Note: Descriptions are shown in the official language in which they were submitted.
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ANTISENSE OLIGOMERS FOR TREATMENT OF POLYCYSTIC KIDNEY DISEASE
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No.
62/267,252, filed
December 14, 2015, which application is incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been
submitted electronically in
ASCII format and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on
December 9, 2016, is named 47991 707 601 SL.txt and is 253,332 bytes in size.
BACKGROUND OF THE INVENTION
[0003] Autosomal dominant polycystic kidney disease (ADPKD), is one of the
most common inherited
renal cystic diseases, conditions characterized by the development of renal
cysts and a variety of
extrarenal manifestations (Tones and Harris, 2009, Kidney International (2009)
76, 149-168). Patients
suffering from ADPKD generally develop end-stage renal disease (ESRD) by age
70, which ultimately
requires interventions such as renal dialysis. The prevalence of ADPKD at
birth is estimated to be
between 1:400 and 1:1,000, affecting about 600,000 people in the US.
[0004] Mutations in either the PKD1 or PKD2 gene have been shown to manifest
as ADPKD, with
mutations in PKD2 being responsible for the late onset form of ADPKD. The PKD1
and PKD2 genes
encode the PC-1 and PC-2 proteins, respectively. These proteins are believed
to be essential to maintain
the differentiated phenotype of the tubular epithelium (Tones and Harris,
2009).
SUMMARY OF THE INVENTION
[0005] Disclosed herein, in some embodiments, are methods of treating
Polycystic Kidney Disease in a
subject in need thereof, by increasing the expression of a target protein or
functional RNA by cells of the
subject, wherein the cells have a retained-intron-containing pre-mRNA (RIC pre-
mRNA), the RIC pre-
mRNA comprising a retained intron, an exon flanking the 5' splice site, an
exon flanking the 3' splice
site, and wherein the RIC pre-mRNA encodes the target protein or functional
RNA, the method
comprising contacting the cells of the subject with an antisense oligomer
(ASO) complementary to a
targeted portion of the RIC pre-mRNA encoding the target protein or functional
RNA, whereby the
retained intron is constitutively spliced from the RIC pre-mRNA encoding the
target protein or functional
RNA, thereby increasing the level of mRNA encoding the target protein or
functional RNA, and
increasing the expression of the target protein or functional RNA in the cells
of the subject.
[0006] Also disclosed herein, in some embodiments, are methods of increasing
expression of a target
protein, wherein the target protein is PC-2, 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 PC-2 protein, the method comprising contacting the cells with an
antisense oligomer (ASO)
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complementary to a targeted portion of the RIC pre-mRNA encoding PC-2 protein,
whereby the retained
intron is constitutively spliced from the RIC pre-mRNA encoding PC-2 protein,
thereby increasing the
level of mRNA encoding PC-2 protein, and increasing the expression of PC-2
protein in the cells.
[0007] In some embodiments of any of the aforementioned methods, the target
protein is PC-2. 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. In some embodiments, the cells are in or
from a subject having a
condition caused by a deficient amount or activity of PC-2 protein.
[0008] In some embodiments of any of the aforementioned methods, 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. 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) a first mutant allele from which (i) the target
protein is produced at a reduced
level compared to production from a wild-type allele, (ii) the target protein
is produced in a form having
reduced function compared to an equivalent wild-type protein, or (iii) the
target protein is not produced,
and (b) a second mutant allele from which (i) the target protein is produced
at a reduced level compared
to production from a wild-type allele, (ii) the target protein is produced in
a form having reduced function
compared to an equivalent wild-type protein, or (iii) the target protein is
not produced, and wherein when
the subject has a first mutant allele (a)(iii), the second mutant allele is
(b)(i) or (b)(ii), and wherein when
the subject has a second mutant allele (b)(iii), the first mutant allele is
(a)(i) or (a)(ii), and wherein the
RIC pre-mRNA is transcribed from either the first mutant allele that is (a)(i)
or (a)(ii), and/or the second
allele that is (b)(i) or (b)(ii). In some embodiments, the target protein is
produced in a form having
reduced function compared to the equivalent wild-type protein. In some
embodiments, the target protein
is produced in a form that is fully-functional compared to the equivalent wild-
type protein.
[0009] In some embodiments of any of the aforementioned methods, 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. In some embodiments,
the targeted portion of the
RIC pre-mRNA is in the retained intron within: (a) the region +6 to +497
relative to the 5' splice site of
the retained intron; or (b) the region -16 to -496 relative to the 3' splice
site of the retained intron. In
some embodiments, the targeted portion of the RIC pre-mRNA is within: (a) the
region +2e to -4e in the
exon flanking the 5' splice site of the retained intron; or (b) the region +2e
to -4e in the exon flanking the
3' splice site of the retained intron. In some embodiments, the targeted
portion of the RIC pre-mRNA is
in the retained intron within: (a) the region -4e to -1,054e relative to the
5' splice site of the retained
intron; (b) the region +6 to +499 relative to the 5' splice site of the
retained intron; (c) the region -16 to -
496 relative to the 3' splice site of the retained intron; or (d) the region
+2e to +1,912e relative to the 3'
splice site of the retained intron. In some embodiments, the target protein is
PC-2.
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[0010] In some embodiments of any of the aforementioned methods, the RIC pre-
mRNA comprises a
sequence with at least about 80%, 85%, 90%, 950, 97%, or 10000 sequence
identity to SEQ ID NO: 2. In
some embodiments, the RIC pre-mRNA is encoded by a genetic sequence with at
least about 80%, 85%,
90%, 950, 97%, or 1000o sequence identity to SEQ ID NO: 1. In some
embodiments, the targeted
portion of the RIC pre-mRNA comprises a sequence with at least 80%, 85%, 90%,
950, 97%, or 1000o
sequence identity to a region comprising at least 8 contiguous nucleic acids
of SEQ ID NO: 281. In some
embodiments, the ASO comprises a sequence that is at least about 80%, 85%,
90%, 950, 97%, or 1000o
complimentary to any one of SEQ ID NOs: 3-280. In some embodiments, the
targeted portion of the RIC
pre-mRNA is within the region -204e to +497 relative to the 5' splice site of
the retained intron 5 or
within the region -496 to +212e relative to the 3' splice site of the retained
intron 5. In some
embodiments, the ASO comprises a sequence that is at least about 80%, 85%,
90%, 950, 97%, or 1000o
complimentary to any one of SEQ ID NOs: 3-280. In some embodiments, the
targeted portion of the RIC
pre-mRNA is in exon 5 within the region -204e to -4e relative to the 5' splice
site of the retained intron 5.
In some embodiments, the ASO comprises a sequence that is at least about 80%,
85%, 90%, 950, 97%, or
1000o complimentary to any one of SEQ ID NOs: 3-43. In some embodiments, the
targeted portion of
the RIC pre-mRNA is in retained intron 5 within the region +6 to +497 relative
to the 5' splice site of the
retained intron. In some embodiments, the ASO comprises a sequence that is at
least about 80%, 85%,
90%, 950, 97%, or 1000o complimentary to any one of SEQ ID NOs:44-140. In some
embodiments, the
targeted portion of the RIC pre-mRNA is in retained intron 5 within the region
-16 to -496 relative to the
3' splice site of the retained intron. In some embodiments, the ASO comprises
a sequence that is at least
about 80%, 85%, 90%, 950, 97%, or 1000o complimentary to any one of SEQ ID
NOs: 141-237. In
some embodiments, the targeted portion of the RIC pre-mRNA is in exon 6 within
the region +2e to
+212e relative to the 3' splice site of the retained intron 5. In some
embodiments, the ASO comprises a
sequence that is at least about 80%, 85%, 90%, 950, 97%, or 1000o
complimentary to any one of SEQ
ID NOs: 238-280.
100111 In some embodiments of any of the aforementioned methods, 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. 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. 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. In some
embodiments, the mRNA
encoding the target protein or functional RNA is a full-length mature mRNA, or
a wild-type mature
mRNA. In some embodiments, the target protein produced is full-length protein,
or wild-type protein. 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
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about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to
about 7-fold, about 2 to about 8-
fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-
fold, about 3 to about 8-fold,
about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold,
about 4 to about 9-fold, at least
about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about
2.5-fold, at least about 3-fold, at
least about 3.5-fold, at least about 4-fold, at least about 5-fold, or at
least about 10-fold, compared to the
total amount of the mRNA encoding the target protein or functional RNA
produced in a control cell. 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.
