Note: Descriptions are shown in the official language in which they were submitted.
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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
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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
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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.
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[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)
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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.
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[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-
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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.
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[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.
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[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,
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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,
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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 -
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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 -
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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.
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[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,
CERS2
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,
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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,
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.
[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
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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,
CERS2
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,
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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,
CERS2
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.
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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.
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[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.
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[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).
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[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
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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.
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[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).
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[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).
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[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.
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[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
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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,
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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
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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
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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.
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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.
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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.
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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
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(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
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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
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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
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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
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[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
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[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.
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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-
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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,
CERS2
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
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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
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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,
CERS2
or NCOA5 isoform sequence. One of skill in the art can determine the
corresponding intron number in
any isoform based on an intron sequence provided herein or using the number
provided in reference to
the one or more mRNA sequence at shown in Table 1 or Table 2. One of skill in
the art also can
determine the sequences of flanking exons in any AMT, ADA, 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,
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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,
CERS2
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,
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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
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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
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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.
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[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
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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
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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,
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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.
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[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.
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[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
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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.
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[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
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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
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(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.
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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,
CERS2
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,
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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,
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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
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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
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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.
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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
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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
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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
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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
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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.
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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
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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%)
AMT 275 NM 000481
Intron 6(15%)
Intron 4(11%)
ADA 100 NM 000022 Intron 11(12%)
Intron 7(N/A)
Intron 5(59%)
Intron 4(22%)
PPDX 5498 NM 000309
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%)
FIMBS 3145 NM 000190
Intron 9(12%)
Intron 3(N/A)
Intron 5(N/A)
Intron 8(N/A)
ACADVL 37 NM 000018
Intron 9(N/A)
Intron 10(15%)
Intron 13(15%)
PC 5091 NM 022172 Intron 16(25%)
