OLIGONUCLEOTIDE THERAPY FOR STARGARDT DISEASE

SEQUENCE LISTING

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 May 12, 2022, is named 51110-711_301_SL.txt and is 317,049 bytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates to the field of oligonucleotides and their use for the treatment of disease. In particular, the disclosure pertains to antisense oligonucleotides that may be used in the treatment of Stargardt disease.

BACKGROUND

ABCA4 (ATP binding cassette subfamily A member 4; entrez gene 24) is a transmembrane lipid transporter expressed in the photoreceptor outer segment, within the disc membranes. It is required to clear the reactive all-trans retinal from the photoreceptor disc lumen.

As part of the light cycle, 11-cis-retinal is generated in the retinal epithelium cells (RPE) and transported to the photoreceptor outer segment, where light triggers isomerization of rhodopsin-bound 11-cis-retinal to all-trans retinal. All-trans retinal can spontaneously flip to the photoreceptor disc membrane cytoplasm-facing side, or it can spontaneously react with phosphatidylethanolamine (PE), a phospholipid that is abundant in the photoreceptor outer segment, to form N-retinylidene-PE. N-retinylidene-PE cannot spontaneously flip, and it would accumulate without a specific transporter.

ABCA4 expression is restricted to photoreceptor cells. RefSeq contains only one curated isoform (NM_000350) comprising 50 exons, which is categorized principal by APPRIS. GENCODE contains one isoform categorized principal by APPRIS (ENST00000370225), which has the same CDS as NM_000350, and two minor isoforms (ENST00000536513, ENST00000649773). NM_000350 can be treated as the only ABCA4 functional isoform.

ABCA4 transports N-retinylidene-PE from the lumen-facing side of the membrane to the cytoplasm-facing side, where it spontaneously dissociates to all-trans retinal and PE. All-trans retinal is then reduced to all-trans retinol by the cytoplasmic enzyme RDH8 and transported back to RPE cells. In addition, ABCA4 transports PE from the lumen-facing to the cytoplasm-facing side of the photoreceptor disc membrane, maintaining the PE concentration lower.

If N-retinylidene-PE accumulates, it can form di-retinoid-pyridinium-PE (A2PE); all-trans retinal can also accumulate and form dimers. Since RPE cells recycle photoreceptor outer segments every 10 days, these compounds end up accumulating in their lysosomes. There, A2PE is hydrolyzed to di-retinoid-pyridinium-ethanolamine (A2E), which can be photoactivated and form highly reactive epoxides. This process is toxic for RPE cells and can lead to cell death. As photoreceptors lose the support of RPE, they can in turn suffer cell death.

The ABCA4 transport reaction follows three main steps: (i) binding of N-retinylidene-PE, binding of ATP, NBD domain dimerization, (ii) using the energy from ATP hydrolysis, change to a conformation that exposes N-retinylidene-PE to the cytoplasmic side and has lower affinity to it, (iii) release of N-retinylidene-PE and ADP, reversal to the original configuration.

Lack of ABCA4 function causes N-retinylidene-PE accumulation, which leads to formation of di-retinoid-pyridinium-PE (A2PE); all-trans retinal can also accumulate and form dimers. Since RPE cells recycle photoreceptor outer segments every 10 days, these compounds end up accumulating in their lysosomes. There, A2PE is hydrolyzed to di-retinoid-pyridinium-ethanolamine (A2E), which can be photoactivated and form highly reactive epoxides. This process is toxic for RPE cells and can lead to cell death. As photoreceptors lose the support of RPE, they can in turn suffer cell death. Higher levels of A2PE accumulation are directly toxic to photoreceptors, and cones are more sensitive than rods.

Pathogenic variants in ABCA4 cause a spectrum of recessive disorders, all characterized by progressive retinal degeneration; the phenotypic severity of the disorder is typically correlated to the extent of loss-of-function imparted by the variants. When both alleles are severely affected by variants severe cone-rod dystrophy may result, with a presentation similar to other forms of retinitis pigmentosa (RP). When one allele is severely affected by a variant while the other is only partially affected cone-rod dystrophy (CRD) may result. When one allele is severely affected by a variant while the other is not or only minorly affected or alternatively both alleles are only partially affected by a variant Stargardt disease (STGD1) may result.

Each disorder follows a progression with retinitis pigmentosa (RP) onset in the 1st decade of life typically progressing to blindness by the 2nd or 3d decade, cone-rod dystrophy (CRD) onset in the 1st decade of life progressing to blindness by mid-adulthood, and Stargardt disease (STGD1) with onset in the 1st or 2nd decade of life following progressive course.

No FDA-approved treatment exists.

Certain human genetic diseases (e.g., caused by genetic aberrations, such as point mutations) may be caused by aberrant splicing. As such, there is a need for a splicing modulator to treat diseases that are caused by aberrant splicing.

SUMMARY

In general, the disclosure provides antisense oligonucleotides and methods of their use in the treatment of conditions associated with incorrect splicing of ABCA4 pre-mRNA (e.g., intron 6 or 36 inclusion, and exon 33 or 40 skipping).

In one aspect, the disclosure provides an antisense oligonucleotide including a nucleobase sequence that is at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) complementary to an ABCA4 pre-mRNA target sequence (e.g., g.107705G>A, g.104307A>G, g.115355G>A, or g.27356G>T mutation in SEQ ID NO: 1). The ABCA4 pre-mRNA target sequence may be disposed in, e.g., a 5′-flanking intron, a 3′-flanking intron, intron, exon, or a combination of an exon and the 5′-flanking or 3′-flanking intron.

In some embodiments, the ABCA4 pre-mRNA target sequence is in exon 6, a 5′-flanking intron adjacent to exon 6, 3′-flanking intron adjacent to exon 6, or a combination of exon 6 and the adjacent 5′-flanking or 3′-flanking intron. In certain embodiments, binding of the antisense oligonucleotide to the ABCA4 pre-mRNA target sequence reduces binding of a splicing factor to an intronic splicing enhancer in an exon, the 5′-flanking intron, the 3′-flanking intron, or a splicing enhancer.

In some embodiments, the ABCA4 pre-mRNA target sequence is in exon 33, a 5′-flanking intron adjacent to exon 33, 3′-flanking intron adjacent to exon 33, or a combination of exon 33 and the adjacent 5′-flanking or 3′-flanking intron. In certain embodiments, the ABCA4 pre-mRNA target sequence reduces the binding of a splicing factor to an intronic splicing silencer in the 5′-flanking intron or 3′-flanking intron.

In some embodiments, the ABCA4 pre-mRNA target sequence is in intron 36. In certain embodiments, the ABCA4 pre-mRNA target sequence reduces the binding of a splicing factor to an intronic splicing enhancer in an intron.

In some embodiments, the ABCA4 pre-mRNA target sequence is in exon 40, a 5′-flanking intron adjacent to exon 40, 3′-flanking intron adjacent to exon 40, or a combination of exon 40 and the adjacent 5′-flanking or 3′-flanking intron. In certain embodiments, the ABCA4 pre-mRNA target sequence reduces the binding of a splicing factor to an intronic splicing silencer in the 5′-flanking or 3′-flanking intron.

In particular embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27362-27419 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In further embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27372-27411 in SEQ ID NO: 1. In yet further embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27377-27397 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In still further embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27383-27402 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In other embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27388-27411 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In other embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27390-27411 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In other embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27396-27414 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions). In other embodiments, the ABCA4 pre-mRNA target sequence includes at least one nucleotide (e.g., 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides) located among positions 27061-27152 in SEQ ID NO: 1 (e.g., the ABCA4 pre-mRNA target sequence is wholly within these positions).

In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 107, 102, 113, 129, 130,133, 134, 269, 270, 329, 333, 336, 337, 342, 343, 393, 422, 433, 438. In some embodiments, the nucleobase sequence is complementary to an aberrant ABCA4 sequence having a mutation in SEQ ID NO: 1 (e.g., a g.107705G>A, g.104307A>G, g.115355G>A, or g.27356G>T mutation in SEQ ID NO: 1).

In further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to any one of SEQ ID NOs: 60-198. In yet further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to any one of SEQ ID NOs: 73-175. In still further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 101-118. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 128-140.

In other embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 157-171. In yet other embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 157-171. In yet further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 165-171. In still other embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 193-196. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 2-16. In certain embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 260-287. In particular embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 316-374 and 463-596. In further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 329-343 and 463-596. In yet further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 390-394. In still further embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 422-423. In some embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 433-434. In certain embodiments, the nucleobase sequence has at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) sequence identity to SEQ ID NO: 438-449.

In yet other embodiments, the antisense oligonucleotide includes at least one modified nucleobase. In still other embodiments, the antisense oligonucleotide includes at least one modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the phosphorothioate linkage is a stereochemically enriched phosphorothioate linkage. In particular embodiments, at least 50% of internucleoside linkages in the antisense oligonucleotide are modified internucleoside linkages. In further embodiments, at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) of internucleoside linkages in the antisense oligonucleotide are modified internucleoside linkage. In yet further embodiments, all internucleoside linkages in the antisense oligonucleotide are modified internucleoside linkages.

In still further embodiments, the antisense oligonucleotide includes at least one modified sugar nucleoside. In some embodiments, at least one modified sugar nucleoside is a 2′-modified sugar nucleoside. In certain embodiments, at least one 2′-modified sugar nucleoside includes a 2′-modification selected from the group consisting of 2′-fluoro, 2′-methoxy, and 2′-methoxyethoxy. In particular embodiments, the 2′-modified sugar nucleoside includes the 2′-methoxyethoxy modification. In further embodiments, at least one modified sugar nucleoside is a bridged nucleic acid. In yet further embodiments, the bridged nucleic acid is a locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), or cEt nucleic acid. In still further embodiments, all nucleosides in the antisense oligonucleotide are modified sugar nucleosides. In some embodiments, the antisense oligonucleotide is a morpholino oligomer.

In certain embodiments, the antisense oligonucleotide further includes a targeting moiety. In particular embodiments, the targeting moiety is covalently conjugated at the 5′-terminus of the antisense oligonucleotide. In further embodiments, the targeting moiety is covalently conjugated at the 3′-terminus of the antisense oligonucleotide. In yet further embodiments, the targeting moiety is covalently conjugated at an internucleoside linkage of the antisense oligonucleotide. In still further embodiments, the targeting moiety is covalently conjugated through a linker (e.g., a cleavable linker). In other embodiments, the linker is a cleavable linker. In yet other embodiments, the targeting moiety includes N-acetylgalactosamine (e.g., is an N-acetylgalactosamine cluster).

In still other embodiments, the antisense oligonucleotide includes at least 12 nucleosides. In some embodiments, the antisense oligonucleotide includes at least 16 nucleosides. In certain embodiments, the antisense oligonucleotide includes a total of 50 nucleosides or fewer (e.g., 30 nucleosides or fewer, or 20 nucleosides or fewer). In particular embodiments, the antisense oligonucleotide includes a total of 16 to 20 nucleosides.

In another aspect, the disclosure provides a pharmaceutical composition including the antisense oligonucleotide of the disclosure and a pharmaceutically acceptable excipient.

In yet another aspect, the disclosure provides a method of increasing the level of exon-containing (e.g., exon 33 or 40-containing) ABCA4 mRNA molecules in a cell expressing an aberrant ABCA4 gene. The method includes contacting the cell with the antisense oligonucleotide of the disclosure.

In yet another aspect, the disclosure provides a method of decreasing the level of intron-containing (e.g., intron 6 or 36-containing) ABCA4 mRNA molecules in a cell expressing an aberrant ABCA4 gene. The method includes contacting the cell with the antisense oligonucleotide of the disclosure.

In some embodiments, the cell is in a subject.

In still another aspect, the disclosure provides a method of treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject having an aberrant ABCA4 gene. The method includes administering a therapeutically effective amount of the antisense oligonucleotide of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof.

In some embodiments, the administering step is performed parenterally. In certain embodiments, the method further includes administering to the subject a therapeutically effective amount of a second therapy for retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease.

In yet further embodiments, the aberrant ABCA4 gene is ABCA4 having a g.107705G>A, g.104307A>G, g.115355G>A, or g.27356G>T mutation in SEQ ID NO: 1.

Recognized herein is the need for compositions and methods for treating diseases that may be caused by abnormal splicing resulting from an underlying genetic aberration. In some cases, antisense nucleic acid molecules, such as oligonucleotides, may be used to effectively modulate the splicing of targeted genes in genetic diseases, in order to alter the gene products produced. This approach can be applied in therapeutics to selectively modulate the expression and gene product composition for genes involved in genetic diseases.

The present disclosure provides compositions and methods that may advantageously use antisense oligonucleotides targeted to and hybridizable with nucleic acid molecules that encode for ABCA4. Such antisense oligonucleotides may target one or more splicing regulatory elements in one or more exons (e.g., exons 6, 33, 40) or introns (e.g., intron 36, 5′-flanking intro or 3′ flanking intron) of ABCA4. These splicing regulatory elements modulate splicing of ABCA4 ribonucleic acid (RNA).

In one aspect, the present disclosure provides an ABCA4 RNA splice-modulating antisense oligonucleotide having a sequence targeted to an exon or an intron adjacent to an exon (e.g., exon 6) of ABCA4. In some embodiments, a genetic aberration of ABCA4 includes the c.768G>T mutation. In some embodiments, the c.768G>T mutation results from ABCA4 chr1: 94564350:C:A [hg19/b37] (g.27356G>T in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements include an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 6 inclusion). In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides.

In one aspect, the present disclosure provides an ABCA4 RNA splice-modulating antisense oligonucleotide having a sequence targeted to an exon or intron adjacent to an exon (e.g., exon 33) of ABCA4. In some embodiments, a genetic aberration of ABCA4 includes the c.4773+3A>G mutation. In some embodiments, the c.4773+3A>G mutation results from ABCA4 chr1: 94487399:T:C [hg19/b37] (g.104307A>G in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements include an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 33 inclusion). In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides.