100121 In some embodiments of any of the aforementioned methods, the antisense
oligomer comprises a
backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate linkage. 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. In some
embodiments, the antisense oligomer comprises at least one modified sugar
moiety. In some
embodiments, each sugar moiety is a modified sugar moiety. 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. 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.
[0013] In some embodiments of any of the aforementioned methods, 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 at least one retained intron, and
wherein the antisense oligomer
binds to the most abundant retained intron in the population of RIC pre-mRNAs.
In some embodiments,
the binding of the antisense oligomer to the most abundant retained intron
induces splicing out of the at
least one retained intron from the population of RIC pre-mRNAs to produce mRNA
encoding the target
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protein or functional RNA. 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. 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. In some embodiments, the method further
comprises assessing PC-2
protein expression.
[0014] In some embodiments of any of the aforementioned methods, the antisense
oligomer binds to a
targeted portion of a PKD2 RIC pre-mRNA, wherein the targeted portion is in a
sequence selected from
SEQ ID NOs: 3-280. In some embodiments, the subject is a human. In some
embodiments, the subject is
a non-human animal. In some embodiments, the subject is a fetus, an embryo, or
a child. In some
embodiments, the cells are ex vivo. In some embodiments, the antisense
oligomer is administered by
intraperitoneal injection, intramuscular injection, subcutaneous injection, or
intravenous injection of the
subject. 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. 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. Disclosed
herein, in some embodiments,
are antisense oligomers as described in any of the aforementioned methods.
[0015] Disclosed herein, in some embodiments, are antisense oligomers
comprising a sequence with at
least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to any one of
SEQ ID NOs: 3-280.
[0016] Also disclosed herein, in some embodiments, are pharmaceutical
compositions comprising any of
the aforementioned antisense oligomers and an excipient.
[0017] Disclosed herein, in some embodiments, are methods of treating a
subject in need thereof, by
administering any of the aforementioned pharmaceutical compositions by
intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous injection.
[0018] Disclosed herein, in some embodiments, are compositions comprising an
antisense oligomer for
use in a method of increasing expression of a target protein or a functional
RNA by cells to treat
Polycystic Kidney Disease in a subject in need thereof, associated with a
deficient protein or deficient
functional RNA, wherein the deficient protein or deficient functional RNA is
deficient in amount or
activity in the subject, wherein the antisense oligomer enhances constitutive
splicing of a retained intron-
containing pre-mRNA (RIC pre-mRNA) encoding the target protein or the
functional RNA, wherein the
target protein is: (a) the deficient protein; or (b) a compensating protein
which functionally augments or
replaces the deficient protein or in the subject; and wherein the functional
RNA is: (c) the deficient RNA;
or (d) 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
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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.
100191 Disclosed herein, in some embodiments, are compositions comprising an
antisense oligomer for
use in a method of treating a condition associated with PC-2 protein in a
subject in need thereof, the
method comprising the step of increasing expression of PC-2 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 PC-2 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 PC-2 protein, thereby increasing the level of mRNA
encoding the PC-2
protein, and increasing the expression of PC-2 protein, in the cells of the
subject. In some embodiments,
the condition is a disease or disorder. In some embodiments, the disease or
disorder is Polycystic Kidney
Disease. In some embodiments, the target protein and RIC pre-mRNA are encoded
by the PKD2 gene.
[0020] In some embodiments of any of the aforementioned compositions, 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. In some embodiments,
the antisense oligomer targets a portion of the RIC pre-mRNA that is in the
retained intron within: (a) the
region +6 to +497 relative to the 5' splice site of the retained intron; or
(b) the region -16 to -496 relative
to the 3' splice site of the retained intron. 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. In some embodiments, the targeted portion of the RIC pre-mRNA
is within: (a) the region
+2e to -4e in the exon flanking the 5' splice site of the retained intron; or
(b) the region +2e to -4e in the
exon flanking the 3' splice site of the retained intron. In some embodiments,
the targeted portion of the
RIC pre-mRNA is within: (a) the region -4e to -1,054e relative to the 5'
splice site of the retained intron;
(b) the region +6 to +499 relative to the 5' splice site of the retained
intron; (c) the region -16 to -496
relative to the 3' splice site of the retained intron; or (d) the region +2e
to +1,912e relative to the 3' splice
site of the retained intron. In some embodiments, the target protein is PC-2.
In some embodiments, the
RIC pre-mRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97%,
or 100% sequence
identity to SEQ ID NO: 2. In some embodiments, the RIC pre-mRNA is encoded by
a genetic sequence
with at least about 80%, 85%, 90%, 95%, 97%, or 100% sequence identity to SEQ
ID NO: 1. 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
SEQ ID NO: 281. In some embodiments, the ASO comprises a sequence that is at
least about 80%, 85%,
90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-280. In some
embodiments, the
targeted portion of the RIC pre-mRNA is within the region -204e to +497
relative to the 5' splice site of
the retained intron 5 or within the region ¨496 to +212e relative to the 3'
splice site of the retained intron
5. In some embodiments, the ASO comprises a sequence that is at least about
80%, 85%, 90%, 95%,
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97%, or 100% complimentary to any one of SEQ ID NOs: 3-280. In some
embodiments, the targeted
portion of the RIC pre-mRNA is in exon 5 within the region -204e to -4e
relative to the 5' splice site of
the retained intron 5. In some embodiments, the ASO comprises a sequence that
is at least about 80%,
85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ ID NOs: 3-43. In
some embodiments,
the targeted portion of the RIC pre-mRNA is in retained intron 5 within the
region +6 to +497 relative to
the 5' splice site of the retained intron. In some embodiments, the ASO
comprises a sequence that is at
least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary to any one of SEQ
ID NOs:44-140. In
some embodiments, the targeted portion of the RIC pre-mRNA is in retained
intron 5 within the region -
16 to -496 relative to the 3' splice site of the retained intron. In some
embodiments, the ASO comprises a
sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% complimentary
to any one of SEQ
ID NOs: 141-237. In some embodiments, the targeted portion of the RIC pre-mRNA
is in exon 6 within
the region +2e to +212e relative to the 3' splice site of the retained intron
5. In some embodiments, the
ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or
100% complimentary to
any one of SEQ ID NOs: 238-280.
[0021] In some embodiments of any of the aforementioned compositions, 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. 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. In some embodiments, the RIC pre-mRNA was produced by partial splicing
from a full-length pre-
mRNA or a wild-type pre-mRNA. In some embodiments, the mRNA encoding the
target protein or
functional RNA is a full-length mature mRNA, or a wild-type mature mRNA. In
some embodiments, the
target protein produced is full-length protein, or wild-type protein. In some
embodiments, the retained
intron is a rate-limiting intron. In some embodiments, the retained intron is
the most abundant retained
intron in said RIC pre-mRNA. In some embodiments, the retained intron is the
second most abundant
retained intron in said RIC pre-mRNA.
[0022] In some embodiments of any of the aforementioned compositions, the
antisense oligomer
comprises a backbone modification comprising a phosphorothioate linkage or a
phosphorodiamidate
linkage. In some embodiments, the antisense oligomer is an antisense
oligonucleotide. 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. In some
embodiments, the antisense oligomer comprises at least one modified sugar
moiety. In some
embodiments, each sugar moiety is a modified sugar moiety. 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
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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.
100231 Disclosed herein, in some embodiments, are pharmaceutical compositions
comprising any of the
aforementioned antisense oligomers and an excipient. Disclosed herein, in some
embodiments, are
methods of treating a subject in need thereof, by administering the
aforementioned pharmaceutical
compositions by intraperitoneal injection, intramuscular injection,
subcutaneous injection, or intravenous
injection.
100241 Disclosed herein, in some embodiments, are pharmaceutical compositions
comprising: an
antisense oligomer that hybridizes to a target sequence of a deficient PKD2
mRNA transcript, wherein the
deficient PKD2 mRNA transcript comprises a retained intron, wherein the
antisense oligomer induces
splicing out of the retained intron from the deficient PKD2 mRNA transcript;
and a pharmaceutical
acceptable excipient. In some embodiments, the deficient PKD2 mRNA transcript
is a PKD2 RIC pre-
mRNA transcript. In some embodiments, the targeted portion of the PKD2 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. In some embodiments,
the PKD2 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 SEQ ID NO: 1. In some embodiments, the PKD2
RIC pre-mRNA
transcript comprises a sequence with at least about 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or 100%
sequence identity to SEQ ID NO: 2. In some embodiments, the antisense oligomer
comprises a backbone
modification comprising a phosphorothioate linkage or a phosphorodiamidate
linkage. In some
embodiments, the antisense oligomer is an antisense oligonucleotide. In some
embodiments,n 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. In some
embodiments, the antisense
oligomer comprises at least one modified sugar moiety. 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,
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. 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 PKD2 RIC pre-mRNA transcript. In
some embodiments, the
targeted portion of the PKD2 RIC pre-mRNA transcript is within SEQ ID NO: 281.
