CA3005090A1 - Compositions and methods for treatment of liver diseases - Google Patents

Abstract

Provided herein are methods and compositions for increasing the expression of a protein, and for treating a subject in need thereof, e.g., a subject with deficient protein expression or a subject having a liver disease.

Description

COMPOSITIONS AND METHODS FOR TREATMENT OF LIVER DISEASES
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No.
62/267,238, filed December 14, 2015, and U.S. Provisional Application No. 62/319,015, filed April 6, 2016, which applications are incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII
format via EFS-Web and is hereby incorporated by reference in its entirety.
Said ASCII copy, created on December 12, 2016, is named 47991-714 601 SL.txt and is 24,385,314 bytes in size. The aforementioned file was created on December 12, 2016, and is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION

[0003] 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.

[0004] 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. Examples of gene products for which increased expression can provide benefit in liver diseases or conditions include AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5.
SUMMARY OF THE INVENTION

[0005] In one aspect, provided herein is a method of treating a liver disease in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site, an exon flanking the 3' splice site, and wherein the RIC pre-mRNA encodes the target protein or functional RNA, the method comprising contacting the cells of the subject with an antisense oligomer (ASO) complementary to a targeted portion of the RIC
pre-mRNA encoding the target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject.

[0006] In some embodiments, 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.

[0007] In one aspect, provided herein is a method of increasing expression of a target protein by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the 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 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, 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-l-alpha albulim proximal factor, 0-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.

[0008] In some embodiments, 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-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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.

[0009] In some embodiments, 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.

[0010] In some embodiments, the cells are in or from a subject having a condition caused by a deficient amount or activity of the target protein.

[0011] In some embodiments, 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.

[0012] In some embodiments, 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 transcribed from either the first mutant allele that is a(i) or a(ii), and/or the second allele that is b(i) or b(ii)

[0013] In some embodiments, the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.

[0014] In some embodiments, the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.

[0015] In some embodiments, 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.

[0016] In some embodiments, 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.

[0017] In some embodiments, 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.

[0018] In some embodiments, 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.

[0019] In some embodiments, 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.

[0020] In some embodiments, 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.

[0021] In some embodiments, the target protein is (a) AMT, (b) ADA, (c) PPDX, (d) UROD, (e) EIMBS, (f) ACADVL, (g) PC, (h) IVD, (i) AP0A5, (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) FIFE, (z) ALMS1, (aa) PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.

[0022] In some embodiments, 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, (i) 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 of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID NOs 32469-34520, (r) any one of SEQ ID
NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ
ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 - 44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ
ID NOs 1780 -1899, or any one of SEQ ID NOs 67282 - 67531.
100231 In some embodiments, 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; (i) SEQ ID NO 78407, 78499, 78420, 78372 or 78397; (j) SEQ ID NO 78489, 78416, 78476, 78352, 78435, 78493, 78423, 78437 or 78449; (k) SEQ ID NO 78354 or 78459; (1) SEQ ID NO 78441, 78392, 78456, 78428, 78491, 78501, 78360, 78429, 78358, 78364, 78475, 78391, 78479, 78401, 78373 or 78450;
(m) SEQ ID NO
78445, 78481, 78379, 78431, 78469, 78408, 78377, 78417, 78387, 78455, 78484 or 78370; (n) SEQ ID
NO 78368, 78350, 78432, 78439 or 78389; (o) SEQ ID NO 78390 or SEQ ID NO
78418, (p) SEQ ID
NO 78462, 78468, 78453, 78361, 78363, 78433, 78438, 78430, 78488, 78405, 78492 or 78427; (q) SEQ
ID NO 78487, 78486 or 78474; (r) SEQ ID NO 78458, (s) SEQ ID NO 78497 or 78426; (t) SEQ ID NO
78425, 78400, 78393, 78351, 78381, 78366, 78457, 78443, 78362 or 78446; (u) SEQ ID NO 78388, 78402, 78471 or 78356; (v) SEQ ID NO 78427, 78383, 78444 or 78394; (w) SEQ ID
NO 78382; (x) SEQ ID NO 78494, 78404, 78371, 78365 or 78353; (y) SEQ ID NO 78419, 78384, 78500, 78424 or 78466; (z) SEQ ID NO 78399, (aa) SEQ ID NO 78414, 78478, 78472, 78359, 78395, 78357, 78374 or 78490; (bb) SEQ ID NO 78436, 78413, 78415, 78398 or 78403; (cc) SEQ ID NO
78349, 78470, 78498, 78406, 78442, 78451, 78396, 78409 or 78378; (dd) SEQ ID NO 78447; or SEQ ID NO
78452.
[0024] In some embodiments, 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, (i) 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 of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID NOs 32469-34520, (r) any one of SEQ ID NOs 51972- 52025, (s) any one of SEQ
ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ
ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 -44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID
NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID
NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ
ID NOs 67282 - 67531.

[0025] In some embodiments, 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, (i) SEQ ID NOs 90 or 91, (j) SEQ ID NOs 85 or 86, (k) SEQ ID NO 32, (1) SEQ ID NOs 115-120 or126-129, (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 97, (q) SEQ ID NOs 83 or 84, (r) SEQ ID NO 114, (s) SEQ ID NO 223, (t) SEQ ID NOs 47-56, (u) SEQ ID
NO 130, (v) SEQ ID
NOs 98-102, (w) SEQ ID NO 103, (x) SEQ ID NOs 80-82, (y) SEQ ID NOs 66-73, (z) SEQ ID NO 39, (aa) SEQ ID NOs 61-65, (bb) SEQ ID NOs 74 or 75, (cc) SEQ ID NOs 106-108, (dd) SEQ ID NOs 35 or 36, or SEQ ID NO 125.
[0026] In some embodiments, 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, (i) 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) SEQ ID NO 5, (aa) SEQ ID NO 10, (bb) SEQ ID NO 12, (cc) SEQ ID NO 24, (dd) SEQ ID NO 3, or (ee) SEQ ID NO 29.
[0027] In some embodiments, 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.
[0028] In some embodiments, 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.
[0029] In some embodiments, the RIC pre-mRNA was produced by partial splicing of a full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.
[0030] In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
[0031] In some embodiments, the target protein produced is full-length protein, or wild-type protein.
[0032] In some embodiments, 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 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.
[0033] In some embodiments, 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 5-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.
[0034] In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
[0035] In some embodiments, 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.
[0036] In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
[0037] In some embodiments, each sugar moiety is a modified sugar moiety.
[0038] In some embodiments, 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, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
[0039] In some embodiments, 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.
[0040] In some embodiments, 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.

[0041] In some embodiments, 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.
[0042] In some embodiments, 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.
[0043] In some embodiments, 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.
[0044] In some embodiments, the condition is a disease or disorder.
[0045] In some embodiments, the disease or disorder is a liver disease.
[0046] In some embodiments, 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.
[0047] In some embodiments, the target protein and the RIC pre-mRNA are encoded by a gene, wherein the gene is AMT, ADA, PPDX, UROD, HMIBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
[0048] In some embodiments, the method further comprises assessing protein expression.
[0049] In some embodiments, the subject is a human.
[0050] In some embodiments, the subject is a non-human animal.
[0051] In some embodiments, the subject is a fetus, an embryo, or a child.
[0052] In some embodiments, the cells are ex vivo.
[0053] In some embodiments, the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.

[0054] In some embodiments, the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence.
[0055] In some embodiments, 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.
[0056] In one aspect, provided herein is an antisense oligomer as used in a method described herein.
[0057] In one aspect, provided herein is an antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs 131-78348.
[0058] In one aspect, provided herein is a pharmaceutical composition comprising an antisense oligomer described herein and an excipient.
[0059] In one aspect, provided herein is a method of treating a subject in need thereof by administering a pharmaceutical composition described herein by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[0060] In one aspect, provided herein is a 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 exon flanking the 3' splice site, and wherein the retained intron is spliced from the RIC pre-mRNA
encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject.
[0061] In some embodiments, 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.
[0062] In one aspect, provided herein is a 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.
[0063] In some embodiments, 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-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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.
[0064] In some embodiments, the condition is a disease or disorder.
[0065] In some embodiments, the disease or disorder is a liver disease.
[0066] In some embodiments, 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.
[0067] In some embodiments, the target protein and RIC pre-mRNA are encoded by a gene, wherein the gene is AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, E1FE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5.
[0068] In some embodiments, 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.
[0069] In some embodiments, 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.
[0070] In some embodiments, 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 [0071] In some embodiments, 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.
[0072] In some embodiments, 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.
[0073] In some embodiments, the target protein is (a) AMT, (b) ADA, (c) PPDX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i) AP0A5, (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) EWE, (z) ALMS1, (aa) PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.
[0074] In some embodiments, 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, (i) 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 of SEQ ID
NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID
NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID NOs 32469-34520, (r) any one of SEQ
ID NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 - 44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID
NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID
NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ ID NOs 67282 - 67531.
100751 In some embodiments, 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; (0 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;
(i) SEQ ID NO 78407, 78499, 78420, 78372 or 78397; (j) SEQ ID NO 78489, 78416, 78476, 78352, 78435, 78493, 78423, 78437 or 78449; (k) SEQ ID NO 78354 or 78459; (1) SEQ ID
NO 78441, 78392, 78456, 78428, 78491, 78501, 78360, 78429, 78358, 78364, 78475, 78391, 78479, 78401, 78373 or 78450; (m) SEQ ID NO 78445, 78481, 78379, 78431, 78469, 78408, 78377, 78417, 78387, 78455, 78484 or 78370; (n) SEQ ID NO 78368, 78350, 78432, 78439 or 78389; (o) SEQ ID NO
78390 or SEQ ID NO
78418, (p) SEQ ID NO 78462, 78468, 78453, 78361, 78363, 78433, 78438, 78430, 78488, 78405, 78492 or 78427; (q) SEQ ID NO 78487, 78486 or 78474; (r) SEQ ID NO 78458, (s) SEQ ID
NO 78497 or 78426; (t) SEQ ID NO 78425, 78400, 78393, 78351, 78381, 78366, 78457, 78443, 78362 or 78446; (u) SEQ ID NO 78388, 78402, 78471 or 78356; (v) SEQ ID NO 78427, 78383, 78444 or 78394; (w) SEQ ID
NO 78382; (x) SEQ ID NO 78494, 78404, 78371, 78365 or 78353; (y) SEQ ID NO
78419, 78384, 78500, 78424 or 78466; (z) SEQ ID NO 78399, (aa) SEQ ID NO 78414, 78478, 78472, 78359, 78395, 78357, 78374 or 78490; (bb) SEQ ID NO 78436, 78413, 78415, 78398 or 78403;
(cc) SEQ ID NO
78349, 78470, 78498, 78406, 78442, 78451, 78396, 78409 or 78378; (dd) SEQ ID
NO 78447; or SEQ ID
NO 78452.
[0076] In some embodiments, 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, (i) 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 of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID NOs 32469-34520, (r) any one of SEQ ID NOs 51972- 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID
NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID
NOs 44371 -44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ ID
NOs 1780 -1899, or any one of SEQ ID NOs 67282 - 67531.
[0077] In some embodiments, 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, (0 SEQ ID NOs 109-112, (g) SEQ ID NOs 87-89, (h) SEQ ID NOs 104 or 105, (i) SEQ ID NOs 90 or 91, (j) SEQ ID NOs 85 or 86, (k) SEQ ID NO 32, (1) SEQ ID NOs 115-120 or126-129, (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 97, (q) SEQ ID NOs 83 or 84, (r) SEQ ID NO 114, (s) SEQ ID NO 223, (t) SEQ ID NOs 47-56, (u) SEQ ID
NO 130, (v) SEQ ID
NOs 98-102, (w) SEQ ID NO 103, (x) SEQ ID NOs 80-82, (y) SEQ ID NOs 66-73, (z) SEQ ID NO 39, (aa) SEQ ID NOs 61-65, (bb) SEQ ID NOs 74 or 75, (cc) SEQ ID NOs 106-108, (dd) SEQ ID NOs 35 or 36, or SEQ ID NO 125.
[0078] In some embodiments, 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, (i) 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) SEQ ID NO 5, (aa) SEQ ID NO 10, (bb) SEQ ID NO 12, (cc) SEQ ID NO 24, (dd) SEQ ID NO 3, or (ee) SEQ ID NO 29.
[0079] In some embodiments, 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.
[0080] In some embodiments, 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.
[0081] In some embodiments, the RIC pre-mRNA was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.
[0082] In some embodiments, the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.
[0083] In some embodiments, the target protein produced is full-length protein, or wild-type protein.
[0084] In some embodiments, the retained intron is a rate-limiting intron.
[0085] In some embodiments, 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.
[0087] In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
[0088] In some embodiments, said antisense oligomer is an antisense oligonucleotide.
[0089] In some embodiments, 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.
[0090] In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
[0091] In some embodiments, each sugar moiety is a modified sugar moiety.
[0092] In some embodiments, 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, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
[0093] In some embodiments, 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.
[0094] In one aspect, provided herein is a pharmaceutical composition comprising an antisense oligomer of any of the compositions described herein, and an excipient.
[0095] In one aspect, provided herein is a method of treating a subject in need thereof by administering a pharmaceutical composition described herein by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[0096] In one aspect, provided herein is a pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient AMT, ADA, PPDX, UROD, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 mRNA transcript, wherein the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript; and a pharmaceutical acceptable excipient.
[0097] In some embodiments, the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
transcript.
[0098] In some embodiments, the targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, 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.
[0099] In some embodiments, the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, 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.
[00100] In some embodiments, the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
or 100% sequence identity to any one of SEQ ID NOs: 32-130.
1001011 In some embodiments, the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.
[00102] In some embodiments, the antisense oligomer is an antisense oligonucleotide.
[00103] In some embodiments, 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.
[00104] In some embodiments, the antisense oligomer comprises at least one modified sugar moiety.
[00105] In some embodiments, 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 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.
[00106] In some embodiments, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript.
[00107] In some embodiments, the targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 RIC pre-mRNA transcript is within a sequence selected from SEQ ID
NOs: 78349-78501.
[00108] In some embodiments, 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.
[00109] In some embodiments, the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 131-78348.
[00110] In some embodiments, the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
[00111] In one aspect, provided herein is a method of inducing processing of a deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript to facilitate removal of a retained intron to produce a fully processed AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript that encodes a functional form of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein, the method comprising: contacting an antisense oligomer to a target cell of a subject; hybridizing the antisense oligomer to the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript, wherein the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript is capable of encoding the functional form of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 protein and comprises at least one retained intron; removing the at least one retained intron from the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript to produce the fully processed AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript that encodes the functional form of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein; and translating the functional form of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein from the fully processed AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript.
[00112] In some embodiments, the retained intron is an entire retained intron.
[00113] In some embodiments, the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
transcript.
[00114] In one aspect, provided herein is a method of treating a subject having a condition caused by a deficient amount or activity of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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.