In one aspect, the present disclosure provides an ABCA4 RNA splice-modulating antisense oligonucleotide having a sequence targeted to an intron (e.g., intron 36) of ABCA4. In some embodiments, a genetic aberration of ABCA4 includes the c.5196+1137G>A mutation. In some embodiments, the c.5196+1137G>A mutation results from ABCA4 chr1: 94484001:C:T [hg19/b37] (g.107705G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements include an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron containing an abnormally spliced intronic sequence (e.g., a pseudo exon). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 36 inclusion). In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides.

In one aspect, the present disclosure provides an ABCA4 RNA splice-modulating antisense oligonucleotide having a sequence targeted to an exon or an intron adjacent to an exon (e.g., exon 40) of ABCA4. In some embodiments, a genetic aberration of ABCA4 includes the c.5714+5G>A mutation. In some embodiments, the c.5714+5G>A mutation results from ABCA4 chr1: 94476351:C:T [hg19/b37] (g.115355G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements include an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 40 inclusion). In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides.

In another aspect, the present disclosure provides a method for modulating splicing of ABCA4 RNA in a cell, tissue, or organ of a subject, including bringing the cell, tissue, or organ in contact with an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 6) of ABCA4. In some embodiments, the genetic aberration of ABCA4 includes the c.768G>T mutation. In some embodiments, the c.768G>T mutation results from ABCA4 chr1: 94564350:C:A [hg19/b37] (g.27356G>T in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 6 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of intron-excluding ABCA4 transcripts (e.g., intron 6-excluding ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an intron-including mutation (e.g., an intron 6-including mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject has or is suspected of having a disease, e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, and the subject is monitored for a progression or regression of the disease in response to bringing the cell, tissue, or organ in contact with the composition.

In another aspect, the present disclosure provides a method for modulating splicing of ABCA4 RNA in a cell, tissue, or organ of a subject, including bringing the cell, tissue, or organ in contact with an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 33) of ABCA4. In some embodiments, the genetic aberration of ABCA4 includes the c.4773+3A>G mutation. In some embodiments, the c.4773+3A>G mutation results from ABCA4 chr1: 94487399:T:C [hg19/b37] (g.104307A>G in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 33 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of exon-including ABCA4 transcripts (e.g., exon 33-including ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an exon-skipping mutation (e.g., an exon 33-skipping mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject has or is suspected of having a disease, e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, and the subject is monitored for a progression or regression of the disease in response to bringing the cell, tissue, or organ in contact with the composition.

In another aspect, the present disclosure provides a method for modulating splicing of ABCA4 RNA in a cell, tissue, or organ of a subject, including bringing the cell, tissue, or organ in contact with an antisense oligonucleotide including one or more sequences targeted to an intron (e.g., intron 36) of ABCA4. In some embodiments, the genetic aberration of ABCA4 includes the c.5196+1137G>A mutation. In some embodiments, the c.5196+1137G>A mutation results from ABCA4 chr1: 94484001:C:T [hg19/b37] (g.107705G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron containing an abnormally spliced intronic sequence (e.g., a pseudo exon). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 36 exclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of intron-excluding ABCA4 transcripts (e.g., intron 36-excluding ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an intron-including mutation (e.g., an intron 36-including mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject has or is suspected of having a disease, e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, and the subject is monitored for a progression or regression of the disease in response to bringing the cell, tissue, or organ in contact with the composition.

In another aspect, the present disclosure provides a method for modulating splicing of ABCA4 RNA in a cell, tissue, or organ of a subject, including bringing the cell, tissue, or organ in contact with an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 40) of ABCA4. In some embodiments, the genetic aberration of ABCA4 includes the c.5714+5G>A mutation. In some embodiments, the c.5714+5G>A mutation results from ABCA4 chr1: 94476351:C:T [hg19/b37] (g.115355G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 40 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of exon-including ABCA4 transcripts (e.g., exon 40-including ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an exon-skipping mutation (e.g., an exon 40-skipping mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject has or is suspected of having a disease, e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, and the subject is monitored for a progression or regression of the disease in response to bringing the cell, tissue, or organ in contact with the composition.

In another aspect, the present disclosure provides a method for treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject, including administering to the subject a therapeutically effective amount of an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 6) of ABCA4. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes the c.768G>T mutation. In some embodiments, the c.768G>T mutation results from ABCA4 chr1: 94564350:C:A [hg19/b37] (g.27356G>T in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon of the genetic aberration of ABCA4 that modulates variant splicing of ABCA4 RNA (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 6 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of intron-excluding ABCA4 transcripts (e.g., intron 6-excluding ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an intron-including mutation (e.g., an intron 6-including mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject is monitored for a progression or regression of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in response to administering to the subject the therapeutically effective amount of the antisense oligonucleotide.

In another aspect, the present disclosure provides a method for treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject, including administering to the subject a therapeutically effective amount of an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 33) of ABCA4. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes the c.4773+3A>G mutation. In some embodiments, the c.4773+3A>G mutation results from ABCA4 chr1: 94487399:T:C [hg19/b37] (g.104307A>G in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon of the genetic aberration of ABCA4 that modulates variant splicing of ABCA4 RNA (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 33 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of exon-including ABCA4 transcripts (e.g., exon 33-including ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an exon-skipping mutation (e.g., an exon 33-skipping mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject is monitored for a progression or regression of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in response to administering to the subject the therapeutically effective amount of the antisense oligonucleotide.

In another aspect, the present disclosure provides a method for treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject, including administering to the subject a therapeutically effective amount of an antisense oligonucleotide including one or more sequences targeted to an intron (e.g., intron 36) of ABCA4. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes the c.5196+1137G>A mutation. In some embodiments, the c.5196+1137G>A mutation results from ABCA4 chr1: 94484001:C:T [hg19/b37] (g.107705G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing enhancer element. In some embodiments, the sequence is targeted to an intron containing an abnormally spliced intronic sequence containing the genetic aberration of ABCA4 that modulates variant splicing of ABCA4 RNA (e.g., a pseudo exon). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in intron exclusion (e.g., intron 36 exclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of intron-excluding ABCA4 transcripts (e.g., intron 36-excluding ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an intron-including mutation (e.g., an intron 36-including mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject is monitored for a progression or regression of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in response to administering to the subject the therapeutically effective amount of the antisense oligonucleotide.

In another aspect, the present disclosure provides a method for treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject, including administering to the subject a therapeutically effective amount of an antisense oligonucleotide including one or more sequences targeted to an exon or intron adjacent to an exon (e.g., exon 40) of ABCA4. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes the c.5714+5G>A mutation. In some embodiments, the c.5714+5G>A mutation results from ABCA4 chr1: 94476351:C:T [hg19/b37] (g.115355G>A in SEQ ID NO: 1). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements. In some embodiments, the one or more splicing regulatory elements are an intronic splicing silencer element. In some embodiments, the sequence is targeted to an intron adjacent to an abnormally spliced exon of the genetic aberration of ABCA4 that modulates variant splicing of ABCA4 RNA (e.g., a flanking intron). In some embodiments, the antisense oligonucleotide modulates variant splicing to yield an increase in exon inclusion (e.g., exon 40 inclusion), e.g., increase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 100%, up to 90%, up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, as compared to the ratio of exon-including ABCA4 transcripts (e.g., exon 40-including ABCA4 transcripts) to the total number of ABCA4 transcript molecules in a cell including ABCA4 gene having an exon-skipping mutation (e.g., an exon 40-skipping mutation) in the absence of a treatment with an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide has a length of 12 to 20 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 30 nucleotides. In some embodiments, the antisense oligonucleotide has a length of 12 to 50 nucleotides. In some embodiments, the subject is monitored for a progression or regression of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in response to administering to the subject the therapeutically effective amount of the antisense oligonucleotide.

In another aspect, the present disclosure provides a pharmaceutical composition for treatment of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease including an antisense oligonucleotide and a pharmaceutically acceptable carrier. The antisense oligonucleotide includes a sequence targeted to an exon or intron adjacent to the abnormally spliced exon. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes c.768G>T. In some embodiments, the c.768G>T mutation results from ABCA4 chr1: 94564350:C:A [hg19/b37] (g.27356G>T in SEQ ID NO: 1).

In another aspect, the present disclosure provides a pharmaceutical composition for treatment of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease including an antisense oligonucleotide and a pharmaceutically acceptable carrier. The antisense oligonucleotide includes a sequence targeted to an exon or intron adjacent to the abnormally spliced exon. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes c.4773+3A>G. In some embodiments, the c.4773+3A>G mutation results from ABCA4 chr1: 94487399:T:C [hg19/b37] (g.104307A>G in SEQ ID NO: 1).

In another aspect, the present disclosure provides a pharmaceutical composition for treatment of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease including an antisense oligonucleotide and a pharmaceutically acceptable carrier. The antisense oligonucleotide includes a sequence targeted to an intron abnormally spliced intron. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes c.5196+1137G>A. In some embodiments, the c.5196+1137G>A mutation results from ABCA4 chr1: 94484001:C:T [hg19/b37] (g.107705G>A in SEQ ID NO: 1).

In another aspect, the present disclosure provides a pharmaceutical composition for treatment of retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease including an antisense oligonucleotide and a pharmaceutically acceptable carrier. The antisense oligonucleotide includes a sequence targeted to an intron adjacent to the abnormally spliced exon. The antisense oligonucleotide modulates splicing of ABCA4 RNA. In some embodiments, the genetic aberration of ABCA4 includes c.5714+5G>A. In some embodiments, the c.5714+5G>A mutation results from ABCA4 chr1: 94476351:C:T [hg19/b37] (g.115355G>A in SEQ ID NO: 1).

Definitions

Various terms used throughout the present description may be read and understood as follows, unless the context indicates otherwise: “or” as used throughout is inclusive, as though written “and/or”; singular articles and pronouns as used throughout include their plural forms, and vice versa; similarly, gendered pronouns include their counterpart pronouns so that pronouns should not be understood as limiting anything described herein to use, implementation, performance, etc. by a single gender; “exemplary” should be understood as “illustrative” or “exemplifying” and not necessarily as “preferred” over other embodiments. Further definitions for terms may be set out herein; these may apply to prior and subsequent instances of those terms, as will be understood from a reading of the present description.

The term “ABCA4” as used herein, generally represents a nucleic acid (e.g., genomic DNA, pre-mRNA, or mRNA) that is translated and, if genomic DNA, first transcribed, in vivo to ABCA4 protein. An exemplary genomic DNA sequence comprising the human ABCA4 gene is given by SEQ ID NO: 1 (NCBI Reference Sequence: NG_009073.1). SEQ ID NO: 1 provides the sequence for the antisense strand of the genomic DNA of ABCA4 (positions 5001-133313 in SEQ ID NO: 1). One of skill in the art will recognize that an RNA sequence typically includes uridines instead of thymidines. The term “ABCA4” as used herein, represents wild-type and mutant versions. An exemplary mutant nucleic acid (e.g., genomic DNA, pre-mRNA, or mRNA) results in ABCA4 protein lacking any of exon 33 or exon 40, or containing an extended exon 6 or pseudo exon.

The term “acyl,” as used herein, generally represents a chemical substituent of formula —C(O)—R, where R is alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, heterocyclyl alkyl, heteroaryl, or heteroaryl alkyl. An optionally substituted acyl is an acyl that is optionally substituted as described herein for each group R.

The term “acyloxy,” as used herein, generally represents a chemical substituent of formula —OR, where R is acyl. An optionally substituted acyloxy is an acyloxy that is optionally substituted as described herein for acyl.

The term “alkane-tetrayl,” as used herein, generally represents a tetravalent, acyclic, straight or branched chain, saturated hydrocarbon group having from 1 to 16 carbons, unless otherwise specified. Alkane-tetrayl may be optionally substituted as described for alkyl.

The term “alkane-triyl,” as used herein, generally represents a trivalent, acyclic, straight or branched chain, saturated hydrocarbon group having from 1 to 16 carbons, unless otherwise specified. Alkane-triyl may be optionally substituted as described for alkyl.

The term “alkanoyl,” as used herein, generally represents a chemical substituent of formula —C(O)—R, where R is alkyl. An optionally substituted alkanoyl is an alkanoyl that is optionally substituted as described herein for alkyl.

The term “alkoxy,” as used herein, generally represents a chemical substituent of formula-OR, where R is a C_1-6alkyl group, unless otherwise specified. An optionally substituted alkoxy is an alkoxy group that is optionally substituted as defined herein for alkyl.

The term “alkyl,” as used herein, generally refers to an acyclic straight or branched chain saturated hydrocarbon group, which, when unsubstituted, has from 1 to 12 carbons, unless otherwise specified. In certain preferred embodiments, unsubstituted alkyl has from 1 to 6 carbons. Alkyl groups are exemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl, and the like, and may be optionally substituted, valency permitting, with one, two, three, or, in the case of alkyl groups of two carbons or more, four or more substituents independently selected from the group consisting of: alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; ═O; ═S; and ═NR′, where R′ is H, alkyl, aryl, or heterocyclyl. In some embodiments, a substituted alkyl includes two substituents (oxo and hydroxy, or oxo and alkoxy) to form a group -L-CO—R, where L is a bond or optionally substituted C_1-11alkylene, and R is hydroxyl or alkoxy. Each of the substituents may itself be unsubstituted or, valency permitting, substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “alkylene,” as used herein, generally represents a divalent substituent that is a monovalent alkyl having one hydrogen atom replaced with a valency. An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.

The term “aryl,” as used herein, generally represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings. Aryl group may include from 6 to 10 carbon atoms. All atoms within an unsubstituted carbocyclic aryl group are carbon atoms. Non-limiting examples of carbocyclic aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, etc. The aryl group may be unsubstituted or substituted with one, two, three, four, or five substituents independently selected from the group consisting of alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; and cyano. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “aryl alkyl,” as used herein, generally represents an alkyl group substituted with an aryl group. The aryl and alkyl portions may be optionally substituted as the individual groups as described herein.