In some
embodiments, the antisense oligomer comprises a nucleotide sequence that is 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:
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3-280. In some embodiments, the antisense oligomer comprises a nucleotide
sequence selected from SEQ
ID NOs: 3-280. In some embodiments, the pharmaceutical composition is
formulated for intrathecal
injection, intracerebroventricular injection, intraperitoneal injection,
intramuscular injection, subcutaneous
injection, or intravenous injection.
[0025] Disclosed herein, in some embodiments, are methods of inducing
processing of a deficient PKD2
mRNA transcript to facilitate removal of a retained intron to produce a fully
processed PKD2 mRNA
transcript that encodes a functional form of a PC-2 protein, the method
comprising: (a) contacting an
antisense oligomer to a target cell of a subject; (b) hybridizing the
antisense oligomer to the deficient
PKD2 mRNA transcript, wherein the deficient PKD2 mRNA transcript is capable of
encoding the
functional form of a PC-2 protein and comprises at least one retained intron;
(c) removing the at least one
retained intron from the deficient PKD2 mRNA transcript to produce the fully
processed PKD2 mRNA
transcript that encodes the functional form of PC-2 protein; and (d)
translating the functional form of PC-2
protein from the fully processed PKD2 mRNA transcript. In some embodiments,
the retained intron is an
entire retained intron. In some embodiments, the deficient PKD2 mRNA
transcript is a PKD2 pre-mRNA
transcript.
[0026] Disclosed herein, in some embodiments, are methods of treating a
subject having a condition
caused by a deficient amount or activity of PC-2 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: 3-280.
INCORPORATION BY REFERENCE
[0027] 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
[0028] 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.
[0029] FIG. 1 depicts an exemplary schematic representation of a retained-
intron-containing (RIC) pre-
mRNA transcript. In FIG.1, 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
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+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.
[0030] FIGS. 2A-B depict a 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. FIG. 2B shows
an example of the same
cell 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.
[0031] FIG. 3 depicts intron-retention in the PKD2 gene with intron 5 shown
in detail. The
identification of intron-retention events in the PKD2 gene using RNA
sequencing (RNAseq) is shown,
visualized in the UCSC genome browser. The upper panel shows the read density
corresponding to the
PKD2 transcript expressed in renal epithelial cells and localized in either
the cytoplasmic (top) or nuclear
fraction (bottom). At the bottom of this panel, a graphic representation of
the PKD2 gene is shown to
scale. The read density is shown as peaks. The highest read density
corresponds to exons (black boxes),
while no reads are observed for the majority of the introns in either cellular
fraction. Higher read density
is detected for intron 5 (pointed by the arrow) in the nuclear fraction
compared to the cytoplasmic fraction
indicating that splicing efficiency of intron 5 is low, resulting in intron
retention. The retained-intron
containing pre-mRNA transcripts are retained in the nucleus and are not
exported out to the cytoplasm.
The read density for intron 5 in renal epithelial cells is shown in detail in
the lower panel.
[0032] FIG. 4 depicts an exemplary PKD2 gene intron 5 (IVS 5) ASO walk. A
graphic representation
of the ASO walk performed for PKD2 IVS 5 targeting sequences immediately
downstream of the 5' splice
site or upstream of the 3' splice site using 2'-0-Me AS0s, PS backbone, is
shown. ASOs were designed
to cover these regions by shifting 5 nucleotides at a time. The PKD2 exon-
intron structure is drawn to
scale.
[0033] FIG. 5 depicts a schematic of the RefSeq Gene for PKD2 corresponding to
NM_000297. The
Percent Intron Retention (PIR) of intron 5 is detailed.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Individual introns in primary transcripts of protein-coding genes
having more than one intron are
spliced from the primary transcript with different efficiencies. In most cases
only the fully spliced mRNA
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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.
[0035] Substantial levels of partially-spliced transcripts of the PKD2 gene,
which encodes the PC-2
protein that is deficient in the debilitating genetic disease, Polycystic
Kidney Disease, have been
discovered in the nucleus of human cells. These PKD2 pre-mRNA species comprise
at least one retained
intron. The present invention provides compositions and methods for
upregulating splicing of one or
more retained PKD2 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 PC-
2 protein levels. These
compositions and methods utilize antisense oligomers (AS0s) that promote
constitutive splicing at an
intron splice site of a retained-intron-containing PKD2 pre-mRNA that
accumulates in the nucleus. Thus,
in embodiments, PC-2 protein is increased using the methods of the invention
to treat a disease caused by
PC-2 deficiency.
[0036] In other embodiments, the methods of the invention are used to increase
PC-2 production to treat
a condition in a subject in need thereof. In embodiments, the subject has
condition in which PC-2 is not
necessarily deficient relative to wild-type, but where an increase in PC-2
mitigates the condition
nonetheless. In embodiments, the condition is a caused by a PC-2
haploinsufficiency.
Polycystic Kidney Disease
[0037] Polycystic Kidney Disease (PKD) is an autosomal dominant multisystem
disorder characterized
by the evolution of renal cysts and a variety of extrarenal manifestations
(Tones and Harris, 2009). The
main clinical and pathological findings are renal disease due to the
development and enlargement of renal
cysts, which results in renal manifestations such an increase in kidney volume
that correlates directly with
the increase in the cyst volume. Other PKD manifestations include
hypertension; endothelial
vasodilation; constrictive nitric oxide synthase activity; polycystic liver
disease; vascular manifestations
including intracranial aneurysms, thoracic aortic dissections and coronary
artery aneurysms; and
progressive renal failure that leads to end-stage renal disease (ESRD) by age
70. While PKD can be
diagnosed in utero or at birth through the use of fetal ultrasonography, PKD
is classically diagnosed later
in life through the detection of renal cysts as determined by renal
ultrasound. The worldwide PKD
prevalence is estimated to be between 1:400 and 1:1000, with a male/female sex
ratio of ¨1.2 (Tones and
Harris, 2009).
[0038] Mutations in either the PKD1 or PKD2 gene have been reported to cause
PKD. Mutations in
PKD1 typically manifest earlier in life than mutations in PKD2 (age at ESRD
54.3 vs. 74.0 years for
PKD1 and PKD2, respectively) and typically result in a more severe disease
state due to the appearance of
cysts at a younger age (Tones and Harris, 2009). Due to this difference in
pathophysiology, the late onset
form of PKD generally arises from mutations in the PKD2 gene. PKD2 encodes the
PC-2 protein, a 968
amino acid protein containing a short N-terminal cytoplasmic region with a
ciliary motif, 6
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transmembrane domains and a short C-terminal portion. The human genomic
sequence of the PKD2 gene
is set forth at NCBI Gene ID 5311, and the protein at UniProtKB/Swiss-Prot:
Q13563-1, described by,
e.g., Mochizuki T, et al., 1996, "PKD2, a gene for polycystic kidney disease
that encodes an integral
membrane protein," Science 272:1339-1342, incorporated by reference herein.
The PKD2 mRNA
sequence is set forth at NCBI Reference Sequence: NM 000297.3.2, described by
Yang Y, etal., 2015,
"Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+
binding properties," J. Bio.
Chem. 290 (16), 10544-10554, both incorporated by reference herein. Mutations
in PKD2 cause late
onset autosomal dominant PKD (ADPKD), the most prevalent of the inherited
renal cystic diseases.
[0039] The PKD2 gene consists of 15 exons and is located on chromosome 4p22.1.
PKD2 mutations in
PKD are spread across the entire protein, with 95 truncating mutations of PKD2
reported in the ADPKD
Mutation Database (maintained by the PKD Foundation, 8330 Ward Parkway, Suite
510, Kansas City,
MO 64114). Because a homozygous deficiency in PKD2 is predicted to be
incompatible with live birth,
haploinsufficiency is the most likely mechanism of ADPKD disease manifestation
(Tones and Harris,
2009). Mutations such as nonsense and insertions/deletions are associated with
the classic ADPKD2
phenotype display functional haploinsufficiency. A PKD missense mutation that
results in expression of
the PC-2-D5 11V protein was predicted to be indistinguishable from wild-type
PC-2 in terms of stability
(Reynolds, etal., 1999, J. Am. Soc. Nephrol. 10: 2342-2351). The PC-2-D5 11V
variant, despite its
stability, was shown to be dysfunctional due to a predicted disruption in its
ability to act as an ion channel.
