. Method according to claim 5, characterized in that the target portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the 5 'end of the NMD exon .
[7]
7. Method according to claim 5, characterized in that the target portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, over 1 nucleotides upstream of the 5 'end of the NMD exon.
[8]
8. Method according to claim 5, characterized in that the target portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the 3 'end of the NMD exon .
[9]
9. Method according to claim 5, characterized in that the target portion is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, over 1 nucleotides downstream of the 3 'end of the NMD exon.
[10]
Method according to claim 5, characterized in that the target portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream from the genomic site selected from the group that consists of: GRCh38 / hg38: chrl 243564388; GRCh38 / hg38: chrl9 13236618; GRCh38 / hg38: chr21 43060012; GRCh38 / hg38: chrl 207775610; GRCh38 / hg38: chrl 196675450; GRCh38 / hg38: chrl5 92998149; GRCh38 / hg38: chrl6 28479765; GRCh38 / hg38: chr6 33183698; GRCh38 / hg38: chr2 227296487; GRCh38 / hg38: chr2 227144833; GRCh38 / hg38: chr2 227015360; GRCh38 / hg38: chrl 207637688; GRCh38 / hg38: chrl9 47835403; GRCh38 / hg38: chrl 59904516; GRCh38 / hg38: chrl 26442335; GRCh38 / hg38: chrl 28230252; GRCh38 / hg38: chr288582824; GRCh38 / hg38: chrl764102804; GRCh38 / hg38: chrl 23798484; GRCh38 / hg38: chrX 109383446; GRCh38 / hg38: chrX 109439175; GRCh38 / hg38: chrl572362466; GRCh38 / hg38: chrl572345776; GRCh38 / hg38: chrl630115645; GRCh38 / hg38: chr2148460219; GRCh38 / hg38: chr2148490695; GRCh38 / hg38: chr2148505761; GRCh38 / hg38: chr649436597; GRCh38
/ hg38: chrl950230825; GRCh38 / hg38: chr675867431; GRCh38 / hg38: chrl731249955; GRCh38 / hg38: chr2229628658; GRCh38 / hg38: chr537048127; GRCh38 / hg38: chrl2100499841; GRCh38 / hg38: chr5177169394; GRCh38 / hg38: chr5177200761; GRCh38 / hg38: chr5177247924; GRCh38 / hg38: chr5177275947; GRCh38 / hg38: chr3193628509; GRCh38 / hg38: chr3193603500; GRCh38 / hg38: chrl3100305751; GRCh38 / hg38: chrl232894778; GRCh38 / hg38: chr2246203575; GRCh38 / hg38: chrl 150327557; GRCh38 / hg38: chrl 150330401; GRCh38 / hg38: chr2165327155; GRCh38 / hg38: chrl251688758; GRCh38 / hg38: chrl251780202; GRCh38 / hg38: chr2166304329; GRCh38 / hg38: chr780794957; GRCh38 / hg38: chr785059541; GRCh38 / hg38: chrll 226081; GRCh38 / hg38: chrl91216268; GRCh38 / hg38: chrl91221621; GRCh38 / hg38: chr633448789; GRCh38 / hg38: chr932551469; and GRCh38 / hg38: chr583544965.