The term “arylene,” as used herein, generally represents a divalent substituent that is an aryl having one hydrogen atom replaced with a valency. An optionally substituted arylene is an arylene that is optionally substituted as described herein for aryl.

The term “aryloxy,” as used herein, generally represents a group —OR, where R is aryl. Aryloxy may be an optionally substituted aryloxy. An optionally substituted aryloxy is aryloxy that is optionally substituted as described herein for aryl.

The term “bicyclic sugar moiety,” as used herein, generally represents a modified sugar moiety including two fused rings. In certain embodiments, the bicyclic sugar moiety includes a furanosyl ring.

The expression “C_x-y,” as used herein, generally indicates that the group, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. If the group is a composite group (e.g., aryl alkyl), C_x-yindicates that the portion, the name of which immediately follows the expression, when unsubstituted, contains a total of from x to y carbon atoms. For example, (C_6-10-aryl)-C_1-6-alkyl is a group, in which the aryl portion, when unsubstituted, contains a total of from 6 to 10 carbon atoms, and the alkyl portion, when unsubstituted, contains a total of from 1 to 6 carbon atoms.

The term “complementary,” as used herein in reference to a nucleobase sequence, generally refers to the nucleobase sequence having a pattern of contiguous nucleobases that permits an oligonucleotide having the nucleobase sequence to hybridize to another oligonucleotide or nucleic acid to form a duplex structure under physiological conditions. Complementary sequences include Watson-Crick base pairs formed from natural and/or modified nucleobases. Complementary sequences can also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs.

The term “contiguous,” as used herein in the context of an oligonucleotide, generally refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

The term “cycloalkyl,” as used herein, generally refers to a cyclic alkyl group having from three to ten carbons (e.g., a C₃-C₁₀cycloalkyl), unless otherwise specified. Cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of bicyclo[p.q.0]alkyl type, in which each of p and q is, independently, 1, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 2, 3, 4, 5, 6, 7, or 8. Alternatively, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, e.g., bicyclo[p.q.r]alkyl, in which r is 1, 2, or 3, each of p and q is, independently, 1, 2, 3, 4, 5, or 6, provided that the sum of p, q, and r is 3, 4, 5, 6, 7, or 8. The cycloalkyl group may be a spirocyclic group, e.g., spiro[p.q]alkyl, in which each of p and q is, independently, 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl, and decalinyl. The cycloalkyl group may be unsubstituted or substituted (e.g., optionally substituted cycloalkyl) with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; amino; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; halo; heterocyclyl; heteroaryl; heterocyclylalkyl; heteroarylalkyl; heterocyclyloxy; heteroaryloxy; hydroxy; nitro; thiol; silyl; cyano; ═O; ═S; —NR′, where R′ is H, alkyl, aryl, or heterocyclyl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “cycloalkylene,” as used herein, generally represents a divalent substituent that is a cycloalkyl having one hydrogen atom replaced with a valency. An optionally substituted cycloalkylene is a cycloalkylene that is optionally substituted as described herein for cycloalkyl.

The term “cycloalkoxy,” as used herein, generally represents a group —OR, where R is cycloalkyl. Cycloalkoxy may be an optionally substituted cycloalkoxy. An optionally substituted cycloalkoxy is cycloalkoxy that is optionally substituted as described herein for cycloalkyl.

The term “duplex,” as used herein, generally represents two oligonucleotides that are paired through hybridization of complementary nucleobases.

The term “exon 6,” as used herein, generally refers to exon 6 of ABCA4 pre-mRNA or genomic DNA which corresponds to positions 27159 to 27356 in SEQ ID NO: 1 (hg19/b37 coordinates chr1:94564350-94564547), or a mutant version thereof (e.g., g.27356G>T in SEQ ID NO: 1).

The term “exon 33,” as used herein, generally refers to exon 33 of ABCA4 pre-mRNA or genomic DNA, e.g. which corresponds to positions 104199 to 104304 in SEQ ID NO: 1 (hg19/b37 coordinates chr1:94487402-94487507), or a mutant version thereof.

The term “exon 40,” as used herein, generally refers to exon 40 of ABCA4 pre-mRNA or genomic DNA, e.g. which corresponds to positions 115221 to 115350 in SEQ ID NO: 1 (hg19/b37 coordinates chr1:94476356-94476485), or a mutant version thereof.

The term “flanking intron,” as used herein, generally refers to an intron that is adjacent to the 5′- or 3′-end of a ABCA4 exon (e.g., exon 6, 33, or 40) or a mutant thereof (e.g. NM_000350.2(ABCA4):c.5714+5G>A [g.115355G>A on SEQ ID NO: 1] or NM_000350.2(ABCA4):c.5196+1137G>A [g.107705G>A on SEQ ID NO: 1]). The flanking intron is a 5′-flanking intron or a 3′-flanking intron. The 5′-flanking intron corresponds to the flanking intron that is adjacent to the 5′-end of the exon (e.g., exon 6, 33, or 40) targeted for inclusion. In some embodiments, the 5′-flanking intron is disposed between exon 5 and exon 6, exon 32 and exon 33, and exon 39 and exon 40 in SEQ ID NO: 1. The 3′-flanking intron corresponds to the flanking intron that is adjacent to the 3′-end of the exon (e.g., exon 6, 33, or 40) targeted for inclusion. In some embodiments, the 3′-flanking intron is disposed between exon 6 and exon 7, exon 33 and exon 34, and exon 40 and exon 41 in SEQ ID NO: 1).

The term “genetic aberration,” as used herein, generally refers to a mutation or variant in a gene. Examples of genetic aberration may include, but are not limited to, a point mutation (single nucleotide variant or single base substitution), an insertion or deletion (indel), a transversion, a translocation, an inversion, or a truncation. An aberrant ABCA4 gene may include one or more mutations causing the splicing of pre-mRNA to: skip an exon in the ABCA4 gene (e.g., exon 33 or 40), include a portion of a flanking intron adjacent to an exon in the ABCA4 gene (e.g., a portion of a flanking intron adjacent to exon 6), or include a pseudo exon (e.g. a pseudo exon located in intro 36).

The term “halo,” as used herein, generally represents a halogen selected from bromine, chlorine, iodine, and fluorine.

The term “heteroalkane-tetrayl,” as used herein generally refers to an alkane-tetrayl group interrupted once by one heteroatom; twice, each time, independently, by one heteroatom; three times, each time, independently, by one heteroatom; or four times, each time, independently, by one heteroatom. Each heteroatom is, independently, O, N, or S. In some embodiments, the heteroatom is O or N. An unsubstituted C_X-Yheteroalkane-tetrayl contains from X to Y carbon atoms as well as the heteroatoms as defined herein. The heteroalkane-tetrayl group may be unsubstituted or substituted (e.g., optionally substituted heteroalkane-tetrayl), as described for heteroalkyl.

The term “heteroalkane-triyl,” as used herein generally refers to an alkane-triyl group interrupted once by one heteroatom; twice, each time, independently, by one heteroatom; three times, each time, independently, by one heteroatom; or four times, each time, independently, by one heteroatom. Each heteroatom is, independently, O, N, or S. In some embodiments, the heteroatom is O or N. An unsubstituted C_X-Yheteroalkane-triyl contains from X to Y carbon atoms as well as the heteroatoms as defined herein. The heteroalkane-triyl group may be unsubstituted or substituted (e.g., optionally substituted heteroalkane-triyl), as described for heteroalkyl.

The term “heteroalkyl,” as used herein, generally refers to an alkyl group interrupted one or more times by one or two heteroatoms each time. Each heteroatom is independently O, N, or S. None of the heteroalkyl groups includes two contiguous oxygen atoms. The heteroalkyl group may be unsubstituted or substituted (e.g., optionally substituted heteroalkyl). When heteroalkyl is substituted and the substituent is bonded to the heteroatom, the substituent is selected according to the nature and valency of the heteroatom. Thus, the substituent bonded to the heteroatom, valency permitting, is selected from the group consisting of ═O, —N(R^N2)₂, —SO₂OR^N3, —SO₂R^N2, —SOR^N3, —COOR^N3, an N protecting group, alkyl, aryl, cycloalkyl, heterocyclyl, or cyano, where each R^N2is independently H, alkyl, cycloalkyl, aryl, or heterocyclyl, and each R^N3is independently alkyl, cycloalkyl, aryl, or heterocyclyl. Each of these substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group. When heteroalkyl is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not Cl, Br, or I. In some embodiments, carbon atoms are found at the termini of a heteroalkyl group. In some embodiments, heteroalkyl is PEG.

The term “heteroalkylene,” as used herein, generally represents a divalent substituent that is a heteroalkyl having one hydrogen atom replaced with a valency. An optionally substituted heteroalkylene is a heteroalkylene that is optionally substituted as described herein for heteroalkyl.

The term “heteroaryl,” as used herein, generally represents a monocyclic 5-, 6-, 7-, or 8-membered ring system, or a fused or bridging bicyclic, tricyclic, or tetracyclic ring system; the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one of the rings is an aromatic ring. Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridinyl, pyrazinyl, pyrimidinyl, qunazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazole), thiazolyl, thienyl, triazolyl, tetrazolyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, etc. The term bicyclic, tricyclic, and tetracyclic heteroaryls include at least one ring having at least one heteroatom as described above and at least one aromatic ring. For example, a ring having at least one heteroatom may be fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring. Examples of fused heteroaryls include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. Heteroaryl may be optionally substituted with one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; ═O; —NR₂, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; —COOR^A, where R^Ais hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and —CON(R^B)₂, where each R^Bis independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl. Each of the substituents may itself be unsubstituted or substituted with unsubstituted substituent(s) defined herein for each respective group.

The term “heteroarylene,” as used herein, generally represents a divalent substituent that is a heteroaryl having one hydrogen atom replaced with a valency. An optionally substituted heteroarylene is a heteroarylene that is optionally substituted as described herein for heteroaryl.

The term “heteroaryloxy,” as used herein, generally refers to a structure —OR, in which R is heteroaryl. Heteroaryloxy can be optionally substituted as defined for heteroaryl.

The term “heterocyclyl,” as used herein, generally represents a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridging 4-, 5-, 6-, 7-, or 8-membered rings, unless otherwise specified, the ring system containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heterocyclyl may be aromatic or non-aromatic. An aromatic heterocyclyl is heteroaryl as described herein. Non-aromatic 5-membered heterocyclyl has zero or one double bonds, non-aromatic 6- and 7-membered heterocyclyl groups have zero to two double bonds, and non-aromatic 8-membered heterocyclyl groups have zero to two double bonds and/or zero or one carbon-carbon triple bond. Heterocyclyl groups have a carbon count of 1 to 16 carbon atoms unless otherwise specified. Certain heterocyclyl groups may have a carbon count up to 9 carbon atoms. Non-aromatic heterocyclyl groups include pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolidinyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, thiazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyranyl, dihydropyranyl, dithiazolyl, etc. The term “heterocyclyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., quinuclidine, tropanes, or diaza-bicyclo[2.2.2]octane. The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another heterocyclic ring. Examples of fused heterocyclyls include 1,2,3,5,8,8a-hexahydroindolizine; 2,3-dihydrobenzofuran; 2,3-dihydroindole; and 2,3-dihydrobenzothiophene. The heterocyclyl group may be unsubstituted or substituted with one, two, three, four or five substituents independently selected from the group consisting of: alkyl; alkoxy; acyloxy; aryloxy; amino; arylalkoxy; cycloalkyl; cycloalkoxy; halogen; heterocyclyl; heterocyclyl alkyl; heteroaryl; heteroaryl alkyl; heterocyclyloxy; heteroaryloxy; hydroxyl; nitro; thiol; cyano; ═O; ═S; —NR₂, where each R is independently hydrogen, alkyl, acyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; —COOR^A, where R^Ais hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl; and —CON(R^B)₂, where each R^Bis independently hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, heterocyclyl, or heteroaryl.

The term “heterocyclyl alkyl,” as used herein, generally represents an alkyl group substituted with a heterocyclyl group. The heterocyclyl and alkyl portions of an optionally substituted heterocyclyl alkyl are optionally substituted as described for heterocyclyl and alkyl, respectively.

The term “heterocyclylene,” as used herein, generally represents a divalent substituent that is a heterocyclyl having one hydrogen atom replaced with a valency. An optionally substituted heterocyclylene is a heterocyclylene that is optionally substituted as described herein for heterocyclyl.

The term “heterocyclyloxy,” as used herein, generally refers to a structure —OR, in which R is heterocyclyl. Heterocyclyloxy can be optionally substituted as described for heterocyclyl.

The term “heteroorganic,” as used herein, generally refers to (i) an acyclic hydrocarbon interrupted one or more times by one or two heteroatoms each time, or (ii) a cyclic hydrocarbon including one or more (e.g., one, two, three, or four) endocyclic heteroatoms. Each heteroatom is independently O, N, or S. None of the heteroorganic groups includes two contiguous oxygen atoms. An optionally substituted heteroorganic group is a heteroorganic group that is optionally substituted as described herein for alkyl.

The term “hydrocarbon,” as used herein, generally refers to an acyclic, branched or acyclic, linear compound or group, or a monocyclic, bicyclic, tricyclic, or tetracyclic compound or group. The hydrocarbon, when unsubstituted, consists of carbon and hydrogen atoms. Unless specified otherwise, an unsubstituted hydrocarbon includes a total of 1 to 60 carbon atoms (e.g., 1 to 16, 1 to 12, or 1 to 6 carbon atoms). An optionally substituted hydrocarbon is an optionally substituted acyclic hydrocarbon or an optionally substituted cyclic hydrocarbon. An optionally substituted acyclic hydrocarbon is optionally substituted as described herein for alkyl. An optionally substituted cyclic hydrocarbon is an optionally substituted aromatic hydrocarbon or an optionally substituted non-aromatic hydrocarbon. An optionally substituted aromatic hydrocarbon is optionally substituted as described herein for aryl. An optionally substituted non-aromatic cyclic hydrocarbon is optionally substituted as described herein for cycloalkyl. In some embodiments, an acyclic hydrocarbon is alkyl, alkylene, alkane-triyl, or alkane-tetrayl. In certain embodiments, a cyclic hydrocarbon is aryl or arylene. In particular embodiments, a cyclic hydrocarbon is cycloalkyl or cycloalkylene.