INCORPORATION BY REFERENCE
[00115] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[00116] The novel features of the invention are set forth with particularity in the appended claims.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.
[00117] 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). In embodiments, 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.
[00118] 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. In the nucleus, a pre-mRNA transcript of a target gene consisting of exons (rectangles) and introns (connecting lines) undergoes splicing to generate an mRNA, and this mRNA is exported to the cytoplasm and translated into target protein. For this target gene, 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.
[00119] 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.

[00120] FIG. 3A depicts a schematic of the RefSeq Genes for AMT corresponding to NM 001164710, NM 00116471,1 NM 001164712, NM 000481 and NR 028435.The Percent Intron Retention (PIR) of the circled intron is shown.
[00121] FIG. 3B depicts an exemplary graph showing the average (n=3) fold change in expression levels of AMT mRNA without intron 4 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 4-exon 5 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00122] 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.The Percent Intron Retention (PIR) of the circled intron is shown.
[00123] 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).
[00124] 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).
[00125] 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).
[00126] FIG. 4D depicts an exemplary graph showing the average (n=3) fold change in expression levels of GALT mRNA without intron 4 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 4¨exon 5 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00127] 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).
[00128] FIG. 4F 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 7, NM 000155).
[00129] 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).
[00130] FIG. 411 depicts an exemplary graph showing the average (n=3) fold change in expression levels of GALT mRNA without intron 8 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 8-exon 9 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.

[00131] FIG. 41 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 9, NM 000155).
[00132] FIG. 4J 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 10, NM 000155).
[00133] 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).
[00134] FIG. 5B depicts an exemplary graph showing the average (n=3) fold change in expression levels of PC mRNA without intron 16 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 16¨exon 17 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00135] FIG. 6A depicts a schematic of the RefSeq Genes for FAH corresponding to FAH:
NM 000137. The Percent Intron Retention (PIR) of the circled intron is shown (FAH intron 1, NM 000137).
[00136] FIG. 6B depicts an exemplary graph showing the average (n=3) fold change in expression levels of FAH mRNA without intron 1 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 1¨exon 2 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00137] FIG. 7A depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD:
NM 006238, NM 177435, NM 001171818, NM 001171819 and NM 001171820. The Percent Intron Retention (PIR) of the circled intron is shown (PPARD intron 3, NM 006238).
[00138] FIG. 7B depicts an exemplary graph showing the average (n=3) fold change in expression levels of PPARD mRNA without intron 3 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs over mock treated cells. Data is normalized to RPL32 expression.
[00139] FIG. 7C depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD:
NM 006238, NM 177435, NM 001171818, NM 001171819 and NM 001171820. The Percent Intron Retention (PIR) of the circled intron is shown (PPARD intron 4, NM 006238).
[00140] FIG. 7D depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD:
NM 006238, NM 177435, NM 001171818, NM 001171819 and NM 001171820. The Percent Intron Retention (PIR) of the circled intron is shown (PPARD intron 5, NM 006238).
[00141] FIG. 7E depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD:
NM 006238, NM 177435, NM 001171818, NM 001171819 and NM 001171820. The Percent Intron Retention (PIR) of the circled intron is shown (PPARD intron 6, NM 006238).

[00142] FIG. 7F depicts an exemplary graph showing the average (n=3) fold change in expression levels of PPARD mRNA without intron 6 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs over mock treated cells. Data is normalized to RPL32 expression.
1001431 FIG. 7G depicts a schematic of the RefSeq Genes for PPARD
corresponding to PPARD:
NM 006238, NM 177435, NM 001171818, NM 001171819 and NM 001171820. The Percent Intron Retention (PIR) of the circled intron is shown (PPARD intron 7, NM 006238).
[00144] 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).
[00145] 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).
[00146] 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).
[00147] 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).
[00148] 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).
[00149] 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).
[00150] 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).
[00151] FIG. 811 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).
[00152] FIG. 9A depicts a schematic of the RefSeq Genes for HMBS corresponding to HMBS:
NM 000190, NM 001258208, NM 001024382 and NM 001258209. The Percent Intron Retention (PIR) of the circled intron is shown (HMBS intron 10, NM 000190).
[00153] FIG. 9B depicts an exemplary graph showing the average (n=3) fold change in expression levels of HMBS mRNA without intron 10 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 10¨exon 11 splice junction) over mock treated cells. Data is normalized to RPL32 expression.
[00154] FIG. 9C depicts a schematic of the RefSeq Genes for HMBS corresponding to HMBS:
NM 000190, NM 001258208, NM 001024382 and NM 001258209. The Percent Intron Retention (PIR) of the circled intron is shown (HMBS intron 11, NM 000190).
[00155] FIG. 10A depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD:
NM 000374 and NR 036510. The Percent Intron Retention (PIR) of the circled intron is shown (UROD
intron 3, NM 0003 74 ).
[00156] FIG. 10B depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD:
NM 000374 and NR 036510. The Percent Intron Retention (PIR) of the circled intron is shown (UROD
intron 4, NM 0003 74 ).
[00157] FIG. 10C depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD:
NM 000374 and NR 036510. The Percent Intron Retention (PIR) of the circled intron is shown (UROD
intron 5 NM 000374).
[00158] FIG. 10D depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD:
NM 000374 and NR 036510. The Percent Intron Retention (PIR) of the circled intron is shown (UROD
intron 6, NM 0003 74 ).
[00159] FIG. 10E depicts a schematic of the RefSeq Genes for UROD
corresponding to UROD:
NM 000374 and NR 036510. The Percent Intron Retention (PIR) of the circled intron is shown (UROD
intron 7, NM 0003 74 ).
[00160] 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).
[00161] FIG. 11B depicts an exemplary graph showing the average (n=3) fold change in expression levels of ALMS1 mRNA without intron 21 in Huh7 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 21¨exon 22 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00162] 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).
[00163] FIG. 12B depicts an exemplary graph showing the average (n=3) fold change in expression levels of ASL mRNA without intron 7 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 7¨exon 8 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.

[00164] FIG. 12C 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 8, NM 000048).
[00165] FIG. 12D depicts an exemplary graph showing the average (n=3) fold change in expression levels of ASL mRNA without intron 8 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 8¨exon 9 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00166] 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).
[00167] FIG. 12F depicts an exemplary graph showing the average (n=3) fold change in expression levels of ASL mRNA without intron 7 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 9¨exon 10 splice junction) over mock treated cells.
Data is normalized to RPL32 expression.
[00168] 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).
[00169] FIG. 13A depicts a schematic of the RefSeq Genes for ATP7B1 corresponding to ATP7B1:
NM 001243182, NM 000053, NM 001005918, NM 001330579 and NM 001330578. The Percent Intron Retention (PIR) of the circled intron is shown (ATP7B1 intron 7, NM
000053).
[00170] FIG. 13B depicts a schematic of the RefSeq Genes for ATP7B1 corresponding to ATP7B1:
NM 001243182, NM 000053, NM 001005918, NM 001330579 and NM 001330578. The Percent Intron Retention (PIR) of the circled intron is shown (ATP7B1 intron 13, NM
000053).
[00171] FIG. 13C depicts an exemplary graph showing the average (n=3) fold change in expression levels of ATP7B1 mRNA without intron 13 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 13¨exon 14 splice junction) over mock treated cells. Data is normalized to RPL32 expression.
[00172] 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).
[00173] 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).
[00174] 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).

23 [00175] FIC. 15C depicts an exemplary graph showing the average (n=3) fold change in expression levels of HSD3B7 mRNA without intron 2 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 2¨exon 3 splice junction) over mock treated cells. Data is normalized to RPL32 expression.
[00176] 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).
[00177] 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).
[00178] FIG. 15F depicts an exemplary graph showing the average (n=3) fold change in expression levels of HSD3B7 mRNA without intron 4 in ARPE-19 cells treated for 24 hrs with 80 nM of the indicated ASOs (spanning the exon 4¨exon 5 splice junction) over mock treated cells. Data is normalized to RPL32 expression.
[00179] 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).
[00180] FIG. 1511 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).
[00181] 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).
[00182] FIG. 17A depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 3, 000309).
[00183] FIG. 17B depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 4, 000309).
[00184] FIG. 17C depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 5, 000309).
[00185] FIG. 17D depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 7, 000309).

24 [00186] FIG. 17E depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 8, 000309).
[00187] FIG. 17F depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 10, 000309).
[00188] FIG. 17G depicts a schematic of the RefSeq Genes for PPDX
corresponding to PPDX:
NM 000309 and NM 001122764. The Percent Intron Retention (PIR) of the circled intron is shown (PPDX intron 12, 000309).
DETAILED DESCRIPTION OF THE INVENTION
[00189] Liver disease is a debilitating condition that results in an estimated 60,000 deaths per year in the United States alone. In many cases, 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.
[00190] Individual 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.
[00191] Substantial levels of partially-spliced transcripts of the gene, which encodes a protein that is deficient in a subset of liver diseases, have been discovered in the nucleus of human cells. These pre-mRNA species comprise at least one retained intron. The present invention provides 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. These compositions and methods utilize antisense oligomers (AS0s) that promote constitutive splicing at an intron splice site of a retained-intron-containing pre-mRNA that accumulates in the nucleus. Thus, in embodiments, protein is increased using the methods of the invention to treat a condition caused by a protein deficiency.
[00192] 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.