[11]
11. Method according to claim 5, characterized in that the target portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides , about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site selected from the group consisting of in: GRCh38 / hg38: chrl 243564388; GRCh38 / hg38: chrl913236618; GRCh38 / hg38: chr2143060012; GRCh38 / hg38: chrl 207775610; GRCh38 / hg38: chrl 196675450; GRCh38 / hg38: chrl592998149; GRCh38 / hg38: chrl628479765; GRCh38 / hg38: chr633183698; GRCh38 / hg38: chr2227296487; GRCh38 / hg38: chr2227144833; GRCh38
/ hg38: chr2227015360; GRCh38 / hg38: chrl 207637688; GRCh38 / hg38: chrl947835403; GRCh38 / hg38: chrl 59904516; GRCh38 / hg38: chrl 26442335; GRCh38 / hg38: chrl 28230252; GRCh38 / hg38: chr288582824; GRCh38 / hg38: chrl764102804; GRCh38 / hg38: chrl 23798484; GRCh38 / hg38: chrX 109383446; GRCh38 / hg38: chrX 109439175; GRCh38 / hg38: chrl572362466; GRCh38 / hg38: chrl572345776; GRCh38 / hg38: chrl630115645; GRCh38 / hg38: chr2148460219; GRCh38 / hg38: chr2148490695; GRCh38 / hg38: chr2148505761; GRCh38 / hg38: chr649436597; GRCh38 / hg38: chrl950230825; GRCh38 / hg38: chr675867431; GRCh38 / hg38: chrl731249955; GRCh38 / hg38: chr2229628658; GRCh38 / hg38: chr537048127; GRCh38 / hg38: chrl2100499841; GRCh38 / hg38: chr5177169394; GRCh38 / hg38: chr5177200761; GRCh38 / hg38: chr5177247924; GRCh38 / hg38: chr5177275947; GRCh38 / hg38: chr3193628509; GRCh38 / hg38: chr3193603500; GRCh38 / hg38: chrl3100305751; GRCh38 / hg38: chrl232894778; GRCh38 / hg38: chr2246203575; GRCh38 / hg38: chrl 150327557; GRCh38 / hg38: chrl 150330401; GRCh38 / hg38: chr2165327155; GRCh38 / hg38: chrl251688758; GRCh38 / hg38: chrl251780202; GRCh38 / hg38: chr2166304329; GRCh38 / hg38: chr780794957; GRCh38 / hg38: chr785059541; GRCh38 / hg38: chrll 226081; GRCh38 / hg38: chrl91216268; GRCh38 / hg38: chrl91221621; GRCh38 / hg38: chr633448789; GRCh38 / hg38: chr932551469; and GRCh38 / hg38: chr583544965.
[12]
12. Method, according to claim 5, characterized by the fact that the target portion is at most about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream from the genomic site selected from the group that consists of: GRCh38 / hg38: chrl 243564285; GRCh38 / hg38: chrl913236449; GRCh38 / hg38: chr2143059730; GRCh38 / hg38: chrl 207775745; GRCh38 / hg38: chrl 196675529; GRCh38 / hg38: chrl592998261; GRCh38 / hg38: chrl628479644; GRCh38 / hg38: chr633183634; GRCh38 / hg38: chr2227296526; GRCh38 / hg38: chr2227144653; GRCh38 / hg38: chr2227015283; GRCh38 / hg38: chrl 207637848; GRCh38 / hg38: chrl947835579; GRCh38 / hg38: chrl 59904366; GRCh38 / hg38: chrl 26442372; GRCh38 / hg38: chrl 28230131; GRCh38 / hg38: chr288582755; GRCh38 / hg38: chrl764102673; GRCh38 / hg38: chrl 23798311; GRCh38 / hg38: chrX 109383365; GRCh38 / hg38: chrX 109439038; GRCh38 / hg38: chrl572362376; GRCh38 / hg38: chrl572345677; GRCh38 / hg38: chrl630115595; GRCh38 / hg38: chr2148460304; GRCh38 / hg38: chr2148490787; GRCh38 / hg38: chr2148505830; GRCh38 / hg38: chr649436522; GRCh38 / hg38: chrl950230999; GRCh38 / hg38: chr675867523; GRCh38 / hg38: chrl731250125; GRCh38 / hg38: chr2229628773; GRCh38 / hg38: chr537048354; GRCh38 / hg38: chrl2100500024; GRCh38 / hg38: chr5177169559; GRCh38 / hg38: chr5177200783; GRCh38 / hg38: chr5177248079; GRCh38 / hg38: chr5177276101; GRCh38 / hg38: chr3193628616; GRCh38 / hg38: chr3193603557; GRCh38 / hg38: chrl3100305834; GRCh38 / hg38: chrl232894516; GRCh38 / hg38: chr2246203752; GRCh38 / hg38: chrl 150327652; GRCh38 / hg38: chrl 150330498; GRCh38 / hg38: chr2165327202; GRCh38 / hg38: chrl251688849; GRCh38 / hg38: chrl251780271; GRCh38 / hg38: chr2166304238; GRCh38 / hg38: chr780794854; GRCh38 / hg38: chr785059498; GRCh38 / hg38: chrll 225673; GRCh38 /
hg38: chrl91216398; GRCh38 / hg38: chrl91221846; GRCh38 / hg38: chr633448868; GRCh38 / hg38: chr932551365; and GRCh38 / hg38: chr583545070.