The terms “hydroxyl” and “hydroxy,” as used interchangeably herein, generally represent —OH.

The term “hydrophobic moiety,” as used herein, generally represents a monovalent group covalently linked to an oligonucleotide backbone, where the monovalent group is a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Non-limiting examples of the monovalent group include ergosterol, stigmasterol, β-sitosterol, campesterol, fucosterol, saringosterol, avenasterol, coprostanol, cholesterol, vitamin A, vitamin D, vitamin E, cardiolipin, and carotenoids. The linker connecting the monovalent group to the oligonucleotide may be an optionally substituted C_1-60hydrocarbon (e.g., optionally substituted C_1-60alkylene) or an optionally substituted C_2-60heteroorganic (e.g., optionally substituted C_2-60heteroalkylene), where the linker may be optionally interrupted with one, two, or three instances independently selected from the group consisting of an optionally substituted arylene, optionally substituted heterocyclylene, and optionally substituted cycloalkylene. The linker may be bonded to an oligonucleotide through, e.g., an oxygen atom attached to a 5′-terminal carbon atom, a 3′-terminal carbon atom, a 5′-terminal phosphate or phosphorothioate, a 3′-terminal phosphate or phosphorothioate, or an internucleoside linkage.

The term “internucleoside linkage,” as used herein, generally represents a divalent group or covalent bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. An internucleoside linkage is an unmodified internucleoside linkage or a modified internucleoside linkage. An “unmodified internucleoside linkage” is a phosphate (—O—P(O)(OH)—O—) internucleoside linkage (“phosphate phosphodiester”). A “modified internucleoside linkage” is an internucleoside linkage other than a phosphate phosphodiester. The two main classes of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, phosphorodithioate linkages, boranophosphonate linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)₂—O—), and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, an internucleoside linkage is a group of the following structure:

embedded image

where

Z is O, S, B, or Se;
Y is —X-L-R1;

each X is independently —O—, —S—, —N(-L-R1)-, or L;

each L is independently a covalent bond or a linker (e.g., optionally substituted C_1-60hydrocarbon linker or optionally substituted C_2-60heteroorganic linker);

each R1 is independently hydrogen, —S—S—R2, —O—CO—R2, —S—CO—R2, optionally substituted C_1-9heterocyclyl, a hydrophobic moiety, or a targeting moiety; and

each R2 is independently optionally substituted C_1-10alkyl, optionally substituted C_2-10heteroalkyl, optionally substituted C_6-10aryl, optionally substituted C_6-10aryl C_1-6alkyl, optionally substituted C_1-9heterocyclyl, or optionally substituted C_1-9heterocyclyl C_1-6alkyl. When L is a covalent bond, R1 is hydrogen, Z is oxygen, and all X groups are —O—, the internucleoside group is known as a phosphate phosphodiester. When L is a covalent bond, R1 is hydrogen, Z is sulfur, and all X groups are —O—, the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are —O—, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphotriester. When Z is sulfur, all X groups are —O—, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphorothioate triester. Non-limiting examples of phosphorothioate triester linkages and phosphotriester linkages are described in US 2017/0037399, the disclosure of which is incorporated herein by reference.

The term “intron 36,” as used herein, generally refers to intron 36 of ABCA4 pre-mRNA or genomic DNA, which corresponds to positions 106569 to 110295 in SEQ ID NO: 1 (hg19/b37 coordinates chr1:94481411-94485137), or a mutant version thereof (e.g., g.34393G>A in SEQ ID NO: 1).

The term “morpholino,” as used herein in reference to a class of oligonucleotides, generally represents an oligomer of at least 10 morpholino monomer units interconnected by morpholino internucleoside linkages. A morpholino includes a 5′ group and a 3′ group. For example, a morpholino may be of the following structure:

embedded image

where

n is an integer of at least 10 (e.g., 12 to 50) indicating the number of morpholino units; each B is independently a nucleobase;

R¹is a 5′ group;

R2 is a 3′ group; and

L is (i) a morpholino internucleoside linkage or, (ii) if L is attached to R², a covalent bond. A 5′ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. A 3′ group in morpholino may be, e.g., hydrogen, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer.

The term “morpholino internucleoside linkage,” as used herein, generally represents a divalent group of the following structure:

embedded image

where

Z is O or S;

X¹is a bond, —CH₂—, or —O—;

X²is a bond, —CH₂—O—, or —O—; and

Y is —NR₂, where each R is independently C_1-6alkyl (e.g., methyl), or both R combine together with the nitrogen atom to which they are attached to form a C_2-9heterocyclyl (e.g., N-piperazinyl);

provided that both X¹and X²are not simultaneously a bond.

The term “nucleobase,” as used herein, generally represents a nitrogen-containing heterocyclic ring found at the 1′ position of the ribofuranose/2′-deoxyribofuranose of a nucleoside. Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e.g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and/or O6-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH₃) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7-deazaguanine, 2-aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

The term “nucleoside,” as used herein, generally represents sugar-nucleobase compounds and groups known in the art (e.g., modified or unmodified ribofuranose-nucleobase and 2′-deoxyribofuranose-nucleobase compounds and groups known in the art). The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified sugar nucleoside is ribofuranose or 2′-deoxyribofuranose having an anomeric carbon bonded to a nucleobase. An unmodified nucleoside is ribofuranose or 2′-deoxyribofuranose having an anomeric carbon bonded to an unmodified nucleobase. Non-limiting examples of unmodified nucleosides include adenosine, cytidine, guanosine, uridine, 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2′-substitution, locking, carbocyclization, or unlocking. A 2′-substitution is a replacement of 2′-hydroxyl in ribofuranose with 2′-fluoro, 2′-methoxy, or 2′-(2-methoxy)ethoxy. A locking modification is an incorporation of a bridge between 4′-carbon atom and 2′-carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

The term “nucleotide,” as used herein, generally represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure —X¹—P(X²)(R¹)₂, where X¹is O, S, or NH, and X²is absent, ═O, or ═S, and each R¹is independently —OH, —N(R²)₂, or —O—CH₂CH₂CN, where each R²is independently an optionally substituted alkyl, or both R²groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

The term “oligonucleotide,” as used herein, generally represents a structure containing 10 or more (e.g., 10 to 50) contiguous nucleosides covalently bound together by internucleoside linkages. An oligonucleotide includes a 5′ end and a 3′ end. The 5′ end of an oligonucleotide may be, e.g., hydroxyl, a targeting moiety, a hydrophobic moiety, 5′ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. The 3′ end of an oligonucleotide may be, e.g., hydroxyl, a targeting moiety, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An oligonucleotide having a 5′-hydroxyl or 5′-phosphate has an unmodified 5′ terminus. An oligonucleotide having a 5′ terminus other than 5′-hydroxyl or 5′-phosphate has a modified 5′ terminus. An oligonucleotide having a 3′-hydroxyl or 3′-phosphate has an unmodified 3′ terminus. An oligonucleotide having a 3′ terminus other than 3′-hydroxyl or 3′-phosphate has a modified 3′ terminus.

The term “oxo,” as used herein, generally represents a divalent oxygen atom (e.g., the structure of oxo may be shown as ═O).

The term “pharmaceutically acceptable,” as used herein, generally refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of an individual (e.g., a human), without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.

The term “protecting group,” as used herein, generally represents a group intended to protect a functional group (e.g., a hydroxyl, an amino, or a carbonyl) from participating in one or more undesirable reactions during chemical synthesis. The term “O-protecting group,” as used herein, represents a group intended to protect an oxygen containing (e.g., phenol, hydroxyl or carbonyl) group from participating in one or more undesirable reactions during chemical synthesis. The term “N-protecting group,” as used herein, represents a group intended to protect a nitrogen containing (e.g., an amino or hydrazine) group from participating in one or more undesirable reactions during chemical synthesis. Commonly used O- and N-protecting groups are disclosed in Wuts, “Greene's Protective Groups in Organic Synthesis,” 4^thEdition (John Wiley & Sons, New York, 2006), which is incorporated herein by reference. Exemplary O- and N-protecting groups include alkanoyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl.

Exemplary O-protecting groups for protecting carbonyl containing groups include, but are not limited to: acetals, acylals, 1,3-dithianes, 1,3-dioxanes, 1,3-dioxolanes, and 1,3-dithiolanes.

Other O-protecting groups include, but are not limited to: substituted alkyl, aryl, and arylalkyl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

Other N-protecting groups include, but are not limited to, chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydroxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropoxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups such as trimethylsilyl, and the like.

The term “pyrid-2-yl hydrazone,” as used herein, generally represents a group of the structure:

embedded image

where each R′ is independently H or optionally substituted C_1-6alkyl. Pyrid-2-yl hydrazone may be unsubstituted (i.e., each R′ is H).

The term “splice site,” as used herein, generally refers to a site in a genome corresponding to an end of an intron that may be involved in a splicing procedure. A splice site may be a 5′ splice site (e.g., a 5′ end of an intron) or a 3′ splice site (e.g., a 3′ end of an intron). A given 5′ splice site may be associated with one or more candidate 3′ splice sites, each of which may be coupled to its corresponding 5′ splice site in a splicing operation.

The term “splicing enhancer,” as used herein, generally refers to motifs with positive effects (e.g., causing an increase) on exon or intron inclusion.

The term “splicing regulatory element,” as used herein, generally refers to an exonic splicing silencer element, an exonic splicing enhancer element, an intronic splicing silencer element, and an intronic splicing enhancer element. An exonic splicing silencer element is a portion of the target pre-mRNA exon that reduces the ratio of transcripts including this exon relative to the total number of the gene transcripts. An intronic splicing silencer element is a portion of the target pre-mRNA intron that reduces the ratio of transcripts including the exon adjacent to the target intron relative to the total number of the gene transcripts. An exonic splicing enhancer element is a portion of the target pre-mRNA exon that increases the ratio of transcripts including this exon relative to the total number of the gene transcripts. An intronic splicing enhancer element is a portion of the target pre-mRNA intron that increases the ratio of transcripts including the exon adjacent to the target intron relative to the total number of the gene transcripts.

The term “splicing silencer,” as used herein, generally refers to motifs with negative effects (e.g., causing a decrease) on exon inclusion.

The term “stereochemically enriched,” as used herein, generally refers to a local stereochemical preference for one enantiomer of the recited group over the opposite enantiomer of the same group. Thus, an oligonucleotide containing a stereochemically enriched internucleoside linkage is an oligonucleotide in which a stereogenic internucleoside linkage (e.g., phosphorothioate) of predetermined stereochemistry is present in preference to a stereogenic internucleoside linkage (e.g., phosphorothioate) of stereochemistry that is opposite of the predetermined stereochemistry. This preference can be expressed numerically using a diastereomeric ratio for the stereogenic internucleoside linkage (e.g., phosphorothioate) of the predetermined stereochemistry. The diastereomeric ratio for the stereogenic internucleoside linkage (e.g., phosphorothioate) of the predetermined stereochemistry is the molar ratio of the diastereomers having the identified stereogenic internucleoside linkage (e.g., phosphorothioate) with the predetermined stereochemistry relative to the diastereomers having the identified stereogenic internucleoside linkage (e.g., phosphorothioate) with the stereochemistry that is opposite of the predetermined stereochemistry. The diastereomeric ratio for the phosphorothioate of the predetermined stereochemistry may be greater than or equal to 1.1 (e.g., greater than or equal to 4, greater than or equal to 9, greater than or equal to 19, or greater than or equal to 39).

The term “subject,” as used herein, generally represents a human or non-human animal (e.g., a mammal) that is suffering from, or is at risk of, disease, disorder, or condition, as determined by a qualified professional (e.g., a doctor or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. A non-limiting example of a disease, disorder, or condition includes retinitis pigmentosa (RP), cone-rod dystrophy (CRD), and Stargardt disease (STGD1) (e.g., retinitis pigmentosa, cone-rod dystrophy, and Stargardt disease associated with skipping an exon in the ABCA4 gene (e.g., exon 33 or 40), the inclusion of a portion of a flanking intron adjacent to an exon in the ABCA4 gene (e.g., a portion of a flanking intron adjacent to exon 6), or the inclusion of a pseudo exon (e.g. a pseudo exon exon located in intro 36).

A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring or a structure that is capable of replacing the furanose ring of a nucleoside. Sugars included in the nucleosides of the disclosure may be non-furanose (or 4′-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the disclosure include β-D-ribose, β-D-2′-deoxyribose, substituted sugars (e.g., 2′, 5′, and bis substituted sugars), 4′-S-sugars (e.g., 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted ribose), bicyclic sugar moieties (e.g., the 2′-O—CH₂-4′ or 2′-O—(CH₂)₂-4′ bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

The term “targeting moiety,” as used herein, generally represents a moiety (e.g., N-acetylgalactosamine or a cluster thereof) that specifically binds or reactively associates or complexes with a receptor or other receptive moiety associated with a given target cell population. An antisense oligonucleotide may contain a targeting moiety. An antisense oligonucleotide including a targeting moiety is also referred to herein as a conjugate. A targeting moiety may include one or more ligands (e.g., 1 to 6 ligands, 1 to 3 ligands, or 1 ligand). The ligand can be an antibody or an antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)). Alternatively, the ligand may be a small molecule (e.g., N-acetylgalactosamine).