[00193] These AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 pre-mRNA species comprise at least one retained intron. The present invention provides compositions and methods for upregulating splicing of one or more retained AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, 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 dehydrogenase, pyruvate carboxylase isovaleryl-CoA
dehydrogenase, apolipoprotein A-V, galactose-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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 protein levels. These compositions and methods can utilize antisense oligomers (AS0s) that promote constitutive splicing at intron splice sites of a retained-intron-containing AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus. Thus, in embodiments, 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-l-alpha albulim proximal factor, protein 0-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 protein can be increased using the methods of the invention to treat a condition caused by 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-l-alpha albulim proximal factor, protein 0-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.
[00194] In some embodiments, disclosed herein are compositions and methods for upregulating splicing of one or more retained AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or 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 AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein levels. These compositions and methods can utilize antisense oligomers (AS0s) that promote constitutive splicing at intron splice sites of a retained-intron-containing AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 pre-mRNA (RIC pre-mRNA) that accumulates in the nucleus. Thus, in embodiments, AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 deficiency.
[00195] In other embodiments, the methods of the invention can be used to increase aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA
dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V, galactose-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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 In embodiments, 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-l-alpha albulim proximal factor, protein 0-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 is not necessarily deficient relative to wild-type, but where an increase in aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA
dehydrogenase, pyruvate carboxylase isovaleryl-CoA dehydrogenase, apolipoprotein A-V, galactose-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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. In embodiments, the condition can be caused by a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 and NCOA5 haploinsufficiency.
[00196] In embodiments, 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. In certain embodiments, wherein the condition that is not necessarily caused by a deficiency of the target protein but is nonetheless treated by increasing production of the target protein using the present methods, the target protein is TRIB1, TGFB1, HAMP, THPO, PNPLA3, PPARD, IL6, CERS2, or NCOA5. In embodiments, the target protein acts on a secondary target to ameliorate or treat the liver condition in the subject. In embodiments, the secondary target protein is deficient in the subject. In embodiments, the secondary target protein is not deficient in the subject.
Glycine Encephalopathy [00197] In some embodiments, the invention described herein can be used to treat the autosomal recessive liver disorder Glycine Encephalopathy (GCE). 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.
[00198] GCE can manifest as a result of deficiency in the glycine cleavage system in the liver.
Deficiency in the protein aminomethyltransferase (AMT) has been implicated in the progression of GCE
and studies have linked AMT deficiency and the progression of GCE. AMT is a component of the glycine cleavage system in the mitochondria of liver cells. The AMT gene, which codes for the AMT
protein, is a 6 kb gene spanning 9 exons located on 3p21.2. Mutations in the AMT gene have been shown to cause the clinical phenotype associated with GCE. In one study, a patient heterozygous for a G269D
mutation. Other studies have examined other missense mutations in AMT that result in the progression of GCE, thereby establishing a positive link between AMT deficiency and the progression of GCE.
Zellweger Syndrome/Heimler Syndrome [00199] In some embodiments, 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.
[00200] In some embodiments, 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.
Adenosine Deaminase Deficiency [00201] In some embodiments, 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.
[00202] Deficiency in the ADA protein results in the clinical manifestations shown in ADA
deficiency. The ADA gene, which is located at 20q13 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. In one study, 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.
Porphyria Variegata [00203] In some embodiments, 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.
[00204] Deficiency in the protoporphyrinogen oxidase (PPDX) protein results in the clinical manifestations shown in VP. The PPDX gene, which is located at 1q23.3 and spans 13 exons, codes for the PPDX protein. Mutations in the PPDX gene resulting in deficient amounts of PPDX protein have been shown to be responsible for the progression of VP. While there exists a rare homozygous form of VP, "classical" VP is characterized by mutations in a single allele of the PPDX gene, thus proceeding via a haploinsufficiency mechanism. Several studies have linked heterozygous mutations in PPDX with the progression of VP, which is a result of diminished levels of PPDX protein found in patients afflicted with VP.
Porphyria Cutanea Tarda [00205] In some embodiments, 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.
[00206] Deficiency in the uroporphyrinogen decarboxylase (UROD) protein results in the clinical manifestations shown in PCT. The UROD gene, which is located at 1p34.1 and spans 10 exons, codes for the UROD protein. Mutations in the UROD gene resulting in deficient amounts of UROD protein have been shown to be responsible for the progression of PCT. In one study, a G381V
mutation in the UROD
gene was shown in a patient with the familial version of PCT, which resulted in diminished levels of the UROD protein. Other studies have also shown correlation between diminished UROD protein levels and the progression of PCT.

Acute Intermittent Porphyria [00207] In some embodiments, 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 dysfunction, and neurologic disturbance that may lead to death.
[00208] Deficiency in the hydroxymethylbilane synthase (HMBS) protein (nonerythroid, or both erythroid and nonerythroid) results in the clinical manifestations shown in AIP. The HMBS gene, which is located at 11q23.3 and spans 15 exons, codes for the HMBS protein. HMBS
also is referred to as porphobilinogen deaminase (PBGD). Mutations in the HMBS gene resulting in deficient amounts of HMBS protein have been shown to be responsible for the progression of AIP. In one study, 19 separate mutations in HMBS were found in 28 families displaying AIP, further providing a link between HMBS
deficiency and AIP.
Very Long Chain Acyl-CoA Dehydrogenase Deficiency [00209] In some embodiments, 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.
[00210] Deficiency in the VLCAD protein results in the clinical manifestations shown in VLCAD
deficiency. The ACADVL gene, which is located at 17p13.1 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.
Pyruvate Carboxylase Deficiency [00211] In some embodiments, 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.
[00212] Deficiency in the pyruvate carboxylase (PC) protein results in the clinical manifestations shown in PC deficiency. The PC gene, which is located at 11q13.2 and spans 19 exons, codes for the PC protein.

The familial inheritance is believed to proceed via an autosomal recessive mechanism, and the carrier frequency is estimated to be as high as 1 in 10 in certain families. Mutations in the PC gene resulting in deficient amounts of PC protein have been shown to be responsible for the progression of PC deficiency.
In one study, missense mutations in the PC gene in patients suffering from type A PC deficiency were discovered, thereby providing a link between a deficiency in PC protein levels and the clinical manifestations associated with PC deficiency.
Isovaleric Acidemia [00213] In some embodiments, the invention described herein can be used to treat the autosomal recessive liver disease isovaleric academia (WA). WA 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 WA include peculiar odor, an aversion to dietary protein and vomiting.
[00214] Deficiency in the isovaleryl-CoA dehydrogenase (IVD) protein results in the clinical manifestations shown in IVA. The IVD gene, which is located at 15q13 and spans 12 exons, codes for the IVD protein. Mutations in the IVD gene resulting in deficient amounts of WD
protein have been shown to be responsible for the progression of IVA deficiency. Several studies have looked at a number of different mutations in IVD that result in IVD deficiency, and have shown that mutations that result in deficiency in the amount of WD protein result in the clinical manifestations seen in WA.
Hyperchylomicronemia/ Hypertriglyceridemia [00215] In some embodiments, 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.
[00216] In some embodiments, 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. Other than elevated triglycerides, patients are generally asymptomatic.
[00217] Deficiency in apolipoprotein A-V (AP0A5) protein results in the clinical manifestations shown in both hyperchylomicronemia and hypertriglyceridemia. The AP0A5 gene, which is located at 11q23.3 and spans 4 exons, codes for the AP0A5 protein. Mutations in the AP0A5 gene resulting in deficient amounts of AP0A5 protein have been shown to be responsible for the progression of both hyperchylomicronemia and hypertriglyceridemia. Several studies have looked at a number of different mutations in AP0A5 that result in both hyperchylomicronemia and hypertriglyceridemia. For example in one study, a S19W mutation in AP0A5 was shown to result in a deficiency in the amount of AP0A5 protein, which manifested as hyperchylomicronemia in a family of patients.