[13]
13. Method according to claim 5, characterized in that the target portion is about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides , about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream from the genomic site selected from the group consisting of in: GRCh38 / hg38: chrl 243564285; GRCh38 / hg38: chrl913236449; GRCh38 / hg38: chr2143059730; GRCh38 / hg38: chrl 207775745; GRCh38 / hg38: chrl 196675529; GRCh38 / hg38: chrl592998261; GRCh38 / hg38: chrl628479644; GRCh38 / hg38: chr633183634; GRCh38 / hg38: chr2227296526; GRCh38 / hg38: chr2227144653; GRCh38 / hg38: chr2227015283; GRCh38 / hg38: chrl 207637848; GRCh38 / hg38: chrl947835579; GRCh38 / hg38: chrl 59904366; GRCh38 / hg38: chrl 26442372; GRCh38 / hg38: chrl 28230131; GRCh38 / hg38: chr288582755; GRCh38 / hg38: chrl764102673; GRCh38 / hg38: chrl 23798311; GRCh38 / hg38: chrX 109383365; GRCh38 / hg38: chrX 109439038; GRCh38 / hg38: chrl572362376; GRCh38 / hg38: chrl572345677; GRCh38 / hg38: chrl630115595; GRCh38 / hg38: chr2148460304; GRCh38 / hg38: chr2148490787; GRCh38 / hg38: chr2148505830; GRCh38 / hg38: chr649436522; GRCh38 / hg38: chrl950230999; GRCh38 / hg38: chr675867523; GRCh38 / hg38: chrl731250125; GRCh38 / hg38: chr2229628773; GRCh38 / hg38: chr537048354; GRCh38 / hg38: chrl2100500024; GRCh38
/ hg38: chr5177169559; GRCh38 / hg38: chr5177200783; GRCh38 / hg38: chr5177248079; GRCh38 / hg38: chr5177276101; GRCh38 / hg38: chr3193628616; GRCh38 / hg38: chr3193603557; GRCh38 / hg38: chrl3100305834; GRCh38 / hg38: chrl232894516; GRCh38 / hg38: chr2246203752; GRCh38 / hg38: chrl 150327652; GRCh38 / hg38: chrl 150330498; GRCh38 / hg38: chr2165327202; GRCh38 / hg38: chrl251688849; GRCh38 / hg38: chrl251780271; GRCh38 / hg38: chr2166304238; GRCh38 / hg38: chr780794854; GRCh38 / hg38: chr785059498; GRCh38 / hg38: chrll 225673; GRCh38 / hg38: chrl91216398; GRCh38 / hg38: chrl91221846; GRCh38 / hg38: chr633448868; GRCh38 / hg38: chr932551365; and GRCh38 / hg38: chr583545070. GRCh38 / hg38: chr932551365; and GRCh38 / hg38: chr583545070. GRCh38 / hg38: chr932551365; and GRCh38 / hg38: chr583545070.
[14]
14. Method, according to claim 3, characterized by the fact that the target portion is located in an intronic region between two canonic exonic regions of the mRNA that encodes the target protein and in which the intronic region contains the NMD exon.
[15]
15. Method according to claim 3, characterized in that the target portion overlaps at least partially with the NMD exon.
[16]
16. Method according to claim 3, characterized in that the target portion overlaps at least partially with an intron upstream or downstream of the NMD exon.
[17]
17. Method according to claim 3, characterized in that the target portion comprises the 5 'NMD exon-intron junction or 5' NMD exon-intron junction.
[18]
18. Method according to claim 3,
characterized by the fact that the target portion is within the NMD exon.
[19]
19. Method according to claim 3, characterized in that the directed portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive nucleotides of the NMD exon.
[20]
20. Method according to claim 1 or 2, characterized in that the mRNA encoding the target protein comprises a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% of sequence identity for a sequence selected from the group consisting of SEQ ID NOs: 135-191.