Thus, even stable variants can cause the phenotype if the nascent activity is
disrupted. The disease is
described, e.g., by OMIM #613095 (Online Mendelian Inheritance in Man, Johns
Hopkins University,
1966-2015), incorporated by reference herein.
Retained Intron Containing Pre-mRNA (RIC Pre-mRNA)
[0040] In embodiments, the methods disclosed herein exploit the presence of
retained-intron-containing
pre-mRNA (RIC pre-mRNA) transcribed from the PKD2 gene and encoding PC-2
protein, in the cell
nucleus. Splicing of the identified PKD2 RIC pre-mRNA species to produce
mature, fully-spliced, PKD2
mRNA, is induced using ASOs that stimulate splicing out of the retained
introns. The resulting mature
PKD2 mRNA can be exported to the cytoplasm and translated, thereby increasing
the amount of PC-2
protein in the patient's cells and alleviating symptoms of Polycystic Kidney
Disease. This method,
described further below, is known as Targeted Augmentation of Nuclear Gene
Output (TANGO).
PKD2 Nuclear Transcripts
[0041] As described herein in the Examples, the PKD2 gene was analyzed for
intron-retention events and
retention of intron 5 was observed. RNA sequencing (RNAseq), visualized in the
UCSC genome
browser, showed PKD2 transcripts expressed in renal epithelial cells and
localized in either the
cytoplasmic or nuclear fraction. In both fractions, reads were not observed
for the majority of the introns.
However, higher read density was detected for intron 5 in the nuclear fraction
compared to the
cytoplasmic fraction indicating that splicing efficiency of intron 5 is low,
resulting in intron retention.
The retained-intron containing pre-mRNA transcripts accumulate primarily in
the nucleus and are not
translated into the PC-2 protein. The read density for intron 5 indicated 18%
intron retention (FIG. 5).
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The percent intron retention (PIR) value for intron 5 was obtained by
averaging four values (23, 13, 22,
and 14), each determined in renal epithelial cells using one of four different
algorithms. Analysis of the
ENCODE data (described by, e.g., Tilgner, etal., 2012, "Deep sequencing of
subcellular RNA fractions
shows splicing to be predominantly co-transcriptional in the human genome but
inefficient for lncRNAs,"
Genome Research 22(9):1616-25) to identify intron retention events did not
identify intron 5 as retained.
[0042] In some embodiments, the ASOs disclosed herein target a RIC pre-mRNA
transcribed from a
PKD2 genomic sequence. In some embodiments, the ASO targets a RIC pre-mRNA
transcript from a
PKD2 genomic sequence comprising retained intron 5. In some embodiments, the
ASO targets a RIC pre-
mRNA transcript of SEQ ID NO: 1. In some embodiments, the ASO targets a RIC
pre-mRNA transcript
of SEQ ID NO: 1 comprising a retained intron 5. In some embodiments, the ASOs
disclosed herein target
a PKD2 RIC pre-mRNA sequence. In some embodiments, the ASO targets a PKD2 RIC
pre-mRNA
sequence comprising a retained intron 5. In some embodiments, the ASO targets
a PKD2 RIC pre-mRNA
sequence according to SEQ ID NO: 2. In some embodiments, the ASO targets a
PKD2 RIC pre-mRNA
sequence according to SEQ ID NO: 2 comprising a retained intron 5. In some
embodiments, the ASOs
disclosed herein target SEQ ID NO: 281. In some embodiments, the ASO has a
sequence according to
any one of SEQ ID NOs: 3-280.
[0043] In some embodiments, the ASO targets exon 5 of a PKD2 RIC pre-mRNA
comprising a retained
intron 5. In some embodiments, the ASO targets an exon 5 sequence upstream (or
5') from the 5' splice
site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO targets an
exon sequence about 4 to about 204 nucleotides upstream (or 5') from the 5'
splice site of a PKD2 RIC
pre-mRNA comprising a retained intron 5. In some embodiments, the ASO has a
sequence according to
any one of SEQ ID NOs: 3-43.
[0044] In some embodiments, the ASO targets intron 5 in a PKD2 RIC pre-mRNA
comprising a retained
intron 5. In some embodiments, the ASO targets an intron 5 sequence downstream
(or 3') from the 5'
splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO
targets an intron 5 sequence about 6 to about 497 nucleotides downstream (or
3') from the 5' splice site of
a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO has a sequence
according to any one of SEQ ID NOs: 44-140.
[0045] In some embodiments, the ASO targets an intron 5 sequence upstream (or
5') from the 3' splice
site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO targets an
intron 5 sequence about 16 to about 496 nucleotides upstream (or 5') from the
3' splice site of a PKD2
RIC pre-mRNA a comprising retained intron 5. In some embodiments, the ASO has
a sequence according
to any one of SEQ ID NOs: 141-237.
[0046] In some embodiments, the ASO targets exon 6 in a PKD2 RIC pre-mRNA
comprising a retained
intron 5. In some embodiments, the ASO targets an exon 6 sequence downstream
(or 3') from the 3'
splice site of a PKD2 RIC pre-mRNA comprising a retained intron 5. In some
embodiments, the ASO
targets an exon 6 sequence about 2 to about 212 nucleotides downstream (or 3')
from the 3' splice site of
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a PKD2 RIC pre-mRNA comprising a retained intron 5. In some embodiments, the
ASO has a sequence
according to any one of SEQ ID NOs: 238-280.
[0047] In embodiments, the targeted portion of the PKD2 RIC pre-mRNA is in
intron 5. The PKD2
intron numbering used herein corresponds to the mRNA sequence at NM 000297.3.
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 retained intron 5 and
subsequently increases PC-2 protein
production. It is understood that the intron numbering may change in reference
to a different PKD2
mRNA isoform sequence. One of skill in the art can determine the corresponding
intron number in any
PKD2 isoform based on an intron sequence provided herein or using the intron
number provided in
reference to the mRNA sequence at NM 000297.3. One of skill in the art also
can determine the
sequences of flanking exons in any PKD2 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 mRNA
sequence at NM 000297.3. In embodiments, the compositions and methods of the
present invention are
used to increase the expression of any known PKD2 isoform, e.g., as described
in the NCBI Gene ID
database at Gene ID 5311 (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), incorporated by reference herein.
PC-2 Protein Expression
[0048] As described above, PC-2 mutations in ADPKD are spread across the
entire protein, with 95 PC-2
truncating mutations having been reported in the ADPKD Mutation Database (PKD
Foundation).
[0049] In embodiments, the methods described herein are used to increase the
production of a functional
PC-2 protein. As used herein, the term "functional" refers to the amount of
activity or function of a PC-2
protein that is necessary to eliminate any one or more symptoms of a treated
condition, e.g., Polycystic
Kidney Disease. In embodiments, the methods are used to increase the
production of a partially
functional PC-2 protein. As used herein, the term "partially functional"
refers to any amount of activity or
function of the PC-2 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.
[0050] In embodiments, the method is a method of increasing the expression of
the PC-2 protein by cells
of a subject having a RIC pre-mRNA encoding the PC-2 protein, wherein the
subject has Polycystic
Kidney Disease caused by a deficient amount of activity of PC-2 protein, and
wherein the deficient
amount of the PC-2 protein is caused by haploinsufficiency of the PC-2
protein. In such an embodiment,
the subject has a first allele encoding a functional PC-2 protein, and a
second allele from which the PC-2
protein is not produced. In another such embodiment, the subject has a first
allele encoding a functional
PC-2 protein, and a second allele encoding a nonfunctional PC-2 protein. In
another such embodiment,
the subject has a first allele encoding a functional PC-2 protein, and a
second allele encoding a partially
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functional PC-2 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 PC-
2 protein), thereby
inducing constitutive splicing of the retained intron from the RIC pre-mRNA,
and causing an increase in
the level of mature mRNA encoding functional PC-2 protein, and an increase in
the expression of the PC-
2 protein in the cells of the subject.
[0051] In embodiments, the subject has a first allele encoding a functional PC-
2 protein, and a second
allele encoding a partially functional PC-2 protein, and the antisense
oligomer binds to a targeted portion
of the RIC pre-mRNA transcribed from the first allele (encoding functional PC-
2 protein) or a targeted
portion of the RIC pre-mRNA transcribed from the second allele (encoding
partially functional PC-2
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 PC-2 protein, and
an increase in the
expression of functional or partially functional PC-2 protein in the cells of
the subject.
