Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/33—Alteration of splicing

Definitions

• liver disease is a debilitating and often fatal group of conditions with an estimated mortality rate of 80%.
• the liver is vital for many functions in the body including, but not limited to clearing the blood of harmful toxins, storing and releasing glucose, production of bile, storage of iron and aiding resistance to infections. Dysfunction of the liver ultimately leads to failure of other major organs and ultimately death. While liver transplantation can often prevent mortality, the odds of receiving a donor liver is typically low.
• liver diseases and conditions associated with the liver While there are a large number of diseases and conditions associated with the liver, a subset of liver diseases have been shown to proceed via a deficiency in the expression of a gene, and in turn, a deficiency in the gene product.
• gene products for which increased expression can provide benefit in liver diseases or conditions include AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5.
• ASO antisense oligomer
• the liver disease is glycine encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine Deaminase deficiency, porphyria variegate, porphyria cutanea tarda, acute intermittent porphyria, very long chain acyl-CoA dehydrogenase deficiency, pyruvate carboxylase deficiency, isovaleric academia, hyperchylomicronemia, hypertriglyceridemia, galactosemia,
• hypercholesterolemia maturity-onset diabetes of the young type 1, maturity-onset diabetes of the young type 2, maturity-onset diabetes of the young type 3, noninsulin-dependent diabetes mellitus, insulin- dependent diabetes mellitus 1, insulin-dependent diabetes mellitus 20, Falconi renotubular syndrome 4 with maturity-onset diabetes of the young, hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome, immunodeficiency 36, agammaglobulinemia 7, lipid metabolism deficiency, liver inflammation, hemochromatosis type 2B, thrombocytopenia, non-alcoholic fatty liver disease, Wilson disease, tyrosinemia type I,
• argininosuccinate lyase deficiency hemochromatosis type I, Alstrom syndrome, congenital bile acid synthesis defect 1, steatohepatitis, insulin resistance, glucose intolerance, type II diabetes or liver cancer.
• a method of increasing expression of a target protein by cells having a retained-intron-containing pre-mRNA comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein
• the method comprising contacting the cells with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA encoding the target protein, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein, thereby increasing the level of mRNA encoding the target protein, and increasing the expression of the target protein in the cells, wherein the target protein is aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase,
• uridylyltransf erase low density lipoprotein receptor adaptor protein 1
• hepatocyte nuclear factor 4-alpha glucokinase
• hepatic nuclear factor- 1 -alpha albulim proximal factor O-glucosyl transferase 1
• phosphatidylinositol 3-kinase regulatory subunit 1 Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary
• hemochromatosis protein alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator activated receptor delta, interleukin 6, ceramide synthase 2 or nuclear receptor coactivator 5.
• the target protein is aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-
• V galactose- 1 -phosphate uridylyl transferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O-glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary hemochromatosis protein, alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator activated receptor delta, interleukin 6, ceramide synthase 2 or nuclear receptor coactivator 5.
• the target protein or the functional RNA is a compensating protein or a compensating functional RNA that functionally augments or replaces a target protein or functional RNA that is deficient in amount or activity in the subject.
• the cells are in or from a subject having a condition caused by a deficient amount or activity of the target protein.
• the deficient amount of the target protein is caused by haploinsufficiency of the target protein, wherein the subject has a first allele encoding a functional target protein, and a second allele from which the target protein is not produced, or a second allele encoding a nonfunctional target protein, and wherein the antisense oligomer binds to a targeted portion of a RIC pre-mRNA transcribed from the first allele.
• the subject has a condition caused by a disorder resulting from a deficiency in the amount or function of the target protein, wherein the subject has a first mutant allele from which the target protein is produced at a reduced level compared to production from a wild-type allele, the target protein is produced in a form having reduced function compared to an equivalent wild- type protein, or the target protein is not produced, and a second mutant allele from which the target protein is produced at a reduced level compared to production from a wild-type allele, the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or the target protein is not produced, and wherein when the subject has a first mutant allele a(iii), the second mutant allele is b(i) or b(ii), and wherein when the subject has a second mutant allele b(iii), the first mutant allele is a(i) or a(ii), and wherein the RIC pre-mRNA is
• the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.
• the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.
• the targeted portion of the RIC pre-mRNA is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
• the targeted portion of the RIC pre-mRNA is in the retained intron within the region +69 relative to the 5' splice site of the retained intron to -79 relative to the 3' splice site of the retained intron.
• the targeted portion of the RIC pre-mRNA is in the retained intron within: the region +6 to +100 relative to the 5' splice site of the retained intron; or the region -16 to -100 relative to the 3 ' splice site of the retained intron.
• the targeted portion of the RIC pre-mRNA is within: the region +2e to -4e in the exon flanking the 5 ' splice site of the retained intron; or the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
• the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 500 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 500 nucleotides upstream of the 3' splice site of the at least one retained intron.
• the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron.
• the target protein is (a) AMT, (b) ADA, (c) PPOX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i) APOA5, (j) GALT, (k) LDLRAP1, (1) HNF4A, (m) GCK, (n) POGLUT1, (o) PIK3R1, (p) HNF1A, (q) TRIB1, (r) TGFB1, (s) HAMP, (t) THPO, (u) PNPLA3, (v) ATP7B, (w) FAH, (x) ASL, (y) HFE, (z) ALMS1, (aa) PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.
• the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to (a) any one of SEQ ID NOs 2813- 3910, (b) any one of SEQ ID NOs 65847-67281, (c) any one of SEQ ID NOs 1900-2599, (d) any one of SEQ ID NOs 926-1779, (e) any one of SEQ ID NOs 37483-38044, (f) any one of SEQ ID NOs 49423- 49969, (g) any one of SEQ ID NOs 35900-36292, (h) any one of SEQ ID NOs 44626-47013, (l) any one of SEQ ID NOs 36293-37482, (j) any one of SEQ ID NOs 34521-35899, (k) any one of SEQ ID NOs 131-925, (1) any one of SEQ ID NOs 58958 - 65846 or 675
• the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of (a) SEQ ID NO 78483, 78465 or 78434; (b) SEQ ID NO 78385, 78482 or 78369; (c) SEQ ID NO 78461, 78473, 78480 or 78421; (d) SEQ ID NO 78410, 78386, 78411, 78460 or 78463; (e) SEQ ID NO 78355, 78467 or 78454; (f) SEQ ID NO 78367, 78376, 78440, 78448, 78477, 78485, 78496, 78422 or 78412; (g) SEQ ID NO 78495, (h) SEQ ID NO 78464, 78375 or 78380; (l) SEQ ID NO 78407, 78407, 78407, 784
• the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to (a) any one of SEQ ID NOs 2813-3910, (b) any one of SEQ ID NOs 65847-67281, (c) any one of SEQ ID NOs 1900-2599, (d) any one of SEQ ID NOs 926-1779, (e) any one of SEQ ID NOs 37483-38044, (f) any one of SEQ ID NOs 49423-49969, (g) any one of SEQ ID NOs 35900-36292, (h) any one of SEQ ID NOs 44626-47013, (l) any one of SEQ ID NOs 36293-37482, (j) any one of SEQ ID NOs 34521-35899, (k) any one of SEQ ID NOs 131-925, (1) any one of SEQ ID NOs 58958 - 65846 or 67532-77374, (m) any one one of SEQ ID NOs 28
• SEQ ID NOs 28 121-124 (c) SEQ ID NOs 37 or 38, (d) SEQ ID NOs 33 or 34, (e) SEQ ID NOs 92-95,
• the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (a) SEQ ID NO 6, (b) SEQ ID NO 28, (c) SEQ ID NO 4, (d) SEQ ID NO 2, (e) SEQ ID NO 19, (f) SEQ ID NO 25, (g) SEQ ID NO 17, (h) SEQ ID NO 23, (l) SEQ ID NO 18, (j) SEQ ID NO 16, (k) SEQ ID NO 1, (1) SEQ ID NO 30, (m) SEQ ID NO 13, (n) SEQ ID NO 7, (o) SEQ ID NO 9, (p) SEQ ID NO 20, (q) SEQ ID NO 15, (r) SEQ ID NO 27, (s) SEQ ID NO 26, (t) SEQ ID NO 8, (u) SEQ ID NO 31, (v) SEQ ID NO 21, (w) SEQ ID NO 22, (x) SEQ ID NO 14, (y) SEQ ID NO 11, (z
• the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating alternative splicing of pre-mRNA transcribed from a gene encoding the functional RNA or target protein.
• the antisense oligomer does not increase the amount of the target protein or the functional RNA by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or the functional RNA.
• the RIC pre-mRNA was produced by partial splicing of a full-length pre- mRNA or partial splicing of a wild-type pre-mRNA.
• the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
• the target protein produced is full-length protein, or wild-type protein.
• the total amount of the mRNA encoding the target protein or functional RNA produced in the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9- fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about
• the total amount of target protein produced by the cell contacted with the antisense oligomer is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5- fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5- fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 1.1 to about 10-fold,
• the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
• the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
• the antisense oligomer comprises at least one modified sugar moiety.
• each sugar moiety is a modified sugar moiety.
• the antisense oligomer consists of from 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,
• 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 RIC pre-mRNA encoding the protein.
• the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the most abundant retained intron in the population of RIC pre-mRNAs.
• the binding of the antisense oligomer to the most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre-mRNAs to produce mRNA encoding the target protein or functional RNA.
• the cell comprises a population of RIC pre-mRNAs transcribed from the gene encoding the target protein or functional RNA, wherein the population of RIC pre-mRNAs comprises two or more retained introns, and wherein the antisense oligomer binds to the second most abundant retained intron in the population of RIC pre-mRNAs.
• the binding of the antisense oligomer to the second most abundant retained intron induces splicing out of the two or more retained introns from the population of RIC pre- mRNAs to produce mRNA encoding the target protein or functional RNA.
• the condition is a disease or disorder.
• the disease or disorder is a liver disease.
• the liver disease is glycine encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine Deaminase deficiency, porphyria variegate, porphyria cutanea tarda, acute intermittent porphyria, very long chain acyl-CoA dehydrogenase deficiency, pyruvate carboxylase deficiency, isovaleric academia, hyperchylomicronemia, hypertriglyceridemia, galactosemia,
• hypercholesterolemia maturity -onset diabetes of the young type 1, maturity -onset diabetes of the young type 2, maturity -onset diabetes of the young type 3, noninsulin-dependent diabetes mellitus, insulin- dependent diabetes mellitus 1, insulin-dependent diabetes mellitus 20, Falconi renotubular syndrome 4 with maturity-onset diabetes of the young, hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome, immunodeficiency 36, agammaglobulinemia 7, lipid metabolism deficiency, liver inflammation, hemochromatosis type 2B, thrombocytopenia, non-alcoholic fatty liver disease, Wilson disease, tyrosinemia type I,
• argininosuccinate lyase deficiency hemochromatosis type I, Alstrom syndrome, congenital bile acid synthesis defect 1, steatohepatitis, insulin resistance, glucose intolerance, type II diabetes or liver cancer.
• the target protein and the RIC pre-mRNA are encoded by a gene, wherein the gene is AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
• the gene is AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
• the method further comprises assessing protein expression.
• the subject is a human.
• the subject is a non-human animal.
• the subject is a fetus, an embryo, or a child.
• the cells are ex vivo.
• the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
• the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site are administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.
• +1 to +6 of the retained intron are identical to the corresponding wild-type sequence.
• the 16 nucleotides at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.
• an antisense oligomer as used in a method described herein.
• an antisense oligomer comprising a sequence with at least about
• a pharmaceutical composition comprising an antisense oligomer described herein and an excipient.
• composition comprising an antisense oligomer for use in a method of increasing expression of a target protein or a functional RNA by cells to treat a liver disease in a subject in need thereof associated with a deficient protein or deficient functional RNA, wherein the deficient protein or deficient functional RNA is deficient in amount or activity in the subject, wherein the antisense oligomer enhances constitutive splicing of a retained intron-containing pre-mRNA (RIC pre- mRNA) encoding the target protein or the functional RNA, wherein the target protein is: the deficient protein; or a compensating protein which functionally augments or replaces the deficient protein or in the subject; and wherein the functional RNA is: the deficient RNA; or a compensating functional RNA which functionally augments or replaces the deficient functional RNA in the subject; wherein the RIC pre-mRNA comprises a retained intron, an exon flanking the 5' splice site and an ex
• the liver disease is glycine encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine Deaminase deficiency, porphyria variegate, porphyria cutanea tarda, acute intermittent porphyria, very long chain acyl-CoA dehydrogenase deficiency, pyruvate carboxylase deficiency, isovaleric academia, hyperchylomicronemia, hypertriglyceridemia, galactosemia,
• hypercholesterolemia maturity-onset diabetes of the young type 1, maturity-onset diabetes of the young type 2, maturity-onset diabetes of the young type 3, noninsulin-dependent diabetes mellitus, insulin- dependent diabetes mellitus 1, insulin-dependent diabetes mellitus 20, Falconi renotubular syndrome 4 with maturity-onset diabetes of the young, hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome, immunodeficiency 36, agammaglobulinemia 7, lipid metabolism deficiency, liver inflammation, hemochromatosis type 2B, thrombocytopenia, non-alcoholic fatty liver disease, Wilson disease, tyrosinemia type I,
• argininosuccinate lyase deficiency hemochromatosis type I, Alstrom syndrome, congenital bile acid synthesis defect 1, steatohepatitis, insulin resistance, glucose intolerance, type II diabetes or liver cancer.