[21]
21. Method according to claim 1 or 2, characterized by the fact that the mRNA encoding the target protein is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity of a sequence selected from the group consisting of SEQ ID NOs: 1-5, 12, 19-21, 25, 26, 28, 30, 33, 35, 38, 40, 41, 44, 45, 51, 53, 55-57, and 192-211.
[22]
22. Method according to claim 3, characterized in that the target portion of the mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 acidic contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs: 135-191.
[23]
23. Method according to claim 1 or 2, characterized by the fact that the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to at least 8 contiguous nucleic acids in a sequence selected from the group consisting of SEQ ID NOs: 135-191.
[24]
24. Method, according to claim 3, characterized by the fact that the targeted portion of the mRNA is within the non-selected sense mediated decay inducer selected from the group consisting of: GRCh38 / hg38: chrl 243564285 243564388; GRCh38 / hg38: chrl9 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chrl 207775610 207775745; GRCh38 / hg38: chrl 196675450 196675529; GRCh38 / hg38: chrl5 92998149 92998261; GRCh38 / hg38: chrl6 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698; GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chrl 207637688 207637848; GRCh38 / hg38: chrl9 47835403 47835579; GRCh38 / hg38: chrl 59904366 59904516; GRCh38 / hg38: chrl 26442335 26442372; GRCh38 / hg38: chrl 28230131 28230252; GRCh38 / hg38: chr2 88582755 88582824; GRCh38 / hg38: chrl7 64102673 64102804; GRCh38 / hg38: chrl 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127
37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chrl 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chrl 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5
177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509
193628616; chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2
100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; GRCh38 / hg38: chr3193603500193603557; GRCh38 / hg38: chrl3100305751100305834; GRCh38 / hg38: chrl23289451632894778; GRCh38 / hg38: chr224620357546203752; GRCh38 / hg38: chrl 150327557150327652; GRCh38 / hg38: chrl 150330401150330498; GRCh38 / hg38: chr2165327155 165327202; GRCh38 / hg38: chrl25168875851688849; GRCh38 / hg38: chrl25178020251780271; GRCh38 / hg38: chr2166304238166304329; GRCh38 / hg38: chr78079485480794957; GRCh38 / hg38: chr78505949885059541; GRCh38 / hg38: chrll
225673226081; GRCh38 / hg38: chrl9 12162681216398; GRCh38 / hg38: chrl912216211221846; GRCh38 / hg38: chr63344878933448868; GRCh38 / hg38: chr93255136532551469; and GRCh38 / hg38: chr58354496583545070.
[25]
25. Method according to claim 3, characterized by the fact that the target portion of the mRNA is upstream or downstream of the senseless mediated RNA decay inducer selected from the group consisting of: GRCh38 / hg38: chrl 243564285243564388 ; GRCh38 / hg38: chrl91323644913236618; GRCh38 / hg38: chr21 4305973043060012; GRCh38 / hg38: chrl 207775610207775745; GRCh38 / hg38: chrl 196675450 196675529; GRCh38 / hg38: chrl59299814992998261; GRCh38 / hg38: chrl62847964428479765; GRCh38 / hg38: chr63318363433183698; GRCh38 / hg38: chr2227296487227296526; GRCh38 / hg38: chr2227144653227144833; GRCh38 / hg38: chr2227015283227015360; GRCh38 / hg38: chrl 207637688207637848; GRCh38 / hg38: chrl94783540347835579; GRCh38 / hg38: chrl 59904366 59904516; GRCh38 / hg38: chrl 2644233526442372; GRCh38 / hg38: chrl 2823013128230252; GRCh38 / hg38: chr28858275588582824; GRCh38 / hg38: chrl76410267364102804; GRCh38 / hg38: chrl 2379831123798484; GRCh38 / hg38: chrX 109383365109383446; GRCh38 / hg38: chrX 109439038109439175; GRCh38 / hg38: chrl57236237672362466; GRCh38 / hg38: chrl572345677 72345776; GRCh38 / hg38: chrl63011559530115645; GRCh38 / hg38: chr2148460219148460304; GRCh38 / hg38: chr2148490695148490787; GRCh38 / hg38: chr2148505761148505830; GRCh38 / hg38: chr64943652249436597; GRCh38 / hg38: chrl95023082550230999; GRCh38 / hg38: chr6