Galactosemia [00218] In some embodiments, 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.
[00219] Deficiency in the galactose-1-phosphate uridylyltransferase (GALT) protein results in the clinical manifestations shown in galactosemia. The GALT gene, which is located at 15q11.2 and spans 11 exons, codes for the GALT protein. Mutations in the GALT gene resulting in deficient amounts of GALT
protein have been shown to be responsible for the progression of galactosemia.
Several studies have looked at a number of different mutations in GALT that result in galactosemia, and have shown that mutations that result in deficiency in the amount of GALT protein result in the clinical manifestations seen in galactosemia. In one study, a M142K mutation was found to decrease the amount of GALT
protein to approximately 4% of the normal level, which provides a positive link between deficiency in the amount of GALT protein and the clinical progression of galactosemia.
Hypercholesterolemia [00220] In some embodiments, 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 atherosclerosis.
[00221] Deficiency in the protein low density lipoprotein receptor adaptor protein 1 (LDLRAP1) results in the clinical manifestations shown in ARH. The LDLRAP1 gene, which is located at 1p36.11 and spans 9 exons, codes for the LDLRAP1 protein. Mutations in the LDLRAP1 gene resulting in deficient amounts of LDLRAP1 protein have been shown to be responsible for the progression of ARH. Several studies have looked at a number of different mutations in LDLRAP1 that result in ARH, and have shown that mutations that result in deficiency in the amount of LDLRAP1protein result in the clinical manifestations seen in ARH. A number of studies examined a conserved nonsense mutation in the LDLRAP1 gene, which resulted in substantial decrease in the amount of LDLRAP1 protein, and hence the clinical manifestations associated with ARH.
Diabetes Mellitus [00222] In some embodiments, 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).
In other embodiments, the invention described herein can be used to treat hyperinsulemic hypoglycemia familial 3 (HEIF3). In other embodiments, 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.
[00223] While there are a number of factors that can contribute to the progression of diabetes, deficiency in the protein hepatocyte nuclear factor 4-alpha (HNF4A) has been shown to correlate to the incidence of MODY1, NIDDM and FRTS4. The HNF4A gene, which is located at 20q13.12 and spans 12 exons, codes for the HNF4A protein. Mutations in the HNF4A gene resulting in deficient amounts of HNF4A
protein have been shown to be responsible for the progression of MODY1, NIDDM
and FRTS4. Several studies have looked at a number of different mutations in HNF4A that result in MODY1, NIDDM and FRTS4 and have shown that mutations that result in deficiency in the amount of HNF4A protein result in the clinical manifestations seen in diabetes. For example, one study demonstrated that a nonsense mutation at Q268 was present in a large population of patients afflicted with MODY1. In another study, the authors concluded that mutations in HNF4A is associated with increased birth weight and macrosomia, which eventually evolves into the hyperinsulinemia seen in patients afflicted with diabetes.
Studies such this and others have positively correlated the deficiency in the amount of expressed HNF4A
protein with the progression of diabetes.
[00224] Deficiency in the protein glucokinase (GCK) has been shown to correlate to the incidence of NIDDM, MODY2, HHF3 and PNDM. The GCK gene, which is located at 7p13 and spans 12 exons, codes for the GCK protein. Mutations in the GCK gene resulting in deficient amounts of GCK protein have been shown to be responsible for the progression of NIDDM, MODY2, HEIF3 and PNDM. Several studies have looked at a number of different mutations in GCK that result in NIDDM, MODY2, HEIF3 and PNDM and have positively correlated the deficiency in the amount of expressed GCK protein with the progression of diabetes.
[00225] Deficiency in the protein hepatic nuclear factor-l-alpha albulim proximal factor (HNF1A) has been shown to correlate to the incidence of MODY3, IDDM20, IDDM1 and NIDDM.
The HNFlA gene, which is located at 12q24.31 and spans 10 exons, codes for the HNFlA protein.
Mutations in the HNF lA
gene resulting in deficient amounts of HNFlA 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 HNFlA that result in MODY3, IDDM20, IDDM1 and NIDDM, and have positively correlated the deficiency in the amount of expressed HNFlA protein with the progression of diabetes.
[00226] Deficiency in the protein nuclear receptor coactivator 5 (NCOA5) has been shown to correlate to the incidence of type II diabetes, glucose intolerance and ultimately liver cancer. The NCOA5 gene, which is located at 20q13.12 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.
Hepatic adenoma [00227] In some embodiments, 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.
[00228] Deficiency in the HNFlA protein can result in the progression of hepatic adenoma. Mutations in the HNFlA gene resulting in deficient amounts of HNFlA protein have been shown to be responsible for the progression of hepatic adenoma.
Dowling-Degos Disease 4 [00229] In some embodiments, 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.
[00230] Deficiency in the protein 0-glucosyltransferase 1 (POGLUT1) results in the clinical manifestations shown in DDD4. The POGLUT1 gene, which is located at 3q13.33 and spans 11 exons, codes for the POGLUT1 protein. Mutations in the POGLUT1 gene resulting in deficient amounts of POGLUT1 protein have been shown to be responsible for the progression of DDD4.
Several studies have looked at a number of different mutations in POGLUT1 that result in DDD4, and have shown that mutations that result in deficiency in the amount of POGLUT1 protein result in the clinical manifestations seen in DDD4.
SHORT Syndrome/Immunodeficiency 36/Agammaglobulinemia 7 [00231] In some embodiments, the invention described herein can be used to treat SHORT syndrome.
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.
[00232] In some embodiments, 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.
[00233] In some embodiments, 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.
[00234] Deficiency in the protein phosphatidylinositol 3-kinase regulatory subunit 1 (PIK3R1) results in the clinical manifestations shown in SHORT syndrome, IM1D36 and AGM7. The PIK3R1 gene, which is located at 5q13.1 and spans 16 exons, codes for the PIK3R1 protein. Mutations in the PIK3R1 gene resulting in deficient amounts of PIK3R1 protein have been shown to be responsible for the progression of SHORT syndrome, IMD36 and AGM7. Several studies have looked at a number of different mutations in PIK3R1 that result in SHORT syndrome, IMD36 and AGM7. In one study, a R649W
mutation in PIK3R1 was found a population of unrelated individuals diagnosed with SHORT
syndrome. The same mutation was witnessed in an affected mother and her two sons, which provided evidence of the autosomal dominant pattern of inheritance of SHORT syndrome.
Lipid Metabolism Dysfunction [00235] In some embodiments, the invention described herein can be used to treat the lipid metabolism deficiency caused by deficiency in the protein Tribbles-1 (TRIB1). Deficiency in the amount of TRIB1 protein, which is encoded by the TRIB1 gene located on 8q24.13 and spans 3 exons, has been shown to be correlated to an increased risk of atherosclerosis. Mice lacking TRIB1 were shown to have diminished adipose tissue mass accompanied by increased lipolysis, which positively linked diminished levels of TRIB1 with dysfunction in lipid metabolism.
[00236] In some embodiments, 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).
Deficiency in the amount of 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.
Liver Inflammation [00237] In some embodiments, the invention described herein can be used to treat the liver inflammation caused by deficiency in the protein transforming growth factor beta-1 (TGFB1).
Deficiency in the amount of TGBF1 protein, which is encoded by the TGBF1 gene located on 19q13.2 and spans 7 exons, has been shown to be correlated to an increased risk of atherosclerosis.
Knockout mice displaying the TGBF1 (-/-) 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.
[00238] In some embodiments, the invention described herein can be used to treat the lipid inflammation caused by deficiency in the protein interleukin 6 (IL6). Deficiency in the amount of IL6 protein, which is encoded by the 1L6 gene located on 7p15.3 and spans 5 exons, has been shown to be correlated to lipid inflammation.
[00239] In some embodiments, the invention described herein can be used to treat the lipid inflammation or steatohepatitis caused by deficiency in the ceramide synthase 2 (CERS2) protein. Deficiency in the amount of CERS2 protein, which is encoded by the CERS2 gene located on 1q21.3 and spans 11 exons, has been shown to be correlated to steatohepatitis and insulin resistance.
Hemochromatosis Type 2B
[00240] In some embodiments, the invention described herein can be used to treat the autosomal recessive liver disease hemochromatosis type 2B (EIFE2B). EIFE2B (otherwise known as iron overload) is characterized by joint pain, fatigue and weakness, which ultimately results in organ damage.
[00241] Deficiency in the protein hepcidin antimicrobial peptide (HAMP) results in the clinical manifestations shown in EIFE2B. The HAMP gene, which is located at 19q13.12 and spans 3 exons, codes for the HAMP protein. Mutations in the HAMP gene resulting in deficient amounts of HAMP
protein have been shown to be responsible for the progression of EIFE2B.
Nonsense mutations have been reported at G93and R56 in patients afflicted with EIFE2B, while missense mutations such as G71D have also been found. These mutations, which result in diminished amounts of the HAMP protein, were proposed to be a direct cause of EIFE2B, thereby providing a direct correlation between diminished levels of HAMP and incidence of EIFE2B.
Thrombocytopenia [00242] In some embodiments, 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.
[00243] Deficiency in the protein thrombopoietin (THPO) has been shown to be correlated to the incidence of thrombocytopenia. 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] In some embodiments, the invention described herein can be used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by the accumulation of excess 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. In some instances, aggregated fatty deposits in a liver can promote an inflammatory response, which can progress to cirrhosis or liver cancer.
[00245] Deficiency in the protein patatin-like phospholipase domain-containing protein 3 (PNPLA3) has been shown to be correlated to the incidence of NAFLD. The PNPLA3 gene, which is located at 22q13 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, G115C, 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 [00246] In some embodiments, the invention described herein can be used to treat the autosomal recessive disorder Wilson disease. 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.
[00247] Deficiency in the protein copper-transporting ATPase 2 (ATP7B) has been shown to be correlated to the incidence of Wilson disease. The ATP 7B gene, which is located at 13q14.3 and spans 21 exons, codes for the ATP7B protein. Mutations in the ATP 7B 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.
Tyrosinemia [00248] In some embodiments, 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 FAH 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 Q279R have been found in populations manifesting symptoms of FAH. These missense mutations were shown to result in decreased levels of FAH, which provides a correlation between the deficiency of the tyrosinemia protein and the progression of FAH.
Argininosuccinate Lyase Deficiency [00250] In some embodiments, 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.
[00251] Deficiency in the protein argininosuccinate lyase (ASL) has been shown to be correlated to the incidence of argininosuccinate lyase deficiency. The ASL gene, which is located at 7q11.21 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 R113Q, 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.
Hemochromatosis Type 1 [00252] In some embodiments, 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.
[00253] 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 565C, 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 [00254] In some embodiments, 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..
[00255] Deficiency in the protein alstrom syndrome protein 1 has been shown to be correlated to the incidence of Alstrom syndrome. The ALMS1 gene, which is located at 2p13.1 and spans 23 exons, codes for the alstrom syndrome protein. Mutations in the ALMS] 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, I1875V, 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.
Congenital Bile Acid Synthesis Defect 1 [00256] In some embodiments, 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.
[00257] 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 16p11.2 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 Gl9S, E 147K, T25 OA and L3 47P 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.
Retained Intron Containing Pre-mRNA (MC Pre-mRNA) [00258] In embodiments, the methods of the present invention can exploit the presence of retained-intron-containing pre-mRNA (RIC pre-mRNA) transcribed from the AMT, ADA, PPDX, UROD, IIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP 1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, RAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS], 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-l-alpha albulim proximal factor, protein 0-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 protein, in the cell nucleus. Splicing of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 RIC pre-mRNA species to produce mature, fully-spliced, AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA, can be induced using ASOs that stimulate splicing out of the retained introns.
The resulting mature AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA can be exported to the cytoplasm and translated, thereby increasing the amount of aminomethyltransferase, adenosine deaminase, protoporphyrinogen oxidase, uroporphyrinogen decarboxylase, hydroxymethylbilane synthase, very long chain acyl-CoA dehydrogenase, pyruvate carboxylase isovaleryl-CoA
dehydrogenase, apolipoprotein A-V, galactose-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-alpha albulim proximal factor, protein 0-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 protein in the patient's cells and alleviating symptoms of the CNS disease or conditions caused by deficiency in each protein. This method, described further below, is known as Targeted Augmentation of Nuclear Gene Output (TANGO).
[00259] AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene can be analyzed for intron-retention events. In some cases, AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EWE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 gene can be analyzed for intron-retention events. RNA sequencing (RNAseq), can be visualized in the UCSC genome browser, and can show AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 transcripts expressed in human liver cells and localized in either the cytoplasmic or nuclear fraction. In some embodiments, the retained-intron containing pre-mRNA transcripts are retained in the nucleus and are not exported out to the cytoplasm [00260] In embodiments, 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. In embodiments, 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 5% to about 65%, about 5% to about 60%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about

25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35%, retention. In embodiments, other ASOs useful for this purpose are identified, using, e.g., methods described herein.
[00261] In embodiments, the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, 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. In embodiments, the targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA is in one or more introns as shown in Table 1 or Table 2. In embodiments, hybridization of an 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein production. It is understood that the intron numbering may change in reference to a different AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL
TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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 Table 1 or Table 2.
[00262] In some embodiments, the ASOs disclosed herein target a RIC pre-mRNA
transcribed from a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence. In some embodiments, the ASOs disclosed herein target a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
sequence.
[00263] In some embodiments, the ASO targets a sequence of a RIC pre-mRNA
transcript encoded by a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence. In some embodiments, the ASO
targets a RIC pre-mRNA transcript encoded by a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 genomic sequence comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, the ASO targets a RIC pre-mRNA encoded by SEQ ID NOs: 1-31.
[00264] In some embodiments, 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: 78349-78501. In some embodiments, the ASO comprises a sequence of any one of SEQ ID NOs:
131-78348.
[00265] In some embodiments, the ASO targets an exon sequence upstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, the ASO targets an exon sequence about 4 to about 1000 nucleotides upstream (or 5') from the 5' splice site of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
[00266] In some embodiments, the ASO targets an intron sequence upstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, the ASO targets an exon sequence about 6 to about 500 nucleotides downstream (or 3') from the 5' splice site of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2.
[00267] In some embodiments, the ASO targets an exon sequence downstream of a retained intron as shown in Table 1 or Table 2 of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA comprising a retained intron as shown in Table 1 or Table 2. In some embodiments, the ASO targets an exon sequence about 2 to about 1000 nucleotides downstream (or 3') from the 3' splice site of a AMT, ADA, PPDX, UROD, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, 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.
Protein Expression [00268] In embodiments, the methods described herein are used to increase the production of a functional protein. As used herein, 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. In embodiments, the methods are used to increase the production of a partially functional protein. As used herein, 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. In some embodiments, 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.
[00269] In embodiments, 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.
In some embodiments, the deficient amount of the protein is caused by haploinsufficiency of the protein. In such an embodiment, the subject has a first allele encoding a functional protein, and a second allele from which the protein is not produced. In another such embodiment, the subject has a first allele encoding a functional protein, and a second allele encoding a nonfunctional protein. In another such embodiment, the subject has a first allele encoding a functional protein, and a second allele encoding a partially functional protein. In any of these embodiments, 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.
[00270] In embodiments, 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.
[00271] In related embodiments, the method is a method of using an ASO to increase the expression of a protein or functional RNA. In embodiments, 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.
[00272] In embodiments, the RIC pre-mRNA transcript that encodes the protein that is causative of the disease or condition is targeted by the ASOs described herein. In some embodiments, a RIC pre-mRNA
transcript that encodes a protein that is not causative of the disease is targeted by the ASOs. For example, 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. In some embodiments, 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).
[00273] In embodiments, the subject has:
a) a first mutant allele from which i) the protein is produced at a reduced level compared to production from a wild-type allele, ii) the protein is produced in a form having reduced function compared to an equivalent wild-type protein, or iii) the protein or functional RNA is not produced; and b) a second mutant allele from which i) the protein is produced at a reduced level compared to production from a wild-type allele, ii) the protein is produced in a form having reduced function compared to an equivalent wild-type protein, or iii) the protein is not produced, and wherein the RIC pre-mRNA is transcribed from the first allele and/or the second allele. In these embodiments, 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. In these embodiments, 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).
[00274] In embodiments, 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.
[00275] In embodiments, 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. In embodiments, 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. In embodiments, 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.
[00276] In embodiments, 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. In embodiments, 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. In embodiments, a subject treated using the methods of the invention has a whole gene deletion, in one allele.
Use of TANGO for Increasing Protein Expression [00277] As described above, in embodiments, Targeted Augmentation of Nuclear Gene Output (TANGO) is used in the methods of the invention to increase expression of a protein. In these embodiments, 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 (AS0s) that are complementary to a targeted portion of the RIC pre-mRNA.
Hybridization of the ASOs 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 the retained intron and subsequently increases target protein production.

[00278] The terms "pre-mRNA," and "pre-mRNA transcript" may be used interchangeably and refer to any pre-mRNA species that contains at least one intron. In embodiments, pre-mRNA or pre-mRNA
transcripts comprise a 5'-7-methylguanosine cap and/or a poly-A tail. In embodiments, pre-mRNA or pre-mRNA transcripts comprise both a 5'-7-methylguanosine cap and a poly-A
tail. In some embodiments, 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).
[00279] As used herein, 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. In some embodiments, the retained intron is the most abundant intron in RIC pre-mRNA encoding the target protein. In embodiments, 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. In embodiments, 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. In embodiments, 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. In embodiments, the targeted region is in a retained intron that is the most abundant intron in a RIC pre-mRNA encoding the protein.
[00280] As used herein, 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.
Thus, as used herein, 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).
[00281] In embodiments of any of the compositions and methods provided herein, "comprising" 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. As used herein, 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).
[00282] In embodiments, 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. For example, 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. In embodiments, 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. In embodiments, 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. In embodiments, fully-spliced (mature) RNA encoding the target protein is thereby produced.
[00283] In embodiments, an ASO is complementary to a targeted region that is within a non-retained intron in a RIC pre-mRNA. In embodiments, 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. In embodiments, 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. As used to identify the location of a region or sequence, "within" is understood to include the residues at the positions recited. For example, a region +6 to +100 includes the residues at positions +6 and +100. In embodiments, fully-spliced (mature) RNA encoding the target protein is thereby produced.
[00284] In embodiments, the retained intron of the RIC pre-mRNA is an inefficiently spliced intron. As used herein, "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. The term "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.
[00285] In embodiments, 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. In embodiments, 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. As used herein, the "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).
Also used herein, a nucleotide position denoted with an "e" indicates the nucleotide is present in the sequence of an exon (e.g., the exon flanking the 5' splice site or the exon flanking the 3' splice site).
[00286] 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. As used herein, "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. In some embodiments, 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.
[00287] As used herein, 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.
[00288] In embodiments, 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.
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.
[00289] In embodiments, 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. In embodiments, 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-fold, or at least about 10-fold, compared to the amount of target protein produced by an control compound. A
control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the RIC pre-mRNA.
[00290] In some embodiments, 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. In some embodiments, 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. In embodiments, 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-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold compared to the amount of mature RNA produced in an untreated cell, e.g., an untreated cell or a cell treated with a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to the targeted portion of the RIC pre-mRNA.
Constitutive Splicing of a Retained Intron from a RIC pre-mRNA
[00291] 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.
In particular, 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.
[00292] 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, as used herein, 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. For example, constitutive splicing of a retained intron, as induced using the methods and antisense oligonucleotides provided herein, 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.
[0001] In embodiments, 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.