[0052] 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 PC-2 protein in
cells of a subject having a RIC pre-mRNA encoding PC-2 protein, wherein the
subject has a deficiency,
e.g., Polycystic Kidney Disease, in the amount or function of a PC-2 protein.
[0053] 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.
[0054] In embodiments, the subject has:
(a) a first mutant allele from which
(i) the PC-2 protein is produced at a reduced level compared to production
from a
wild-type allele,
(ii) the PC-2 protein is produced in a form having reduced function
compared to an
equivalent wild-type protein, or
(iii) the PC-2 protein or functional RNA is not produced; and
(b) a second mutant allele from which
(i) the PC-2 protein is produced at a reduced level compared to production
from a
wild-type allele,
(ii) the PC-2 protein is produced in a form having reduced function
compared to an
equivalent wild-type protein, or
(iii) the PC-2 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 PC-2 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).
[0055] In embodiments, the level of mRNA encoding PC-2 protein is increased
1.1 to 10-fold, when
compared to the amount of mRNA encoding PC-2 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 PKD2 RIC pre-mRNA.
[0056] In embodiments, the condition caused by a deficient amount or activity
of PC-2 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 PC-
2 protein is not a condition
caused by alternative or aberrant splicing of any retained intron in a RIC pre-
mRNA encoding the PC-2
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 in the pre-mRNA.
[0057] In embodiments, a subject treated using the methods of the invention
expresses a partially
functional PC-2 protein from one allele, wherein the partially functional PC-2
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 PC-2 protein
from one allele, wherein the nonfunctional PC-2 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 PKD2 whole gene deletion, in one
allele.
[0058] In embodiments, the subject has a PC-2 missense mutation selected from
M1K, P24L, R28P,
A35D, R6ON, S8OL, Q107D, R119H, G121A, G135V, A147V, A190T, V262M, W292C,
R306Q,
L314V, R322W, R322Q, R325P, R325Q, C331S, S332A, Y324C, S349P, A356P, A384P,
G390S,
W414G, G418V, T419A, R420G, A421S, R440S,1448K, I452V, F482C, Y487H, D5 11V,
V516L,
L517R, V519M, A552P, I556V, N578D, N580K, M583I, A615T, F629S, C632R, R638C,
L656W,
L715I, I758V, R798C, M800L, S804N, R807Q, R848Q, D886G, R893G, V909I, D919N,
T931M, R945H
or S949F. In embodiments, the subject has a PC-2 deletion mutation selected
from EX l_EX13del,
IVS2 3'(ABCG2)del8Okb*, IVS2 3'(ABCG2)de198kb, IVS4+1452 IVS5-965de15722,
S378de1, F605de1,
IVS9_31de128kb, 2182_2183delAG, L736_N737de12 or R878de1. In embodiments, the
subject has PC-2
duplication mutation Ex3dup*. In embodiments, a subject having any PC-2
mutation known in the art and
described in the literature, e.g., by Chang, etal., 2005, Ren Fail 27: 95-100,
is treated using the methods
and compositions of the present invention.
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Use of TANGO for Increasing PC-2 Protein Expression
[0059] 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 PC-2
protein. In these embodiments, a
retained-intron-containing pre-mRNA (RIC pre-mRNA) encoding PC-2 protein is
present in the nucleus
of a cell. Cells having a PKD2 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 PC-2
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.
[0060] 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).
[0061] 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 PC-2 protein. In
embodiments, the most retained intron in a RIC pre-mRNA encoding the PC-2
protein is intron 5.
[0062] 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
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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 10000, about 5% to about 95%, about 5%
to about 90%, about
5% to about 85%, about 5% to about 80%, about 5% to about '75%, about 5% to
about 70%, about 5% to
about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about
50%, about 5% to about
450, about 5% to about 40%, about 5% to about 350, about 5% to about 30%,
about 5% to about 25%,
about 5% to about 20%, about 5% to about 15%, about 10% to about 1000o, 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 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 1000o,
about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about
15% to about 80%,
about 1500 to about 75%, about 150o to about 70%, about 150o to about 65%,
about 150o 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 1000o,
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 550, about 20% to about 500o, about 20% to about 45%, about
20% to about 40%,
about 20% to about 35%, about 20% to about 30%, about 25% to about 1000o,
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 550
,
about 25% to about 500o, about 25% to about 45%, about 25% to about 40%, or
about 25% to about 35%,
retention.
[0063] 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).
[0064] In embodiments of any of the compositions and methods provided herein,
"comprising" may be
replaced with "consisting essentially of" or "consisting of." The phrase
"consisting essentially of' is used
herein to require the specified feature(s) (e.g., nucleobase sequence) as well
as those which do not
materially affect the character or function of the claimed invention. As used
herein, the term "consisting"
is used to indicate the presence of the recited feature (e.g., nucleobase
sequence) alone (so that in the case
of an antisense oligomer consisting of a specified nucleobase sequence, the
presence of additional,
unrecited nucleobases is excluded).
[0065] 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
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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.
[0066] 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.
[0067] 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 the PKD2 gene in the published reference genome
deposited in the NCBI
repository of biological and scientific information. As used herein, the "wild-
type sequence" refers to the
canonical sequence for the PKD2 gene found at NCBI Gene ID 5311. Also used
herein, a nucleotide
position denoted with an "e" indicates the nucleotide is present in the
sequence of an exon (e.g., the exon
flanking the 5' splice site or the exon flanking the 3' splice site).
[0068] The methods involve contacting cells with an ASO that is complementary
to a portion of a pre-
mRNA encoding PC-2 protein, resulting in increased expression of PC-2. 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 any of the ASOs described herein. 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.
[0069] 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.
[0070] In embodiments, contacting a cell that expresses a PKD2 RIC pre-mRNA
with an ASO that is
complementary to a targeted portion of the PKD2 RIC pre-mRNA transcript
results in a measurable
increase in the amount of the PC-2 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
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known method, for example, Western blotting, flow cytometry,
immunofluorescence microscopy, and
ELISA.
100711 In embodiments, contacting cells with an ASO that is complementary to a
targeted portion of a
PKD2 RIC pre-mRNA transcript results in an increase in the amount of PC-2
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 PC-2 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 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.
[0072] In some embodiments, contacting cells with an ASO that is complementary
to a targeted portion
of a PKD2 RIC pre-mRNA transcript results in an increase in the amount of mRNA
encoding PC-2,
including the mature mRNA encoding the target protein. In some embodiments,
the amount of mRNA
encoding PC-2 protein, or the mature mRNA encoding the PC-2 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 PC-2 protein, or the mature mRNA encoding PC-2
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 PKD2 RIC pre-mRNA.
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Constitutive Splicing of a Retained Intron from a RIC pre-mRNA
[0073] The methods and antisense oligonucleotide compositions provided herein
are useful for increasing
the expression of PC-2 protein in cells, for example, in a subject having
Polycystic Kidney Disease caused
by a deficiency in the amount or activity of PC-2 protein, by increasing the
level of mRNA encoding PC-2
protein, or the mature mRNA encoding PC-2 protein. In particular, the methods
and compositions as
described herein induce the constitutive splicing of a retained intron from a
PKD2 RIC pre-mRNA
transcript encoding PC-2 protein, thereby increasing the level of mRNA
encoding PC-2 protein, or the
mature mRNA encoding PC-2 protein and increasing the expression of PC-2
protein.
[0074] 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.
[0075] In embodiments, constitutive splicing correctly removes a retained
intron from a PKD2
RIC pre-mRNA, wherein the PKD2 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.
[0076] In some embodiments, constitutive splicing of a retained intron from a
PKD2 RIC pre-mRNA
encoding PC-2 protein correctly removes a retained intron from a PKD2 RIC pre-
mRNA encoding PC-2
protein, wherein the PKD2 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.
[0077] In other embodiments, constitutive splicing correctly removes a
retained intron from a PKD2 RIC
pre-mRNA, wherein the PKD2 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.
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[0078] "Correct removal" of the retained intron by constitutive splicing
refers to removal of the entire
intron, without removal of any part of an exon.
[0079] In embodiments, an antisense oligomer as described herein or used in
any method described
herein does not increase the amount of mRNA encoding PC-2 protein or the
amount of PC-2 protein by
modulating alternative splicing or aberrant splicing of a pre-mRNA transcribed
from the PKD2 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 PC-2 protein in the methods of the invention.