7586743175867523; GRCh38 / hg38: chrl73124995531250125; GRCh38 / hg38: chr2229628658 29628773; GRCh38 / hg38: chr53704812737048354; GRCh38 / hg38: chrl2100499841100500024; GRCh38 / hg38: chr5177169394177169559; GRCh38 / hg38: chr5177200761177200783; GRCh38 / hg38: chr5177247924177248079; GRCh38 / hg38: chr5177275947177276101; GRCh38 / hg38: chr3 193628509193628616; GRCh38 / hg38: chr3193603500193603557; GRCh38 / hg38: chrl3100305751 100305834; GRCh38 / hg38: chrl23289451632894778; GRCh38 / hg38: chr224620357546203752; GRCh38 / hg38: chrl 150327557150327652; GRCh38 / hg38: chrl 150330401150330498; GRCh38 / hg38: chr2165327155165327202; GRCh38 / hg38: chrl25168875851688849; GRCh38 / hg38: chrl2 5178020251780271; GRCh38 / hg38: chr2166304238166304329; GRCh38 / hg38: chr780794854 80794957; GRCh38 / hg38: chr78505949885059541; GRCh38 / hg38: chrll 225673226081; GRCh38 / hg38: chrl912162681216398; GRCh38 / hg38: chrl912216211221846; GRCh38 / hg38: chr6 3344878933448868; GRCh38 / hg38: chr93255136532551469; and GRCh38 / hg38: chr583544965 83545070.
[26]
26. Method according to claim 3, characterized in that the target portion of the mRNA comprises an exon-intron junction of the exon selected from the group consisting of: GRCh38 / hg38: chrl 243564285 243564388; GRCh38 / hg38: chrl9 13236449 13236618; GRCh38 / hg38: chr21 43059730 43060012; GRCh38 / hg38: chrl 207775610 207775745; GRCh38 / hg38: chrl 196675450 196675529; GRCh38 / hg38: chrl5 92998149 92998261; GRCh38 / hg38: chrl6 28479644 28479765; GRCh38 / hg38: chr6 33183634 33183698;
GRCh38 / hg38: chr2 227296487 227296526; GRCh38 / hg38: chr2 227144653 227144833; GRCh38 / hg38: chr2 227015283 227015360; GRCh38 / hg38: chrl 207637688 207637848; GRCh38 / hg38: chrl9 47835403 47835579; GRCh38 / hg38: chrl 59904366 59904516; GRCh38 / hg38: chrl 26442335 26442372; GRCh38 / hg38: chrl 28230131 28230252; GRCh38 / hg38: chr2 88582755 88582824; GRCh38 / hg38: chrl7 64102673 64102804; GRCh38 / hg38: chrl 23798311 23798484; GRCh38 / hg38: chrX 109383365 109383446; GRCh38 / hg38: chrX 109439038 109439175; GRCh38 / hg38: chrl5 72362376 72362466; GRCh38 / hg38: chrl5 72345677 72345776; GRCh38 / hg38: chrl6 30115595 30115645; GRCh38 / hg38: chr2 148460219 148460304; GRCh38 / hg38: chr2 148490695 148490787; GRCh38 / hg38: chr2 148505761 148505830; GRCh38 / hg38: chr6 49436522 49436597; GRCh38 / hg38: chrl9 50230825 50230999; GRCh38 / hg38: chr6 75867431 75867523; GRCh38 / hg38: chrl7 31249955 31250125; GRCh38 / hg38: chr22 29628658 29628773; GRCh38 / hg38: chr5 37048127 37048354; GRCh38 / hg38: chrl2 100499841 100500024; GRCh38 / hg38: chr5 177169394 177169559; GRCh38 / hg38: chr5 177200761 177200783; GRCh38 / hg38: chr5 177247924 177248079; GRCh38 / hg38: chr5 177275947 177276101; GRCh38 / hg38: chr3 193628509 193628616; GRCh38 / hg38: chr3 193603500 193603557; GRCh38 / hg38: chrl3 100305751 100305834; GRCh38 / hg38: chrl2 32894516 32894778; GRCh38 / hg38: chr22 46203575 46203752; GRCh38 / hg38: chrl 150327557 150327652; GRCh38 / hg38: chrl 150330401 150330498; GRCh38 / hg38: chr2 165327155 165327202; GRCh38 / hg38: chrl2 51688758 51688849; GRCh38 / hg38: chrl2 51780202 51780271; GRCh38 / hg38: chr2 166304238 166304329; GRCh38 / hg38: chr7 80794854 80794957; GRCh38 / hg38: chr7 85059498 85059541;