IVD 3712 NM 002225 Intron 7(22%)
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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)
GALT 2592 NM 000155
Intron 3(N/A)
Intron 4(N/A)
Intron 8(N/A)
Intron 9(N/A)
Intron 1(12%)
LDLRAP1 26119 NM 015627
Intron 8(12%)
GCK 2645 No reads No reads
POTGLUT1 56983 NM 152305 Intron 1(18%)
Intron 4(47%)
POTGLUT1 56983 NR 024265
Intron 5(44%)
Intron 3(7%)
PIK3R1 5295 NM 181523
Intron 9(11%)
HNF 1 A 6927 No reads No reads
Intron 2(48%)
TRIB1 10221 NM 025195
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%)
PNPLA3 80339 NM 025225
Intron 5(16%)
Intron 7(41%)
Intron 7(N/A)
ATP7B 540 NM 000053
Intron 13(8%)
FAH 2184 NM 000137 Intron l(N/A)
Intron 7(24%)
Intron 8(N/A)
ASL 435 NM 000048
Intron 9(23%)
Intron 16(16%)
FIFE 3077 NM 000410 Intron 2(N/A)
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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%)
IL6 3569 NM 000600
Intron 3(10%)
Intron 4(N/A)
Intron 1(74%)
Intron 2(25%)
HSD3B7 80270 NM 001142777
Intron 3(14%)
Intron 4(22%)
Intron 5(12%)
HSD3B7 80270 NM 025193
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
926 - 964 3 78410
965 - 1059 4 78386
UROD: NM 000374
1060 - 1150 5 78411
SEQ ID NO. 33
1151 -1279 6 78460
UROD 1280 - 1352 7 78463
SEQ ID NO. 2 1353 - 1391 3 78410
1392 - 1486 4 78386
UROD: NR 036510
1487 - 1577 5 78411
SEQ ID NO. 34
1578 - 1706 6 78460
1707 - 1779 7 78463
CERS2 CERS2: NM 022075
1780 - 1839 6 78447
SEQ ID NO. 3 SEQ ID NO. 35
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CERS2: NM 181746
1840 - 1899 6 78447
SEQ ID NO. 36
1900 - 1959 8 78461
PPDX: NM 000309 1960 - 2060 5 78473
SEQ ID NO. 37 2161 - 2110 12 78480
PPDX 2111 -2249 4 78421
SEQ ID NO. 4 2250 - 2309 8 78461
PPDX: NM 001122764 2310 - 2410 5 78473
SEQ ID NO. 38 2411 - 2460 12 78480
2461 -2599 4 78421
ALMS1 ALMS1: NM 015120
2600 - 2812 21 78399
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
3911 -4092 1 78368
POGLUT1: NM 152305
4093 - 4333 4 78350
SEQ ID NO. 45
POGLUT1 4334 - 4559 5 78432
SEQ ID NO. 7 4560 - 4731 1 78368
POGLUT1: NR 024265
4732 - 5010 4 78439
SEQ ID NO. 46
5011 -5264 5 78389
5265 - 5525 2 78425
THPO: NM 001289998 5526 - 5616 3 78400
SEQ ID NO. 47 5617 - 5704 4 78393
THPO 5705 - 5943 5 78351
SEQ ID NO. 8 5944 - 6244 6 78381
6245 - 6473 1 78366
THPO: NM 001177598
6474 - 6564 2 78400
SEQ ID NO. 48
6565 - 6652 3 78393
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6653 -6891 4 78351
6892 - 7192 5 78457
7193 - 7453 2 78425
7454 - 7544 3 78400
THPO: NM 001290022
7545 - 7632 4 78393
SEQ ID NO. 49
7633 -7870 5 78351
7871 - 8172 6 78443
8173 - 8433 2 78425
8434 - 8524 3 78400
THPO: NM 001290003
8525 - 8612 4 78393
SEQ ID NO. 50
8613 - 8851 5 78351
8852 - 9152 6 78381
9153 - 9381 1 78366
9382 - 9472 2 78400
THPO: NM 001289997
9473 - 9560 3 78393
SEQ ID NO. 51
9561 - 9799 4 78351
9800 - 9888 5 78362
9889 - 10117 1 78366
10118 - 10208 2 78400
THPO: NM 000460
10208 - 10296 3 78393
SEQ ID NO. 52
10297 - 10535 4 78351
10536- 10836 5 78381
10837 - 11065 1 78366
11066 - 11156 2 78400
THPO: NM 001177597
11157 - 11244 3 78393
SEQ ID NO. 53
11245 - 11483 4 78351
11484 - 11784 5 78443
11785 - 12005 1 78446
12006 - 12096 2 78400
THPO: NM 001290028
12097 - 12184 3 78393
SEQ ID NO. 54
12185 - 12423 4 78351
12424 - 12724 5 78381
12725 - 12985 2 78425
THPO: NM 001290027 12986 - 13076 3 78400
SEQ ID NO. 55 13077 - 13164 4 78393
13165 - 13403 5 78351
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13404 - 13492 6 78362
13493 - 13753 2 78425
13754 - 13844 3 78400
THPO: NM 001290026
13845 - 13932 4 78393
SEQ ID NO. 56
13933 - 14171 5 78351
14172 - 14472 6 78457
PIK3R1: NM 181523 14473 - 14544 9 78390
SEQ ID NO. 57 14545 - 14660 3 78418
PIK3R1: NM 181524
14661 - 14732 3 78390
PIK3R1 SEQ ID NO. 58
SEQ ID NO. 9 PIK3R1: NM 181504
14733 - 14804 3 78390
SEQ ID NO. 59
PIK3R1: NM 001242466
14805 - 14876 2 78390
SEQ ID NO. 60
14877 - 15133 3 78414
PPARD: NM 006238 15134 - 15376 4 78478
SEQ ID NO. 61 15377 - 15628 5 78472
15629 - 15788 6 78357
15789 - 16479 7 78395
16480 - 16736 3 78414
PPARD: NM 177435
16737 - 16979 4 78478
SEQ ID NO. 62
16980 - 17231 5 78472
17232 - 17512 6 78359
17513 - 18203 8 78395
PPARD 18204 - 18460 4 78414
PPARD: NM 001171818
SEQ ID NO. 10 18461 - 18703 5 78478
SEQ ID NO. 63
18704 - 18955 6 78472
18956 - 19115 7 78357
19116 - 19345 2 78374
19346 - 19588 3 78478
PPARD: NM 001171819
19589 - 19840 4 78472
SEQ ID NO. 64