[00293] In some embodiments, 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. In these embodiments, 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.
[00294] In other embodiments, 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. In these embodiments, 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.
[00295] "Correct removal" of the retained intron by constitutive splicing refers to removal of the entire intron, without removal of any part of an exon.
[00296] In embodiments, 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. In embodiments, 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. In embodiments, 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.
[00297] In embodiments, 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.

[00298] In embodiments, the mRNA encoding a protein is a full-length mature mRNA, or a wild-type mature mRNA. In these embodiments, 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.
Antisense Oligomers [00299] One aspect of the present disclosure is a composition comprising antisense oligomers that enhances splicing by binding to a targeted portion of an RIC pre-mRNA. As used herein, the terms "ASO" and "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 complementarity to bind the target sequence and enhancing splicing at a splice site). 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. Any antisense oligomers known in the art, for example in PCT Application No.
PCT/US2014/054151, published as WO 2015/035091, titled "Reducing Nonsense-Mediated mRNA
Decay," can be used to practice the methods described herein.
[00300] In some embodiments, ASOs "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a RIC pre-mRNA. Typically such hybridization occurs with a 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. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
[00301] 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. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target 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. In this example, 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).
[00302] 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 hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA
transcript. For example, an ASO
can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.
[00303] 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). The ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "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.
[00304] 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. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethoylcytosine.

[00305] The ASOs described herein also comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and refer to the connection between monomers of the ASO. In naturally occurring oligonucleotides, 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, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. See e.g., LaPlanche et al., Nucleic Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucleic Acids Res. 16:3209 (1988), Zon et al., Anti Cancer Drug Design 6:539 (1991);
Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, 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.
In some embodiments, the backbone modification is a phosphorothioate linkage. In some embodiments, the backbone modification is a phosphoramidate linkage.
[00306] In embodiments, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is random. In embodiments, the stereochemistry at each of the phosphorus internucleotide linkages of the ASO backbone is controlled and is not random. For example, 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. In embodiments, an ASO used in the methods of the invention comprises an ASO having phosphorus internucleotide linkages that are not random. In embodiments, a composition used in the methods of the invention comprises a pure diastereomeric ASO. In embodiments, 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%.
[00307] In embodiments, the ASO has a nonrandom mixture of Rp and Sp configurations at its phosphorus internucleotide 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). In embodiments, 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%
Rp. In embodiments, 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 30% to about 100% Rp, about 35% to about 100% Rp, about 40% to about 100% Rp, about 45% to about 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 100% Rp, about 20% to about 80% Rp, about 25% to about 75% Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about 45% to about 55% Rp, with the remainder Sp.
[00308] In embodiments, 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. In embodiments, 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.
[00309] 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. In some embodiments, the sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2'4'-constrained 2'0-methyloxyethyl (cM0E) modifications. In some embodiments, the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, 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 Oligonucleotides for Exon Skipping and Related Drug Applications," Nucleic Acid Therapeutics 24(1):
37-47, incorporated by reference for this purpose herein.
[00310] In some examples, 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." In some examples, 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).
Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."
[00311] In some embodiments, 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). 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. For example, 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.
[00312] In some embodiments, the ASOs are comprised of 2'-0-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. 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.

1003131 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.
1003141 Unless specified otherwise, 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.
Similarly, the right-hand end or direction of a nucleic acid sequence (single or double stranded) is the 3' end or direction. Generally, a region or sequence that is 5' to a reference point in a nucleic acid is referred to as "upstream," and a region or sequence that is 3' to a reference point in a nucleic acid is referred to as "downstream."
Generally, the 5' direction or end of an mRNA is where the initiation or start codon is located, while the 3' end or direction is where the termination codon is located. In some aspects, 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. For example, a reference point (e.g., an exon-exon junction in mRNA) may be designated as the "zero" site, and a nucleotide that is directly adjacent and upstream of the reference point is designated "minus one,"
e.g., "-1," while a nucleotide that is directly adjacent and downstream of the reference point is designated "plus one," e.g., "+1."
1003151 In other embodiments, the ASOs are complementary to (and bind to) a targeted portion of an AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, or NCOA5 RIC pre-mRNA (e.g., the direction designated by positive numbers relative to the 5' splice site) (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, 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. In some embodiments, 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). In some embodiments, the ASOs may be complementary to a targeted portion of an AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, 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.
In some aspects, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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.
[00316] In some embodiments, the ASOs are complementary to a targeted region of an AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA (e.g., in the direction designated by negative numbers) (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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. In some embodiments, 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). In some embodiments, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, 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. In some aspects, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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.
[00317] In embodiments, the targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, 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.
[00318] In some embodiments, the ASOs are complementary to a targeted portion of an AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the exon flanking the 5' splice site (upstream) of the retained intron (FIG. 1). In some embodiments, the ASOs are complementary to a targeted portion of the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA
that is within the region +2e to -4e in the exon flanking the 5' splice site of the retained intron. In some embodiments, 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, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA that is within the region -4e to-100e, -4e to -90e, -4e to -80e, -4e to -70e, -4e to -60e, -4e to -50e, -4 to -40e, -4e to -30e, or -4e to -20e relative to the 5' splice site of the retained intron.
[00319] In some embodiments, 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). In some embodiments, the ASOs are complementary to a targeted portion to the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, 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. In some embodiments, 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, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIM, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, 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 +20e relative to the 3' splice site of the retained intron. The ASOs may be of any length suitable for specific binding and effective enhancement of splicing. In some embodiments, the ASOs consist of 8 to 50 nucleobases. For example, 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. In some embodiments, the ASOs consist of more than 50 nucleobases. In some embodiments, 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 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, 12 to 15 nucleobases, 13 to 50 nucleobases, 13 to 40 nucleobases, 13 to 35 nucleobases, 13 to 30 nucleobases, 13 to 25 nucleobases, 13 to 20 nucleobases, 14 to 50 nucleobases, 14 to 40 nucleobases, 14 to 35 nucleobases, 14 to 30 nucleobases, 14 to 25 nucleobases, 14 to 20 nucleobases, 15 to 50 nucleobases, 15 to 40 nucleobases, 15 to 35 nucleobases, 15 to 30 nucleobases, 15 to 25 nucleobases, 15 to 20 nucleobases, 20 to 50 nucleobases, 20 to 40 nucleobases, 20 to 35 nucleobases, 20 to 30 nucleobases, 20 to 25 nucleobases, 25 to 50 nucleobases, 25 to 40 nucleobases, 25 to 35 nucleobases, or 25 to 30 nucleobases in length. In some embodiments, 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. In some embodiments, the ASOs are 20 nucleotides in length. In some embodiments, the ASOs are 19 nucleotides in length. In some embodiments, the ASOs are 18 nucleotides in length. In some embodiments, the ASOs are 17 nucleotides in length. In some embodiments, the ASOs are 16 nucleotides in length. In some embodiments, the ASOs are 15 nucleotides in length. In some embodiments, the ASOs are 14 nucleotides in lengthAn some embodiments, the ASOs are 13 nucleotides in length. In some embodiments, the ASOs are 12 nucleotides in length. In some embodiments, the ASOs are 11 nucleotides in length. In some embodiments, the ASOs are 10 nucleotides in length.
[00320] In some embodiments, 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.
[00321] In embodiments, 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. Such 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.
Oligonucleotides comprising lipophilic moieties, and preparation methods have been described in the published literature. In embodiments, 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. 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. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S.
Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated by reference herein.
[00322] In some embodiments, the nucleic acid to be targeted by an ASO is an RIC pre-mRNA
expressed in a cell, such as a eukaryotic cell. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cell is in a subject. In some embodiments, the cell is isolated from a subject. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a condition or disease-relevant cell or a cell line. In some embodiments, the cell is in vitro (e.g., in cell culture).
Pharmaceutical Compositions [00323] Pharmaceutical 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. In embodiments, 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.
[00324] Pharmaceutically acceptable 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. Other pharmaceutically acceptable 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, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative 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.
[00325] In embodiments, 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.
In embodiments, 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. In embodiments, 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).
[00326] 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. In embodiments, 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. In embodiments, 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. In embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in drug products, formulations and emulsions is well known in the art. In embodiments, 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.
In embodiments, the penetration enhancers is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating nonsurfactant.
[00327] In embodiments, the pharmaceutical formulation comprises multiple antisense oligonucleotides.
In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent. In embodiments, 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. 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.
[00328] In embodiments, the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In embodiments, 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. In embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration are described, e.g., in U.S.
Pat. No. 4,866,042, "Method for the delivery of genetic material across the blood brain barrier," U.S. Pat.
No. 6,294,520, "Material for passage through the blood-brain barrier," and U.S. Pat. No. 6,936,589, "Parenteral delivery systems,"
each incorporated herein by reference.
[00329] In embodiments, 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. Such 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.
Oligonucleotides comprising lipophilic moieties, and preparation methods have been described in the published literature. In embodiments, 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. 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. In embodiments, the conjugate is attached to the 3' end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, e.g., in U.S.
Pat. No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," incorporated by reference herein.
Treatment of Subjects [00330] Any of the 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. In embodiments, the individual is a human. In embodiments, the individual is a fetus, an embryo, or a child. In other embodiments, the individual may be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered to a cell ex vivo.
[00331] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or disorder. In some embodiments, the individual has a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of having the disease, such as any of the diseases described herein. In some embodiments, 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. For example, an individual may be at an increased risk of having such a disease or disorder because of family history of the disease. Typically, 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).
[00332] 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. In embodiments, a fetus is treated in utero, e.g., by administering the ASO composition to the fetus directly or indirectly (e.g., via the mother).
Methods of identifying additional ASOs that enhance splicing [00333] Also within the scope of the present invention are methods for identifying (determining) additional ASOs that enhance splicing of an RIC pre-mRNA, specifically at the target intron. 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. In some embodiments, 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.
[00334] A round of screening, referred to as an ASO "walk" may be performed using ASOs that have been designed to hybridize to a target region of a pre-mRNA. For example, 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). For example, 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. In embodiments, 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.
[00335] 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) 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. In some embodiments, 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.
[00336] 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.
[00337] 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.
[00338] As described for the ASO walk above, 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. In some embodiments, 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.
[00339] 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. Following administration, 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.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
EXAMPLES
[00340] The present invention will be more specifically illustrated by the following Examples. However, it should be understood that the present invention is not limited by these examples in any manner.
Example 1: Identification of intron retention events in AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, GALT, LDLRAP1, POGLUT1, PIK3R1, TRIB1, TGFB1, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS!, PPARD, IL6, HSD3B7, CERS2 and NCOA5 transcripts by RNAseq using next generation sequencing [00341] Whole transcriptome shotgun sequencing was carried out using next generation sequencing to reveal a snapshot of transcripts produced by the AMT, ADA, PPDX, UROD, TIMBS, ACADVL, PC, IVD, GALT, LDLRAP1, POGLUT1, PIK3R1, TRIB1, TGFB1, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS], PPARD, IL6, HSD3B7, CERS2 and NCOA5 genes described herein to identify intron-retention events.