GRCh38 / hg38: chapter 225673 226081; GRCh38 / hg38: chrl9 1216268 1216398; GRCh38 / hg38: chrl9 1221621 1221846; GRCh38 / hg38: chr6 33448789 33448868; GRCh38 / hg38: chr9 32551365 32551469; and GRCh38 / hg38: chr5 83544965 83545070. GRCh38 / hg38: chrl 150330401 150330498; GRCh38 / hg38: chr2 165327155 165327202; GRCh38 / hg38: chrl2 51688758 51688849; GRCh38 / hg38: chrl2 51780202 51780271; GRCh38 / hg38: chr2 166304238 166304329; GRCh38 / hg38: chr7 80794854 80794957; GRCh38 / hg38: chr7 85059498 85059541; GRCh38 / hg38: chapter 225673 226081; GRCh38 / hg38: chrl9 1216268 1216398; GRCh38 / hg38: chrl9 1221621 1221846; GRCh38 / hg38: chr6 33448789 33448868; GRCh38 / hg38: chr9 32551365 32551469; and GRCh38 / hg38: chr5 83544965 83545070. GRCh38 / hg38: chrl 150330401 150330498; GRCh38 / hg38: chr2 165327155 165327202; GRCh38 / hg38: chrl2 51688758 51688849; GRCh38 / hg38: chrl2 51780202 51780271; GRCh38 / hg38: chr2 166304238 166304329; GRCh38 / hg38: chr7 80794854 80794957; GRCh38 / hg38: chr7 85059498 85059541; GRCh38 / hg38: chapter 225673 226081; GRCh38 / hg38: chrl9 1216268 1216398; GRCh38 / hg38: chrl9 1221621 1221846; GRCh38 / hg38: chr6 33448789 33448868; GRCh38 / hg38: chr9 32551365 32551469; and GRCh38 / hg38: chr5 83544965 83545070. chrl9 1221621 1221846; GRCh38 / hg38: chr6 33448789 33448868; GRCh38 / hg38: chr9 32551365 32551469; and GRCh38 / hg38: chr5 83544965 83545070. chrl9 1221621 1221846; GRCh38 / hg38: chr6 33448789 33448868; GRCh38 / hg38: chr9 32551365 32551469; and GRCh38 / hg38: chr5 83544965 83545070.
[27]
27. Method according to claim 1 or 2, characterized in that the target protein produced is a complete protein or a wild-type protein.
[28]
28. Method according to claim 1 or 2,
characterized by the fact that the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA that encodes the target protein.
[29]
29. Method according to claim 28, characterized in that the exclusion of the NMD exon from the processed mRNA encoding the target protein in the cell in contact with the therapeutic agent is increased by about 1.1 to about 10 times , about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to the exclusion of the NMD exon from the processed mRNA that encodes the target protein in a control cell.
[30]
30. Method according to claim 28, characterized in that the therapeutic agent increases the level of the processed mRNA that encodes the target protein in the cell.
[31]
31. The method of claim 28,
characterized by the fact that the level of processed mRNA that encodes the target protein produced in the cell in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times , about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to a level of the processed mRNA that encodes the target protein in a control cell.
[32]
32. Method according to claim 28, characterized in that the therapeutic agent increases the expression of the target protein in the cell.
[33]
33. Method according to claim 28, characterized in that a level of the target protein produced in the cell in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times,
about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to a level of the target protein produced in a cell control.
[34]
34. Method according to claim 2, characterized by the fact that the disease or condition is induced by a loss of function mutation in the target protein.