• composition comprising an antisense oligomer for use in a method of treating a condition associated with a target protein in a subject in need thereof, the method comprising the step of increasing expression of the target protein by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA) comprising a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3' splice site of the retained intron, and wherein the RIC pre-mRNA encodes the target protein, the method comprising contacting the cells with the antisense oligomer, whereby the retained intron is constitutively spliced from the RIC pre- mRNA transcripts encoding the target protein, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein, in the cells of the subject.
• RIC pre-mRNA retained-intron-containing pre-mRNA
• the target protein is aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A- V, galactose- 1 -phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O-glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoiet
• the condition is a disease or disorder.
• the disease or disorder is a liver disease.
• the liver disease is glycine encephalopathy, Zellweger syndrome, Heimler syndrome, Adenosine Deaminase deficiency, porphyria variegate, porphyria cutanea tarda, acute intermittent porphyria, very long chain acyl-CoA dehydrogenase deficiency, pyruvate carboxylase deficiency, isovaleric academia, hyperchylomicronemia, hypertriglyceridemia, galactosemia,
• hypercholesterolemia maturity-onset diabetes of the young type 1, maturity-onset diabetes of the young type 2, maturity-onset diabetes of the young type 3, noninsulin-dependent diabetes mellitus, insulin- dependent diabetes mellitus 1, insulin-dependent diabetes mellitus 20, Falconi renotubular syndrome 4 with maturity-onset diabetes of the young, hyperinsulemic hypoglycemia familial 3, permanent neonatal diabetes mellitus, hepatic adenoma, Dowling-Degos disease 4, SHORT syndrome, immunodeficiency 36, agammaglobulinemia 7, lipid metabolism deficiency, liver inflammation, hemochromatosis type 2B, thrombocytopenia, non-alcoholic fatty liver disease, Wilson disease, tyrosinemia type I, argininosuccinate lyase deficiency, hemochromatosis type I, Alstrom syndrome, congenital bile acid synthesis defect 1, steatohepatitis, insulin resistance, glucose intolerance, type II diabetes or liver cancer.
• the target protein and RIC pre-mRNA are encoded by a gene, wherein the gene is AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
• the gene is AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, H
• the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within the region +6 relative to the 5' splice site of the retained intron to -16 relative to the 3' splice site of the retained intron.
• the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within: the region +6 to +100 relative to the 5' splice site of the retained intron; or the region -16 to -100 relative to the 3' splice site of the retained intron.
• the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 500 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 500 nucleotides upstream of the 3' splice site of the at least one retained intron
• the antisense oligomer targets a portion of the RIC pre-mRNA that is within the region about 100 nucleotides downstream of the 5' splice site of the at least one retained intron, to about 100 nucleotides upstream of the 3' splice site of the at least one retained intron.
• the targeted portion of the RIC pre-mRNA is within: the region +2e to -4e in the exon flanking the 5 ' splice site of the retained intron; or the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
• the target protein is (a) AMT, (b) ADA, (c) PPOX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i) APOA5, (j) GALT, (k) LDLRAPl, (1) HNF4A, (m) GCK, (n) POGLUTl, (o) PIK3R1, (p) HNF1A, (q) TRIB1, (r) TGFB1, (s) HAMP, (t) THPO, (u) PNPLA3, (v) ATP7B, (w) FAH, (x) ASL, (y) HFE, (z) ALMS1, (aa) PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.
• the targeted portion of the RIC pre-mRNA comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complimentary to (a) any one of SEQ ID NOs 2813-3910, (b) any one of SEQ ID NOs 65847-67281, (c) any one of SEQ ID NOs 1900-2599, (d) any one of SEQ ID NOs 926-1779, (e) any one of SEQ ID NOs 37483-38044, (f) any one of SEQ ID NOs 49423-49969, (g) any one of SEQ ID NOs 35900-36292, (h) any one of SEQ ID NOs 44626-47013, (l) any one of SEQ ID NOs 36293-37482, (j) any one of SEQ ID NOs 34521-35899, (k) any one of SEQ ID NOs 131-925, (1) any one of SEQ ID NOs 58958
• the targeted portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a region comprising at least 8 contiguous nucleic acids of (a) SEQ ID NO 78483, 78465 or 78434; (b) SEQ ID NO 78385, 78482 or 78369; (c) SEQ ID NO 78461, 78473, 78480 or 78421; (d) SEQ ID NO 78410, 78386, 78411, 78460 or 78463; (e) SEQ ID NO 78355, 78467 or 78454; (f) SEQ ID NO 78367, 78376, 78440, 78448, 78477, 78485, 78496, 78422 or 78412; (g) SEQ ID NO 78495, (h) SEQ ID NO 78464, 78375 or 78380; (l) SEQ ID NO 78495, (h
• the ASO comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (a) any one of SEQ ID NOs 2813-3910, (b) any one of SEQ ID NOs 65847-67281, (c) any one of SEQ ID NOs 1900-2599, (d) any one of SEQ ID NOs 926- 1779, (e) any one of SEQ ID NOs 37483-38044, (f) any one of SEQ ID NOs 49423-49969, (g) any one of SEQ ID NOs 35900-36292, (h) any one of SEQ ID NOs 44626-47013, (l) any one of SEQ ID NOs 36293-37482, (j) any one of SEQ ID NOs 34521-35899, (k) any one of SEQ ID NOs 131-925, (1) any one of SEQ ID NOs 58958- 65846 or 67532
• the RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of (a) SEQ ID NOs 41-44, (b) SEQ ID NOs 28 121-124, (c) SEQ ID NOs 37 or 38, (d) SEQ ID NOs 33 or 34, (e) SEQ ID NOs 92-95, (f) SEQ ID NOs 109-112, (g) SEQ ID NOs 87-89, (h) SEQ ID NOs 104 or 105, (l) SEQ ID NOs 90 or 91, (j) SEQ ID NOs 85 or 86, (k) SEQ ID NO 32, (1) SEQ ID NOs 115-120 orl26-129, (m) SEQ ID NOs 76-78, (n) SEQ ID NOs 45 or 46, (o) SEQ ID NOs 57-60, (p) SEQ ID NOs 96 or
• the RIC pre-mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to (a) SEQ ID NO 6, (b) SEQ ID NO 28, (c) SEQ ID NO 4, (d) SEQ ID NO 2, (e) SEQ ID NO 19, (f) SEQ ID NO 25, (g) SEQ ID NO 17, (h) SEQ ID NO 23, (l) SEQ ID NO 18, (j) SEQ ID NO 16, (k) SEQ ID NO 1, (1) SEQ ID NO 30, (m) SEQ ID NO 13, (n) SEQ ID NO 7, (o) SEQ ID NO 9, (p) SEQ ID NO 20, (q) SEQ ID NO 15, (r) SEQ ID NO 27, (s) SEQ ID NO 26, (t) SEQ ID NO 8, (u) SEQ ID NO 31, (v) SEQ ID NO 21, (w) SEQ ID NO 22, (x) SEQ ID NO 14, (y) SEQ ID NO 11, (z
• the antisense oligomer does not increase the amount of target protein or functional RNA by modulating alternative splicing of the pre-mRNA transcribed from a gene encoding the target protein or functional RNA.
• the antisense oligomer does not increase the amount of the functional RNA or functional protein by modulating aberrant splicing resulting from mutation of the gene encoding the target protein or functional RNA.
• the RIC pre-mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.
• the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
• the target protein produced is full-length protein, or wild-type protein.
• the retained intron is a rate-limiting intron.
• said retained intron is the most abundant retained intron in said RIC pre- mRNA. [0086] In some embodiments, the retained intron is the second most abundant retained intron in said RIC pre-mRNA.
• the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
• said antisense oligomer is an antisense oligonucleotide.
• the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
• the antisense oligomer comprises at least one modified sugar moiety.
• each sugar moiety is a modified sugar moiety.
• the antisense oligomer consists of from 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,
• 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 the targeted portion of the RIC pre-mRNA encoding the protein.
• compositions described herein comprising an antisense oligomer of any of the compositions described herein, and an excipient.
• a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript, wherein the deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, P
• the deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFl A, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcnpt is a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, H
• ACADVL ACADVL
• PC IVD
• APOA5 GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript is in the retained intron within the region +500 relative to the 5' splice site of the retained intron to -500 relative to the 3' spliced site of the retained intron.
• the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre- mRNA transcript is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1-31.
• the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
• the antisense oligomer is an antisense oligonucleotide.
• the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-0-methyl, a 2'-Fluoro, or a 2'-0-methoxyethyl moiety.
• the antisense oligomer comprises at least one modified sugar moiety.
• the antisense oligomer comprises from 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
• 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 AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFl A, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript.
• ACADVL ACADVL
• PC IVD
• the antisense oligomer comprises a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 131-78348.
• the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 131-78348.
• the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
• ACADVL PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNF1A, TRIBl,
• TGFBl HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein and comprises at least one retained intron; removing the at least one retained intron from the deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
• LDLRAPl HNF4A, GCK, POGLUTl, PIK3R1, HNF1A, TRIBl, TGFBl, HAMP, THPO, PNPLA3,
• ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript to produce the fully processed AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
• LDLRAPl HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3,
• ATP7B ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript that encodes the functional form of AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5,
• GALT GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO,
• GALT GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO,
• LDLRAPl HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3,
• ATP7B ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript.
• the retained intron is an entire retained intron.
• the deficient AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, H
• a method of treating a subject having a condition caused by a deficient amount or activity of AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein comprising administering to the subject an antisense oligomer comprising a nucleotide sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 131-78348.
• FIG. 1 depicts a schematic representation of an exemplary retained-intron-containing (RIC) pre-mRNA transcript.
• the 5' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -3e to -le and +1 to +6 (numbers labeled "e” are exonic and unlabeled numbers are intronic).
• the 3' splice site consensus sequence is indicated with underlined letters (letters are nucleotides; upper case: exonic portion and lower case: intronic portion) from -15 to -1 and +le (numbers labeled "e” are exonic and unlabeled numbers are intronic).
• Intronic target regions for ASO screening comprise nucleotides +6 relative to the 5' splice site of the retained intron (arrow at left) to -16 relative to the 3' splice site of the retained intron (arrow at right).
• intronic target regions for ASO screening comprise nucleotides +6 to +100 relative to the 5' splice site of the retained intron and -16 to -100 relative to the 3' splice site of the retained intron.
• Exonic target regions comprise nucleotides +2e to -4e in the exon flanking the 5' splice site of the retained intron and +2e to -4e in the exon flanking the 3' splice site of the retained intron.
• n or N denote any nucleotide
• y denotes pyrimidine.
• the sequences shown represent consensus sequences for mammalian splice sites and individual introns and exons need not match the consensus sequences at every position.
• FIG. 2A depicts an exemplary schematic representation of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
• FIG. 2A shows a cell divided into nuclear and cytoplasmic compartments.
• a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this rnRNA is exported to the cytoplasm and translated into target protein.
• the splicing of intron 1 is inefficient and a retained intron-containing (RIC) pre-mRNA accumulates primarily in the nucleus, and if exported to the cytoplasm, is degraded, leading to no target protein production.
• RIC retained intron-containing
• FIG. 2B depicts an exemplary schematic representation of the Targeted Augmentation of Nuclear Gene Output (TANGO) approach.
• FIG. 2B shows an example of the same cell as in FIG. 2A divided into nuclear and cytoplasmic compartments. Treatment with an antisense oligomer (ASO) promotes the splicing of intron 1 and results in an increase in mRNA, which is in turn translated into higher levels of target protein.
• FIG. 3A depicts a schematic of the RefSeq Genes for AMT corresponding to NM 001164710,
• FIG. 3C depicts a schematic of the RefSeq Genes for AMT corresponding to NM_001164710, NM 00116471,1 NM_001164712, NM_000481 and NR_028435.
• PIR Percent Intron Retention
• FIG. 4A depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• NM_000155 and NM_001258332 The Percent Intron Retention (PIR) of the circled intron is shown (GALT intron 2, NM_000155).
• FIG. 4B depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• NM_000155 and NM_001258332 The Percent Intron Retention (PIR) of the circled intron is shown (GALT intron 3, NM_000155).
• FIG. 4C depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• NM_000155 and NM_001258332 The Percent Intron Retention (PIR) of the circled intron is shown (GALT intron 4, NM_000155).
• FIG. 4E depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• NM_000155 and NM_001258332 The Percent Intron Retention (PIR) of the circled intron is shown (GALT intron 5, NM_000155).
• FIG. 4F depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• FIG. 4G depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• NM_000155 and NM_001258332 The Percent Intron Retention (PIR) of the circled intron is shown (GALT intron 8, NM_000155).