[0080] In embodiments, the methods described herein is a method wherein the
PKD2 RIC pre-mRNA
was produced by partial splicing of a wild-type PKD2 pre-mRNA. In embodiments,
the method is a
method wherein the PKD2 RIC pre-mRNA was produced by partial splicing of a
full-length wild-type
PKD2 pre-mRNA. In embodiments, the PKD2 RIC pre-mRNA was produced by partial
splicing of a full-
length PKD2 pre-mRNA. In these embodiments, a full-length PKD2 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.
[0081] In embodiments, the mRNA encoding PC-2 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 PC-2 protein encoded by the wild-type mature mRNA.
Ant/sense Oligomers
[0082] One aspect of the present disclosure is a composition comprising
antisense oligomers that
enhances splicing by binding to a targeted portion of a PKD2 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 hybridize to a target nucleic acid
(e.g., a PKD2 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
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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.
[0083] 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.
[0084] Oligomers, such as oligonucleotides, are "complementary" to one another
when hybridization
occurs in an antiparallel configuration between two single-stranded
polynucleotides. A double-stranded
polynucleotide can be "complementary" to another polynucleotide, if
hybridization can occur between
one of the strands of the first polynucleotide and the second. Complementarity
(the degree to which one
polynucleotide is complementary with another) is quantifiable in terms of the
proportion (e.g., the
percentage) of bases in opposing strands that are expected to form hydrogen
bonds with each other,
according to generally accepted base-pairing rules. The sequence of an
antisense oligomer (ASO) need
not be 100% complementary to that of its target nucleic acid to hybridize. In
certain embodiments, ASOs
can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a
target region within the
target nucleic acid sequence to which they are targeted. For example, an ASO
in which 18 of 20
nucleobases of the oligomeric compound are complementary to a target region,
and would therefore
specifically hybridize, would represent 90 percent complementarity. In this
example, the remaining
noncomplementary nucleobases may be clustered together or interspersed with
complementary
nucleobases and need not be contiguous to each other or to complementary
nucleobases. Percent
complementarity of an ASO with a region of a target nucleic acid can be
determined routinely using
BLAST programs (basic local alignment search tools) and PowerBLAST programs
known in the art
(Altschul, etal., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome
Res., 1997, 7, 649-656).
[0085] 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.
[0086] 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
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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. Patent No. 5,656,612,
U.S. Patent Publication No. 2012/0190728, and Dias and Stein, Mol. Cancer
Ther. 2002, 347-355, herein
incorporated by reference in their entirety.
[0087] 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-me thylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-
hydroxymethoylcytosine.
[0088] 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, etal.,
Nucleic Acids Res.
14:9081 (1986); Stec, etal., J. Am. Chem. Soc. 106:6077 (1984), Stein, etal.,
Nucleic Acids Res.
16:3209 (1988), Zon, etal., Anti Cancer Drug Design 6:539 (1991); Zon, etal.,
Oligonucleotides and
Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford
University Press, Oxford England
(1991)); Stec, etal., 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.
[0089] 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 disclosure,
including, but not limited to, any of the ASOs set forth herein in Table 1,
comprises an ASO having
phosphorus internucleotide linkages that are not random. In embodiments, a
composition used in the
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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
10000, about 90% to about
1000o, about 91% to about 1000o, about 92% to about 1000o, about 93% to about
1000o, about 94% to
about 1000o, about 95% to about 1000o, about 96% to about 1000o, about 97% to
about 1000o, about 98%
to about 10000 , or about 99% to about 1000o.
[0090] 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, etal., 2014, "Synthesis, biophysical properties and biological activity
of second generation
antisense oligonucleotides containing chiral phosphorothioate linkages,"
Nucleic Acids Research 42(22):
13456-13468, incorporated herein by reference). In embodiments, an ASO used in
the methods of the
invention, including, but not limited to, any of the ASOs set forth herein in
Table 1, comprises about 5-
1000o Rp, at least about 50 Rp, at least about 10% Rp, at least about 1500 Rp,
at least about 20% Rp, at
least about 25% Rp, at least about 30% Rp, at least about 350 Rp, at least
about 40% Rp, at least about
45% Rp, at least about 5000 Rp, at least about 550 Rp, at least about 60% Rp,
at least about 65% Rp, at
least about 70% Rp, at least about 750 Rp, at least about 80% Rp, at least
about 85% Rp, at least about
90% Rp, or at least about 950 Rp, with the remainder Sp, or about 100% Rp. In
embodiments, an ASO
used in the methods of the invention, including, but not limited to, any of
the ASOs set forth herein in
Table 1, 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 450 to about 100% Rp, about 5000 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 750 to about 100% Rp, about 80% to about 100% Rp, about 85% to about
100% Rp, about
90% to about 100% Rp, or about 950 to about 100% Rp, about 20% to about 80%
Rp, about 25% to
about 750 Rp, about 30% to about 70% Rp, about 40% to about 60% Rp, or about
450 to about 55% Rp,
with the remainder Sp.
[0091] In embodiments, an ASO used in the methods of the invention, including,
but not limited to, any
of the ASOs set forth herein in Table 1, 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 350 Sp, at least about 40% Sp, at least about 450 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 750 Sp, at
least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least
about 950 Sp, with the
remainder Rp, or about 100% Sp. In embodiments, an ASO used in the methods of
the invention,
including, but not limited to, any of the ASOs set forth herein in Table 1,
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 350 to about 100% Sp, about 40% to about
100% Sp, about 450 to
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about 10000 Sp, about 50% to about 1000o Sp, about 55% to about 1000o Sp,
about 60% to about 1000o
Sp, about 65% to about 1000o Sp, about 70% to about 1000o Sp, about 75% to
about 1000o Sp, about 80%
to about 1000o Sp, about 85% to about 1000o Sp, about 90% to about 1000o Sp,
or about 95% to about
1000o 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.
[0092] Any of the ASOs described herein may contain a sugar moiety that
comprises ribose or
deoxyribose, as present in naturally occurring nucleotides, or a modified
sugar moiety or sugar analog,
including a morpholine ring. Non-limiting examples of modified sugar moieties
include 2' substitutions
such as 2'-0-methyl (2'-0-Me), 2'-0-methoxyethyl (2'MOE), 2'-0-aminoethyl,
2'F; N3'->P5'
phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-
guanidinidium, 2'-0-
guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In
some embodiments, the
sugar moiety modification is selected from 2'-0-Me, 2'F, and 2'MOE. In some
embodiments, the sugar
moiety modification is an extra bridge bond, such as in a locked nucleic acid
(LNA). In some
embodiments the sugar analog contains a morpholine ring, such as
phosphorodiamidate morpholino
(PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or
2'deoxyribofuransyl
modification. In some embodiments, the sugar moiety comprises 2'4'-constrained
2'0-methyloxyethyl
(cM0E) modifications. In some embodiments, the sugar moiety comprises cEt 2',
4' constrained 2'-0
ethyl BNA modifications. In some embodiments, the sugar moiety comprises
tricycloDNA (tcDNA)
modifications. In some embodiments, the sugar moiety comprises ethylene
nucleic acid (ENA)
modifications. In some embodiments, the sugar moiety comprises MCE
modifications. Modifications are
known in the art and described in the literature, e.g., by Jarver, etal.,
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.
[0093] 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."
[0094] 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 moipholino (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
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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.
[0095] 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, etal., J Pharmacol Exp Ther. 2001;
296(3):898-904.
[0096] 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.
[0097] 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., "4," while a
nucleotide that is directly adjacent and downstream of the reference point is
designated "plus one," e.g.,
,.+1
[0098] In some embodiments, the ASOs are complementary to (and bind to) a
targeted portion of a
PKD2 RIC pre-mRNA that is downstream (in the 3' direction) of the 5' splice
site of the retained intron in
a PKD2 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 PKD2 RIC pre-
mRNA that is within the region of about +6 to about +500 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 a PKD2 RIC pre-mRNA that is
within the region
between nucleotides +6 and +497 relative to the 5' splice site of the retained
intron. In some aspects, the
ASOs are complementary to a targeted portion that is within the region about
+6 to about +500, about +6
to about +490, about +6 to about +480, about +6 to about +470, about +6 to
about +460, about +6 to about
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+450, about +6 to about +440, about +6 to about +430, about +6 to about +420,
about +6 to about +410,
about +6 to about +400, about +6 to about +390, about +6 to about +380, about
+6 to about +370, about
+6 to about +360, about +6 to about +350, about +6 to about +340, about +6 to
about +330, about +6 to
about +320, about +6 to about +310, about +6 to about +300, about +6 to about
+290, about +6 to about
+280, about +6 to about +270, about +6 to about +260, about +6 to about +250,
about +6 to about +240,
about +6 to about +230, about +6 to about +220, about +6 to about +210, about
+6 to about +200, about
+6 to about +190, about +6 to about +180, about +6 to about +170, about +6 to
about +160, about +6 to
about +150, about +6 to about +140, about +6 to about +130, about +6 to about
+120, about +6 to about
+110, about +6 to about +100, about +6 to about +90, about +6 to about +80,
about +6 to about +70,
about +6 to about +60, about +6 to about +50, about +6 to about +40, about +6
to about +30, or about +6
to about +20 relative to 5' splice site of the retained intron.