For this purpose, 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/hg19 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 High 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/gku1177) and the coverage and number of reads were inferred by the peak signals. The height of the peaks indicates the level of expression given by the density of the reads in a particular region.
A schematic representation of the gene was provided by the UCSC genome browser so that peaks could be matched to the exonic and intronic regions. Based on this display, introns were identified that have high read density in the nuclear fraction of THLE-3 cells, but have very low to no reads in the cytoplasmic fraction of these cells (see Table 1 and 2 and FIGs. for percent intron retention (PIR) data obtained).
This indicated that these introns were retained and that the intron-containing transcripts remain in the nucleus, and suggested that these retained RIC pre-mRNAs are non-productive, as they were not exported out to the cytoplasm.
Example 2: Identification of intron retention events in AP0A5, HNF4A, GCK, HNF1A, HAMP, and THPO transcripts by RNAseq using next generation sequencing [00342] Whole transcriptome shotgun sequencing was carried out using next generation sequencing to reveal a snapshot of transcripts, e.g., those produced by the AP0A5, HNF4A, GCK, HNF1A, HA/VIP, and THPO genes described herein, to identify intron-retention events. For this purpose, 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/hg19 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 High 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/gku1177) 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. A schematic representation of the gene was provided by the UCSC genome browser so that peaks could be matched to the exonic and intronic regions. Based on this display, introns were identified that have high read density in the nuclear fraction of THLE-3 cells, but have very low to no reads in the cytoplasmic fraction of these cells (see Table 1 and 2 and FIGs.
for percent intron retention (PIR) data obtained). This indicated that these introns were retained and that the intron-containing transcripts remained in the nucleus, and suggested that these retained RIC pre-mRNAs were non-productive, as they were not exported out to the cytoplasm.
Example 3: Design of ASO-walk targeting a retained intron [00343] 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.
Table 1 RNA Accession Gene Symbol Gene ID
Retained Intron (Percent Intron Retention) Number Intron 4(39%) Intron 6(15%) Intron 4(11%) ADA 100 NM 000022 Intron 11(12%) Intron 7(N/A) Intron 5(59%) Intron 4(22%) Intron 8(18%) Intron 12(18%) Intron 3(20%) Intron 4(17%) UROD 7389 NM 000374 Intron 5(14%) Intron 6(19%) Intron 7(18%) Intron 8(15%) Intron 9(12%) Intron 3(N/A) Intron 5(N/A) Intron 8(N/A) Intron 9(N/A) Intron 10(15%) Intron 13(15%) PC 5091 NM 022172 Intron 16(25%) IVD 3712 NM 002225 Intron 7(22%) ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ Intron 9(13%) Intron 11(23%) AP0A5 116519 No reads No reads Intron 5(13%) Intron 7(21%) Intron 10(15%) Intron 2(N/A) Intron 3(N/A) Intron 4(N/A) Intron 8(N/A) Intron 9(N/A) Intron 1(12%) Intron 8(12%) GCK 2645 No reads No reads POTGLUT1 56983 NM 152305 Intron 1(18%) Intron 4(47%) Intron 5(44%) Intron 3(7%) Intron 9(11%) HNF 1 A 6927 No reads No reads Intron 2(48%) Intron 1(N/A) TGFB1 7040 NM 000660 Intron 4(N/A) HAMP 57817 No reads No reads THPO 7066 No reads No reads Intron 1(8%) Intron 4(34%) Intron 5(16%) Intron 7(41%) Intron 7(N/A) Intron 13(8%) FAH 2184 NM 000137 Intron l(N/A) Intron 7(24%) Intron 8(N/A) Intron 9(23%) Intron 16(16%) FIFE 3077 NM 000410 Intron 2(N/A) ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ ALMS1 7840 NM 015120 Intron 21(60%) Intron 3(20%) Intron 4(18%) PPARD 5467 NM 006238 Intron 5(12%) Intron 6(17%) Intron 7(N/A) Intron 1(N/A) Intron 2(10%) Intron 3(10%) Intron 4(N/A) Intron 1(74%) Intron 2(25%) Intron 3(14%) Intron 4(22%) Intron 5(12%) Intron 6(24%) CERS2 29956 NM 022075 Intron 6(13%) NCOA5 57727 NM 020967 Intron 2(22%) Table 2 Target Gene Pre-mRNA ASOs Retained Sequence SEQ ID NO. SEQ ID NO. SEQ ID NOs. Intron SEQ ID NO.
LDLRAP1 LDLRAP1: NM 015627 131 - 682 8 78354 SEQ ID NO. 1 SEQ ID NO. 32 683 - 925 1 78459 UROD: NM 000374 SEQ ID NO. 33 SEQ ID NO. 2 1353 - 1391 3 78410 UROD: NR 036510 SEQ ID NO. 34 CERS2 CERS2: NM 022075 SEQ ID NO. 3 SEQ ID NO. 35 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ CERS2: NM 181746 SEQ ID NO. 36 PPDX: NM 000309 1960 - 2060 5 78473 SEQ ID NO. 37 2161 - 2110 12 78480 SEQ ID NO. 4 2250 - 2309 8 78461 PPDX: NM 001122764 2310 - 2410 5 78473 SEQ ID NO. 38 2411 - 2460 12 78480 ALMS1 ALMS1: NM 015120 SEQ ID NO. 5 SEQ ID NO. 39 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 POGLUT1: NM 152305 SEQ ID NO. 45 SEQ ID NO. 7 4560 - 4731 1 78368 POGLUT1: NR 024265 SEQ ID NO. 46 THPO: NM 001289998 5526 - 5616 3 78400 SEQ ID NO. 47 5617 - 5704 4 78393 SEQ ID NO. 8 5944 - 6244 6 78381 THPO: NM 001177598 SEQ ID NO. 48 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ THPO: NM 001290022 SEQ ID NO. 49 THPO: NM 001290003 SEQ ID NO. 50 THPO: NM 001289997 SEQ ID NO. 51 THPO: NM 000460 SEQ ID NO. 52 THPO: NM 001177597 SEQ ID NO. 53 THPO: NM 001290028 SEQ ID NO. 54 THPO: NM 001290027 12986 - 13076 3 78400 SEQ ID NO. 55 13077 - 13164 4 78393 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ THPO: NM 001290026 SEQ ID NO. 56 PIK3R1: NM 181523 14473 - 14544 9 78390 SEQ ID NO. 57 14545 - 14660 3 78418 PIK3R1: NM 181524 PIK3R1 SEQ ID NO. 58 SEQ ID NO. 9 PIK3R1: NM 181504 SEQ ID NO. 59 PIK3R1: NM 001242466 SEQ ID NO. 60 PPARD: NM 006238 15134 - 15376 4 78478 SEQ ID NO. 61 15377 - 15628 5 78472 PPARD: NM 177435 SEQ ID NO. 62 PPARD: NM 001171818 SEQ ID NO. 10 18461 - 18703 5 78478 SEQ ID NO. 63 PPARD: NM 001171819 SEQ ID NO. 64 PPARD: NM 001171820 SEQ ID NO. 65 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ HFE: NM 139007 SEQ ID NO. 66 HFE: NM 139006 SEQ ID NO. 67 HFE: NM 139004 SEQ ID NO. 68 HFE: NM 139003 FIFE SEQ ID NO. 69 SEQ ID NO. 11 FIFE: NM 001300749 SEQ ID NO. 70 HFE: NM 139009 SEQ ID NO. 71 HFE: NM 139008 SEQ ID NO. 72 HFE: NM 000410 SEQ ID NO. 73 1L6: NM 001318095 SEQ ID NO. 74 SEQ ID NO. 12 1L6: 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 SEQ ID NO. 13 26432 - 26674 8 78417 GCK: NM 000162 27435 - 27652 2 78379 SEQ ID NO. 77 27653 - 27880 3 78431 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ GCK: NM 033507 SEQ ID NO. 78 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

SEQ ID NO. 14 __________________________________________________________________ ASL: NM 001024944 31804 - 31843 6 78365 SEQ ID NO. 81 31844 - 31986 14 78371 ASL: NM 001024946 SEQ ID NO. 82 TRIB1: NM 025195 32469 - 32870 1 78487 TRIB1 SEQ ID NO. 83 32871 - 33566 2 78486 SEQ ID NO. 15 TRIB 1: NM 001282985 33567 - 33824 1 78474 SEQ ID NO. 84 33825 - 34520 2 78486 GALT GALT: NM 000155 SEQ ID NO. 16 SEQ ID NO. 85 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ GALT: NM 001258332 35424 - 35518 5 78435 SEQ ID NO. 86 35519 - 35567 6 78493 PC: NM 000920 SEQ ID NO. 87 35900 - 36030 17 78495 PC
PC: NM 001040716 SEQ ID NO. 17 36031 -36161 18 78495 SEQ ID NO. 88 PC: NM 022172 SEQ ID NO. 89 AP0A5: NM 052968 SEQ ID NO. 90 SEQ ID NO. 18 36891 -36994 1 78372 AP0A5: NM 001166598 SEQ ID NO. 91 HMBS: NM 000190 37483 - 37554 10 78355 SEQ ID NO. 92 37555 - 37632 11 78467 HMBS: NM 001258208 FIMBS SEQ ID NO. 93 SEQ ID NO. 19 HMB5: NM 001024382 37764 - 37835 10 78355 SEQ ID NO. 94 37836 - 37913 11 78467 HMBS: NM 001258209 SEQ ID NO. 95 HNF 1 A HNF1A: NM 000545 SEQ ID NO. 20 SEQ ID NO. 96 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ HNF1A: NM 001306179 SEQ ID NO. 97 ATP7B: NM 001243182 42106 - 42367 8 78427 SEQ ID NO. 98 42368 - 42615 14 78383 ATP7B: NM 000053 42616 - 42863 13 78383 SEQ ID NO. 99 42864 - 43125 7 78427 ATP7B ATP7B: NM 001005918 SEQ ID NO. 21 SEQ ID NO. 100 ATP7B: NM 001330579 43374 - 43621 11 78383 SEQ ID NO. 101 43622 - 43886 5 78444 ATP7B: NM 001330578 43887 - 44134 12 78383 SEQ ID NO. 102 44135 -44370 7 78394 FAH FAH: NM 000137 SEQ ID NO. 22 SEQ ID NO. 103 IVD: NM 001159508 SEQ ID NO. 104 SEQ ID NO. 23 45820 - 45969 9 78464 IVD: NM 002225 SEQ ID NO. 105 HSD3B7 HSD3B7: NM 001142777 SEQ ID NO. 24 SEQ ID NO. 106 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ HSD3B7: NM 001142778 SEQ ID NO. 107 HSD3B7: NM 025193 48799 - 48879 3 78498 SEQ ID NO. 108 48880 -48924 4 78406 ACADVL: NM 001270448 49566 - 49697 8 78448 SEQ ID NO. 109 49698 - 49800 9 78477 ACADVL: NM 001270447 50113 - 50244 10 78448 ACADVL
SEQ ID NO. 110 50245 - 50347 11 78477 SEQ ID NO. 25 ACADVL: NM 000018 50660 - 50791 9 78448 SEQ ID NO. 111 50792 - 50894 10 78477 ACADVL: NM 001033859 51064 - 51158 2 78412 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ SEQ ID NO. 112 51159 - 51212 4 78376 HAMP HAMP: NM 021175 51670 - 51921 1 78497 SEQ ID NO. 26 SEQ ID NO. 113 51922 - 51971 2 78426 TGFB 1 TGFB1: NM 000660 SEQ ID NO. 27 SEQ ID NO. 114 HNF4A: NM 001287184 SEQ ID NO. 115 HNF4A: NM 001287183 HNF4A SEQ ID NO. 116 SEQ ID NO. 30 HNF4A: NM 175914 SEQ ID NO. 117 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ HNF4A: NM 001030004 SEQ ID NO. 118 HNF4A: NM 001287182 SEQ ID NO. 119 HNF4A: NM 001030003 SEQ ID NO. 120 HNF4A: NM 000457 SEQ ID NO. 126 HNF4A: NM 001258355 70194 - 70436 1 78373 SEQ ID NO. 127 70437 - 70648 2 78450 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ HNF4A: NM 178850 SEQ ID NO. 128 HNF4A: NM 178849 SEQ ID NO. 129 ADA: NM 001322051 65847 - 66014 10 78482 SEQ ID NO. 121 66015 -66192 4 78385 ADA: NM 000022 66193 - 66360 11 78482 ADA SEQ ID NO. 122 66361 - 66538 4 78385 SEQ ID NO. 28 ADA: NM 001322050 66539 - 66706 10 78482 SEQ ID NO. 123 66707 - 66935 4 78369 ADA: NR 136160 66936 - 67103 10 78482 SEQ ID NO. 124 67104 - 67281 4 78385 NCOA5 NCOA5: NM 020967 SEQ ID NO. 29 SEQ ID NO. 125 PNPLA3 PNPLA3: NM 025225 77375 -77659 1 78388 SEQ ID NO. 31 SEQ ID NO. 130 77670 - 77891 4 78402 ____ WO 2017/106283 _______________________________________ PCT/US2016/066564 __ Example 4: Improved splicing efficiency via ASO-targeting of a retained intron increases AMT
transcript levels [00344] To determine whether an increase in expression of AMT 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 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. Ct values from AMT targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several AMT
targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the AMT gene using ASOs led to an increase in AMT gene expression.
Example 5: Improved splicing efficiency via ASO-targeting of a retained intron increases GALT
transcript levels [00345] To determine whether an increase in expression of GALT 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 GALT 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. 4C and D; and FIG. 4G and 4H). Taqman qPCR results showed that several targeting ASOs increase GALT gene transcript level compared to the mock-transfected. Ct values from GALT targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several GALT targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the GALT gene using ASOs led to an increase in GALT
gene expression.
Example 6: Improved splicing efficiency via ASO-targeting of a retained intron increases PC
transcript levels [00346] To determine whether an increase in expression of PC 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 PC 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. 5A and B). Taqman qPCR results showed that several targeting ASOs increase PC gene transcript level compared to the mock-transfected. Ct values from PC targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells.
Results of this analysis indicated that several PC targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the PC gene using ASOs led to an increase in PC gene expression.
Example 7: Improved splicing efficiency via ASO-targeting of a retained intron increases FAH
transcript levels [00347] To determine whether an increase in expression of FAH 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 FAH 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. 6A and B). Taqman qPCR results showed that several targeting ASOs increase FAH gene transcript level compared to the mock-transfected. Ct values from FAH targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several FAH
targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the FAH gene using ASOs led to an increase in FAH gene expression.
Example 8: Improved splicing efficiency via ASO-targeting of a retained intron increases PPARD
transcript levels [00348] To determine whether an increase in expression of PPARD 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 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. Ct values from PPARD targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several PPARD targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the PPARD gene using ASOs led to an increase in PPARD gene expression.