[35]
35. Method according to claim 34, characterized by the fact that the disease or condition is associated with the haploinsufficiency of a gene that encodes the target protein and in which the subject has a first allele that encodes a functional target protein and a second an allele from which the target protein is not produced or produced at a reduced level, or a second allele that encodes a non-functional target protein or a partially functional target protein.
[36]
36. Method, according to claim 35, characterized by the fact that the disease or condition is selected from the group consisting of: Sotos syndrome 1;
Beckwith-Wiedemann syndrome; Familial hemiplegic migraine, 1; Episodic ataxia, type 2; Epileptic encephalopathy initiated in childhood; Wagner's syndrome 1; Optical atrophy type 1; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Neurofibromatosis type 1; Early childhood epileptic encephalopathy, 11; Benign familial infantile seizures, 3; Cognitive impairment with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Benign familial infantile seizures, 5; Path (CNS); 16pl deletion syndrome ; Autosomal dominant mental retardation 1; Retinitis pigmentosa 18; Retinitis pigmentosa 31; Autosomal dominant deafness 13; Retinal dystrophy-2 of the cone; Autosomal dominant deafness 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Autosomal dominant deafness 22; Neurofibromatosis type 2; Autosomal dominant mental retardation 5; Generalized epilepsy, with more febrile seizures, type 7; and febrile, familial convulsions, 3B.
[37]
37. Method, according to claim 34, characterized by the fact that the disease or condition is associated with an autosomal recessive mutation of a gene that encodes the target protein, in which the subject has a first allele that encodes from which : (i) the target protein is not produced or produced at a reduced level compared to a wild type allele; or (ii) the target protein produced is not functional or partially functional compared to a wild-type allele, and a second allele from which:
(iii) the target protein is produced at a reduced level compared to a wild type allele and the target protein produced is at least partially functional compared to a wild type allele; or (iv) the target protein produced is partially functional compared to a wild type allele.
[38]
38. Method, according to claim 37, characterized by the fact that the disease or condition is selected from the group consisting of: Alport's syndrome; Ceroid, neuronal lipofuscinosis, 3; Galactose epimerase deficiency; Homocystinuria, types responsive to B6 and unresponsive; Malonic Methyl Aciduria; Propionic acidemia; Retinitis pigmentosa 59; Tay-Sachs disease; Insensitivity to congenital pain; and HSAN2D, autosomal recessive.
[39]
39. Method according to claim 34, characterized in that the therapeutic agent promotes the exclusion of the NMD exon from the processed mRNA that encodes the target protein and increases the expression of the target protein in the cell.
[40]
40. Method according to claim 1 or 2, characterized in that the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA encoding the target protein.
[41]
41. Method according to claim 40, characterized in that the exclusion of the NMD exon from the processed mRNA that encodes the target protein in the cell in contact with the therapeutic agent is reduced by about 1.1 to about 10 times , about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to 8 times, about 3 to 9 times, about 4 to 7 times, about 4 to 8 times, about 4 to 9 times, at least 1.1 times, at least at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to excluding NMD exon from mRNA processed protein that encodes the target protein in a control cell.
[42]
42. Method according to claim 40, characterized in that the therapeutic agent decreases the level of the processed mRNA that encodes the target protein in the cell.
[43]
43. Method according to claim 40, characterized in that the level of the processed mRNA that encodes the target protein in the cell in contact with the therapeutic agent is reduced by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1 , 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8-fold, about 1.1 to about 9 times, about 2 to about 5 times , about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 fold, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about about 2.5 times, at least about 3-fold, at least about 3.5 times, at least about 4 times, at least about 5 times or at least about 10 times, compared to a level of the processed mRNA encoding the target protein in a control cell.
[44]
44. Method according to claim 40, characterized in that the therapeutic agent decreases the expression of the target protein in the cell.
[45]
45. Method according to claim 40, characterized in that a level of the target protein produced in the cell in contact with the therapeutic agent is reduced by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9-fold, at least about 1.1-fold, at least about 1.5 times, at least about 2 times, at least about 2.5 times, p at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to a level of the target protein produced in a cell of control.