100991 In some embodiments, the ASOs are complementary to (and bind to) a
targeted portion of a
PKD2 RIC pre-mRNA that is upstream (in the 5' direction) of the 5' splice site
of the retained intron in a
PKD2 RIC pre-mRNA (e.g., the direction designated by negative numbers relative
to the 5' splice site)
(FIG. 1). In some embodiments, the ASOs are complementary to a targeted
portion of the PKD2 RIC pre-
mRNA that is within the region of about -4e to about -210e relative to the 5'
splice site of the retained
intron. In some embodiments, the ASO is not complementary to nucleotides -le
to -3e relative to the 5'
splice site (the first three nucleotides located upstream of the 5' splice
site). In some embodiments, the
ASOs may be complementary to a targeted portion of a PKD2 RIC pre-mRNA that is
within the region
between nucleotides -4e and about -204e relative to the 5' splice site of the
retained intron. In some
aspects, the ASOs are complementary to a targeted portion that is within the
region about -4e to about -
210e, about -4e to about -200e, about -4e to about -190e, about -4e to about -
180e, about -4e to about -
170e, about -4e to about -160e, about -4e to about -150e, about -4e to about -
140e, about -4e to about -
130e, about -4e to about -120e, about -4e to about -110e, about -4e to about -
100e, about -4e to about -
90e, about -4e to about -80e, about -4e to about -70e, about -4e to about -
60e, about -4e to about -50e,
about -4e to about -40e, about -4e to about -30e, or about -4e to about -20e
relative to 5' splice site of the
retained intron.
1001001ln some embodiments, the ASOs are complementary to a targeted region of
a PKD2 RIC pre-
mRNA that is upstream (in the 5' direction) of the 3' splice site of the
retained intron in a PKD2 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 PKD2 RIC pre-mRNA that is
within the region of about -
16 to about -500 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 PKD2 RIC pre-mRNA that is within the region -16 to -496 relative to the
3' splice site of the
retained intron. In some aspects, the ASOs are complementary to a targeted
portion that is within the
region about -16 to about -500, about -16 to about -490, about -16 to about -
480, about -16 to about -470,
about -16 to about -460, about -16 to about -450, about -16 to about -440,
about -16 to about -430, about -
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16 to about -420, about -16 to about -410, about -16 to about -400, about -16
to about -390, about -16 to
about -380, about -16 to about -370, about -16 to about -360, about -16 to
about -350, about -16 to about -
340, about -16 to about -330, about -16 to about -320, about -16 to about -
310, about -16 to about -300,
about -16 to about -290, about -16 to about -280, about -16 to about -270,
about -16 to about -260, about -
16 to about -250, about -16 to about -240, about -16 to about -230, about -16
to about -220, about -16 to
about -210, about -16 to about -200, about -16 to about -190, about -16 to
about -180, about -16 to about -
170, about -16 to about -160, about -16 to about -150, about -16 to about -
140, about -16 to about -130,
about -16 to about -120, about -16 to about -110, about -16 to about -100,
about -16 to about -90, about -
16 to about -80, about -16 to about -70, about -16 to about -60, about -16 to
about -50, about -16 to about -
40, or about -16 to about -30 relative to 3' splice site of the retained
intron.
1001011ln some embodiments, the ASOs are complementary to a targeted region of
a PKD2 RIC pre-
mRNA that is downstream (in the 3' direction) of the 3' splice site of the
retained intron in a PKD2 RIC
pre-mRNA (e.g., in the direction designated by positive numbers) (FIG. 1). In
some embodiments, the
ASOs are complementary to a targeted portion of the PKD2 RIC pre-mRNA that is
within the region of
about +2e to about +220e relative to the 3' splice site of the retained
intron. In some embodiments, the
ASO is not complementary to nucleotides +le relative to the 3' splice site
(the first nucleotide located
downstream of the 3' splice site). In some embodiments, the ASOs may be
complementary to a targeted
portion of a PKD2 RIC pre-mRNA that is within the region between nucleotides
+2e and +212e relative
to the 3' splice site of the retained intron. In some aspects, the ASOs are
complementary to a targeted
portion that is within the region about +2e to about +220e, about +2e to about
+210e, about +2e to about
+200e, about +2e to about +190e, about +2e to about +180e, about +2e to about
+170e, about +2e to
about +160e, about +2e to about +150e, about +2e to about +140e, about +2e to
about +130e, about +2e
to about +120e, about +2e to about +110e, about +2e to about +100e, about +2e
to about +90e, about +2e
to about +80e, about +2e to about +70e, about +2e to about +60e, about +2e to
about +50e, about +2e to
about +40e, about +2e to about +30e, about +2e to about +20e, or about +2e to
about +10e relative to 3'
splice site of the retained intron.
1001021 In embodiments, the targeted portion of the PKD2 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.
[00103] 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,
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
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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 18 nucleotides in length. In some embodiments, the
ASOs are 15 nucleotides
in length. In some embodiments, the ASOs are 25 nucleotides in length.
[00104] 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.
[00105] 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.
[00106] In some embodiments, the nucleic acid to be targeted by an ASO is a
PKD2 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).
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Pharmaceutical Compositions
[00107] 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.
[00108] Pharmaceutically acceptable salts are suitable for use in contact with
the tissues of humans and
lower animals without undue toxicity, irritation, allergic response, etc., and
are commensurate with a
reasonable benefit/risk ratio. (See, e.g., S. M. Berge, et al., J.
Pharmaceutical Sciences, 66: 1-19 (1977),
incorporated herein by reference for this purpose). The salts can be prepared
in situ during the final
isolation and purification of the compounds, or separately by reacting the
free base function with a
suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid
addition salts are salts of
an amino group formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, phosphoric
acid, sulfuric acid and perchloric acid or with organic acids such as acetic
acid, oxalic acid, maleic acid,
tartaric acid, citric acid, succinic acid or malonic acid or by using other
documented methodologies such
as ion exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate,
fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,
2-hydroxy-
ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate,
maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,
pamoate, pectinate, persulfate, 3-
phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,
succinate, sulfate, tartrate,
thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline
earth metal salts include sodium, lithium, potassium, calcium, magnesium, and
the like. Further
pharmaceutically acceptable salts include, when appropriate, nontoxic
ammonium, quaternary
ammonium, and amine cations formed using counterions such as halide,
hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
[00109] 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).
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[00110] 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 enhancer is a surfactant, fatty acid, bile salt, chelating agent,
or non-chelating nonsurfactant.
[00111] 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.
[00112] In embodiments, the antisense oligonucleotides are linked or
conjugated with agents that
provide desirable pharmaceutical or pharmacodynamic properties. In
embodiments, the antisense
oligonucleotide is coupled to a substance, known in the art to promote
penetration or transport across the
blood-brain barrier, e.g., an antibody to the transferrin receptor. In
embodiments, the antisense
oligonucleotide is linked with a viral vector, e.g., to render the antisense
compound more effective or
increase transport across the blood-brain barrier. In embodiments, osmotic
blood brain barrier disruption
is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+)
galactose, D(+) lactose, D(+) xylose,
dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+)
arabinose, D(-) arabinose,
cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-)
ribose, adonitol, D(+)
arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose,
and L(-) lyxose, or amino acids,
e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine,
glutamic acid, glycine, histidine,
leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine,
valine, and taurine. Methods and
materials for enhancing blood brain barrier penetration are described, e.g.,
in U.S. Pat. No. 4,866,042,
"Method for the delivery of genetic material across the blood brain barrier,"
U.S. Pat. No. 6,294,520,
"Material for passage through the blood-brain barrier," and U.S. Pat. No.
6,936,589, "Parenteral delivery
systems," each incorporated herein by reference.
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[00113] 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
[00114] Any of the compositions provided herein may be administered to an
individual. "Individual"
may be used interchangeably with "subject" or "patient." An individual may be
a mammal, for example a
human or animal such as a non-human primate, a rodent, a rabbit, a rat, a
mouse, a horse, a donkey, a
goat, a cat, a dog, a cow, a pig, or a sheep. In embodiments, the individual
is a human. In embodiments,
the individual is a fetus, an embryo, or a child. In other embodiments, the
individual may be another
eukaryotic organism, such as a plant. In some embodiments, the compositions
provided herein are
administered to a cell ex vivo.