The term “therapeutically effective amount,” as used herein, generally represents the quantity of an antisense oligonucleotide of the disclosure necessary to ameliorate, treat, or at least partially arrest the symptoms of a disease or disorder (e.g., to increase the level of ABCA4 mRNA molecules including the otherwise skipped exon (e.g., exon 33 or 40) or to increase the level of ABCA4 mRNA molecules excluding otherwise included intronic mRNA (e.g. flanking intronic sequence of exon 6 or a pseudo exon located within intron 36). Amounts effective for this use may depend, e.g., on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide of the disclosure reduces the plasma triglycerides level, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, or up to 20%, as compared to the plasma triglycerides level prior to the administration of an antisense oligonucleotide. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide of the disclosure reduces or maintains the plasma triglyceride levels in the subject to 300 mg/dL or less, 250 mg/dL or less, 200 mg/dL or less, or to 150 mg/dL or less. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide of the disclosure reduces the plasma low density lipoprotein (LDL-C) level, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%; e.g., up to 80%, up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, or up to 20%, as compared to the LDL-C level prior to the administration of an antisense oligonucleotide. In some embodiments, a therapeutically effective amount of an antisense oligonucleotide of the disclosure reduces or maintains the plasma LDL-C levels in the subject to less than 300 mg/dL, less than 250 mg/dL, less than 200 mg/dL, less than 190 mg/dL, less than 160 mg/dL, less than 150 mg/dL, less than 130 mg/dL, or less than 100 mg/dL. Lipid levels can be assessed using plasma lipid analyses or tissue lipid analysis. In plasma lipid analysis, blood plasma can be collected, and total plasma free cholesterol levels can be measured using, for example colorimetric assays with a COD-PAP kit (Wako Chemicals), total plasma triglycerides can be measured using, for example, a Triglycerides/GB kit (Boehringer Mannheim), and/or total plasma cholesterol can be determined using a Cholesterol/HP kit (Boehringer Mannheim). In tissue lipid analysis, lipids can be extracted, for example, from liver, spleen, and/or small intestine samples (e.g., using the method in Folch et al. J Biol. Chem 226: 497-505 (1957)). Total tissue cholesterol concentrations can be measured, for example, using O-phthalaldehyde.

The term “thiocarbonyl,” as used herein, generally represents a C(═S) group. Non-limiting example of functional groups containing a “thiocarbonyl” includes thioesters, thioketones, thioaldehydes, thioanhydrides, thioacyl chlorides, thioamides, thiocarboxylic acids, and thiocarboxylates.

The term “thioheterocyclylene,” as used herein, generally represents a divalent group —S—R′—, where R′ is a heterocyclylene as defined herein.

The term “thiol,” as used herein, generally represents an —SH group.

The term “triazolocycloalkenylene,” as used herein, generally refers to the heterocyclylenes containing a 1,2,3-triazole ring fused to an 8-membered ring, all of the endocyclic atoms of which are carbon atoms, and bridgehead atoms are sp²-hybridized carbon atoms. Triazocycloalkenylenes can be optionally substituted in a manner described for heterocyclyl.

The term “triazoloheterocyclylene,” as used herein, generally refers to the heterocyclylenes containing a 1,2,3-triazole ring fused to an 8-membered ring containing at least one heteroatom. The bridgehead atoms in triazoloheterocyclylene are carbon atoms. Triazoloheterocyclylenes can be optionally substituted in a manner described for heterocyclyl.

Enumeration of positions within oligonucleotides and nucleic acids, as used herein and unless specified otherwise, starts with the 5′-terminal nucleoside as 1 and proceeds in the 3′-direction.

The compounds described herein, unless otherwise noted, encompass isotopically enriched compounds (e.g., deuterated compounds), tautomers, and all stereoisomers and conformers (e.g. enantiomers, diastereomers, E/Z isomers, atropisomers, etc.), as well as racemates thereof and mixtures of different proportions of enantiomers or diastereomers, or mixtures of any of the foregoing forms as well as salts (e.g., pharmaceutically acceptable salts).

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

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. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B shows the c.768G>T variant leads to exon 6 extension in ABCA4 c.768G>T mutant minigene. FIG. TA is a schematic of the ABCA4 c.768G>T mutant minigene. FIG. 1B shows RT-PCR analysis of HEK293T and ARPE19 cells transfected with ABCA4 wild-type and c.768G>T mutant minigenes. Exon 6 inclusion (337 bp) and extension (371 bp) fragments are indicated by solid arrowheads for both wildtype minigene (WT) and c.768G>T (Mut) variant minigenes. 50 bp DNA ladder is shown for size reference.

FIGS. 2A-2B shows the c.4773+3A>G variant leads to exon 33 skipping in ABCA4 c.4773+3A>G mutant minigene. FIG. 2A is a schematic of the ABCA4 c.4773+3A>G mutant minigene. FIG. 2B shows RT-PCR analysis of HEK293T and ARPE19 cells transfected with ABCA4 wild-type and c.4773+3A>G mutant minigenes. Exon 33 inclusion (169 bp) and exclusion (69 bp) fragments are indicated by solid arrowheads for both wildtype minigene (WT) and c.4773+3A>G (Mut) variant minigenes. 50 bp DNA ladder is shown for size reference.

FIGS. 3A-3B shows the c.5196+1137G>A variant leads to intron 36 pseudo exon (36.1) inclusion in ABCA4 c.5196+1137G>A mutant minigene. FIG. 3A is a schematic of the ABCA4 c.5196+1137G>A mutant minigene. FIG. 3B shows RT-PCR analysis of HEK293T and ARPE19 cells transfected with ABCA4 wild-type and c.5196+1137G>A mutant minigenes. Pseudo exon 36.1 inclusion (173 bp) and exclusion (103 bp) fragments are indicated by solid arrowheads for both wildtype minigene (WT) and c.5196+1137G>A (Mut) variant minigenes. 50 bp DNA ladder is shown for size reference.

FIGS. 4A-4B shows the c.5714+5G>A variant leads to exon 40 skipping in ABCA4 c.5714+5G>A mutant minigene. FIG. 4A is a schematic of the ABCA4 c.5714+5G>A mutant minigene. FIG. 4B shows RT-PCR analysis of HEK293T and ARPE19 cells transfected with ABCA4 wild-type and c.5714+5G>A mutant minigenes. Exon 40 inclusion (318 bp) and exclusion (188 bp) fragments are indicated by solid arrowheads for both wildtype minigene (WT) and c.5714+5G>A (Mut) variant minigenes. 50 bp DNA ladder is shown for size reference.

DETAILED DESCRIPTION

In general, the present disclosure provides antisense oligonucleotides, compositions, and methods that target an ABCA4 exon (e.g., exon 6, 33, or 40) or a flanking intron (e.g. intron 36). Surprisingly, the inventors have found that altering ABCA4 gene splicing to promote inclusion of an otherwise skipped exon (e.g., exon 33, or 40) or the exclusion of otherwise included intronic RNA (e.g. intronic RNA in a flanking intron adjacent to exon 6 or intronic RNA associated with a pseudo exon in intron 36) in the transcript of splice variants may be used to treat retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, and antisense oligonucleotides may be used to alter splicing of the ABCA4 gene to include the otherwise skipped exon (e.g., exon 33, or 40) or the exclusion of otherwise included intronic RNA (e.g. intronic RNA in a flanking intron adjacent to exon 6 or intronic RNA associated with a pseudo exon in intron 36). The antisense oligonucleotides of the disclosure may modulate splicing of ABCA4 pre-mRNA to increase the level of ABCA4 mRNA molecules having the otherwise skipped exon (e.g., exon 33, or 40) or ABCA4 mRNA molecules excluding otherwise included intronic RNA (e.g. intronic RNA in a flanking intron adjacent to exon 6 or intronic RNA associated with a pseudo exon in intron 36). Accordingly, the antisense oligonucleotides may be used to treat retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject in need of a treatment therefor. Typically, an antisense oligonucleotide includes a nucleobase sequence at least 70% (e.g., at least 80%, at least 90%, at least 95%, or 100%) complementary to a ABCA4 pre-mRNA sequence in a 5′-flanking intron, a 3′-flanking intron, a combination of an exon (e.g., exon 6, 33, 40) and a 5′-flanking or 3′-flanking intron (e.g., a 5′-flanking or 3′-flanking intron adjacent to exon 6, 33, 40), or an intron (e.g. intron 36).

Genetic variants may correspond to changes or modifications in transcription and/or splicing. RNA is initially transcribed from DNA as pre-mRNA, with protein-coding and 5′UTR/3′UTR exons separated by introns. Splicing generally refers to the molecular process, carried out by the spliceosome complexes that may remove introns and adjoins exons, producing a mature mRNA sequence, which is then scanned and translated to protein by the ribosome. The molecular reaction catalyzed by the spliceosome may comprise (i) nucleophilic attack of the branch site adenosine 2′OH onto the outmost base of the intronic donor dinucleotide, with consequent release of the outmost exonic donor base 3′OH; and (ii) nucleophilic attack of the exonic donor 3′OH onto the outmost exonic acceptor base, with consequent release of the intron lariat and the spliced exons.

Splicing sequence changes can include the following categories: (a) alteration of a splice site (denominated canonical splice site) or exon recognition sequence required for the proper composition of a gene product, and (b) activation and utilization of an incorrect splice site (denominated cryptic splice site), or incorrect recognition of intronic sequence as an exon (denominated pseudo exon). Both (a) and (b) may result in the improper composition of a gene product. The splice site recognition signal may be required for spliceosome assembly and can comprise the following structures: (i) highly conserved intronic dinucleotide (AG, GT) immediately adjacent to the exon-intron boundary, and (ii) consensus sequence surrounding the intronic dinucleotide (often delimited to 3 exonic and 6 intronic nucleotides for the donor site, 3 exonic and 20 intronic nucleotides for the acceptor site) and branch site (variable position on the intronic acceptor side), both with lower conservation and more sequence variety.

In addition to splice site recognition, the exon recognition signal may comprise a plethora of motifs recognized by splicing factors and other RNA binding proteins, some of which may be ubiquitously expressed and some of which may be tissue specific. These motifs may be distributed over the exon body and in the proximal intronic sequence. The term “splicing enhancer” refers to motifs with positive effects (e.g., causing an increase) on exon inclusion, and the term “splicing silencer” refers to motifs with negative effects (e.g., causing a decrease) on exon inclusion. The exon recognition signal may be particularly important for correct splicing in the presence of weak consensus sequence. When a variant weakens the splice site recognition, the exon can be skipped and/or a nearby cryptic splice site which is already fairly strong can be used. In the presence of short introns, full intron retention is also a possible outcome. In particular, alteration of the intronic dinucleotide often results in splicing alteration, whereas consensus sequence alteration may be, on average, less impactful and more context-dependent. When the exon recognition signal is weakened, exon skipping may be a more likely outcome, but cryptic splice site use is also possible, especially in the presence of a very weak consensus sequence. Variants can also strengthen a weak cryptic splice site in proximity of the canonical splice site, and significantly increase its usage resulting in improper splicing and incorrect gene product (with effects including amino acid insertion/deletion, frameshift, and stop-gain).

Antisense oligonucleotides can be used to modulate gene splicing (e.g., by targeting splicing regulatory elements of the gene).

Antisense oligonucleotides may comprise splice-switching oligonucleotides (SSOs), which may modulate splicing by steric blockage, preventing the spliceosome assembly or the binding of splicing factors and RNA binding proteins. Blocking binding of specific splicing factors or RNA binding proteins that have an inhibitory effect may be used to produce increased exon inclusion (e.g. exon 33, or 40 inclusion). Blocking binding of specific splicing factors or RNA binding proteins that enhance cryptic splice site utilization may be used to decrease intron inclusion (e.g., the inclusion intronic RNA in a flanking intron adjacent to exon 6 or intronic RNA associated with a pseudo exon in intron 36). Specific steric blocker antisense oligonucleotide chemistries may include the modified RNA chemistry with phosphorothioate backbone (PS) with a sugar modification (e.g., 2′-modification) and phosphorodiamidate morpholino (PMO). Exemplary PS backbone sugar modifications may include 2′-O-methyl (2′OMe) and 2′-O-methoxyethyl (2′-MOE), which is also known as 2′-methoxyethoxy. Other nucleotide modifications may be used, for example, for the full length of the oligonucleotide or for specific bases. The oligonucleotides can be covalently conjugated to a targeting moiety (e.g., a GalNAc cluster), or to a peptide (e.g., a cell penetrating peptide), or to another molecular or multimolecular group (e.g., a hydrophobic moiety or neutral polymer) different from the rest of the oligonucleotide. Antisense oligonucleotides may be used as a single stereoisomer or a combination of stereoisomers.

The ABCA4 gene (ATP binding cassette subfamily A member 4; entrez gene 24) may play an important role in the pathogenicity of retinitis pigmentosa, cone-rod dystrophy, and Stargardt disease. ABCA4 is a transmembrane lipid transporter expressed in the photoreceptor outer segment, within the disc membranes. It is required to clear the reactive all-trans retinal from the photoreceptor disc lumen. Lack of ABCA4 function causes N-retinylidene-PE accumulation, which leads to formation of di-retinoid-pyridinium-PE (A2PE); all-trans retinal can also accumulate and form dimers. Since RPE cells recycle photoreceptor outer segments every 10 days, these compounds end up accumulating in their lysosomes. There, A2PE is hydrolyzed to di-retinoid-pyridinium-ethanolamine (A2E), which can be photoactivated and form highly reactive epoxides. This process is toxic for RPE cells and can lead to cell death. As photoreceptors lose the support of RPE, they can in turn suffer cell death. Higher levels of A2PE accumulation are directly toxic to photoreceptors, and cones are more sensitive than rods.