[46]
46. Method according to claim 2, characterized by the fact that the disease or condition is induced by a gain-of-function mutation in the target protein
[47]
47. Method according to claim 46, characterized in that the subject has an allele from which the target protein is produced at an increased level or an allele that encodes a mutant target protein that exhibits increased activity in the cell.
[48]
48. Method according to claim 46, characterized in that the therapeutic agent inhibits the exclusion of the NMD exon from the processed mRNA encoding the target protein and decreases the expression of the target protein in the cell.
[49]
49. Method according to claim 40, characterized in that the target protein comprises SCN8A.
[50]
50. Method according to claim 49, characterized in that the disease or condition comprises a disease of the central nervous system.
[51]
51. Method according to claim 50, characterized by the fact that the disease or condition comprises epilepsy.
[52]
52. Method according to claim 51, characterized by the fact that the disease or condition comprises Dravet's syndrome.
[53]
53. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a skeleton modification comprising a phosphorothioate bond or a phosphorodiamidate bond.
[54]
54. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-fluoro or a 2'-0-methoxyethyl moiety.
[55]
55. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar fraction.
[56]
56. Method according to claim 55, characterized in that each sugar portion is a modified sugar portion.
[57]
57. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases , 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases , 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases , 11 to 35 nucleobases, 11 to 30 nucleobases,
11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 25 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[58]
58. Method according to claim 3, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%, complement the target portion of the mRA.
[59]
59. Method according to claim 1 or 2, characterized in that the method further comprises assessing the level of mRNA or the level of expression of the target protein.
[60]
60. Method, according to claim 2, characterized by the fact that the subject is a human.
[61]
61. Method according to claim 2, characterized by the fact that the subject is a non-human animal.
[62]
62. Method, according to claim 2, characterized by the fact that the subject is a fetus, an embryo or a child.
[63]
63. Method according to claim 1 or 2, characterized by the fact that the cells are ex vivo.
[64]
64. Method, according to claim 2, characterized by the fact that the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal or intravenous injection of the subject.
[65]
65. Method, according to claim 2, characterized by the fact that the method further comprises administering a second therapeutic agent to the subject.
[66]
66. Method according to claim 65, characterized in that the second therapeutic agent is a small molecule.
[67]
67. Method according to claim 65, characterized in that the second therapeutic agent is an antisense oligomer.
[68]
68. Method according to claim 65, characterized in that the second therapeutic agent corrects intron retention.
[69]
69. Method, according to claim 2, characterized by the fact that the disease or condition is selected from the group consisting of: 16pl 1.2 exclusion syndrome; Alport's syndrome; Arrhythmogenic right ventricular dysplasia 9; Ceroid, neuronal lipofuscinosis, 3; Cognitive impairment with or without cerebellar ataxia; Early childhood epileptic encephalopathy, 13; Benign familial infantile seizures, 5; Retinal dystrophy-2 of the cone; Cornelia de Lange; Autosomal dominant deafness 13; Autosomal dominant deafness 4A; Peripheral neuropathy, myopathy, hoarseness and hearing loss; Generalized epilepsy, with more febrile seizures, type 7; Familial febrile seizures 3B; Insensitivity to congenital pain; HSAN2D, autosomal recessive; Epileptic encephalopathy initiated in childhood; Early childhood epileptic encephalopathy, 1 1; Benign familial infantile seizures, 3; Galactose epimerase deficiency; Homocystinuria, types responsive to B6 and unresponsive; Autosomal dominant mental retardation 1; Autosomal dominant mental retardation 5; Malonic Methyl Aciduria; Familial hemiplegic migraine, 1; Episodic ataxia, type 2; NASH; Neurofibromatosis type 1; Neurofibromatosis type 2; Optical atrophy type 1; Propionic acidemia; Retinitis pigmentosa 18; Sotos Syndrome 1; Beckwith-Wiedemann syndrome; Tay-Sachs disease; and Wagner's syndrome 1.

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法律状态:
2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762575924P| true| 2017-10-23|2017-10-23|

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US62/575,924|2017-10-23|

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US201862667200P| true| 2018-05-04|2018-05-04|

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PCT/US2018/057165|WO2019084050A1|2017-10-23|2018-10-23|Antisense oligomers for treatment of non-sense mediated rna decay based conditions and diseases|

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