Example 9: Improved splicing efficiency via ASO-targeting of a retained intron increases HMBS
transcript levels [00349] To determine whether an increase in expression of EIMBS 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 HMBS 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. 9A and B). Taqman qPCR results showed that several targeting ASOs increase EIMBS gene transcript level compared to the mock-transfected. Ct values from EIMBS targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several EIMBS
targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the HMBS gene using ASOs led to an increase in EIMBS gene expression.
Example 10: Improved splicing efficiency via ASO-targeting of a retained intron increases ALMS1 transcript levels [00350] To determine whether an increase in expression of ALMS1 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 ALMS1 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. 11A and B). Taqman qPCR results showed that several targeting ASOs increase ALMS1 gene transcript level compared to the mock-transfected. Ct values from ALMS1 targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR
product from mock-treated cells. Results of this analysis indicated that several ALMS1 targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the ALMS1 gene using ASOs led to an increase in ALMS1 gene expression.
Example 11: Improved splicing efficiency via ASO-targeting of a retained intron increases ASL
transcript levels [00351] To determine whether an increase in expression of ASL 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 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. Ct values from ASL targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several ASL
targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression.
Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the ASL gene using ASOs led to an increase in ASL gene expression.
Example 12: Improved splicing efficiency via ASO-targeting of a retained intron increases ATP7B
transcript levels [00352] To determine whether an increase in expression of ATP7B 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 ATP7B 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. 13B and C). Taqman qPCR results showed that several targeting ASOs increase ATP7B gene transcript level compared to the mock-transfected. Ct values from ATP7B
targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR
product from mock-treated cells. Results of this analysis indicated that several ATP7B targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the ATP7B gene using ASOs led to an increase in ATP7B
gene expression.
Example 13: Improved splicing efficiency via ASO-targeting of a retained intron increases HSD3B7 transcript levels [00353] 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.
Experiments were performed using 80 nM ASOs for 24 hrs (FIG. 15B and C, FIG. 15E and F). Taqman qPCR
results showed that several targeting ASOs increase HSD3B7 gene transcript level compared to the mock-transfected. Ct values from HSD3B7 targeting-ASO-transfected cells are normalized to RPL32 and plotted relative to the normalized qPCR product from mock-treated cells. Results of this analysis indicated that several HSD3B7 targeting ASOs increase gene transcript levels. These results show that inducing splicing of a retained intron in the gene using ASOs leads to an increase in gene expression. Altogether, these results show that improving the splicing efficiency of a rate limiting intron in the HSD3B7 gene using ASOs led to an increase in HSD3B7 gene expression.
Example 14: Improved splicing efficiency via ASO-targeting of a retained intron increases transcript levels [00354] To determine whether an increase in expression of a target gene could be acheived by improving intron splicing efficiency with ASOs we used the method described herein. 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. Cells were transfected using Lipofectamine 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. To quantify the amount of splicing at the intron of interest, quantitative PCR
was carried out using Taqman assays with probes spanning the corresponding exon-exon 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^-(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). Several 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.

Claims (110)

What is claimed is:

1. A method of treating a liver disease in a subject in need thereof by increasing the expression of a target protein or functional RNA by cells of the subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-mRNA), the RIC pre-mRNA comprising a retained intron, an exon flanking the 5' splice site, an exon flanking the 3' splice site, and wherein the RIC pre-mRNA
encodes the target protein or functional RNA, the method comprising contacting the cells of the subject with an antisense oligomer (ASO) complementary to a targeted portion of the RIC pre-mRNA
encoding the target protein or functional RNA, whereby the retained intron is constitutively spliced from the RIC pre-mRNA encoding the target protein or functional RNA, thereby increasing the level of mRNA encoding the target protein or functional RNA, and increasing the expression of the target protein or functional RNA in the cells of the subject.

2. The method of claim 1, wherein 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.

3. A method of increasing expression of a target protein by cells having a retained-intron-containing pre-mRNA (RIC pre-mRNA), the 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 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, 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-l-alpha albulim proximal factor, 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.

4. The method of claim 1 or 2, wherein 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-l-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.

5. The method of claim 1 or 2, wherein 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.

6. The method of claim 3, wherein the cells are in or from a subject having a condition caused by a deficient amount or activity of the target protein.

7. The method of any of claims 1 to 6, wherein 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.

8. The method of any of claims 1 to 6, wherein 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) a first mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii)the target protein is not produced, and (b) a second mutant allele from which (i) the target protein is produced at a reduced level compared to production from a wild-type allele, (ii) the target protein is produced in a form having reduced function compared to an equivalent wild-type protein, or (iii)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 transcribed from either the first mutant allele that is a(i) or a(ii), and/or the second allele that is b(i) or b(ii)

9. The method of claim 8, wherein the target protein is produced in a form having reduced function compared to the equivalent wild-type protein.

10. The method of claim 8, wherein the target protein is produced in a form that is fully-functional compared to the equivalent wild-type protein.

11. The method of any of claims 1 to 10, wherein 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.

12. The method of any of claims 1 to 10, wherein 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.

13. The method of any one of claims 1 to 10, wherein the targeted portion of the RIC pre-mRNA is in the retained intron within:
(a) the region +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -100 relative to the 3' splice site of the retained intron.

14. The method of any one of claims 1 to 10, wherein the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.

15. The method of any one of claims 1 to 10, wherein 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.

16. The method of any one of claims 1 to 10, wherein 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.

17. The method of any one of claims 1 to 16, wherein the target protein is (a) AMT, (b) ADA, (c) PPDX, (d) UROD, (e) EIMBS, (f) ACADVL, (g) PC, (h) IVD, (i) AP0A5, (j) GALT, (k) LDLRAP1, (l) HNF4A, (m) GCK, (n) POGLUT1, (o) PIK3R1, (p) EINF1A, (q) TRIBL (r) TGFB1, (s) HAMP, (t) THPO, (u) PNPLA3, (v) ATP7B, (w) FAH, (x) ASL, (y) FIFE, (z) ALMS1, (aa) PPARD, (bb) IL6, (cc) HSD3B7, (dd) CERS2, or (ee) NCOA5.

18. The method of claim 17, wherein 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, (i) 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, (l) any one of SEQ ID NOs 58958 - 65846 or 67532-77374, (m) any one of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID
NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ
ID NOs 32469-34520, (r) any one of SEQ ID NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID
NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 - 44625, (x) any one of SEQ
ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ
ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ
ID NOs 67282 -67531.

19. The method of claim 17 or 18, wherein 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; (i) SEQ ID NO 78407, 78499, 78420, 78372 or 78397; (j) SEQ ID
NO 78489, 78416, 78476, 78352, 78435, 78493, 78423, 78437 or 78449; (k) SEQ ID NO 78354 or 78459; (1) SEQ ID NO 78441, 78392, 78456, 78428, 78491, 78501, 78360, 78429, 78358, 78364, 78475, 78391, 78479, 78401, 78373 or 78450; (m) SEQ ID NO 78445, 78481, 78379, 78431, 78469, 78408, 78377, 78417, 78387, 78455, 78484 or 78370; (n) SEQ ID NO 78368, 78350, 78432, 78439 or 78389; (o) SEQ ID NO 78390 or SEQ ID NO 78418, (p) SEQ ID NO 78462, 78468, 78453, 78361, 78363, 78433, 78438, 78430, 78488, 78405, 78492 or 78427; (q) SEQ ID NO 78487, 78486 or 78474; (r) SEQ ID NO 78458, (s) SEQ ID NO 78497 or 78426; (t) SEQ ID NO 78425, 78400, 78393, 78351, 78381, 78366, 78457, 78443, 78362 or 78446; (u) SEQ ID NO 78388, 78402, 78471 or 78356; (v) SEQ ID NO 78427, 78383, 78444 or 78394; (w) SEQ ID NO 78382; (x) SEQ ID NO
78494, 78404, 78371, 78365 or 78353; (y) SEQ ID NO 78419, 78384, 78500, 78424 or 78466; (z) SEQ ID NO 78399, (aa) SEQ ID NO 78414, 78478, 78472, 78359, 78395, 78357, 78374 or 78490;
(bb) SEQ ID NO 78436, 78413, 78415, 78398 or 78403; (cc) SEQ ID NO 78349, 78470, 78498, 78406, 78442, 78451, 78396, 78409 or 78378; (dd) SEQ ID NO 78447; or SEQ ID NO
78452.

20. The method of any one of claims 17 to 19, wherein 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, (i) 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, (l) any one of SEQ ID NOs 58958 - 65846 or 67532-77374, (m) any one of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID
NOs 32469-34520, (r) any one of SEQ ID NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 - 44625, (x) any one of SEQ ID
NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ
ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ
ID NOs 67282 -67531.

21. The method of any one of claims 17 to 20, wherein 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, (i) SEQ ID NOs 90 or 91, (j) SEQ ID NOs 85 or 86, (k) SEQ ID NO 32, (l) SEQ ID NOs 115-120 or126-129, (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 97, (q) SEQ ID NOs 83 or 84, (r) SEQ ID NO 114, (s) SEQ ID NO
223, (t) SEQ ID
NOs 47-56, (u) SEQ ID NO 130, (v) SEQ ID NOs 98-102, (w) SEQ ID NO 103, (x) SEQ ID NOs 80-82, (y) SEQ ID NOs 66-73, (z) SEQ ID NO 39, (aa) SEQ ID NOs 61-65, (bb) SEQ ID
NOs 74 or 75, (cc) SEQ ID NOs 106-108, (dd) SEQ ID NOs 35 or 36, or SEQ ID NO 125.

22. The method of any one of claims 17 to 21, wherein 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, (i) SEQ ID NO 18, (j) SEQ ID
NO 16, (k) SEQ ID NO 1, (l) 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) SEQ ID
NO 5, (aa) SEQ ID NO 10, (bb) SEQ ID NO 12, (cc) SEQ ID NO 24, (dd) SEQ ID NO
3, or (ee) SEQ ID NO 29.

23. The method of any one of claims 1 to 22, wherein 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.

24. The method of any one of claims 1 to 23, wherein 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.

25. The method of any one of claims 1 to 24, wherein the RIC pre-mRNA was produced by partial splicing of a full-length pre-mRNA or partial splicing of a wild-type pre-mRNA.

26. The method of any one of claims 1 to 25, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

27. The method of any one of claims 1 to 26, wherein the target protein produced is full-length protein, or wild-type protein.

28. The method of any one of claims 1 to 27, wherein 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 3.5-fold, at least about 4-fold, at least about 5-fold, or at least about 10-fold, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell.

29. The method of any one of claims 1 to 28, wherein 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 5-fold, or at least about 10-fold, compared to the total amount of target protein produced by a control cell.

30. The method of any one of claims 1 to 29, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

31. The method of any one of claims 1 to 30, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety.

32. The method of any one of claims 1 to 31, wherein the antisense oligomer comprises at least one modified sugar moiety.

33. The method of claim 32, wherein each sugar moiety is a modified sugar moiety.

34. The method of any one of claims 1 to 33, wherein 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, to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

35. The method of any one of claims 1 to 34, wherein 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.

36. The method of any one of claims 1 to 35, wherein 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.

37. The method of claim 36, whereby 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.

38. The method of any one of claims 1 to 35, wherein 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.

39. The method of claim 38, whereby 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.

40. The method of any one of claims 6 to 39, wherein the condition is a disease or disorder.

41. The method of claim 40, wherein the disease or disorder is a liver disease.

42. The method of claim 41, wherein 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.

43. The method of claim 42, wherein the target protein and the RIC pre-mRNA
are encoded by a gene, wherein the gene is AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP , THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMS], PPARD, IL6, HSD3B7, CERS2 or NCOA5.