• FIG. 41 depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• FIG. 4J depicts a schematic of the RefSeq Genes for GALT corresponding to GALT:
• FIG. 5A depicts a schematic of the RefSeq Genes for PC corresponding to PC: NM 000920, NM_022172 and NM_001040716.
• the Percent Intron Retention (PIR) of the circled intron is shown (PC intron 16, NM_022172).
• FIG. 6A depicts a schematic of the RefSeq Genes for FAH corresponding to FAH:
• FIG. 7A depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
• FIG. 7C depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
• FIG. 7D depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
• FIG. 7E depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
• FIG. 7G depicts a schematic of the RefSeq Genes for PPARD corresponding to PPARD:
• FIG. 8A depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 3, NM 000018).
• FIG. 8B depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 5, NM 000018).
• FIG. 8C depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 8, NM 000018).
• FIG. 8D depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 9, NM 000018).
• FIG. 8E depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 10, NM 000018).
• FIG. 8F depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 13, NM 000018).
• FIG. 8G depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 18, NM 000018).
• FIG. 8H depicts a schematic of the RefSeq Genes for ACADVL corresponding to ACADVL: NM_001270448, NM_001270447, NM_000018 and NM_001033859.
• the Percent Intron Retention (PIR) of the circled intron is shown (ACADVL intron 19, NM 000018).
• FIG. 9A depicts a schematic of the RefSeq Genes for HMBS corresponding to HMBS:
• PIR Percent Intron Retention
• FIG. 9C depicts a schematic of the RefSeq Genes for HMBS corresponding to HMBS:
• PIR Percent Intron Retention
• FIG. 10A depicts a schematic of the RefSeq Genes for UROD corresponding to UROD:
• FIG. 10B depicts a schematic of the RefSeq Genes for UROD corresponding to UROD:
• FIG. IOC depicts a schematic of the RefSeq Genes for UROD corresponding to UROD:
• FIG. 10D depicts a schematic of the RefSeq Genes for UROD corresponding to UROD:
• FIG. 10E depicts a schematic of the RefSeq Genes for UROD corresponding to UROD:
• FIG. 11A depicts a schematic of the RefSeq Genes for ALMS1 corresponding to ALMS1 : NM 015120.
• the Percent Intron Retention (PIR) of the circled intron is shown (ALMS1 intron 21, NM_015120).
• FIG. 12A depicts a schematic of the RefSeq Genes for ASL corresponding to ASL:
• NM_000048 NM_001024943, NM_001024944 and NM_001024946.
• the Percent Intron Retention (PIR) of the circled intron is shown (ASL intron 7, NM_000048).
• FIG. 12C depicts a schematic of the RefSeq Genes for ASL corresponding to ASL:
• FIG. 12E depicts a schematic of the RefSeq Genes for ASL corresponding to ASL:
• NM_000048 NM_001024943, NM_001024944 and NM_001024946.
• the Percent Intron Retention (PIR) of the circled intron is shown (ASL intron 9, NM 000048).
• FIG. 12G depicts a schematic of the RefSeq Genes for ASL corresponding to ASL:
• NM_000048 NM_001024943, NM_001024944 and NM_001024946.
• the Percent Intron Retention (PIR) of the circled intron is shown (ASL intron 16, NM 000048).
• FIG. 13A depicts a schematic of the RefSeq Genes for ATP7B1 corresponding to ATP7B1 :
• Intron Retention (PIR) of the circled intron is shown (ATP7B1 intron 7, NM 000053).
• FIG. 13B depicts a schematic of the RefSeq Genes for ATP7B 1 corresponding to ATP7B 1 :
• Intron Retention (PIR) of the circled intron is shown (ATP7B1 intron 13, NM 000053).
• FIG. 14 depicts a schematic of the RefSeq Genes for HFE corresponding to HFE: NM 139007, NM_139006, NM_139004, NM_139003, NM_001300749, NM_139009, NM_139008, and NM_000410.
• the Percent Intron Retention (PIR) of the circled intron is shown (HFE intron 2, NM 000410).
• FIG. 15A depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 1, NM_001142777).
• FIG. 15B depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 1, NM_001142777).
• FIG. 15D depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 3, NM_001142777).
• FIG. 15E depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 4, NM_001142777).
• FIG. 15G depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 5, NM_025193).
• FIG. 15H depicts a schematic of the RefSeq Genes for HSD3B7 corresponding to HSD3B7: NM_001142777, NM_001142778 and NM_025193.
• the Percent Intron Retention (PIR) of the circled intron is shown (HSD3B7 intron 6, NM_025193).
• FIG. 16 depicts a schematic of the RefSeq Genes for NCOA5 corresponding to NCOA5: NM 020967.
• the Percent Intron Retention (PIR) of the circled intron is shown (NCOA5 intron2, NM_020967).
• FIG. 17A depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: NM 000309 and NM OOl 122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 3, 000309).
• FIG. 17B depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: NM 000309 and NM OOl 122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 4, 000309).
• FIG. 17C depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: NM 000309 and NM OOl 122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 5, 000309).
• FIG. 17D depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: NM 000309 and NM OOl 122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 7, 000309).
• FIG. 17E depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: M 000309 and NM 001122764.
• the Percent Intron Retention (PIR) of the circled intron is shown
• FIG. 17F depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: M 000309 and NM 001122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 10, 000309).
• FIG. 17G depicts a schematic of the RefSeq Genes for PPOX corresponding to PPOX: M 000309 and NM 001122764.
• the Percent Intron Retention (PIR) of the circled intron is shown (PPOX intron 12, 000309).
• liver disease is a debilitating condition that results in an estimated 60,000 deaths per year in the United States alone.
• the only hope for those suffering from liver failure is a liver transplant, though the donor pool is only estimated to be approximately 4,000. Therefore, the odds of receiving a transplant is low, and there are few treatments available to ameliorate the condition for those unable to receive a transplant. Therefore, there exists a need for compositions and methods for treating liver diseases.
• introns in primary transcripts of protein-coding genes having one or more than one intron are spliced from the primary transcript with different efficiencies. In most cases only the fully spliced mRNA is exported through nuclear pores for subsequent translation in the cytoplasm. Unspliced and partially spliced transcripts are detectable in the nucleus. It is generally thought that nuclear accumulation of transcripts that are not fully spliced is a mechanism to prevent the accumulation of potentially deleterious mRNAs in the cytoplasm that may be translated to protein. For some genes, splicing of the least efficient intron is a rate-limiting post-transcriptional step in gene expression, prior to translation in the cytoplasm.
• compositions and methods for upregulating splicing of one or more retained introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated protein levels.
• compositions and methods utilize antisense oligomers (ASOs) that promote constitutive splicing at an intron splice site of a retained-intron-containing pre-mRNA that accumulates in the nucleus.
• ASOs antisense oligomers
• Liver diseases that can be treated by the invention described herein are diseases where a subject is deficient in a gene product, where deficiency in a gene product causes the liver disease.
• AMT AMT
• ADA PPOX
• UROD UROD
• HMBS HMBS
• ACADVL PC
• IVD APOA5
• GALT GALT
• LDLRAP1 LDLRAP1
• HNF4A HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 pre-mRNA species compnse at least one retained intron.
• the present invention provides compositions and methods for upregulating splicing of one or more retained AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 and NCOA5 introns that are rate-limiting for the nuclear stages of gene expression to increase steady-state production of fully-spliced, mature mRNA, and thus, translated aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA dehydr
• compositions and methods can utilize antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a retained-intron-containing AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus.
• ASOs antisense oligomers
• aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V, galactose- 1- phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O- glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like
• uridylyltransferase low density lipoprotein receptor adaptor protein 1
• hepatocyte nuclear factor 4-alpha glucokinase
• hepatic nuclear factor- 1 -alpha albulim proximal factor protein O-glucosyltransferase 1
• phosphatidylinositol 3-kinase regulatory subunit 1 Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary
• hemochromatosis protein alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator activated receptor delta, interleukin 6, ceramide synthase 2 or nuclear receptor coactivator 5 deficiency.
• compositions and methods can utilize antisense oligomers (ASOs) that promote constitutive splicing at intron splice sites of a retained-intron-containing AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 pre- mRNA (RIC pre-mRNA) that accumulates in the nucleus.
• ASOs antisense oligomers
• AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein can be increased using the methods of the invention to treat a condition caused by AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL
• the methods of the invention can be used to increase
• uridylyltransferase low density lipoprotein receptor adaptor protein 1
• hepatocyte nuclear factor 4-alpha glucokinase
• hepatic nuclear factor- 1 -alpha albulim proximal factor protein O-glucosyltransferase 1
• phosphatidylinositol 3-kinase regulatory subunit 1 Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary
• hemochromatosis protein alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator activated receptor delta, interleukin 6, ceramide synthase 2 or nuclear receptor coactivator 5 production to treat a condition in a subject in need thereof.
• the subject has a condition in which aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA
• dehydrogenase pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V, galactose- 1- phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O- glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain- containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary hemochromatosis protein, alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7,
• uridylyltransferase low density lipoprotein receptor adaptor protein 1
• hepatocyte nuclear factor 4-alpha glucokinase
• hepatic nuclear factor- 1 -alpha albulim proximal factor protein O-glucosyltransferase 1
• phosphatidylinositol 3-kinase regulatory subunit 1 Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary
• hemochromatosis protein alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7, peroxisome proliferator activated receptor delta, interleukin 6, ceramide synthase 2 or nuclear receptor coactivator 5 mitigates the condition nonetheless.
• the condition can be caused by a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 haploinsufficiency.
• the described compositions and methods are used to treat a subject or patient having a liver condition that is caused by a deficiency in the target protein. In embodiments the described compositions and methods are used to treat a subject or patient having a liver condition that is not caused by a deficiency in the target protein. In embodiments, the subject or patient having a liver condition can benefit from increased production of the target protein by supplementing normal production of the target protein. In related embodiments, the subject or patient having a liver condition can benefit from increased production of the target protein by increasing mature mRNA production and/or supplementing normal production of the target protein.
• the target protein is TRIBl, TGFBl, HAMP, THPO, PNPLA3,
• the target protein acts on a secondary target to ameliorate or treat the liver condition in the subject.
• the secondary target protein is deficient in the subject.
• the secondary target protein is not deficient in the subject.
• the invention described herein can be used to treat the autosomal recessive liver disorder Glycine Encephalopathy (GCE).
• GCE autosomal recessive liver disorder Glycine Encephalopathy
• the predominant phenotype of GCE is the neonatal phenotype, which manifests early in life. Symptoms of this phenotype include lethargy, hypotonia, myoclonic jerks, seizures, mental retardation, apnea and often death. In other instances, GCE manifests in childhood, with symptoms including mild mental retardation, delirium, chorea, and vertical gaze palsy. The late onset form of GCE results in spastic diplegia and optic atrophy, but typically does not result in mental retardation or seizures.
• GCE can manifest as a result of deficiency in the glycine cleavage system in the liver.
• AMT protein aminomethyltransferase
• the invention described herein can be used to treat the autosomal recessive liver disorder Zellweger Syndrome.
• Zellweger Syndrome is a severe peroxisomal biogenesis disorder characterized by severe neurologic dysfunction, craniofacial abnormalities, and liver dysfunction. Patients afflicted with the classic Zellweger Syndrome phenotype typically die within the first year of life.
• the invention described herein can be used to treat the autosomal recessive liver disorder Heimler syndrome.
• Heimler syndrome is the mildest form the peroxisomal biogenesis disorders characterized by sensorineural hearing loss, enamel hypoplasia of the secondary dentition and nail abnormalities.
• the invention described herein can be used to treat the autosomal recessive liver disease Adenosine Deaminase (ADA) deficiency.
• ADA deficiency generally manifests in infancy, and is generally fatal, though a small subset of patients display a late onset form of the disease that is generally milder than the infantile form. Patients afflicted with the late onset form of ADA deficiency typically show gradual immunologic deterioration, which leads to a number of secondary infections.
• ADA deficiency in the ADA protein results in the clinical manifestations shown in ADA deficiency.
• the ADA gene which is located at 20ql3 and spans 10 exons, codes for the ADA protein. Mutations in the ADA gene resulting in deficient amounts of ADA protein have been shown to be responsible for the progression of ADA deficiency.
• a pair of children diagnosed with ADA deficiency were examined. Both children were found to have diminished levels of ADA protein, while both of the parents were found to have intermediate levels of the ADA protein. This finding supported the recessive pattern of inheritance proposed for the disease, and provides a positive link between diminished ADA protein and the clinical manifestations of ADA deficiency.
• the invention described herein can be used to treat the liver disease porphyria variegate (VP).
• VP is characterized by cutaneous manifestations, such as increased photosensitivity, blistering, skin fragility, and postinflammatory hyperpigmentation. Additional manifestations include abdominal pain, dark urine, and neuropsychiatric symptoms that characterize the acute hepatic porphyrias.