Recognizing a need for effective splicing modulation therapies for diseases such as retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease, the present disclosure provides ABCA4 splice-modulating antisense oligonucleotides comprising sequences targeted to an intron adjacent to an abnormally spliced exon (e.g., exon 6, 33, or 40) of ABCA4 or an abnormally spliced intron (e.g. intron 36). In some embodiments, the antisense oligonucleotide has a sequence targeted to one or more splicing regulatory elements which may be located in an intron adjacent to an abnormally spliced exon (e.g., exon 6, 33, or 40) of ABCA4 or alternatively splicing regulatory elements which may be located in an intron next to a pseudo exon (e.g. intron 36). The present disclosure also provides methods for modulating splicing of ABCA4 RNA in a cell, tissue, or organ of a subject by bringing the cell, tissue, or organ in contact with an antisense oligonucleotide of the disclosure. An ABCA4 splice-modulating antisense oligonucleotide may comprise a nucleobase sequence targeted to a splicing regulatory element of an intron adjacent to an abnormally spliced exon (e.g., exon 6, 33, or 40) of ABCA4 or alternatively splicing regulatory elements which may be located in an intron next to a pseudo exon (e.g. intron 36). In addition, the present disclosure provides a method for treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject by administering to the subject a therapeutically effective amount of an oligonucleotide of the disclosure. An ABCA4 splice-modulating antisense oligonucleotide may comprise a sequence targeted to a splicing regulatory element of or an intron adjacent to an abnormally spliced exon (e.g., exon 6, 33, or 40) of ABCA4 or alternatively splicing regulatory elements which may be located in an intron next to a pseudo exon (e.g. intron 36).

Splicing regulatory elements may include, for example, exonic splicing silencer elements or intronic splicing silencer elements. The antisense oligonucleotides may comprise sequences targeted to an intron adjacent to the exon (e.g., 33, or 40) of ABCA4 which modulates variant splicing of ABCA4 RNA. The modulation of splicing may result in an increase in exon inclusion (e.g. exon 33, or 40 inclusion). Antisense oligonucleotides may comprise a total of 8 to 50 nucleotides (e.g. 8 to 16 nucleotides, 8 to 20 nucleotides, 12 to 20 nucleotides, 12 to 30 nucleotides, or 12 to 50 nucleotides).

Additional splicing regulatory elements may include, for example, cryptic splice sites which are intronic mRNA sequences that have the potential to interact with the spliceosome. Cryptic splice sites may be activated by a variant and lead to the inclusion of a pseudo exon in the fully processed mRNA (e.g. the inclusion of a pseudo exon located in intron 36) or the elongation of an exon to include flanking intronic sequence in the fully processed (e.g. the inclusion of flanking intronic sequence in exon 6). The antisense oligonucleotides may comprise sequences targeted to an intron containing a pseudo exon (e.g. intron 36), or an exon or an intron adjacent to the exon which is mispliced (e.g. exon 6) of ABCA4 which modulates variant splicing of ABCA4 RNA. The modulation of splicing may result in a decrease in intronic sequence inclusion (e.g., partial intron 36 or 6 inclusion). Antisense oligonucleotides may comprise a total of 8 to 50 nucleotides (e.g., 8 to 16 nucleotides, 8 to 20 nucleotides, 12 to 20 nucleotides, 12 to 30 nucleotides, or 12 to 50 nucleotides).

Genetic aberrations of the ABCA4 gene may play an important role in pathogenicity. In particular, ABCA4 chr1:94484001:C:T [hg19/b37], chr1:94487399:T:C [hg19/b37], chr1:94476351:C:T [hg19/b37], and chr1:94564350:C:A [hg19/b37] genetic aberrations (g.107705G>A, g.104307A>G, g.115355G>A, g.27356G>T mutants of SEQ ID NO: 1, respectively), may result in NM_000350.2 (ABCA4) mRNA changes c.5196+1137G>A, c.4773+3A>G, c.5714+5G>A, and cDNA change c.768G>T respectively. Intronic variants c.5196+1137G>A, c.4773+3A>G, c.5714+5G>A are non-coding and c.768G>T results in no change in the protein sequence at amino acid position 256 (Val) in exon 6. Genome coordinates may be expressed, for example, with respect to human genome reference hg19/b37. For example, these variants have been reported as pathogenic in patients with retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease. Exemplary variants which have been reported or predicted to be pathogenic in patients with retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease variants are listed in Table 1.

TABLE 1

Genomic_
mRNA coordinate

Genomic_coordinate
coordinate
(protein sequence

[hg19/b37]
[SEQ ID NO: 1]
change) [NM_000350.2]

chr1:94466425:C:A
g.125281G > T
c.6446G > T (p.Arg2149Leu)

chr1:94466602:C:T
g.125104G > A
c.6342G > A (p.Val2114=)

chr1:94526295:C:T
g.65411G > A
c.1958G > A (p.Arg653His)

chr1:94528683:T:C
g.63023A > G
c.1745A > G (p. Asn582Ser)

chr1:94476378:G:A
g.115328C > T
c.5692C > T (p.Arg1898Cys)

chr1:94480241:G:A
g.111465C > T
c.5318C > T (p.Ala1773Val)

chr1:94487443:C:T
g.104263G > A
c.4732G > A (p.Gly1578Arg)

chr1:94496008:C:T
g.95698G > A
c.4328G > A (p.Arg1443His)

chr1:94496610:C:T
g.95096G > A
c.4195G > A (p.Glu1399Lys)

chr1:94528819:G:A
g.62887C > T
c.1609C > T (p.Arg537Cys)

chr1:94473791:C:T
g.117915G > A
c.5898G > A (p.Glu1966=)

chr1:94476351:C:T
g.115355G > A
c.5714 + 5G > A

chr1:94487269:C:T
g.104437G > A
c.4775G > A (p.Gly1592Asp)

chr1:94487399:T:C
g.104307A > G
c.4773 + 3A > G

chr1:94496547:C:T
g.95159G > A
c.4253 + 5G > A

chr1:94496548:G:A
g.95158C > T
c.4253 + 4C > T

chr1:94510164:C:T
g.81542G > A
c.3050 + 5G > A

chr1:94543248:C:T
g.48458G > A
c.1552G > A (p.Glu518Lys)

chr1:94564350:C:A
g.27356G > T
c.768G > T (p.Val256=)

chr1:94586533:T:G
g.5173A > C
c.66 + 3A > C

chr1:94484001:C:T
g.107705G > A
c.5196 + 1137G > A

chr1:94566773:T:C
g.24933A > G
c.570 + 1798A > G

These exemplary genetic aberrations may be targeted with antisense oligonucleotides to increase levels of exon inclusion (e.g., exon 33, or 40 inclusion) or decrease intronic sequence inclusion (e.g., partial intron 36 or 6 inclusion) of ABCA4.

Different antisense oligonucleotides can be combined for increasing an exon inclusion (e.g., exon 33, or 40 inclusion), or decreasing intronic sequence inclusion (e.g., partial intron 36 or 6 inclusion) of ABCA4. A combination of two antisense oligonucleotides may be used in a method of the disclosure, such as two antisense oligonucleotides, three antisense oligonucleotides, four different antisense oligonucleotides, or five different antisense oligonucleotides targeting the same or different regions or “hotspots.”

An antisense oligonucleotide according to the disclosure may be indirectly administered using suitable techniques and methods known in the art. It may for example be provided to an individual or a cell, tissue or organ of the individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In an embodiment, there is provided a viral based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of an antisense oligonucleotide as identified herein. Accordingly, the present disclosure provides a viral vector expressing an antisense oligonucleotide according to the disclosure.

An antisense oligonucleotide according to the disclosure may be directly administered using suitable techniques and methods known in the art, e.g., using conjugates described herein.

Conjugates

Oligonucleotides of the disclosure may include an auxiliary moiety, e.g., a targeting moiety, hydrophobic moiety, cell penetrating peptide, or a polymer. An auxiliary moiety may be present as a 5′ terminal modification (e.g., covalently bonded to a 5′-terminal nucleoside), a 3′ terminal modification (e.g., covalently bonded to a 3′-terminal nucleoside), or an internucleoside linkage (e.g., covalently bonded to phosphate or phosphorothioate in an internucleoside linkage).

Targeting Moieties

An oligonucleotide of the disclosure may include a targeting moiety.

A targeting moiety is selected based on its ability to target oligonucleotides of the disclosure to a desired or selected cell population that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) for the selected targeting moiety. For example, an oligonucleotide of the disclosure could be targeted to hepatocytes expressing asialoglycoprotein receptor (ASGP-R) by selecting a targeting moiety containing N-acetylgalactosamine (GalNAc).

A targeting moiety may include one or more ligands (e.g., 1 to 9 ligands, 1 to 6 ligands, 1 to 3 ligands, 3 ligands, or 1 ligand). The ligand may target a cell expressing asialoglycoprotein receptor (ASGP-R), IgA receptor, HDL receptor, LDL receptor, or transferrin receptor. Non-limiting examples of the ligands include N-acetylgalactosamine, glycyrrhetinic acid, glycyrrhizin, lactobionic acid, lactoferrin, IgA, or a bile acid (e.g., lithocholyltaurine or taurocholic acid).

The ligand may be a small molecule, e.g., a small molecules targeting a cell expressing asialoglycoprotein receptor (ASGP-R). A non-limiting example of a small molecule targeting an asialoglycoprotein receptor is N-acetylgalactosamine. Alternatively, the ligand can be an antibody or an antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)).

A targeting moiety may be -LinkA(-T)_p, where LinkA is a multivalent linker, each T is a ligand (e.g., asialoglycoprotein receptor-targeting ligand (e.g., N-acetylgalactosamine)), and p is an integer from 1 to 9. When each T is N-acetylgalactosamine, the targeting moiety is referred to as a galactosamine cluster. Galactosamine clusters that may be used in oligonucleotides of the disclosure are known in the art. Non-limiting examples of the galactosamine clusters that may be included in the oligonucleotides of the disclosure are provided in U.S. Pat. Nos. 5,994,517; 7,491,805; 9,714,421; 9,867,882; 9,127,276; US 2018/0326070; US 2016/0257961; WO 2017/100461; and in Sliedregt et al., J. Med. Chem., 42:609-618, 1999. Ligands other than GalNAc may also be used in clusters, as described herein for galactosamine clusters.

Targeting moiety -LinkA(-T)_pmay be a group of formula (I):

-Q¹-Q²([-Q³-Q⁴-Q⁵]_s-Q⁶-T)_p, (I)

where

each s is independently an integer from 0 to 20 (e.g., from 0 to 10), where the repeating units are the same or different;

Q¹is a conjugation linker (e.g., [-Q³-Q⁴-Q⁵]_s-Q^C- where Q^Cis optionally substituted C_2-12heteroalkylene (e.g., a heteroalkylene containing —C(O)—N(H)—, —N(H)—C(O)—, —S(O)₂—N(H)—, —N(H)—S(O)₂—, or —S—S—), optionally substituted C_1-12thioheterocyclylene

embedded image

optionally substituted C_1-12heterocyclylene (e.g., 1,2,3-triazole-1,4-diyl or

embedded image

cyclobut-3-ene-1,2-dione-3,4-diyl, pyrid-2-yl hydrazone, optionally substituted C_6-16triazoloheterocyclylene (e.g.,

embedded image

optionally substituted C_8-16triazolocycloalkenylene

embedded image

or a dihydropyridazine group (e.g., trans-

embedded image

Q²is a linear group (e.g., [-Q³-Q⁴-Q⁵]_s-), if p is 1, or a branched group (e.g., [-Q³-Q⁴-Q⁵]_s-Q⁷([-Q³-Q⁴-Q⁵]_s-(Q⁷)_p1)_p2, where p1 is 0, 1, or 2, and p2 is 0, 1, 2, or 3), if p is an integer from 2 to 9;

each Q³and each Q⁶is independently absent, —CO—, —NH—, —O—, —S—, —SO₂—, —OC(O)—, —C(O)O—, —NHC(O)—, —C(O)NH—, —CH₂—, —CH₂NH—, —NHCH₂—, —CH₂O—, or —OCH₂—; each Q⁴is independently absent, optionally substituted C_1-12alkylene, optionally substituted C_2-12alkenylene, optionally substituted C_2-12alkynylene, optionally substituted C_2-12heteroalkylene, optionally substituted C₆-10 arylene, optionally substituted C_1-9heteroarylene, or optionally substituted C_1-9heterocyclylene;

each Q⁵is independently absent, —CO—, —NH—, —O—, —S—, —SO₂—, —CH₂—, —C(O)O—, —OC(O)—, —C(O)NH—, —NH—C(O)—, —NH—CH(R^a)—C(O)—, —C(O)—CH(R^a)—NH—, —OP(O)(OH)O—, or —OP(S)(OH)O—;

each Q⁷is independently optionally substituted hydrocarbon or optionally substituted heteroorganic (e.g., C_1-6alkane-triyl, optionally substituted C_1-6alkane-tetrayl, optionally substituted C_2-6heteroalkane-triyl, or optionally substituted C_2-6heteroalkane-tetrayl); and

each R^ais independently H or an amino acid side chain;

provided that at least one of Q³, Q⁴, and Q⁵is present.

In some instances, for each occurrence of [-Q³-Q⁴-Q⁵]_s-, at least one of Q³, Q⁴, and Q⁵is present.

In some instances, Q⁷may be a structure selected from the group consisting of:

embedded image

where R^Ais H or oligonucleotide, X is O or S, Y is O or NH, and the remaining variables are as described for formula (I).

Group -LinkA- may include a poly(alkylene oxide) (e.g., polyethylene oxide, polypropylene oxide, poly(trimethylene oxide), polybutylene oxide, poly(tetramethylene oxide), and diblock or triblock co-polymers thereof). In some embodiments, -LinkA- includes polyethylene oxide (e.g., poly(ethylene oxide) having a molecular weight of less than 1 kDa).