19841 -20000 5 78357
20001 -20691 6 78395
20692 - 20958 3 78490
PPARD: NM 001171820
20959 - 21118 4 78357
SEQ ID NO. 65
21119 - 21809 5 78395
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HFE: NM 139007
21810 - 22083 1 78419
SEQ ID NO. 66
HFE: NM 139006
22084 - 22227 2 78384
SEQ ID NO. 67
HFE: NM 139004
22228 - 22513 2 78500
SEQ ID NO. 68
HFE: NM 139003
22514 - 22793 2 78424
FIFE SEQ ID NO. 69
SEQ ID NO. 11 FIFE: NM 001300749
22794 - 22937 2 78384
SEQ ID NO. 70
HFE: NM 139009
22938 - 23067 2 78466
SEQ ID NO. 71
HFE: NM 139008
23068 - 23341 1 78419
SEQ ID NO. 72
HFE: NM 000410
23342 - 23485 2 78384
SEQ ID NO. 73
23486 - 23706 1 78436
1L6: NM 001318095
23707 - 23891 2 78413
SEQ ID NO. 74
23892 - 24222 3 78415
IL6
24223 - 24305 1 78398
SEQ ID NO. 12
1L6: NM 000600 24306 - 24541 2 78403
SEQ ID NO. 75 24542 - 24726 3 78413
24727 - 25057 4 78415
25058 - 25285 1 78445
25286 - 25495 2 78481
25496 - 25713 3 78379
25714 - 25941 4 78431
GCK: NM 033508 25942 - 26144 5 78469
SEQ ID NO. 76 26145 - 26197 6 78408
GCK 26198 - 26431 7 78377
SEQ ID NO. 13 26432 - 26674 8 78417
26675 - 26882 9 78387
26883 - 27143 10 78455
27144 - 27434 1 78484
GCK: NM 000162 27435 - 27652 2 78379
SEQ ID NO. 77 27653 - 27880 3 78431
27881 -28083 4 78469
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28084 - 28136 5 78408
28137 - 28370 6 78377
28371 - 28613 7 78417
28614 - 28821 8 78387
28822 - 29082 9 78455
29083 - 29328 1 78370
29329 - 29546 2 78379
29547 - 29774 3 78431
29775 -29977 4 78469
GCK: NM 033507
29978 - 30030 5 78408
SEQ ID NO. 78
30031 - 30264 6 78377
30265 - 30507 7 78417
30508 - 30715 8 78387
30716 - 30976 9 78455
30977 - 31094 8 78494
ASL: NM 000048 31095 - 31169 9 78404
SEQ ID NO. 79 31170 - 31312 16 78371
31313 - 31352 7 78365
31353 - 31427 8 78404
ASL: NM 001024943 31428 - 31570 15 78371
SEQ ID NO. 80 31571 - 31610 6 78365
ASL
31611 - 31728 7 78494
SEQ ID NO. 14
__________________________________________________________________
31729 - 31803 8 78404
ASL: NM 001024944 31804 - 31843 6 78365
SEQ ID NO. 81 31844 - 31986 14 78371
31987 - 32104 7 78494
32105 - 32247 14 78371
ASL: NM 001024946
32248 - 32393 6 78353
SEQ ID NO. 82
32394 - 32468 7 78404
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
34521 -34601 2 78489
GALT GALT: NM 000155
34602 - 34634 3 78416
SEQ ID NO. 16 SEQ ID NO. 85
34635 - 34685 4 78476
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34686 - 34740 5 78352
34741 -34835 7 78435
34836 - 34884 8 78493
34885 - 34988 9 78423
34989 - 35216 10 78437
35217 - 35368 2 78449
35369 - 35423 3 78352
GALT: NM 001258332 35424 - 35518 5 78435
SEQ ID NO. 86 35519 - 35567 6 78493
35568 - 35671 7 78423
35672 - 35899 8 78437
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
36162 - 36292 16 78495
SEQ ID NO. 89
36293 - 36397 1 78407
AP0A5: NM 052968
36398 - 36455 2 78499
SEQ ID NO. 90
AP0A5 36456 - 36890 3 78420
SEQ ID NO. 18 36891 -36994 1 78372
AP0A5: NM 001166598
36995 - 37047 2 78397
SEQ ID NO. 91
37048 - 37482 3 78420
HMBS: NM 000190 37483 - 37554 10 78355
SEQ ID NO. 92 37555 - 37632 11 78467
HMBS: NM 001258208
37633 - 37763 10 78454
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
37914 - 38044 10 78454
SEQ ID NO. 95
38045 - 38361 1 78453
38362 - 38621 2 78361
HNF 1 A HNF1A: NM 000545
38622 - 38778 3 78363
SEQ ID NO. 20 SEQ ID NO. 96
38779 - 39030 4 78433
39031 - 39107 5 78438
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39108 - 39309 6 78430
39310 - 39543 7 78488
39544 - 39598 8 78462
39599 - 40073 9 78468
40074 - 40390 1 78453
40391 -40650 2 78361
40651 -40807 3 78363
40808 - 41509 4 78433
HNF1A: NM 001306179
41510 - 41136 5 78438
SEQ ID NO. 97
41137 - 41338 6 78430
41339 - 41572 7 78488
41573 - 41628 8 78405
41629 - 42105 9 78492
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
43126 - 43373 9 78383
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
44371 - 44625 1 78382
SEQ ID NO. 22 SEQ ID NO. 103
44626 - 44775 8 78464
IVD: NM 001159508
44776 - 45598 10 78375
SEQ ID NO. 104
IVD 45599 - 45819 6 78380
SEQ ID NO. 23 45820 - 45969 9 78464
IVD: NM 002225
45970 - 46792 11 78375
SEQ ID NO. 105
46793 - 47013 7 78380
47014 - 47115 1 78349
47116 - 47244 2 78470
HSD3B7 HSD3B7: NM 001142777
47245 - 47325 3 78498
SEQ ID NO. 24 SEQ ID NO. 106
47326 - 47370 4 78406
47371 -47825 5 78442
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47826 - 47924 1 78451
47925 - 48021 2 78396
HSD3B7: NM 001142778