• PPOX protoporphyrinogen oxidase
• the invention described herein can be used to treat the autosomal dominant liver disease porphyria cutanea tarda (PCT).
• PCT is characterized by light sensitive dermatitis and excretion of uroporphyrin in urine.
• UROD uroporphyrinogen decarboxylase
• the invention described herein can be used to treat the autosomal dominant liver disease acute intermittent porphyria (AIP).
• AIP is characterized by defects in the biosynthesis of heme.
• Clinical manifestations of AIP include abdominal pain, gastrointestinal pain, gastrointestinal pain, gastrointestinal pain, gastrointestinal pain, gastrointestinal pain, etc.
• HMBS hydroxymethylbilane synthase
• PBGD porphobilinogen deaminase
• the invention described herein can be used to treat the autosomal recessive liver disease, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency.
• VLCAD is characterized by nonketotic hypoglycemia, cardiorespiratory arrest, hepatomegaly, cardiomegaly, and hypotonia, which are believed to be manifestations resulting from a defect in mitochondrial fatty acid oxidation.
• VLCAD deficiency results in the clinical manifestations shown in VLCAD deficiency.
• the ACADVL gene which is located at 17p 13.1 and spans 20 exons, codes for the VLCAD protein. Mutations in the ACADVL gene resulting in deficient amounts of VLCAD protein have been shown to be responsible for the progression of VLCAD deficiency. In one study, 2 patients displaying VLCAD deficiency were found to have a 105 bp deletion in the ACADVL gene. In another study, 21 different missense mutations were found in 18 children displaying VLCAD deficiency. In aggregate, studies such as these have shown a positive link between deficiency in VLCAD and the clinical manifestations seen in patients displaying VLCAD deficiency.
• the invention described herein can be used to treat the autosomal recessive liver disease pyruvate carboxylase (PC) deficiency.
• PC deficiency is categorized into 3 phenotypic subtypes. Patients with type A, which is more prevalent in North America, display lactic academia and psychomotor retardation. Patients with type B, which is more severe than type A and is more prevalent in France and the United Kingdom, display increased serum lactate, ammonia, citrulline and lysine, and intracellular redox disturbance with a higher incidence of mortality. Type C is the more milder form of PC deficiency and is generally benign.
• PC pyruvate carboxylase
• the invention described herein can be used to treat the autosomal recessive liver disease isovaleric academia (rVA).
• rVA is categorized into 2 phenotypic subtypes: an acute and chronic subtype.
• the acute subtype leads to massive metabolic acidosis and ultimately death.
• the chronic subtype results in periods of attacks of severe ketoacidosis followed by asymptomatic periods.
• Clinical manifestations of rVA include peculiar odor, an aversion to dietary protein and vomiting.
• IVD isovaleryl-CoA dehydrogenase
• the invention described herein can be used to treat the autosomal dominant liver disease hyperchylomicronemia.
• Hyperchylomicronemia is characterized by increased amounts of chylomicrons and very low density lipoprotein (VLDL) and decreased LDL and high density lipoprotein (HDL) in the plasma.
• VLDL very low density lipoprotein
• HDL high density lipoprotein
• the invention described herein can be used to treat the autosomal dominant liver disease hypertriglyceridemia.
• Patients afflicted with hypertriglyceridemia generally have normal levels of cholesterol while displaying elevated levels of triglycerides.
• patients are generally asymptomatic.
• APOA5 apolipoprotein A-V
• the APOA5 gene which is located at l lq23.3 and spans 4 exons, codes for the APOA5 protein. Mutations in the APOA5 gene resulting in deficient amounts of APOA5 protein have been shown to be responsible for the progression of both
• the invention described herein can be used to treat the autosomal recessive liver disease galactosemia.
• Galactosemia generally manifests during neonatal development, and is characterized by jaundice, hepatosplenomegaly, hepatocellular insufficiency, food intolerance, hypoglycemia, renal tubular dysfunction, muscle hypotonia, sepsis, and cataracts. Overtime, patients afflicted with galactosemia experience mental retardation, verbal dyspraxia, motor abnormalities, and hypergonadotropic hypogonadism.
• GALT galactose- 1 -phosphate uridylyl transferase
• the invention described herein can be used to treat the autosomal recessive liver disease hypercholesterolemia (ARH).
• ARH is characterized by severely elevated plasma low density lipoprotein (LDL) cholesterol, tuberous and tendon xanthomata and premature
• LDLRAPl protein low density lipoprotein receptor adaptor protein 1
• the LDLRAPl gene which is located at lp36.11 and spans 9 exons, codes for the LDLRAPl protein. Mutations in the LDLRAPl gene resulting in deficient amounts of LDLRAPl protein have been shown to be responsible for the progression of ARH.
• Several studies have looked at a number of different mutations in LDLRAPl that result in ARH, and have shown that mutations that result in deficiency in the amount of LDLRAPl protein result in the clinical manifestations seen in ARH.
• a number of studies examined a conserved nonsense mutation in the LDLRAPl gene, which resulted in substantial decrease in the amount of LDLRAPl protein, and hence the clinical manifestations associated with ARH.
• the invention described herein can be used to treat maturity-onset diabetes of the young type 1 (MODY1). In other embodiments, the invention described herein can be used to treat maturity-onset diabetes of the young type 2 (MODY2). In other embodiments, the invention described herein can be used to treat maturity-onset diabetes of the young type 3 (MODY3). In other embodiments, the invention described herein can be used to treat noninsulin-dependent diabetes mellitus (NIDDM). In other embodiments, the invention described herein can be used to treat insulin-dependent diabetes mellitus 1 (IDDM1). In other embodiments, the invention described herein can be used to treat insulin-dependent diabetes mellitus 20 (IDDM20). In other embodiments, the invention described herein can be used to treat Falconi renotubular syndrome 4 with maturity-onset diabetes of the young (FRTS4).
• NIDDM noninsulin-dependent diabetes mellitus
• IDDM1 insulin-dependent diabetes mellitus 1
• IDDM20 insulin-dependent diabetes mellitus 20
• the invention described herein
• the invention described herein can be used to treat hyperinsulemic hypoglycemia familial 3 (HHF3).
• HHF3 hyperinsulemic hypoglycemia familial 3
• the invention described herein can be used to treat permanent neonatal diabetes mellitus (PNDM).
• Diabetes mellitus are a group of metabolic diseases characterized by high blood sugar, with symptoms including frequent urination, increased thirst, increased hunger, diabetic ketoacidosis, cardiovascular disease, stroke, chronic kidney failure, foot ulcers and damage to the eyes.
• HNF4A hepatocyte nuclear factor 4-alpha
• GCK protein glucokinase
• Deficiency in the protein hepatic nuclear factor- 1 -alpha albulim proximal factor has been shown to correlate to the incidence of MODY3, IDDM20, IDDM1 and NIDDM.
• the HNFIA gene which is located at 12q24.31 and spans 10 exons, codes for the FINFl A protein. Mutations in the HNFIA gene resulting in deficient amounts of FINFl A protein have been shown to be responsible for the progression of MODY3, IDDM20, IDDM1 and NIDDM.
• Several studies have looked at a number of different mutations in HNFIA that result in MODY3, IDDM20, IDDM1 and NIDDM, and have positively correlated the deficiency in the amount of expressed HNFIA protein with the progression of diabetes.
• NCOA5 protein nuclear receptor coactivator 5
• the NCOA5 gene which is located at 20ql3.12 and spans 8 exons, codes for the NCOA5 protein. Mutations in the NCOA5 gene resulting in deficient amounts of NCOA5 protein have been shown to be responsible for the progression of diabetes.
• Several studies have looked at a number of different mutations in NCOA5 that result in diabetes, and have positively correlated the deficiency in the amount of expressed NCOA5 protein with the progression of diabetes.
• the invention described herein can be used to treat the autosomal dominant liver disease hepatic adenoma.
• Hepatic adenoma is an uncommon benign liver tumor with a very low risk of malignant transformation. Patients afflicted with hepatic adenoma are generally asymptomatic unless the tumor begins to hemorrhage, which could lead to hypotension, tachycardia and diaphoresis.
• HNFIA protein can result in the progression of hepatic adenoma. Mutations in the HNFIA gene resulting in deficient amounts of HNFl A protein have been shown to be responsible for the progression of hepatic adenoma.
• the invention described herein can be used to treat the autosomal dominant liver disease Dowling-Degos disease 4 (DDD4).
• DDD4 is characterized by retricular pigmentation that presents in adult life, particularly in the folds of the skin. While the manifestations affect somatic cells, the pigmentation results from dysfunction of the liver.
• POGLUTl O-glucosyltransferase 1
• SHORT syndrome is an acronym for the clinical conditions that are associated with the condition, including short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, rieger anomaly and teething delay.
• Other symptoms characteristic of SHORT syndrome include a triangular face, small chin with a dimple, loss of fat under the skin, abnormal position of the ears, hearing loss and delayed speech.
• the invention described herein can be used to treat the autosomal dominant disease immunodeficiency 36 (IMD36).
• IMD36 is characterized by impaired B-cell function, hypogammaglobulinemia and recurrent infections.
• the invention described herein can be used to treat the autosomal recessive disease agammaglobulinemia 7 (AGM7).
• AGM7 is characterized by impaired B-cell function, hypogammaglobulinemia and recurrent infections.
• AGM7 is an immunodeficiency disease characterized by low serum antibodies and low circulating B cells, which results in recurrent infections.
• PIK3R1 protein phosphatidylinositol 3 -kinase regulatory subunit 1
• the invention described herein can be used to treat the lipid metabolism deficiency caused by deficiency in the protein Tribbles-1 (TRIBl).
• TRIBl Tribbles-1
• Mice lacking TRIBl were shown to have diminished adipose tissue mass accompanied by increased lipolysis, which positively linked diminished levels of TRIBl with dysfunction in lipid metabolism.
• the invention described herein can be used to treat the lipid metabolism deficiency caused by deficiency in the protein peroxisome proliferator activated receptor delta (PPARD).
• PPARD protein peroxisome proliferator activated receptor delta
• PPARD protein which is encoded by the PPARD gene located on chromosome 6 and spans 8 exons, has been shown to be correlated to increased lipid metabolism dysfunction.
• the invention described herein can be used to treat the liver inflammation caused by deficiency in the protein transforming growth factor beta-1 (TGFB1).
• TGBFl protein which is encoded by the TGBF1 gene located on 19ql3.2 and spans 7 exons, has been shown to be correlated to an increased risk of atherosclerosis.
• Knockout mice displaying the TGBFl (-/-) genotype were shown to develop severe liver inflammation due to CD4(+) T-cell mediated inflammation. Such studies provide a positive correlation between deficiency in the amount of TGBF1 protein and increased incidence of liver inflammation.
• the invention described herein can be used to treat the lipid inflammation caused by deficiency in the protein interleukin 6 (IL6).
• IL6 protein interleukin 6
• the invention described herein can be used to treat the lipid inflammation or steatohepatitis caused by deficiency in the ceramide synthase 2 (CERS2) protein.
• CERS2 ceramide synthase 2
• the invention described herein can be used to treat the autosomal recessive liver disease hemochromatosis type 2B (HFE2B).
• HFE2B alsowise known as iron overload
• HFE2B is characterized by joint pain, fatigue and weakness, which ultimately results in organ damage.
• HAMP protein hepcidin antimicrobial peptide
• the invention described herein can be used to treat the autosomal dominant disease thrombocytopenia.
• thrombocytopenia is characterized by a decrease in the amount of thrombocytes in the blood. While many cases of thrombocytopenia are asymptomatic, some patients experience external bleeding such as nose bleeds, malaise, fatigue and general weakness.
• THPO thrombopoietin
• the THPO gene which is located at 3q27.1 and spans 6 exons, codes for the THPO protein. Mutations in the THPO gene resulting in deficient amounts of THPO protein have been shown to be responsible for the progression of thrombocytopenia. In one study, a single nucleotide deletion at 3252 was seen in 3 generations of a family afflicted with thrombocytopenia. This study correlated the deficiency of THPO protein levels as a result of the deletion with the incidence of thrombocytopenia.
• Non-alcoholic Fatty Liver Disease [00244]
• the invention described herein can be used to treat non-alcoholic fatty liver disease (NAFLD).
• NAFLD non-alcoholic fatty liver disease
• triglycerides in the liver, which can be associated with adverse metabolic consequences such as insulin resistance and
• dyslipidemia Factors that can influence the progression of NAFLD include obesity, diabetes, and insulin resistance.
• aggregated fatty deposits in a liver can promote an inflammatory response, which can progress to cirrhosis or liver cancer.
• PNPLA3 patatin-like phospholipase domain-containing protein 3
• the PNPLA3 gene which is located at 22ql3 and spans 9 exons, codes for the PNPLA3 protein. Mutations in the PNPLA3 gene resulting in deficient amounts of PNPLA3 protein have been shown to be responsible for the progression of NAFLD.
• Polymorphisms such as C99G, Gl 15C, I148M, T216P and K434E have been found in populations manifesting symptoms of NAFLD. These missense mutations were shown to result in decreased levels of PNPLA3, which provides a correlation between the deficiency of the PNPLA3 protein and the progression of NAFLD.