44. The method of any one of claims 1 to 43, wherein the method further comprises assessing protein expression.

45. The method of any one of claims 1 to 44, wherein the subject is a human.

46. The method of any one of claims 1 to 44, wherein the subject is a non-human animal.

47. The method of any one of claims 1 to 45, wherein the subject is a fetus, an embryo, or a child.

48. The method of any one of claims 1 to 46, wherein the cells are ex vivo.

49. The method of any one of claims 1 to 46, wherein the antisense oligomer is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject.

50. The method of any one of claims 1 to 49, wherein the 9 nucleotides at -3e to -le of the exon flanking the 5' splice site and +1 to +6 of the retained intron, are identical to the corresponding wild-type sequence.

51. The method of any one of claims 1 to 50, wherein the 16 nucleotides at -15 to -1 of the retained intron and +1e of the exon flanking the 3' splice site are identical to the corresponding wild-type sequence.

52. An antisense oligomer as used in a method of any one of claims 1 to 51.

53. An antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs 131-78348.

54. A pharmaceutical composition comprising the antisense oligomer of claim 52 or 53 and an excipient.

55. A method of treating a subject in need thereof by administering the pharmaceutical composition of claim 54 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

56. A 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:
(a) the deficient protein; or (b) a compensating protein which functionally augments or replaces the deficient protein or in the subject;
and wherein the functional RNA is:
(a) the deficient RNA; or (b) 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 exon flanking the 3' splice site, and wherein the retained intron is spliced from the RIC pre-mRNA
encoding the target protein or the functional RNA, thereby increasing production or activity of the target protein or the functional RNA in the subject.

57. The composition of claim 56, wherein 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.

58. A 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.

59. The composition of any one of claims 56 to 58, wherein 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-l-phosphate uridylyltransferase, low density lipoprotein receptor adaptor protein 1, hepatocyte nuclear factor 4-alpha, glucokinase, hepatic nuclear factor-l-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.

60. The composition of claim 58, wherein the condition is a disease or disorder.

61. The composition of claim 60, wherein the disease or disorder is a liver disease.

62. The composition of claim 61, wherein 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.

63. The composition of claim 61 or 62, wherein the target protein and RIC pre-mRNA are encoded by a gene, wherein the gene is AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, AP0A5, GALT, LDLRAP 1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, HFE, ALMSl, PPARD, IL6, HSD3B7, CERS2 or NCOA5.

64. The composition of any one of claims 56 to 63, wherein 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.

65. The composition of any one of claims 56 to 63, wherein the antisense oligomer targets a portion of the RIC pre-mRNA that is in the retained intron within:
(a) the region +6 to +100 relative to the 5' splice site of the retained intron; or (b) the region -16 to -100 relative to the 3' splice site of the retained intron.

66. The composition of any one of claims 56 to 63, wherein 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

67. The composition of any one of claims 56 to 63, wherein 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.

68. The composition of any one of claims 56 to 63, wherein the targeted portion of the RIC pre-mRNA is within:
(a) the region +2e to -4e in the exon flanking the 5' splice site of the retained intron; or (b) the region +2e to -4e in the exon flanking the 3' splice site of the retained intron.

69. The composition of any one of claims 56 to 68, wherein the target protein is (a) AMT, (b) ADA, (c) PPDX, (d) UROD, (e) HMBS, (f) ACADVL, (g) PC, (h) IVD, (i) APOA5, (j) GALT, (k) LDLRAP1, (l) 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.

70. The composition of claim 69, wherein 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, (i) 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, (l) any one of SEQ ID NOs or 67532-77374, (m) any one of SEQ ID NOs 25058-30976, (n) any one of SEQ ID
NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ ID NOs 32469-34520, (r) any one of SEQ ID NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 -44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ ID NOs 67282 - 67531.

71. The composition of claim 69 or 70, wherein 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; (i) SEQ ID NO 78407, 78499, 78420, 78372 or 78397; (j) SEQ ID
NO 78489, 78416, 78476, 78352, 78435, 78493, 78423, 78437 or 78449; (k) SEQ ID
NO 78354 or 78459; (1) SEQ ID NO 78441, 78392, 78456, 78428, 78491, 78501, 78360, 78429, 78358, 78364, 78475, 78391, 78479, 78401, 78373 or 78450; (m) SEQ ID NO 78445, 78481, 78379, 78431, 78469, 78408, 78377, 78417, 78387, 78455, 78484 or 78370; (n) SEQ ID NO 78368, 78350, 78432, 78439 or 78389; (o) SEQ ID NO 78390 or SEQ ID NO 78418, (p) SEQ ID NO 78462, 78468, 78453, 78361, 78363, 78433, 78438, 78430, 78488, 78405, 78492 or 78427; (q) SEQ ID NO
78487, 78486 or 78474; (r) SEQ ID NO 78458, (s) SEQ ID NO 78497 or 78426; (t) SEQ ID NO
78425, 78400, 78393, 78351, 78381, 78366, 78457, 78443, 78362 or 78446; (u) SEQ ID NO 78388, 78402, 78471 or 78356; (v) SEQ ID NO 78427, 78383, 78444 or 78394; (w) SEQ ID NO 78382; (x) SEQ ID NO
78494, 78404, 78371, 78365 or 78353; (y) SEQ ID NO 78419, 78384, 78500, 78424 or 78466; (z) SEQ ID NO 78399, (aa) SEQ ID NO 78414, 78478, 78472, 78359, 78395, 78357, 78374 or 78490;
(bb) SEQ ID NO 78436, 78413, 78415, 78398 or 78403; (cc) SEQ ID NO 78349, 78470, 78498, 78406, 78442, 78451, 78396, 78409 or 78378; (dd) SEQ ID NO 78447; or SEQ ID NO
78452.

72. The composition of any one of claims 69 to 71, wherein 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, (i) 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 of SEQ ID NOs 25058-30976, (n) any one of SEQ ID NOs 3911-5264, (o) any one of SEQ ID NOs 14473 -14876, (p) any one of SEQ ID NOs 38045 -42105, (q) any one of SEQ
ID NOs 32469-34520, (r) any one of SEQ ID NOs 51972 - 52025, (s) any one of SEQ ID NOs 51670-51971, (t) any one of SEQ ID NOs 5265 -14472, (u) any one of SEQ ID NOs 77375-78348, (v) any one of SEQ ID NOs 42106 -44370, (w) any one of SEQ ID NOs 44371 -44625, (x) any one of SEQ ID NOs 30977-32468, (y) any one of SEQ ID NOs 21810-23485, (z) any one of SEQ ID NOs 2600-2812, (aa) any one of SEQ ID NOs 14877 -21809, (bb) any one of SEQ ID NOs 23486-25057, (cc) any one of SEQ ID NOs 47014 -49422, (dd) any one of SEQ ID NOs 1780 -1899, or any one of SEQ ID NOs 67282 - 67531.

73. The composition of any one of claims 69 to 72, wherein 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, (i) SEQ ID NOs 90 or 91, (j) SEQ ID NOs 85 or 86, (k) SEQ ID
NO 32, (l) SEQ ID
NOs 115-120 or126-129, (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 97, (q) SEQ ID NOs 83 or 84, (r) SEQ ID NO 114, (s) SEQ
ID NO 223, (t) SEQ ID NOs 47-56, (u) SEQ ID NO 130, (v) SEQ ID NOs 98-102, (w) SEQ ID NO 103, (x) SEQ ID
NOs 80-82, (y) SEQ ID NOs 66-73, (z) SEQ ID NO 39, (aa) SEQ ID NOs 61-65, (bb) SEQ ID NOs 74 or 75, (cc) SEQ ID NOs 106-108, (dd) SEQ ID NOs 35 or 36, or SEQ ID NO 125.

74. The composition of any one of claims 69 to 73, wherein 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, (i) SEQ ID NO 18, (j) SEQ ID
NO 16, (k) SEQ ID NO 1, (l) 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) SEQ ID
NO 5, (aa) SEQ ID NO 10, (bb) SEQ ID NO 12, (cc) SEQ ID NO 24, (dd) SEQ ID NO
3, or (ee) SEQ ID NO 29.

75. The composition of any one of claims 56 to 74, wherein 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.

76. The composition of any one of claims 56 to 75, wherein 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.

77. The composition of any one of claims 56 to 76, wherein the RIC pre-mRNA
was produced by partial splicing from a full-length pre-mRNA or a wild-type pre-mRNA.

78. The composition of any one of claims 56 to 77, wherein the mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a wild-type mature mRNA.

79. The composition of any one of claims 56 to 78, wherein the target protein produced is full-length protein, or wild-type protein.

80. The composition of any one of claims 56 to 79, wherein the retained intron is a rate-limiting intron.

81. The composition of any one of claims 56 to 80 wherein said retained intron is the most abundant retained intron in said RIC pre-mRNA.

82. The composition of any one of claims 56 to 80, wherein the retained intron is the second most abundant retained intron in said RIC pre-mRNA.

83. The composition of any one of claims 56 to 82, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

84. The composition of any one of claims 56 to 83 wherein said antisense oligomer is an antisense oligonucleotide.

85. The composition of any one of claims 56 to 84, wherein 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.

86. The composition of any one of claims 56 to 85, wherein the antisense oligomer comprises at least one modified sugar moiety.

87. The composition of claim 86, wherein each sugar moiety is a modified sugar moiety.

88. The composition of any one of claims 56 to 87, wherein 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, to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

89. The composition of any one of claims 56 to 88, wherein 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.

90. A pharmaceutical composition comprising the antisense oligomer of any of the compositions of claims 56 to 89, and an excipient.

91. A method of treating a subject in need thereof by administering the pharmaceutical composition of claim 90 by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

92. A pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a deficient AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript, wherein the deficient AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA
transcript comprises a retained intron, wherein the antisense oligomer induces splicing out of the retained intron from the deficient AMT, ADA, PPDX, UROD, FIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript; and a pharmaceutical acceptable excipient.

93. The pharmaceutical composition of claim 92, wherein the deficient AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript.

94. The pharmaceutical composition of claim 92 or 93, wherein the targeted portion of the AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, 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.

95. The pharmaceutical composition of claim 92 or 93, wherein the AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIBL TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, 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.

96. The pharmaceutical composition of claim 92 or 93, wherein the AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ
ID NOs: 32-130.

97. The pharmaceutical composition of claim 92, wherein the antisense oligomer comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage.

98. The pharmaceutical composition of claim 92, wherein the antisense oligomer is an antisense oligonucleotide.

99. The pharmaceutical composition of claim 92, wherein the antisense oligomer comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O-methoxyethyl moiety.

100. The pharmaceutical composition of claim 92, wherein the antisense oligomer comprises at least one modified sugar moiety.

101. The pharmaceutical composition of claim 92, wherein 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 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases.

102. The pharmaceutical composition of claim 92 or 93, wherein 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, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA transcript.

103. The pharmaceutical composition of claim 92 or 93, wherein the targeted portion of the AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC pre-mRNA transcript is within a sequence selected from SEQ ID NOs: 78349-78501.

104. The pharmaceutical composition of claim 92, wherein 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.

105. The pharmaceutical composition of claim 92, wherein the antisense oligomer comprises a nucleotide sequence selected from SEQ ID NOs: 131-78348.

106. The pharmaceutical composition of any one of the claims 92-105, wherein the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

107. A method of inducing processing of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, WD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript to facilitate removal of a retained intron to produce a fully processed AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript that encodes a functional form of a AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein, the method comprising:
(a) contacting an antisense oligomer to a target cell of a subject;
(b) hybridizing the antisense oligomer to the AMT, ADA, PPDX, UROD, HIVIBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, TIIPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript, wherein the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is capable of encoding the functional form of a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein and comprises at least one retained intron;
(c) removing the at least one retained intron from the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, EIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript to produce the fully processed AMT, ADA, PPDX, UROD, IIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript that encodes the functional form of AMT, ADA, PPDX, UROD, EIMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein; and (d) translating the functional form of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 protein from the fully processed AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript.

108. The method of claim 107, wherein the retained intron is an entire retained intron.

109. The method of claim 107 or 108, wherein the AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 mRNA transcript is a AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, EINF4A, GCK, POGLUT1, PIK3R1, HNF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, PPARD, IL6, HSD3B7, CERS2 or NCOA5 RIC
pre-mRNA transcript.

110. A method of treating a subject having a condition caused by a deficient amount or activity of AMT, ADA, PPDX, UROD, HMBS, ACADVL, PC, IVD, APOA5, GALT, LDLRAP1, HNF4A, GCK, POGLUT1, PIK3R1, EINF1A, TRIB1, TGFB1, HAMP, THPO, PNPLA3, ATP7B, FAH, ASL, FIFE, ALMS1, 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.