[00115] 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).
[00116] 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 are
affected by Polycystic
Kidney Disease, with the kidney being the most significantly affected tissue.
The ASOs of the present
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invention may be administered to patients parenterally, for example, by
intraperitoneal injection,
intramuscular injection, subcutaneous injection, or intravenous injection.
[00117] Subjects are evaluated for response to treatment using any appropriate
markers. In
embodiments, subjects with kidney disease are evaluated for response to
treatment by measuring specific
markers for kidney disease, including creatinine, creatinine clearance, blood
pressure, 24-hour urine
volume, 24-hour urine protein, vWAg and platelet aggregation by arachidonic
acid.
Methods of Identifying Additional ASOs that Enhance Splicing
[00118] Also within the scope of the present invention are methods for
identifying (determining)
additional ASOs that enhance splicing of a PKD2 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.
[00119] A round of screening, referred to as an ASO "walk" may be performed
using ASOs that have
been designed to hybridize to a target region of a pre-mRNA. For example, the
ASOs used in the ASO
walk can be tiled every 5 nucleotides from approximately 100 nucleotides
upstream of the 5' splice site of
the retained intron (e.g., a portion of sequence of the exon located upstream
of the target/retained intron)
to approximately 100 nucleotides downstream of the 5' splice site of the
target/retained intron and/or from
approximately 100 nucleotides upstream of the 3' splice site of the retained
intron to approximately 100
nucleotides downstream of the 3' splice site of the target/retained intron
(e.g., a portion of sequence of the
exon located downstream of the target/retained intron). For example, a first
ASO of 15 nucleotides in
length may be designed to specifically hybridize to nucleotides +6 to +20
relative to the 5' splice site of
the target/retained intron. A second ASO is designed to specifically hybridize
to nucleotides +11 to +25
relative to the 5' splice site of the target/retained intron. ASOs are
designed as such spanning the target
region of the pre-mRNA. In embodiments, the ASOs can be tiled more closely,
e.g., every 1, 2, 3, or 4
nucleotides. Further, the ASOs can be tiled from 100 nucleotides downstream of
the 5' splice site, to 100
nucleotides upstream of the 3' splice site. In some embodiments, the ASOs can
be tiled from about 210
nucleotides upstream of the 5' splice site, to about 500 nucleotides
downstream of the 5' splice site. In
some embodiments, the ASOs can be tiled from about 500 nucleotides upstream of
the 3' splice site, to
about 220 nucleotides downstream of the 3' splice site.
[00120] 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
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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.
[00121] 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.
[00122] 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.
[00123] As described for the ASO walk above, the ASO micro-walk is performed
by delivering one or
more ASOs, or a control ASO (an ASO with a scrambled sequence, sequence that
is not expected to
hybridize to the target region), for example by transfection, into a disease-
relevant cell line that expresses
the target pre-mRNA. The splicing-inducing effects of each of the ASOs may be
assessed by any method
known in the art, for example by reverse transcriptase (RT)-PCR using primers
that span the splice
junction, as described herein (see "Identification of intron-retention
events"). A reduction or absence of
the RT-PCR product produced using the primers spanning the splice junction in
ASO-treated cells as
compared to in control ASO-treated cells indicates that splicing of the target
intron has been enhanced. In
some embodiments, the splicing efficiency, the ratio of spliced to unspliced
pre-mRNA, the rate of
splicing, or the extent of splicing may be improved using the ASOs described
herein. The amount of
protein or functional RNA that is encoded by the target pre-mRNA can also be
assessed to determine
whether each ASO achieved the desired effect (e.g., enhanced protein
production). Any method known in
the art for assessing and/or quantifying protein production, such as Western
blotting, flow cytometry,
immunofluorescence microscopy, and ELISA, can be used.
[00124] 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
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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.
EXAMPLES
[00125] The following examples provide illustrative embodiments of the
invention. One of ordinary
skill in the art will recognize the numerous modifications and variations that
may be performed without
altering the spirit or scope of the disclosure. Such modifications and
variations are encompassed within
the scope of the disclosure. The Examples do not in any way limit the
disclosure described herein.
Example 1: Identification of intron retention events in PKD2 transcripts by
RNAseq using next
generation sequencing
[00126] Whole transcriptome shotgun sequencing was carried out using next
generation sequencing to
reveal a snapshot of transcripts produced by the PKD2 gene to identify intron-
retention events. For this
purpose, polyA+ RNA from nuclear and cytoplasmic fractions of renal 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 sequencing results for PKD2 are shown in FIG. 3.
Briefly, FIG. 3 shows
the mapped reads 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,
etal., 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 can be 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 PKD2 (drawn to scale) is provided by the
UCSC genome browser
(below the read signals) so that peaks can be matched to PKD2 exonic and
intronic regions. Based on this
display, we identified one intron (intron 5, as indicated) that has high read
density in the nuclear fraction
of HCN, but very low to no reads in the cytoplasmic fraction of these cells
(as shown for intron 5 in the
bottom diagram of FIG. 3). This indicates that intron 5 is retained and that
the intron-5 containing
transcripts remain in the nucleus, suggesting that this retained PKD2 RIC pre-
mRNAs is non-productive,
as it is not exported out to the cytoplasm.
Example 2: Design of ASO-walk targeting intron 5 of PKD2
[00127] An ASO walk was designed to target intron 5 using the method described
herein (FIG. 4; Table
1, SEQ ID NOS: 3 to 280). A region immediately upstream and downstream of the
5' splice site of intron
5, spanning nucleotides +497 to -204e, and a region immediately upstream and
downstream of the 3'
splice site of intron 5, spanning nucleotides -496to +212e were utilized to
design ASOs to target retained
intron 5 PKD2 RIC pre-mRNAs. Table 1 lists exemplary ASOs that were designed
and their target
sequences. From this design, 2'-0-Me RNA, PS backbone, 18-mer ASOs shifted by
5-nucleotide
intervals can be produced and utilized to target PKD2 RIC pre-mRNAs to
increase PC-2 protein
production.
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Table 1. List of ASOs targeting the PKD2 gene
Gene Pre-mRNA ASOs Retained Target
Sequence
SEQ ID NO: SEQ ID NO: Intron SEQ ID NO:
PKD2 PKD2:NM 000297
SEQ ID NOs: 3-280 Intron 5 SEQ ID NO: 281
SEQ ID NO: 1 SEQ ID NO: 2
Example 3: Improved splicing efficiency via ASO-targeting of PKD2 intron 5
increases transcript
levels
[00128] To determine whether an increase in PKD2 expression could be achieved
by improving splicing
efficiency of PKD2 intron 5 using ASOs, the method described herein can be
used. Cell lines of interest
(e.g., 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)) are mock-transfected, or transfected with the targeting ASOs
described in Table 1.
Cells are transfected using Lipofectamine RNAiMax transfection reagent (Thermo
Fisher) according to
manufacturer's specifications. Briefly, ASOs are plated in 96-well tissue
culture plates and combined
with RNAiMax diluted in Opti-MEM. Cells are detached using trypsin,
resuspended in full medium, and
approximately 25,000 cells are added to the ASO-transfection mixture.
Transfection experiments are
carried out in triplicate plate replicates. Final ASO concentration is 80 nM.
Media is changed 6h post-
transfection, and cells are harvested at 24h, using the Cells-to-Ct lysis
reagent, supplemented with DNAse
(Thermo Fisher), according to manufacturer's specifications. cDNA is generated
with Cells-to-Ct RT
reagents (Thermo Fisher) according to manufacturer's specifications. To
quantify the amount of splicing
at the intron of interest, quantitative PCR is carried out using Taqman assays
with probes spanning the
corresponding exon-exon junction (Thermo Fisher), listed in Table 1. Taqman
assays are carried out
according to manufacturer's specifications, on a QuantStudio 7 Flex Real-Time
PCR system (Thermo
Fisher). Target gene assay values are 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 is plotted. ASOs
identified as increasing the target
gene expression by a threshold amount imply an increase in splicing at that
target intron. Together with
whole transcriptome data confirming retention of the target intron (FIG. 3),
these results confirm that
ASOs can improve the splicing efficiency of a rate limiting intron.
[00129] 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
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claims define the scope of the invention and that methods and structures
within the scope of these claims
and their equivalents be covered thereby.
38