• Wilson disease is characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neurologic abnormalities. Wilson disease may present itself in a patient with tiredness, increased bleeding tendency or confusion (due to hepatic encephalopathy) and portal hypertension.
• ATP7B protein copper-transporting ATPase 2
• T e ATP7B gene which is located at 13ql4.3 and spans 21 exons, codes for the ATP7B protein. Mutations in the ATP7B gene resulting in deficient amounts of ATP7B protein have been shown to be responsible for the progression of Wilson disease. Mutations in ATP7B such as L492S, Y532H, G591D, R616Q and G626A have been found in populations manifesting symptoms of Wilson disease. These missense mutations were shown to result in decreased levels of ATP7B, which provides a correlation between the deficiency of the ATP7B protein and the progression of Wilson disease.
• the invention described herein can be used to treat tyrosinemia.
• Tyrosinemia is characterized by progressive liver disease and a secondary renal tubular dysfunction leading to hypophosphatemic rickets. Onset varies from infancy to adolescence. In the most acute form patients present with severe liver failure within weeks after birth, whereas rickets may be the major symptom in chronic tyrosinemia. Untreated, patients die from cirrhosis or hepatocellular carcinoma at a young age [00249] Deficiency in the protein fumarylacetoacetase (FAH) has been shown to be correlated to the incidence of tyrosinemia. The i3 ⁇ 4H gene, which is located at 15q25.1 and spans 14 exons, codes for the
• FAH protein Mutations in the FAH gene resulting in deficient amounts of FAH protein have been shown to be responsible for the progression of FAH.
• Polymorphisms such as N16I, A134D, C193R, D233V and
• the invention described herein can be used to treat the autosomal recessive disorder argininosuccinate lyase deficiency.
• Argininosuccinate lyase deficiency is characterized by mental and physical retardation, liver enlargement, skin lesions, dry and brittle hair showing trichorrhexis nodosa microscopically and fluorescing red, convulsions, and episodic unconsciousness.
• ASL protein argininosuccinate lyase
• the ASL gene which is located at 7ql 1.21 and spans 17 exons, codes for the ASL protein. Mutations in the ASL gene resulting in deficient amounts of ASL protein have been shown to be responsible for the progression of argininosuccinate lyase deficiency. Polymorphisms such as Rl 13Q, V178M, R182Q, R236W, Q286R and R456W have been found in populations manifesting symptoms of argininosuccinate lyase deficiency. These missense mutations were shown to result in decreased levels of ASL, which provides a correlation between the deficiency of the ASL protein and the progression of argininosuccinate lyase deficiency.
• the invention described herein can be used to treat the autosomal recessive disorder hemochromatosis type 1.
• Hemochromatosis type 1 is characterized by iron overload. Excess iron is deposited in a variety of organs leading to their failure, and resulting in serious illnesses including cirrhosis, hepatomas, diabetes, cardiomyopathy, arthritis, and hypogonadotropic
• hypogonadism Severe effects of the disease usually do not appear until after decades of progressive iron loading.
• Deficiency in the hereditary hemochromatosis protein has been shown to be correlated to the incidence of hemochromatosis type 1.
• the HFE gene which is located at 6p22.2 and spans 6 exons, codes for the hereditary hemochromatosis protein. Mutations in the ASL gene resulting in deficient amounts of hereditary hemochromatosis protein have been shown to be responsible for the progression of hemochromatosis type 1. Polymorphisms such as S65C, Q127H, A176V, C282Y, Q283P and V295A have been found in populations manifesting symptoms of hemochromatosis type 1. These missense mutations were shown to result in decreased levels of hereditary hemochromatosis protein, which provides a correlation between the deficiency of the hereditary hemochromatosis protein and the progression of hemochromatosis type 1. Alstrom Syndrome
• the invention described herein can be used to treat the autosomal recessive disorder Alstrom syndrome.
• Alstrom syndrome is characterized by progressive cone-rod retinal dystrophy, neurosensory hearing loss, early childhood obesity and diabetes mellitus type 2.
• Dilated cardiomyopathy, acanthosis nigricans, male hypogonadism, hypothyroidism, developmental delay and hepatic dysfunction can also be associated with the syndrome..
• Deficiency in the protein alstrom syndrome protein 1 has been shown to be correlated to the incidence of Alstrom syndrome.
• the ALMS 1 gene which is located at 2p 13.1 and spans 23 exons, codes for the alstrom syndrome protein. Mutations in the ALMS1 gene resulting in deficient amounts of alstrom syndrome protein 1 have been shown to be responsible for the progression of Alstrom syndrome.
• Polymorphisms such as V671G, G1412A, II 875V, S2111R, D2672H and K3434E have been found in populations manifesting symptoms of Alstrom syndrome. These missense mutations were shown to result in decreased levels of alstrom syndrome protein 1, which provides a correlation between the deficiency of the alstrom syndrome protein 1 and the progression of Alstrom syndrome.
• the invention described herein can be used to treat the autosomal recessive disorder congenital bile acid synthesis defect 1 (CBAS1).
• CBAS1 is a primary defect in bile synthesis leading to progressive liver disease.
• Clinical features include neonatal jaundice, severe intrahepatic cholestasis, cirrhosis.
• Deficiency in the protein 3 beta-hydroxysteroid dehydrogenase type 7 (3BHS7) has been shown to be correlated to the incidence of CBAS1.
• the HSD3B7 gene which is located at 16pl 1.2 and spans 6 exons, codes for the 3BHS7 protein. Mutations in the HSD3B7 gene resulting in deficient amounts of 3BHS7 protein have been shown to be responsible for the progression of CBAS1. Polymorphisms such as G19S, E147K, T250A and L347P have been found in populations manifesting symptoms of CBAS1. These missense mutations were shown to result in decreased levels of 3BHS7, which provides a correlation between the deficiency of the 3BHS7 protein and the progression of CBAS1.
• RIC Pre-mRNA Retained Intron Containing Pre-mRNA
• the methods of the present invention can exploit the presence of retained- intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLPAPl, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene and encoding aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA
• dehydrogenase pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V, galactose- 1- phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O- glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain- containing protein 3, copper-transporting ATPase 2, fumarylacetoacetase, argininosuccinate lyase, hereditary hemochromatosis protein, alstrom syndrome protein 1, 3 beta-hydroxysteroid dehydrogenase type 7,
• dehydrogenase apolipoprotein A-V, galactose- 1 -phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor- 1 -alpha albulim proximal factor, protein O-glucosyltransferase 1, phosphatidylinositol 3 -kinase regulatory subunit 1, Tribbles-1, transforming growth factor beta-1, hemochromatosis type 2B, thrombopoietin, patatin-like phospholipase domain-containing protein 3, copper-transporting ATPase 2,
• AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene can be analyzed for intron-retention events.
• AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene can be analyzed for intron-retention events.
• RNA sequencing can be visualized in the UCSC genome browser, and can show AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A,
• HFE HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 transcripts expressed in human liver cells and localized in either the cytoplasmic or nuclear fraction.
• the retained-intron containing pre-mR A transcripts are retained in the nucleus and are not exported out to the cytoplasm
• a retained intron is an intron that is identified as a retained intron 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%, retention.
• a retained intron is an intron that is identified as a retained intron 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
• the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 intron numbering corresponds to the one or more mRNA sequences at shown in Table 1 or Table 2.
• THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA is in one or more introns as shown in Table 1 or Table 2.
• ASO to the targeted portion of the RIC pre-mRNA results in enhanced splicing at the splice site (5' splice site or 3' splice site) of one or more retained introns as shown in Table 1 or Table 2 and subsequently increases AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl,
• HNF4A HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH,
• ACADVL PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl,
• TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 isoform sequence One of skill in the art can determine the corresponding intron number in any isoform based on an intron sequence provided herein or using the number provided in reference to the one or more mRNA sequence at shown in Table 1 or Table 2.
• One of skill in the art also can determine the sequences of flanking exons in any AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC,
• HAMP HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or
• NCOA5 isoform for targeting using the methods of the invention, based on an intron sequence provided herein or using the intron number provided in reference to the one or more mRNA sequence at shown in
• the ASOs disclosed herein target a RIC pre-mRNA transcribed from a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence.
• the ASOs disclosed herein target a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA sequence.
• the ASO targets a sequence of a RIC pre-mRNA transcript encoded by a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence.
• the ASO targets a RIC pre-mRNA transcript encoded by a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or
• NCOA5 genomic sequence comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets a RIC pre-mRNA encoded by SEQ ID NOs: 1-31.
• the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 32-130. In some embodiments, the ASO targets a RIC pre-mRNA transcript of SEQ ID NO: 32-130 comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, the ASOs target SEQ ID NO: 32-130.
• the ASO comprises a sequence of any one of SEQ ID NOs:
• the ASO targets an exon sequence upstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence upstream (or 5') from the 5' splice site of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence about 4 to about 1000 nucleotides upstream (or 5') from the 5' splice site of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an intron sequence upstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre- mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an intron sequence downstream (or 3') from the 5' splice site of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence about 6 to about 500 nucleotides downstream (or 3') from the 5' splice site of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence downstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre- rriRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence downstream (or 3') from the 3' splice site of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the ASO targets an exon sequence about 2 to about 1000 nucleotides downstream (or 3') from the 3' splice site of a AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
• the methods described herein are used to increase the production of a functional protein.
• the term “functional” refers to the amount of activity or function of a protein that is necessary to eliminate any one or more symptoms of a treated condition.
• the methods are used to increase the production of a partially functional protein.
• the term “partially functional” refers to any amount of activity or function of the protein that is less than the amount of activity or function that is necessary to eliminate or prevent any one or more symptoms of a disease or condition.
• a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 90%, or at least 95% less activity relative to the fully functional protein or RNA.
• the method is a method of increasing the expression of the protein by cells of a subject having a RIC pre-mRNA encoding the protein, wherein the subject has a condition described herein caused by a deficient amount of activity of a protein described herein.
• the deficient amount of the protein is caused by haploinsufficiency of the protein.
• the subject has a first allele encoding a functional protein, and a second allele from which the protein is not produced.
• the subject has a first allele encoding a functional protein, and a second allele encoding a nonfunctional protein.
• the subject has a first allele encoding a functional protein, and a second allele encoding a partially functional protein.
• the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele (encoding functional protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding functional protein, and an increase in the expression of the protein in the cells of the subject.
• the subject has a first allele encoding a functional protein, and a second allele encoding a partially functional protein, and the antisense oligomer binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or a targeted portion of the RIC pre-mRNA transcribed from the second allele (encoding partially functional protein), thereby inducing constitutive splicing of the retained intron from the RIC pre-mRNA, and causing an increase in the level of mature mRNA encoding the protein, and an increase in the expression of functional or partially functional protein in the cells of the subject.
• the method is a method of using an ASO to increase the expression of a protein or functional RNA.
• an ASO is used to increase the expression of a protein described herein in cells of a subject having a RIC pre-mRNA encoding the protein, wherein the subject has a deficiency in the amount or function of the protein.
• the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein.
• a RIC pre-mRNA transcript that encodes a protein that is not causative of the disease is targeted by the ASOs.
• a disease that is the result of a mutation or deficiency of a first protein in a particular pathway may be ameliorated by targeting a RIC pre-mRNA that encodes a second protein, thereby increasing production of the second protein.
• the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which is causative of the disease or condition).
• the subject has:
• the protein is produced at a reduced level compared to production from a wild- type allele
• the protein is produced in a form having reduced function compared to an
• the protein is produced at a reduced level compared to production from a wild- type allele
• the protein is produced in a form having reduced function compared to an
• the RIC pre-mRNA is transcribed from the first allele and/or the second allele.
• the ASO binds to a targeted portion of the RIC pre-mRNA transcribed from the first allele or the second allele, thereby inducing constitutive splicing of the retained intron from the RIC pre- mRNA, and causing an increase in the level of mRNA encoding a protein and an increase in the expression of the target protein or functional RNA in the cells of the subject.
• the target protein or functional RNA having an increase in expression level resulting from the constitutive splicing of the retained intron from the RIC pre-mRNA is either in a form having reduced function compared to the equivalent wild-type protein (partially-functional), or having full function compared to the equivalent wild-type protein (fully-functional).
• the level of mRNA encoding a protein described herein is increased 1.1 to 10- fold, when compared to the amount of mRNA encoding the protein that is produced in a control cell, e.g., one that is not treated with the antisense oligomer or one that is treated with an antisense oligomer that does not bind to the targeted portion of the RIC pre-mRNA.
• the condition caused by a deficient amount or activity of a protein is not a condition caused by alternative or aberrant splicing of the retained intron to which the ASO is targeted.
• the condition caused by a deficient amount or activity of the protein is not a condition caused by alternative or aberrant splicing of any retained intron in a RIC pre-mRNA encoding the protein.
• alternative or aberrant splicing may occur in a pre-mRNA transcribed from the gene, however the compositions and methods of the invention do not prevent or correct this alternative or aberrant splicing.