Hydrophobic Moieties

Advantageously, an oligonucleotide including a hydrophobic moiety may exhibit superior cellular uptake, as compared to an oligonucleotide lacking the hydrophobic moiety. Oligonucleotides including a hydrophobic moiety may therefore be used in compositions that are substantially free of transfecting agents. A hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently linked to the oligonucleotide backbone (e.g., 5′-terminus). Non-limiting examples of the monovalent group include ergosterol, stigmasterol, β-sitosterol, campesterol, fucosterol, saringosterol, avenasterol, coprostanol, cholesterol, vitamin A, vitamin D, vitamin E, cardiolipin, and carotenoids. The linker connecting the monovalent group to the oligonucleotide may be an optionally substituted C_1-60hydrocarbon (e.g., optionally substituted C_1-60alkylene) or an optionally substituted C_2-60heteroorganic (e.g., optionally substituted C_2-60heteroalkylene), where the linker may be optionally interrupted with one, two, or three instances independently selected from the group consisting of an optionally substituted arylene, optionally substituted heterocyclylene, and optionally substituted cycloalkylene. The linker may be bonded to an oligonucleotide through, e.g., an oxygen atom attached to a 5′-terminal carbon atom, a 3′-terminal carbon atom, a 5′-terminal phosphate or phosphorothioate, a 3′-terminal phosphate or phosphorothioate, or an internucleoside linkage.

Cell Penetrating Peptides

One or more cell penetrating peptides (e.g., from 1 to 6 or from 1 to 3) can be attached to an oligonucleotide disclosed herein as an auxiliary moiety. The CPP can be linked to the oligonucleotide through a disulfide linkage, as disclosed herein. Thus, upon delivery to a cell, the CPP can be cleaved intracellularly, e.g., by an intracellular enzyme (e.g., protein disulfide isomerase, thioredoxin, or a thioesterase) and thereby release the polynucleotide.

CPPs are known in the art (e.g., TAT or Args (SEQ ID NO: 462)) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2, 43-51). Specific examples of CPPs including moieties suitable for conjugation to the oligonucleotides disclosed herein are provided, e.g., in WO 2015/188197; the disclosure of these CPPs is incorporated by reference herein.

CPPs are positively charged peptides that are capable of facilitating the delivery of biological cargo to a cell. It is believed that the cationic charge of the CPPs is essential for their function. Moreover, the transduction of these proteins does not appear to be affected by cell type, and these proteins can efficiently transduce nearly all cells in culture with no apparent toxicity. In addition to full-length proteins, CPPs have also been used successfully to induce the intracellular uptake of DNA, antisense polynucleotides, small molecules, and even inorganic 40 nm iron particles suggesting that there is considerable flexibility in particle size in this process.

In one embodiment, a CPP useful in the methods and compositions of the disclosure includes a peptide featuring substantial alpha-helicity. It has been discovered that transfection is optimized when the CPP exhibits significant alpha-helicity. In another embodiment, the CPP includes a sequence containing basic amino acid residues that are substantially aligned along at least one face of the peptide. A CPP useful in the disclosure may be a naturally occurring peptide or a synthetic peptide.

Polymers

An oligonucleotide of the disclosure may include covalently attached neutral polymer-based auxiliary moieties. Neutral polymers include poly(C_1-6alkylene oxide), e.g., poly(ethylene glycol) and poly(propylene glycol) and copolymers thereof, e.g., di- and triblock copolymers. Other examples of polymers include esterified poly(acrylic acid), esterified poly(glutamic acid), esterified poly(aspartic acid), poly(vinyl alcohol), poly(ethylene-co-vinyl alcohol), poly(N-vinyl pyrrolidone), poly(ethyloxazoline), poly(alkylacrylates), poly(acrylamide), poly(N-alkylacrylamides), poly(N-acryloylmorpholine), poly(lactic acid), poly(glycolic acid), poly(dioxanone), poly(caprolactone), styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyurethane, N-isopropylacrylamide polymers, and poly(N,N-dialkylacrylamides). Exemplary polymer auxiliary moieties may have molecular weights of less than 100, 300, 500, 1000, or 5000 Da (e.g., greater than 100 Da). Other polymers are known in the art.

Nucleobase Modifications

Oligonucleotides of the disclosure may include one or more modified nucleobases. Unmodified nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e.g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and/or O6-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

The replacement of cytidine with 5-methylcytidine can reduce immunogenicity of oligonucleotides, e.g., those oligonucleotides having CpG units.

The replacement of one or more guanosines with, e.g., 7-deazaguanosine or 6-thioguanosine, may inhibit the antisense activity reducing G tetraplex formation within antisense oligonucleotides.

Sugar Modifications

Oligonucleotides of the disclosure may include one or more sugar modifications in nucleosides. Nucleosides having an unmodified sugar include a sugar moiety that is a furanose ring as found in ribonucleosides and 2′-deoxyribonucleosides.

Sugars included in the nucleosides of the disclosure may be non-furanose (or 4′-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six-membered ring). Alternative sugars may also include sugar surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the disclosure include β-D-ribose, β-D-2′-deoxyribose, substituted sugars (e.g., 2′, 5′, and bis substituted sugars), 4′-S-sugars (e.g., 4′-S-ribose, 4′-S-2′-deoxyribose, and 4′-S-2′-substituted ribose), bridged sugars (e.g., the 2′-O—CH₂-4′ or 2′-O—(CH₂)₂-4′ bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

Typically, a sugar modification may be, e.g., a 2′-substitution, locking, carbocyclization, or unlocking. A 2′-substitution is a replacement of 2′-hydroxyl in ribofuranose with 2′-fluoro, 2′-methoxy, or 2′-(2-methoxy)ethoxy. A locking modification is an incorporation of a bridge between 4′-carbon atom and 2′-carbon atom of ribofuranose. Nucleosides having a sugar with a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides.

Internucleoside Linkage Modifications

Oligonucleotides of the disclosure may include one or more internucleoside linkage modifications. The two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages, methylphosphonates, and phosphoramidate. Non-limiting examples of non-phosphorus internucleoside linkages include methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)₂—O—), and N,N′-dimethylhydrazine (—CH2-N(CH₃)—N(CH₃)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are known in the art.

Internucleoside linkages may be stereochemically enriched. For example, phosphorothioate-based internucleoside linkages (e.g., phosphorothioate diester or phosphorothioate triester) may be stereochemically enriched. The stereochemically enriched internucleoside linkages including a stereogenic phosphorus are typically designated S_Por R_Pto identify the absolute stereochemistry of the phosphorus atom. Within an oligonucleotide, S_Pphosphorothioate indicates the following structure:

embedded image

Within an oligonucleotide, R_Pphosphorothioate indicates the following structure:

embedded image

The oligonucleotides of the disclosure may include one or more neutral internucleoside linkages. Non-limiting examples of neutral internucleoside linkages include phosphotriesters, phosphorothioate triesters, methylphosphonates, methylenemethylimino (5′-CH₂—N(CH₃)—O-3′), amide-3 (5′-CH₂—C(═O)—N(H)-3′), amide-4 (5′-CH₂—N(H)—C(═O)-3′), formacetal (5′-O—CH₂—O-3′), and thioformacetal (5′-S—CH₂—O-3′). Further neutral internucleoside linkages include nonionic linkages including siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester, and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65).

Terminal Modifications

Oligonucleotides of the disclosure may include a terminal modification, e.g., a 5′-terminal modification or a 3′-terminal modification.

The 5′ end of an oligonucleotide may be, e.g., hydroxyl, a hydrophobic moiety, a targeting moiety, 5′ cap, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, diphosphrodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer. An unmodified 5′-terminus is hydroxyl or phosphate. An oligonucleotide having a 5′ terminus other than 5′-hydroxyl or 5′-phosphate has a modified 5′ terminus.

The 3′ end of an oligonucleotide may be, e.g., hydroxyl, a targeting moiety, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, disphorodithioate, triphosphorodithioate, phosphonate, phosphoramidate, a cell penetrating peptide, an endosomal escape moiety, or a neutral organic polymer (e.g., polyethylene glycol). An unmodified 3′-terminus is hydroxyl or phosphate. An oligonucleotide having a 3′ terminus other than 3′-hydroxyl or 3′-phosphate has a modified 3′ terminus.

The terminal modification (e.g., 5′-terminal modification) may be, e.g., a targeting moiety as described herein.

The terminal modification (e.g., 5′-terminal modification) may be, e.g., a hydrophobic moiety as described herein.

Complementarity

In some embodiments, oligonucleotides of the disclosure are complementary to an ABCA4 target sequence over the entire length of the oligonucleotide. In other embodiments, oligonucleotides are at least 99%, 95%, 90%, 85%, 80%, or 70% complementary to the ABCA4 target sequence. In further embodiments, oligonucleotides are at least 80% (e.g., at least 90% or at least 95%) complementary to the ABCA4 target sequence over the entire length of the oligonucleotide and include a nucleobase sequence that is fully complementary to a ABCA4 target sequence. The nucleobase sequence that is fully complementary may be, e.g., 6 to 20, 10 to 18, or 18 to 20 contiguous nucleobases in length.

An oligonucleotide of the disclosure may include one or more (e.g., 1, 2, 3, or 4) mismatched nucleobases relative to the target nucleic acid. In certain embodiments, a splice-switching activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, the off-target selectivity of the oligonucleotides may be improved.

Methods for Preparing Compositions

The present disclosure provides methods for preparing or generating compositions provided herein. A nucleic acid molecule, such as an oligonucleotide, comprising a targeted sequence may be generated, for example, by various nucleic acid synthesis approaches. For example, a nucleic acid molecule comprising a sequence targeted to a splice site may be generated by oligomerization of modified and/or unmodified nucleosides, thereby producing DNA or RNA oligonucleotides. Antisense oligonucleotides can be prepared, for example, by solid phase synthesis. Such solid phase synthesis can be performed, for example, in multi-well plates using equipment available from vendors such as Applied Biosystems (Foster City, Calif.). It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. Oligonucleotides may be subjected to purification and/or analysis using methods known to those skilled in the art. For example, analysis methods may include capillary electrophoresis (CE) and electrospray-mass spectroscopy.

Pharmaceutical Compositions

An oligonucleotide of the disclosure may be included in a pharmaceutical composition. A pharmaceutical composition typically includes a pharmaceutically acceptable diluent or carrier. A pharmaceutical composition may include (e.g., consist of), e.g., a sterile saline solution and an oligonucleotide of the disclosure. The sterile saline is typically a pharmaceutical grade saline. A pharmaceutical composition may include (e.g., consist of), e.g., sterile water and an oligonucleotide of the disclosure. The sterile water is typically a pharmaceutical grade water. A pharmaceutical composition may include (e.g., consist of), e.g., phosphate-buffered saline (PBS) and an oligonucleotide of the disclosure. The sterile PBS is typically a pharmaceutical grade PBS.

Pharmaceutical compositions may include one or more oligonucleotides and one or more excipients. Excipients may be selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

Pharmaceutical compositions including an oligonucleotide encompass any pharmaceutically acceptable salts of the oligonucleotide. Pharmaceutical compositions including an oligonucleotide, upon administration to a subject (e.g., a human), are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligonucleotides. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs include one or more conjugate group(s) attached to an oligonucleotide, wherein the one or more conjugate group(s) is cleaved by endogenous enzymes within the body.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligonucleotide, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. DNA complexes with mono- or poly-cationic lipids may form, e.g., without the presence of a neutral lipid. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to fat tissue. A lipid moiety may be, e.g., selected to increase distribution of a pharmaceutical agent to muscle tissue.

Pharmaceutical compositions may include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. Certain organic solvents such as dimethylsulfoxide may be used.

Pharmaceutical compositions may include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present disclosure to specific tissues or cell types. For example, pharmaceutical compositions may include liposomes coated with a targeting moiety as described herein.

Pharmaceutical compositions may include a co-solvent system. Certain co-solvent systems include, e.g., benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. Such co-solvent systems may be used, e.g., for hydrophobic compounds. A non-limiting example of a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Pharmaceutical compositions may be prepared for administration by injection or infusion (e.g., intravenous, subcutaneous, intramuscular, intrathecal, intracerebroventricular, intravitreal etc.). A pharmaceutical composition may include, e.g., a carrier and may be formulated, e.g., in aqueous solution, e.g., water or physiologically compatible buffers, e.g., Hanks's solution, Ringer's solution, or physiological saline buffer. Other ingredients may also be included (e.g., ingredients that aid in solubility or serve as preservatives). Injectable suspensions may be prepared, e.g., using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection may be, e.g., suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain excipients (e.g., suspending, stabilizing and/or dispersing agents). Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, e.g., sesame oil, synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), and liposomes.

Methods of the Disclosure

The disclosure provides methods of using oligonucleotides of the disclosure.

A method of the disclosure may be a method of increasing the level of an exon-containing (e.g., exon 33 or 40-containing) ABCA4 mRNA molecules in a cell expressing an aberrant ABCA4 gene by contacting the cell with an antisense oligonucleotide of the disclosure.

A method of the disclosure may be a method of decreasing the level of an intron-containing (e.g., partial intron 6 or 36-containing) ABCA4 mRNA molecules in a cell expressing an aberrant ABCA4 gene by contacting the cell with an antisense oligonucleotide of the disclosure.

A method of the disclosure may be a method of treating retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease in a subject having an aberrant ABCA4 gene by administering a therapeutically effective amount of an antisense oligonucleotide of the disclosure or a pharmaceutical composition of the disclosure to the subject in need thereof.

The oligonucleotide of the disclosure or the pharmaceutical composition of the disclosure may be administered to the subject using methods known in the art. For example, the oligonucleotide of the disclosure or the pharmaceutical composition of the disclosure may be administered parenterally (e.g., intravenously, intramuscularly, subcutaneously, transdermally, intranasally, intravitreally, or intrapulmonarily) to the subject.

Dosing is typically dependent on a variety of factors including, e.g., severity and responsiveness of the disease state to be treated. The treatment course may last, e.g., from several days to several years, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Thus, optimum dosages, dosing methodologies and repetition rates can be established as needed. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage may be from 0.01 μg to 1 g per kg of body weight, and may be given once or more daily, weekly, monthly, bimonthly, trimonthly, every six months, annually, or biannually. Frequency of dosage may vary. Repetition rates for dosing may be established, for example, based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 1 g per kg of body weight, e.g., once daily, twice daily, three times daily, every other day, weekly, biweekly, monthly, bimonthly, trimonthly, every six months, annually or biannually.