48022 - 48102 3 78498
SEQ ID NO. 107
48103 - 48147 4 78406
48148 - 48602 5 78442
48603 - 48701 1 78451
48702 - 48798 2 78396
HSD3B7: NM 025193 48799 - 48879 3 78498
SEQ ID NO. 108 48880 -48924 4 78406
48925 - 48989 5 78409
48990 - 49422 6 78378
49423 - 49458 2 78367
49459 - 49512 4 78376
49513 - 49565 7 78440
ACADVL: NM 001270448 49566 - 49697 8 78448
SEQ ID NO. 109 49698 - 49800 9 78477
49801 -49846 12 78485
49847 - 49890 17 78496
49891 -49969 18 78422
49970 - 50005 4 78367
50006 - 50059 6 78376
50060 - 50112 9 78440
ACADVL: NM 001270447 50113 - 50244 10 78448
ACADVL
SEQ ID NO. 110 50245 - 50347 11 78477
SEQ ID NO. 25
50348 - 50393 14 78485
50394 - 50437 19 78496
50438 - 50516 20 78422
50517 - 50552 3 78367
50553 - 50606 5 78376
50607 - 50659 8 78440
ACADVL: NM 000018 50660 - 50791 9 78448
SEQ ID NO. 111 50792 - 50894 10 78477
50895 - 50940 13 78485
50941 - 50984 18 78496
50985 - 51063 19 78422
ACADVL: NM 001033859 51064 - 51158 2 78412
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SEQ ID NO. 112 51159 - 51212 4 78376
51213 - 51265 7 78440
51266 - 51397 8 78448
51398 - 51500 9 78477
51501 -51546 12 78485
51547 - 51590 17 78496
51591 - 51669 18 78422
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
51972 - 52025 4 78458
SEQ ID NO. 27 SEQ ID NO. 114
52026 - 52267 2 78392
52268 - 52499 3 78456
52500 - 52699 4 78428
HNF4A: NM 001287184
52700 - 52877 5 78491
SEQ ID NO. 115
52878 - 53111 6 78501
53112 - 53348 7 78360
53349 - 53676 8 78441
53677 - 53918 2 78392
53919 - 54150 3 78456
54151 - 54350 4 78428
54351 -54528 5 78491
HNF4A: NM 001287183
54529 - 54762 6 78501
HNF4A SEQ ID NO. 116
54763 - 54999 7 78360
SEQ ID NO. 30
55000 - 55255 8 78429
55256 - 55508 9 78358
55509 - 56331 10 78364
56332 - 56544 1 78475
56545 - 56776 2 78456
56777 - 56976 3 78428
56977 - 57154 4 78491
HNF4A: NM 175914
57155 - 57388 5 78501
SEQ ID NO. 117
57389 - 57625 6 78360
57626 - 57881 7 78429
57882 - 58134 8 78358
58135 - 58957 9 78364
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58958 - 59170 1 78475
59171 -59402 2 78456
59403 - 59602 3 78428
HNF4A: NM 001030004
59603 - 59780 4 78491
SEQ ID NO. 118
59781 - 60014 5 78501
60015 - 60251 6 78360
60252 - 60579 7 78441
60580 - 60821 2 78392
60822 - 61053 3 78456
61054 - 61253 4 78428
61254 - 61431 5 78491
HNF4A: NM 001287182
61432 - 61665 6 78501
SEQ ID NO. 119
61666 - 61902 7 78360
61903 - 62158 8 78429
62159 - 62408 9 78391
62409 - 63227 10 78479
63228 - 63440 1 78475
63441 -63672 2 78456
63673 - 63872 3 78428
63873 - 64050 4 78491
HNF4A: NM 001030003
64051 - 64284 5 78501
SEQ ID NO. 120
64285 - 64521 6 78360
64522 - 64777 7 78429
64778 - 65027 8 78391
65028 - 65846 9 78479
67532 - 67780 1 78401
67781 - 68012 2 78456
68013 - 68212 3 78428
68213 - 68390 4 78491
HNF4A: NM 000457
68391 - 68624 5 78501
SEQ ID NO. 126
68625 - 68861 6 78360
68862 - 69117 7 78429
69118 - 69370 8 78358
69371 -70193 9 78364
HNF4A: NM 001258355 70194 - 70436 1 78373
SEQ ID NO. 127 70437 - 70648 2 78450
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70649 - 70880 3 78456
70881 - 71080 4 78428
71081 - 71258 5 78491
71259 - 71492 6 78501
71493 - 71729 7 78360
71730 - 71985 8 78429
71986 - 72238 9 78358
72239 - 73061 10 78364
73062 - 73310 1 78401
73311 - 73542 2 78456
73543 - 73742 3 78428
HNF4A: NM 178850
73743 - 73920 4 78491
SEQ ID NO. 128
73921 - 74154 5 78501
74155 - 74391 6 78360
74392 - 74719 7 78441
74720 - 74968 1 78401
74969 - 75200 2 78456
75201 -75400 3 78428
75401 -75578 4 78491
HNF4A: NM 178849
75579 - 75812 5 78501
SEQ ID NO. 129
75813 - 76049 6 78360
76050 - 76305 7 78429
76306 - 76555 8 78391
76556 - 77374 9 78479
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
67282 - 67531 2 78452
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
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77892 - 78118 5 78471
78119 - 78348 7 78356
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,
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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.
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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
CA 03005090 2018-05-10
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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
86
CA 03005090 2018-05-10
WO 2017/106283 PCT/US2016/066564
[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.
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