• a subject treated using the methods of the invention expresses a partially functional protein from one allele, wherein the partially functional protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, or a partial gene deletion.
• a subject treated using the methods of the invention expresses a nonfunctional protein from one allele, wherein the nonfunctional protein is caused by a frameshift mutation, a nonsense mutation, a missense mutation, a partial gene deletion, in one allele.
• a subject treated using the methods of the invention has a whole gene deletion, in one allele.
• TANGO is used in the methods of the invention to increase expression of a protein.
• a retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding a protein is present in the nucleus of a cell.
• Cells having a RIC pre-mRNA that comprises a retained intron, an exon flanking the 5' splice site, and an exon flanking the 3' splice site, encoding the protein are contacted with antisense oligomers (ASOs) that are complementary to a targeted portion of the RIC pre-mRNA.
• ASOs antisense oligomers
• pre-mRNA and “pre-mRNA transcript” may be used interchangeably and refer to any pre-mRNA species that contains at least one intron.
• pre-mRNA or pre-mRNA transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail.
• pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A tail.
• the pre-mRNA transcript does not comprise a 5'-7-methylguanosine cap and/or a poly-A tail.
• a pre-mRNA transcript is a non-productive messenger RNA (mRNA) molecule if it is not translated into a protein (or transported into the cytoplasm from the nucleus).
• a "retained-intron-containing pre-mRNA” (“RIC pre-mRNA”) is a pre-mRNA transcript that contains at least one retained intron.
• the RIC pre-mRNA contains a retained intron, an exon flanking the 5' splice site of the retained intron, an exon flanking the 3 ' splice site of the retained intron, and encodes the target protein.
• An "RIC pre-mRNA encoding a target protein” is understood to encode the target protein when fully spliced.
• a “retained intron” is any intron that is present in a pre- mRNA transcript when one or more other introns, such as an adjacent intron, encoded by the same gene have been spliced out of the same pre-mRNA transcript.
• the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein.
• the retained intron is the most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
• an antisense oligomer targeted to the most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
• a mature mRNA encoding the target protein is thereby produced.
• the terms "mature mRNA,” and “fully-spliced mRNA,” are used interchangeably herein to describe a fully processed mRNA encoding a target protein (e.g., mRNA that is exported from the nucleus into the cytoplasm and translated into target protein) or a fully processed functional RNA.
• the term "productive mRNA,” also can be used to describe a fully processed mRNA encoding a target protein.
• the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the protein.
• the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature (e.g. in the case of an antisense oligomer, a defined nucleobase sequence) but not the exclusion of any other features.
• the term “comprising” is inclusive and does not exclude additional, unrecited features (e.g. in the case of an antisense oligomer, the presence of additional, unrecited nucleobases).
• compositions and methods comprising
• consisting may be replaced with “consisting essentially of " or “consisting of.”
• the phrase “consisting essentially of is used herein to require the specified feature(s) (e.g. nucleobase sequence) as well as those which do not materially affect the character or function of the claimed invention.
• the term “consisting” is used to indicate the presence of the recited feature (e.g. nucleobase sequence) alone (so that in the case of an antisense oligomer consisting of a specified nucleobase sequence, the presence of additional, unrecited nucleobases is excluded).
• the targeted region is in a retained intron that is the second most abundant intron in a RIC pre-mRNA encoding a protein described herein.
• the second most abundant retained intron may be targeted rather than the most abundant retained intron due to the uniqueness of the nucleotide sequence of the second most abundant retained intron, ease of ASO design to target a particular nucleotide sequence, and/or amount of increase in protein production resulting from targeting the intron with an ASO.
• the retained intron is the second most abundant intron in a population of RIC pre-mRNAs transcribed from the gene encoding the target protein in a cell, wherein the population of RIC pre-mRNAs comprises two or more retained introns.
• an antisense oligomer targeted to the second most abundant intron in the population of RIC pre-mRNAs encoding the target protein induces splicing out of two or more retained introns in the population, including the retained intron to which the antisense oligomer is targeted or binds.
• fully-spliced (mature) RNA encoding the target protein is thereby produced.
• an ASO is complementary to a targeted region that is within a non-retained intron in a RIC pre-mRNA.
• the targeted portion of the RIC pre-mRNA is within: the region +6 to +100 relative to the 5' splice site of the non-retained intron; or the region -16 to -100 relative to the 3 ' splice site of the non-retained intron.
• the targeted portion of the RIC pre- mRNA is within the region +100 relative to the 5' splice site of the non-retained intron to -100 relative to the 3' splice site of the non-retained intron.
• RNA encoding the target protein is thereby produced.
• the retained intron of the RIC pre-mRNA is an inefficiently spliced intron.
• "inefficiently spliced” may refer to a relatively low frequency of splicing at a splice site adjacent to the retained intron (5' splice site or 3' splice site) as compared to the frequency of splicing at another splice site in the RIC pre-mRNA.
• inefficiently spliced may also refer to the relative rate or kinetics of splicing at a splice site, in which an "inefficiently spliced" intron may be spliced or removed at a slower rate as compared to another intron in a RIC pre-mRNA.
• the 9-nucleotide sequence at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron is identical to the corresponding wild-type sequence.
• the 16 nucleotide sequence at -15 to -1 of the retained intron and +le of the exon flanking the 3' splice site is identical to the corresponding wild-type sequence.
• wild-type sequence refers to the nucleotide sequence for a gene in the published reference genome deposited in the NCBI repository of biological and scientific information (operated by National Center for Biotechnology Information, National Library of Medicine, 8600 Rockville Pike, Bethesda, MD USA 20894).
• a nucleotide position denoted with an "e” indicates the nucleotide is present in the sequence of an exon
• the methods involve contacting cells with an ASO that is complementary to a portion of a pre- mRNA encoding a protein described herein, resulting in increased expression of the protein.
• contacting or administering to cells refers to any method of providing an ASO in immediate proximity with the cells such that the ASO and the cells interact.
• a cell that is contacted with an ASO will take up or transport the ASO into the cell.
• the method involves contacting a condition or disease- associated or condition or disease-relevant cell with an ASO.
• the ASO may be further modified or attached (e.g., covalently attached) to another molecule to target the ASO to a cell type, enhance contact between the ASO and the condition or disease-associated or condition or disease- relevant cell, or enhance uptake of the ASO.
• the term “increasing protein production” or “increasing expression of a target protein” means enhancing the amount of protein that is translated from an mRNA in a cell.
• a “target protein” may be any protein for which increased expression/production is desired.
• contacting a cell that expresses a RIC pre-mRNA with an ASO that is complementary to a targeted portion of the RIC pre-mRNA transcript results in a measurable increase in the amount of a protein (e.g., a target protein) encoded by the pre-mRNA.
• a protein e.g., a target protein
• Methods of measuring or detecting production of a protein will be evident to one of skill in the art and include any known method, for example, Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA.
• contacting cells with an ASO that is complementary to a targeted portion of an RIC pre-mRNA transcript results in an increase in the amount of a protein produced by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
• the total amount of a protein produced by the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-
• contacting cells with an ASO that is complementary to a targeted portion of an RIC pre-mRNA transcript results in an increase in the amount of mRNA encoding a protein, including the mature mRNA encoding the target protein.
• the amount of mRNA encoding a protein, or the mature mRNA encoding the protein is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 1000%, compared to the amount of the protein produced by a cell in the absence of the ASO/absence of treatment.
• the total amount of the mRNA encoding a protein, or the mature mRNA encoding a protein produced in the cell to which the antisense oligomer was contacted is increased about 1.1 to about 10-fold, about 1.5 to about 10-fold, about 2 to about 10-fold, about 3 to about 10-fold, about 4 to about 10-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-
• the methods and antisense oligonucleotide compositions provided herein are useful for increasing the expression of a protein in cells, for example, in a subject having a condition described herein caused by a deficiency in the amount or activity of a protein described herein, by increasing the level of mRNA encoding the protein, or the mature mRNA encoding the protein.
• the methods and compositions as described herein induce the constitutive splicing of a retained intron from an RIC pre-mRNA transcript encoding the protein, thereby increasing the level of mRNA encoding the protein, or the mature mRNA encoding the protein and increasing the expression of the protein.
• Constitutive splicing of a retained intron from a RIC pre-mRNA correctly removes the retained intron from the RIC pre-mRNA, wherein the retained intron has wild-type splice sequences.
• Constitutive splicing does not encompass splicing of a retained intron from a RIC pre-mRNA transcribed from a gene or allele having a mutation that causes alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene or allele.
• constitutive splicing of a retained intron does not correct aberrant splicing in or influence alternative splicing of a pre-mRNA to result in an increased expression of a target protein or functional RNA.
• constitutive splicing correctly removes a retained intron from an RIC pre- mRNA, wherein the RIC pre-mRNA is transcribed from a wild-type gene or allele, or a polymorphic gene or allele, that encodes a fully-functional target protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
• constitutive splicing of a retained intron from an RIC pre-mRNA encoding a protein correctly removes a retained intron from an RIC pre-mRNA encoding the protein, wherein the RIC pre-mRNA is transcribed from a gene or allele from which the target gene or functional RNA is produced at a reduced level compared to production from a wild-type allele, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
• the correct removal of the constitutively spliced retained intron results in production of target protein or functional RNA that is functional when compared to an equivalent wild- type protein or functional RNA.
• constitutive splicing correctly removes a retained intron from an RIC pre- mRNA, wherein the RIC pre-mRNA is transcribed from a gene or allele that encodes a target protein or functional RNA produced in a form having reduced function compared to an equivalent wild-type protein or functional RNA, and wherein the gene or allele does not have a mutation that causes alternative splicing or aberrant splicing of the retained intron.
• the correct removal of the constitutively spliced retained intron results in production of partially functional target protein, or functional RNA that is partially functional when compared to an equivalent wild-type protein or functional RNA.
• an antisense oligomer as described herein or used in any method described herein does not increase the amount of mRNA encoding a protein or the amount of a protein by modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed from the gene.
• Modulation of alternative splicing or aberrant splicing can be measured using any known method for analyzing the sequence and length of RNA species, e.g., by RT-PCR and using methods described elsewhere herein and in the literature.
• modulation of alternative or aberrant splicing is determined based on an increase or decrease in the amount of the spliced species of interest of at least 10% or 1.1 -fold.
• modulation is determined based on an increase or decrease at a level that is at least 10% to 100% or 1.1 to 10-fold, as described herein regarding determining an increase in mRNA encoding the protein in the methods of the invention.
• the method is a method wherein the RIC pre-mRNA was produced by partial splicing of a wild-type pre-mRNA. In embodiments, the method is a method wherein the RIC pre-mRNA was produced by partial splicing of a full-length wild-type pre-mRNA. In embodiments, the RIC pre- mRNA was produced by partial splicing of a full-length pre-mRNA. In these embodiments, a full-length pre-mRNA may have a polymorphism in a splice site of the retained intron that does not impair correct splicing of the retained intron as compared to splicing of the retained intron having the wild-type splice site sequence.
• the mRNA encoding a protein is a full-length mature mRNA, or a wild-type mature mRNA.
• a full-length mature mRNA may have a polymorphism that does not affect the activity of the target protein or the functional RNA encoded by the mature mRNA, as compared to the activity of the protein encoded by the wild-type mature mRNA.
• composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of an RIC pre-mRNA.
• ASO antisense oligomer
• antisense oligomer are used interchangeably and refer to an oligomer such as a
• polynucleotide comprising nucleobases, that hybridizes to a target nucleic acid ⁇ e.g., an RIC pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U).
• the ASO may have exact sequence complementary to the target sequence or near complementarity ⁇ e.g., sufficient
• ASOs are designed so that they bind (hybridize) to a target nucleic acid ⁇ e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid).
• Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre- mRNA or transcriptome, such that the likelihood the ASO will bind other sites and cause "off-target” effects is limited.
• PCT/US2014/054151 published as WO 2015/035091, titled “Reducing Nonsense-Mediated mRNA Decay,” can be used to practice the methods described herein.
• ASOs "specifically hybridize” to or are “specific” to a target nucleic acid or a targeted portion of a RIC pre-mRNA.
• hybridization occurs with a Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
• Tm substantially greater than 37°C, preferably at least 50°C, and typically between 60°C to approximately 90°C.
• Such hybridization preferably corresponds to stringent hybridization conditions.
• the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
• Oligomers such as oligonucleotides, are "complementary" to one another 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 of the strands of the first polynucleotide and the second.
• Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion ⁇ e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
• ASO antisense oligomer
• ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least
• nucleic acid sequence to which they are targeted For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
• the remaining noncomplementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
• Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
• An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the
• an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript.
• an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.
• the ASOs described herein comprise nucleobases that are complementary to nucleobases present in a target portion of a RIC pre-mRNA.
• the term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• the ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding.
• nucleotides includes deoxyribonucleotides and ribonucleotides.
• modified nucleotides includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides.
• Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described herein will be evident to one of skill in the art and can be found, for example, in U.S. Patent No. 8,258,109 B2, U.S. Pat. No. 5,656,612, U.S. Pat. Pub. No. 2012/0190728, and Dias and Stein, Mol. Cancer Ther. 2002, 1, 347-355, herein incorporated by reference in their entirety.
• the nucleobase of an ASO may be any naturally occurring, unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.
• modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5- hydroxymethoylcytosine.
• the ASOs described herein also comprise a backbone structure that connects the components of an oligomer.
• backbone structure and "oligomer linkages” may be used interchangeably and refer to the connection between monomers of the ASO.
• the backbone comprises a 3 '-5' phosphodiester linkage connecting sugar moieties of the oligomer.
• the backbone structure or oligomer linkages of the ASOs described herein may include (but are not limited to) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
• the backbone structure of the ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups.
• PNA peptide nucleic acid
• the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
• the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus intemucleotide linkages of the ASO backbone is controlled and is not random.
• U.S. Pat. App. Pub. No. 2014/0194610 "Methods for the Synthesis of Functionalized Nucleic Acids," incorporated herein by reference, describes methods for independently selecting the handedness of chirality at each phosphorous atom in a nucleic acid oligomer.
• an ASO used in the methods of the invention comprises an ASO having phosphorus intemucleotide linkages that are not random.
• a composition used in the methods of the invention comprises a pure diastereomeric ASO.
• a composition used in the methods of the invention comprises an ASO that has diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
• the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus intemucleotide linkages. For example, it has been suggested that a mix of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan, et al., 2014, "Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages," Nucleic Acids Research 42(22): 13456-13468, incorporated herein by reference).
• an ASO used in the methods of the invention comprises about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15%
• Rp at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60%
• Rp at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, with the remainder Sp, or about 100%
• an ASO used in the methods of the invention comprises about 10% to about 100%
• Rp about 15% to about 100% Rp, about 20% to about 100% Rp, about 25% to about 100% Rp, about
• Rp 100% Rp, about 50% to about 100% Rp, about 55% to about 100% Rp, about 60% to about 100% Rp, about 65% to about 100% Rp, about 70% to about 100% Rp, about 75% to about 100% Rp, about 80% to about 100% Rp, about 85% to about 100% Rp, about 90% to about 100% Rp, or about 95% to about
• Rp 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about
• an ASO used in the methods of the invention 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, 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 remainder Rp, or about 100% Sp.
• an ASO used in the methods of the invention 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 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 remainder Rp.
• Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring.
• Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-0- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars.
• the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE.
• the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA).
• LNA locked nucleic acid
• the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO).
• PMO phosphorodiamidate morpholino
• the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification.
• the sugar moiety comprises 2'4' -constrained 2'0-methyloxyethyl
• the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications.
• the sugar moiety comprises tricycloDNA (tcDNA) modifications.
• the sugar moiety comprises ethylene nucleic acid (ENA) modifications.
• the sugar moiety comprises MCE modifications. Modifications are known in the art and described in the literature, e.g., by Jarver, et al., 2014, "A Chemical View of
• each monomer of the ASO is modified in the same way, for example each linkage of the backbone of the ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'0-methyl modification.
• Such modifications that are present on each of the monomer components of an ASO are referred to as "uniform modifications.”
• a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholinos).
• the ASO comprises one or more backbone modification. In some embodiments, the ASO comprises one or more sugar moiety modification. In some embodiments, the ASO comprises one or more backbone modification and one or more sugar moiety modification. In some embodiments, the ASO comprises 2'MOE modifications and a phosphorothioate backbone. In some embodiments, the ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, the ASO comprises a peptide nucleic acid (PNA).
• PMO phosphorodiamidate morpholino
• PNA peptide nucleic acid
• any of the ASOs or any component of an ASO ⁇ e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO.
• an ASO or one or more component of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (i.e., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and modulate the half-life of the ASO.
• the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE)
• ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein. See e.g., Geary et al, J Pharmacol Exp Ther. 2001; 296(3):890-7; Geary et al, J Pharmacol Exp Ther. 2001; 296(3):898-904. [00313] Methods of synthesizing ASOs will be known to one of skill in the art. Alternatively or in addition, ASOs may be obtained from a commercial source.
• the left-hand end of single-stranded nucleic acid e.g., pre-mRNA transcript, oligonucleotide, ASO, etc.
• sequences is the 5' end and the left-hand direction of single or double-stranded nucleic acid sequences is referred to as the 5' direction.
• the right-hand end or direction of a nucleic acid sequence is the 3' end or direction.
• 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.”
• nucleotides that are upstream of a reference point in a nucleic acid may be designated by a negative number, while nucleotides that are downstream of a reference point may be designated by a positive number.
• a reference point e.g., an exon-exon junction in mRNA
• a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one,” e.g., while a nucleotide that is directly adjacent and downstream of the reference point is designated “plus one,” e.g.,
• the ASOs are complementary to (and bind to) a targeted portion of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNF1A, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is downstream (in the 3 ' direction) of the 5' splice site of the retained intron in an AMT, ADA, PPOX, UROD, HMBS,
• ACADVL ACADVL
• PC IVD
• APOA5 GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA (e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1).
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region +6 to +100 relative to the 5' splice site of the retained intron.
• the ASO is not complementary to nucleotides +1 to +5 relative to the 5' splice site (the first five nucleotides located downstream of the 5' splice site).
• the ASOs may be complementary to a targeted portion of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFB 1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region between nucleotides +6 and +50 relative to the 5' splice site of the retained intron.
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP,
• THPO THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region +6 to +500, +6 to +400, +6 to +300, +6 to +200, or +6 to +90, +6 to
• +80 +6 to +70, +6 to +60, +6 to +50, +6 to +40, +6 to +30, or +6 to +20 relative to 5' splice site of the retained intron.
• the ASOs are complementary to a targeted region of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is upstream (5' relative) of the 3 ' splice site of the retained intron in an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, TH
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region -16 to -500, -16 to -400, -16 to -300, -6 to -200, or -16 to -100 relative to the 3 ' splice site of the retained intron.
• the ASO is not complementary to nucleotides -1 to -15 relative to the 3 ' splice site (the first 15 nucleotides located upstream of the 3 ' splice site).
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region -16 to -50 relative to the 3 ' splice site of the retained intron.
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region -16 to -90, -16 to -80, -16 to -70, -16 to -60, -16 to -50, -16 to -40, or -16 to -30 relative to 3 ' splice site of the retained intron.
• the targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIBl, TGFBl, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA is within the region +100 relative to the 5' splice site of the retained intron to - 100 relative to the 3 ' splice site of the retained intron.
• the ASOs are complementary to a targeted portion of an AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1,
• the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT,
• LDLRAPl HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIB1, TGFB1, HAMP, THPO, PNPLA3,
• the ASOs are not complementary to nucleotides -le to -3e relative to the 5' splice site of the retained intron. In some embodiments, the ASOs are complementary to a targeted portion of the
• AMT AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK,
• the ASOs are complementary to a targeted portion of an RIC pre-mRNA that is within the exon flanking the 3' splice site (downstream) of the retained intron (FIG. 1).
• the ASOs are complementary to a targeted portion to the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.
• the ASOs are not complementary to nucleotide +le relative to the 3' splice site of the retained intron. In some embodiments, the ASOs are complementary to a targeted portion of the AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAPl, HNF4A, GCK, POGLUTl, PIK3R1, HNFIA, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region+2e to +100e, +2e to +90e, +2e to +80e, +2e to +70e, +2e to +60e, +2e to +50e, +2e to +40e, +2e to +30e, or +2 to +
• the ASOs may be of any length suitable for specific binding and effective enhancement of splicing.
• the ASOs consist of 8 to 50 nucleobases.
• the ASO may be 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.
• the ASOs consist of more than 50 nucleobases.
• the ASO is from 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
• the ASOs are 30 nucleotides in length. In some embodiments, the ASOs are 29 nucleotides in length. In some embodiments, the ASOs are 28 nucleotides in length. In some embodiments, the ASOs are 27 nucleotides in length. In some embodiments, the ASOs are 26 nucleotides in length. In some embodiments, the ASOs are 25 nucleotides in length. In some embodiments, the ASOs are 24 nucleotides in length. In some embodiments, the ASOs are 23 nucleotides in length. In some embodiments, the ASOs are 22 nucleotides in length. In some embodiments, the ASOs are 21 nucleotides in length.
• two or more ASOs with different chemistries but complementary to the same targeted portion of the RIC pre-mRNA are used. In some embodiments, two or more ASOs that are complementary to different targeted portions of the RIC pre-mRNA are used.
• the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
• moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
• the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose ⁇ e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.
• a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose ⁇ e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon
• Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
• Linkers can include a bivalent or trivalent branched linker.
• the conjugate is attached to the 3' end of the antisense oligonucleotide.
• the nucleic acid to be targeted by an ASO is an RIC pre-mRNA expressed in a cell, such as a eukaryotic cell.
• the term "cell" may refer to a population of cells.
• the cell is in a subject.
• the cell is isolated from a subject.
• the cell is ex vivo.
• the cell is a condition or disease-relevant cell or a cell line.
• the cell is in vitro ⁇ e.g., in cell culture).
• compositions or formulations comprising the 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 published literature.
• a pharmaceutical composition or formulation for treating a subject comprises an effective amount of any antisense oligomer as described above, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof, and a pharmaceutically acceptable diluent.
• the antisense oligomer of a pharmaceutical formulation may further comprise a pharmaceutically acceptable excipient, diluent or carrier.
• salts are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et al., J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose.
• the salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting the free base function with a suitable organic acid.
• Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
• inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
• organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange.
• salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate,
• alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
• Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
• the compositions are formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
• the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media.
• Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
• the suspension may also contain stabilizers.
• a pharmaceutical formulation or composition of the present invention includes, but is not limited to, a solution, emulsion, microemulsion, foam or liposome- containing formulation (e.g., cationic or noncationic liposomes).
• the pharmaceutical composition or formulation of the present invention may comprise one or more penetration enhancer, carrier, excipients or other active or inactive ingredients as appropriate and well known to those of skill in the art or described in the published literature.
• liposomes also include sterically stabilized liposomes, e.g., liposomes comprising one or more specialized lipids. These specialized lipids result in liposomes with enhanced circulation lifetimes.
• a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
• a surfactant is included in the pharmaceutical formulation or compositions.
• the present invention employs a penetration enhancer to effect the efficient delivery of the antisense oligonucleotide, e.g., to aid diffusion across cell membranes and /or enhance the permeability of a lipophilic drug.
• the penetration enhancers is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant.
• the pharmaceutical formulation comprises multiple antisense oligonucleotides.
• the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.
• the antisense oligonucleotide is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of agents by administration of an adenovirus vector to motor neurons in muscle tissue is described in U.S. Pat. No. 6,632,427, "Adenoviral -vector- mediated gene transfer into medullary motor neurons," incorporated herein by reference.
• vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra
• Delivery of vectors directly to the brain e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, is described, e.g., in U.S. Pat. No. 6,756,523, "Adenovirus vectors for the transfer of foreign genes into cells of the central nervous system particularly in brain," incorporated herein by reference.
• the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
• the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor.
• the antisense oligonucleotide is linked with a viral vector, e.g., to render the antisense compound more effective or increase transport across the blood-brain barrier.
• the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, e.g., a targeting moiety or other conjugate that enhances the activity or cellular uptake of the oligonucleotide.
• moieties include, but are not limited to, a lipid moiety, e.g., as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, or adamantane acetic acid.
• the antisense oligonucleotide is conjugated with a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.
• a moiety including, but not limited to, an abasic nucleotide, a polyether, a polyamine, a polyamide, a peptides, a carbohydrate, e.g., N- acetylgalactosamine (GalNAc), N-Ac-Glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), a lipid, or a polyhydrocarbon compound.
• Conjugates can be linked to one or more of any nucleotides comprising the antisense oligonucleotide at any of several positions on the sugar, base or phosphate group, as understood in the art and described in the literature, e.g., using a linker.
• Linkers can include a bivalent or trivalent branched linker.
• the conjugate is attached to the 3' end of the antisense oligonucleotide.
• compositions provided herein may be administered to an individual.
• “Individual” may be used interchangeably with “subject” or "patient.”
• An individual may be a mammal, for example a human or animal such as a non-human primate, a rodent, a rabbit, a rat, a mouse, a horse, a donkey, a goat, a cat, a dog, a cow, a pig, or a sheep.
• the individual is a human.
• the individual is a fetus, an embryo, or a child.
• the individual may be another eukaryotic organism, such as a plant.
• the compositions provided herein are administered to a cell ex vivo.
• the compositions provided herein are administered to an individual as a method of treating a disease or disorder.
• the individual has a genetic disease, such as any of the diseases described herein.
• the individual is at risk of having the disease, such as any of the diseases described herein.
• the individual is at increased risk of having a disease or disorder caused by insufficient amount of a protein or insufficient activity of a protein. If an individual is "at an increased risk" of having a disease or disorder caused insufficient amount of a protein or insufficient activity of a protein, the method involves preventative or prophylactic treatment.
• an individual may be at an increased risk of having such a disease or disorder because of family history of the disease.
• individuals at an increased risk of having such a disease or disorder benefit from prophylactic treatment (e.g., by preventing or delaying the onset or progression of the disease or disorder).
• Suitable routes for administration of ASOs of the present invention may vary depending on cell type to which delivery of the ASOs is desired. Multiple tissues and organs can be affected in a condition described herein, with the liver being the most significantly affected tissue.
• the ASOs of the present invention may be administered to patients parenterally, for example, by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. In embodiments, delivery is to the heart or liver.
• a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
• ASOs that specifically hybridize to different nucleotides within the target region of the pre-mRNA may be screened to identify (determine) ASOs that improve the rate and/or extent of splicing of the target intron.
• the ASO may block or interfere with the binding site(s) of a splicing repressor(s)/silencer.
• Any method known in the art may be used to identify (determine) an ASO that when hybridized to the target region of the intron results in the desired effect (e.g., enhanced splicing, protein or functional RNA production). These methods also can be used for identifying ASOs that enhance splicing of the retained intron by binding to a targeted region in an exon flanking the retained intron, or in a non-retained intron. An example of a method that may be used is provided below.
• a round of screening may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
• the ASOs used in the ASO walk can be tiled every 5 nucleotides from approximately 100 nucleotides upstream of the 5' splice site of the retained intron (e.g., a portion of sequence of the exon located upstream of the target/retained intron) to approximately 100 nucleotides downstream of the 5' splice site of the target/retained intron and/or from approximately 100 nucleotides upstream of the 3 ' splice site of the retained intron to approximately 100 nucleotides downstream of the 3 ' splice site of the target/retained intron (e.g., a portion of sequence of the exon located downstream of the target/retained intron).
• a first ASO of 15 nucleotides in length may be designed to specifically hybridize to nucleotides +6 to +20 relative to the 5' splice site of the target/retained intron.
• a second ASO is designed to specifically hybridize to nucleotides +11 to +25 relative to the 5' splice site of the target/retained intron.
• ASOs are designed as such spanning the target region of the pre-mRNA.
• the ASOs can be tiled more closely, e.g., every 1 , 2, 3, or 4 nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of the 5' splice site, to 100 nucleotides upstream of the 3 ' splice site.
• One or more ASOs, or a control ASO are delivered, for example by transfection, into a disease- relevant cell line that expresses the target pre-mRNA (e.g., the RIC pre-mRNA described elsewhere herein).
• the splicing-inducing effects of each of the ASOs may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
• a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
• the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
• the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry,
• a second round of screening referred to as an ASO "micro-walk” may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA.
• the ASOs used in the ASO micro-walk are tiled every 1 nucleotide to further refine the nucleotide acid sequence of the pre- mRNA that when hybridized with an ASO results in enhanced splicing.
• Regions defined by ASOs that promote splicing of the target intron are explored in greater detail by means of an ASO "micro-walk", involving ASOs spaced in 1-nt steps, as well as longer ASOs, typically 18-25 nt.
• the ASO micro-walk is performed by delivering one or more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that is not expected to hybridize to the target region), 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 may be assessed by any method known in the art, for example by reverse transcriptase (RT)-PCR using primers that span the splice junction, as described herein (see “Identification of intron-retention events").
• a reduction or absence of the RT-PCR product produced using the primers spanning the splice junction in ASO-treated cells as compared to in control ASO-treated cells indicates that splicing of the target intron has been enhanced.
• the splicing efficiency, the ratio of spliced to unspliced pre-mRNA, the rate of splicing, or the extent of splicing may be improved using the ASOs described herein.
• the amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO achieved the desired effect (e.g., enhanced protein production). Any method known in the art for assessing and/or quantifying protein production, such as Western blotting, flow cytometry, immunofluorescence microscopy, and ELISA, can be used.
• ASOs that when hybridized to a region of a pre-mRNA result in enhanced splicing and increased protein production may be tested in vivo using animal models, for example transgenic mouse models in which the full-length human gene has been knocked-in or in humanized mouse models of disease.
• Suitable routes for administration of ASOs may vary depending on the disease and/or the cell types to which delivery of the ASOs is desired.
• ASOs may be administered, for example, by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
• the cells, tissues, and/or organs of the model animals may be assessed to determine the effect of the ASO treatment by for example evaluating splicing (efficiency, rate, extent) and protein production by methods known in the art and described herein.
• the animal models may also be any phenotypic or behavioral indication of the disease or disease severity.
• Example 1 Identification of intron retention events in AMT, ADA, PPOX, UROD, HMBS, ACADVL, PC, IVD, GALT, LDLRAP1, POGLUT1, PIK3R1, TRIB1, TGFB1, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 transcripts by RNAseq using next generation sequencing
• RNA from nuclear and cytoplasmic fractions of THLE-3 (human liver epithelial) cells was isolated and cDNA libraries constructed using Illumina's TruSeq Stranded mRNA library Prep Kit. The libraries were pair-end sequenced resulting in 100-nucleotide reads that were mapped to the human genome (Feb. 2009, GRCh37/hgl9 assembly). The mapped reads were visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for
• Example 2 Identification of intron retention events in APOA5, HNF4A, GCK, HNFIA, HAMP, and THPO transcripts by RNAseq using next generation sequencing
• transcriptome shotgun sequencing was carried out using next generation sequencing to reveal a snapshot of transcripts, e.g., those produced by the APOA5, HNF4A, GCK, HNFIA, HAMP, and THPO genes described herein, to identify intron-retention events.
• polyA+ RNA from nuclear and cytoplasmic fractions of THLE-3 (human liver epithelial) cells was isolated and cDNA libraries constructed using Illumina's TruSeq Stranded mRNA library Prep Kit. The libraries were pair- end sequenced resulting in 100-nucleotide reads that were mapped to the human genome (Feb. 2009, GRCh37/hgl9 assembly).
• the mapped reads were visualized using the UCSC genome browser (operated by the UCSC Genome Informatics Group (Center for Biomolecular Science & Engineering, University of California, Santa Cruz, 1156 Hgh Street, Santa Cruz, CA 95064) and described by, e.g., Rosenbloom, et al, 2015, "The UCSC Genome Browser database: 2015 update," Nucleic Acids Research 43, Database Issue, doi: 10.1093/nar/gkul 177) and the coverage and number of reads were inferred by the peak signals. The height of the peaks indicated the level of expression given by the density of the reads in a particular region.
• Example 3 Design of ASO-walk targeting a retained intron
• An ASO walk was designed to target a retained intron using the method described herein.
• a region immediately downstream of the intron 5' splice site e.g. , spanning nucleotides +6 to +69 and a region immediately upstream of intron 3 ' splice site, e.g. , spanning nucleotides -16 to -79 of the intron was targeted with 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by 5-nucleotide intervals.
• Table 1 lists retained introns in genes of interest in THLE-3 cells.
• Table 2 lists exemplary ASOs that were designed and their target sequences.
• AMT NM_001164710 2813 -3027 3 78483 SEQ ID NO.40 3028 -3110 5 78465
• AMT NM_001164711 3111 -3227 3 78434 SEQ ID NO.41 3228 -3310 5 78465
• AMT AMT NM_001164712 3311 -3427 4 78434 SEQ ID NO.6 SEQ ID NO.42 3428 -3510 6 78465
• AMT NM_000481 3511 -3627 4 78434 SEQ ID NO.43 3628 -3710 6 78465
• AMT NR_028435 3711 -3827 4 78434 SEQ ID NO.44 3828 -3910 6 78465
• THPO NM_001289998 5526 -5616 3 78400 SEQ ID NO.47 5617 -5704 4 78393
• THPO NM_001290027 12986 - 13076 3 78400 SEQ ID NO. 55 13077 - 13164 4 78393
• IL6 NM_000600 24306 -24541 2 78403 SEQ ID NO.75 24542 - 24726 3 78413
• GCK NM_033508 25942 -26144 5 78469 SEQ ID NO.76 26145 -26197 6 78408
• GCK NM_000162 27435 - 27652 2 78379 SEQ ID NO.77 27653 -27880 3 78431
• ASL NM_000048 31095 -31169 9 78404 SEQ ID NO.79 31170-31312 16 78371
• ASL NM_001024943 31428 -31570 15 78371 SEQ ID NO.80 31571 -31610 6 78365
• ASL NM_001024944 31804 -31843 6 78365 SEQ ID NO.81 31844 -31986 14 78371
• TRIBl SEQ ID NO.83 32871 -33566 2 78486 SEQ ID NO.15 TRIBl: NM_001282985 33567 -33824 1 78474
• HMBS NM_000190 37483 -37554 10 78355 SEQ ID NO.92 37555 -37632 11 78467
• HMBS NM_001258208
• HNF1A HNF1A: NM 000545
• ATP7B NM_000053 42616 -42863 13 78383 SEQ ID NO.99 42864 -43125 7 78427
• Example 4 Improved splicing efficiency via ASO-targeting of a retained intron increases AMT transcript levels
• ARPE-19 cells were mock-transfected, or transfected with AMT targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 3A and B). Taqman qPCR results showed that several targeting ASOs increase AMT gene transcript level compared to the mock-transfected.
• Example 5 Improved splicing efficiency via ASO-targeting of a retained intron increases GALT transcript levels
• Example 6 Improved splicing efficiency via ASO-targeting of a retained intron increases PC transcript levels
• Example 7 Improved splicing efficiency via ASO-targeting of a retained intron increases FAH transcript levels
• Example 8 Improved splicing efficiency via ASO-targeting of a retained intron increases PPARD transcript levels
• ARPE-19 cells were mock-transfected, or transfected with PPARD targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 7A and B, FIG. 7E and F). Taqman qPCR results showed that several targeting ASOs increase PPARD gene transcript level compared to the mock-transfected.
• HMBS HMBS gene transcript level compared to the mock-transfected.
• Example 10 Improved splicing efficiency via ASO-targeting of a retained intron increases ALMSl transcript levels
• ARPE-19 cells were mock-transfected, or transfected with ALMSl targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 11 A and B). Taqman qPCR results showed that several targeting ASOs increase ALMSl gene transcript level compared to the mock-transfected.
• Example 11 Improved splicing efficiency via ASO-targeting of a retained intron increases ASL transcript levels
• ARPE-19 cells were mock-transfected, or transfected with ASL targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents. Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 12A and B, FIG. 12C and D, FIG. 12E and F). Taqman qPCR results showed that several targeting ASOs increase ASL gene transcript level compared to the mock-transfected.
• Example 12 Improved splicing efficiency via ASO-targeting of a retained intron increases ATP7B transcript levels
• Example 13 Improved splicing efficiency via ASO-targeting of a retained intron increases
• HSD3B7 To determine whether an increase in expression of HSD3B7 could be achieved by improving splicing efficiency of a retained intron using ASOs, the methods described herein were used.
• ARPE-19 cells were mock-transfected, or transfected with HSD3B7 targeting ASOs, or a non-targeting ASO control, independently, using RNAiMAX (Invitrogen) delivery reagents.
• RNAiMAX Invitrogen
• Example 14 Improved splicing efficiency via ASO-targeting of a retained intron increases transcript levels [00354]
• ARPE-19 cells a human retinal epithelium cell line (American Type Culture Collection (ATCC), USA), or Huh-7, a human hepatoma cell line (NIBIOHN, Japan), or SK-N-AS, a human neuroblastoma cell line (ATCC) were mock-transfected, or transfected with the targeting ASOs described in FIGs. 3-17 and Tables 1 and 2.
• RNAiMax transfection reagent (Thermo Fisher) according to vendor's specifications. Briefly, ASOs were plated in 96-well tissue culture plates and combined with RNAiMax diluted in Opti-MEM. Cells were detached using trypsin and resuspended in full medium, and approximately 25,000 cells were added the ASO-transfection mixture. Transfection experiments were carried out in triplicate plate replicates. Final ASO concentration was 80 nM. Media was changed 6h post-transfection, and cells harvested at 24h, using the Cells-to-Ct lysis reagent, supplemented with DNAse (Thermo Fisher), according to vendor's specifications.
• cDNA was generated with Cells-to-Ct RT reagents (Thermo Fisher) according to vendor's specifications.
• quantitative PCR was carried out using Taqman assays with probes spanning the corresponding exon-ex on junction (Thermo Fisher) of the reetained intron listed in Tables 1 and 2.
• Taqman assays were carried out according to vendor's specifications, on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher).
• Target gene assay values were normalized to RPL32 (deltaCt) and plate-matched mock transfected samples (delta-delta Ct), generating fold-change over mock quantitation (2 A -(delta- deltaCt). Average fold-change over mock of the three plate replicates was plotted (FIG. 3B, FIG. 4D, FIG. 4H, FIG. 5B, FIG. 6B, FIG. 7F, FIG. 9B, FIG. 11B, FIG. 12B, FIG. 12D, FIG. 12F, FIG. 13C, FIC. 15C and FIG. 15F).
• ASOs were identified that increase the target gene expression, implying an increase in splicing at that target intron. Together with whole transcriptome data showing retention of the target intron (FIG. 3-17), these results confirm that ASOs can improve the splicing efficiency of a rate limiting intron.

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