EXAMPLES

The following materials, methods, and examples are illustrative only and not intended to be limiting.

Materials and Methods

In general, the practice of the present disclosure employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, and recombinant DNA technology. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992).

Oligonucleotides. All antisense oligonucleotides used were obtained from Integrated DNA Technologies Inc. (USA). All bases in the antisense oligonucleotides were 2′-O-methoxyethyl-modified (MOE) with a full phosphorothioate backbone.

Cell culture. HEK293T cells were grown in Iscove's Modified Dulbecco's Medium (Gibco) supplemented with 10% (v/v) Cosmic Calf Serum (HyClone), 2 mM L-Glutamine (Gibco) and 1% antibiotics (100-U/ml penicillin G and 100-ug/ml streptomycin, Gibco) in a humidified incubator at 37° C. with 5% CO2. Upon reaching confluency the HEK293T cells were passaged by washing with Phosphate-Buffered Saline followed by Trypsin (Gibco) dissociation and plated in 10 to 20-fold dilution. ARPE19 cells were grown in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12; Gibco) with 10% (v/v) Fetal Bovine Serum (Gibco) and 1% antibiotics (100-U/ml penicillin G and 100-ug/ml streptomycin, Gibco). Upon reaching confluency the ARPE19 cells were passaged by washing with Phosphate-Buffered Saline followed by TrypLE (Gibco) dissociation and plated in a culture flask in 2 to 4-fold dilution.

Transfection of cells with minigene plasmids. HEK293T cells were seeded at 75000 cells per well in 24 well plates using Iscove's Modified Dulbecco's Medium (IMDM; Gibco) supplemented with 10% (v/v) Cosmic Calf Serum (HyClone) and 2 mM L-glutamine (Gibco) and incubated at 37° C. and 5% CO2 overnight. ARPE19 cells were seeded at 100,000 cells per well in 24 well plates using DMEM/F-12 (Gibco) with 10% Fetal Bovine Serum (Gibco). Plasmid transfection mixes were made by combining 250 ng of plasmid diluted in 25 μl Opti-MEM (Gibco) with 1 of P3000 reagent (Invitrogen). 25 μl of Opti-MEM along with 1.5 μl Lipofectamine 3000 reagent was added to the diluted DNA mix and incubated at room temperature for 10-15 minutes. 50 μl of the transfection mix was added to the cells and incubated at 37° C. and 5% CO2 overnight.

Co-transfection of cells with minigene plasmids and antisense oligonucleotides. Minigene plasmids were transfected into HEK293T cells or ARPE19 cells. HEK293T cells were seeded at 75000 cells per well in 24 well plates using IMDM supplemented with 10% Cosmic Calf Serum and 2 mM L-glutamine and incubated at 37° C. and 5% CO2 overnight. ARPE19 cells were seeded at 100,000 cells per well in 24 well plates using DMEM/F-12 (Gibco) with 10% Fetal Bovine Serum (Gibco). Plasmid transfection mixes were made by combining 250 ng of plasmid diluted in 25 μl Opti-MEM with 1 of P3000 reagent (Invitrogen). 25 μl of Opti-MEM along with 1.5 μl Lipofectamine 3000 reagent was added to the diluted DNA mix and incubated at room temperature for 10-15 minutes. 50 μl of the transfection mix was added to the cells and incubated at 37° C. and 5% CO2 overnight. 24 hours after plasmid transfection, cells were transfected with antisense oligonucleotides at absolute amounts of 150 pmol per well. For this, 150 pmol antisense oligonucleotide was mixed with 25 μl Opti-MEM and 1 μl P3000 mix to make the DNA mix. 25 μl Opti-MEM and 1.5 μl Lipofectamine 3000 was added to the DNA mix and incubated for 10-15 minutes at room temperature. Next, media was removed for the transfected cells and 500 μl of fresh IMDM (Gibco) with 10% Cosmic Calf Serum and 2 mM L-glutamine was added to each well. Subsequently, 50 μl of the antisense oligo mix was added to each well and incubated for 48 hrs hours at 37° C. and 5% CO2.

RNA isolation. RNA was isolated using ZymoResearch Magnetic Bead Kit or QIAGEN RNeasy kit, according to manufacturer's instructions.

RT-PCR analysis. First-strand cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher), according to manufacturer's instructions. Target-specific fragments were amplified by PCR using the primers listed in Table 2. PCR reactions contained 5 μl first-strand cDNA product, 0.4 μM forward primer, 0.4 μM reverse primer, 300 μM of each dNTP, 25 mM Tricine, 7.0% Glycerol (m/v), 1.6% DMSO (m/v), 2 mM MgCl2, 85 mM NH4-acetate (pH8.7), and 1 unit Taq DNA polymerase (FroggaBio) in a total volume of 25 μL. Fragments were amplified by a touchdown PCR program (95° C. for 120 sec; 10 cycles of 95° C. for 20 sec, 68° C. for 30 sec with a decrement of 1° C. per cycle, and 72° C. for 60 sec; followed by 20 cycles of 95° C. for 20 sec, 58° C. for 30 sec, and 72° C. for 60 sec; 72° C. for 180 sec).

TABLE 2

SEQ

Sequence
ID

Exon
Variant
Primers
(5′>3′)
NO:

40
c.5714+5G>A
P1009
GATTACAAGGAT
450

GACGACGATAAG

P1986
TCTTCATCAACA
451

ATGGGCTCC

6
c.768G>T
P863
ATGGGCCTGTCT
452

GACTCAG

P868
TCATTCCTCCCC
453

AAGATCTCAGA

36.1
c.5196+1137G>A
P1979
GTTTATCAGTGG
454

AGTGAGCCC

P1980
GATGAAGATGCC
455

CACCACC

33
c.4773+3A>G
P995
GTTCTGGGTCAA
456

TGAACAGAG

P1978
GAAATCAGGTAT
457

TTCTTTAGAGGCC

Capillary electrophoresis. Samples were analyzed using a LabChip GX Touch Nucleic Acid Analyzer using a DNA 1K Hi Sensitivity LabChip and associated reagents according to manufacturer's recommendations (GE).

Minigene plasmids. Minigene plasmids for variants c.5714+5G>A, c.768G>T, and c.5196+1137G>A were synthesized by Genscript (NJ, USA). For variant c.4773+3A>G, PCR amplification was used to obtain the sequences from ARPE19 genomic DNA. To generate the ABCA4 exon 33 wildtype minigene, PCR reactions were performed with primers ATGTTCTGGGTCAATGAACAGAGGT (SEQ ID NO: 458) and CTATCAGGTATTTCTTTAGAGGCCTC (SEQ ID NO: 459) using the Q5 High-Fidelity DNA Polymerase (NEB), according to manufacturer's protocol. To generate the ABCA4 c.4773+3A>G mutant minigene, the ABCA4 exon 33 wildtype minigene PCR product was used as a template for overlap PCR. For this, PCR was performed using with the primers ATCATGAATGTGAGCGGGgtGtgtaaacagactggagatttgagtag (SEQ ID NO: 460) and aaatctccagtctgtttacaCacCCCGCTCACATTCATGATC (SEQ ID NO: 461) using the Q5 High-Fidelity DNA Polymerase (NEB), according to manufacturer's protocol to create two fragments. Overlap PCR was performed to create the minigene insert using the Phusion High-Fidelity DNA Polymerase (NEB) under the following cycling conditions: (98° C. for 30 sec; 15 cycles of 98° C. for 10 sec, 60° C. for 30 sec and 72° C. for 120 sec; followed by 20 cycles of cycles of 98° C. for 10 sec, 72° C. for 150 sec; 72° C. for 120 sec). PCR fragments were cloned into CMV containing expression vector.

Example 1 the Splicing of ABCA4 is Disrupted in the c.768G>T Variant and can be Partially Rescued Through the Use of Antisense Oligonucleotides

To confirm partial intron 6 inclusion (i.e. exon 6 extension) in the chr1: 94564350:C:A [hg19/b37] (c.768G>T) variant, wild type and variant containing minigenes were constructed containing exons 5-7 and the corresponding introns, 5 and 6 (FIG. 1A). Minigenes were then transfected into HEK293T and ARPE19 cells to examine the effect of the c.768G>T variant on splicing. As seen in FIG. 1B, wildtype minigenes showed intron 6 exclusion, represented by the 337 bp band. C.768G>T mutants, however, showed partial intron 6 inclusion (i.e. exon 6 extension) indicating the chr1: 94564350:C:A [hg19/b37] mutation induces partial intron 6 inclusion.

To examine the ability of antisense oligonucleotides to promote intron 6 exclusion in the c.768G>T variant the minigenes above were co-transfected with antisense oligonucleotides having sequences set forth in SEQ ID Nos: 2-207 (see Tables 3 and 4). Antisense oligonucleotides were tiled along exon 6 and the surrounding introns. Antisense oligonucleotides were cotransfected with the mutant minigene containing the c.768G>T variant in ARPE19 (Table 3) and HEK293T (Table 4) cells. RT-PCR was conducted to analyze the effect on the splicing of the minigene. Samples were measured by capillary electrophoresis. These results were quantified and are set forth in Tables 3 and 4. Observing Table 3 and 4 it is clear that targeting the intronic regions surrounding exon 6 reduces intron 6 inclusion in c.768G>T variant minigenes (high percent spliced in/correctly (PSI) and change in PSI as compared to mutant PSI (dPSI)). These observations also suggest antisense oligonucleotides targeting certain regions or “hotspots” in intron 6 (positions 27362-27419 in SEQ ID NO: 1; chr1: 94564287-94564344), e.g., those complementary to a nucleobase sequence in SEQ ID Nos: 60-198 and 207, may be particularly useful in the treatment of retinal disease associated with partial intron 6 inclusion (i.e. exon 6 extension) (e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease caused by the c.768G>T mutation).

TABLE 3

Start

on

SEQ

Start

SEQ
Stop on

ID
DG

Chr1
End Chr1
ID
SEQ ID

NO:
ID
PSI
Sequence
[hg19/b37]
[hg19/b37]
NO: 1
NO: 1
length
dPSI

2
4128
0.02431697
ATACCT
94564626
94564645
27061
27080
20
0.00628733

TGTGTT

ACATGG

CG

3
4073
0.11405178
GGGAAT
94564622
94564638
27068
27084
17
0.09602214

ACCTTG

TGTTA

4
4141
0.15732851
AGAACC
94564615
94564635
27071
27091
21
0.13929887

TGGGAA

TACCTT

GTG

5
4114
0.01903612
CTAACC
94564606
94564624
27082
27100
19
0.00100648

CACAGA

ACCTGG

G

6
4129
0.00522592
CCACGT
94564599
94564618
27088
27107
20
−0.0128037

CCTAAC

CCACAG

AA

7
4130
0.02776844
GAAAGA
94564590
94564609
27097
27116
20
0.0097388

CACCCA

CGTCCT

AA

8
4095
0.02402144
TAGGAA
94564587
94564604
27102
27119
18
0.00599179

AGACAC

CCACGT

9
4074
0.02229321
GGTAGG
94564585
94564601
27105
27121
17
0.00426357

AAAGAC

ACCCA

10
4115
0.0201467
CCCTGT
94564579
94564597
27109
27127
19
0.00211706

GGTAGG

AAAGAC

A

11
4096
0.01513791
CTGCCC
94564576
94564593
27113
27130
18
−0.0028917

TGTGGT

AGGAAA

12
4075
0.01842179
AACTGC
94564574
94564590
27116
27132
17
0.00039215

CCTGTG

GTAGG

13
4076
0.01700658
GAAACT
94564572
94564588
27118
27134
17
-0.0010231

GCCCTG

TGGTA

14
4097
0.02153469
CTAGAA
94564569
94564586
27120
27137
18
0.00350505

ACTGCC

CTGTGG

15
4131
0.01829548
GGCAAC
94564562
94564581
27125
27144
20
0.00026584

ACTAGA

AACTGC

CC

16
4142
0.0197163
GGAGAA
94564554
94564574
27132
27152
21
0.00168666

GAGGCA

ACACTA

GAA

17
4098
0.01724193
CAGGGA
94564551
94564568
27138
27155
18
−0.0007877

GAAGAG

GCAACA

18
4077
0.02141061
ACTGCA
94564547
94564563
27143
27159
17
0.00338097

GGGAGA

AGAGG

19
4132
0.0033317
GAGCGA
94564541
94564560
27146
27165
20
−0.0146979

ACTGCA

GGGAGA

AG

20
4133
0.01705203
TCCATG
94564536
94564555
27151
27170
20
−0.0009776

AGCGAA

CTGCAG

GG

21
4134
0.01805005
GGGACT
94564531
94564550
27156
27175
20
2.0406E-05

CCATGA

GCGAAC

TG

22
4078
0.01914936
TCCGGG
94564528
94564544
27162
27178
17
0.00111972

ACTCCA

TGAGC

23
4143
0.01964017
AGCGCC
94564519
94564539
27167
27187
21
0.00161053

AGGTCC

GGGACT

CCA

24
4144
0.01859193
GTCCTTC
94564512
94564532
27174
27194
21
0.00056229

AGCGCC

AGGTCC

GG

25
4145
0.02145499
CAGGCG
94564504
94564524
27182
27202
21
0.00342535

ATGTCC

TTCAGC

GCC

26
4116
0.02012965
CCTCGC
94564496
94564514
27192
27210
19
0.00210001

TGCAGG

CGATGT

C

27
4099
0.02319291
GGAGGG
94564490
94564507
27199
27216
18
0.00516327

CCTCGC

TGCAGG

28
4100
0.03543829
GCTCCA
94564484
94564501
27205
27222
18
0.01740865

GGAGGG

CCTCGC

29
4079
0.02046444
GCGCTC
94564482
94564498
27208
27224
17
0.0024348

CAGGAG

GGCCT

30
4101
0.01793776
TGAAGC
94564478
94564495
27211
27228
18
−9.188E-05

GCTCCA

GGAGGG

31
4135
0.01365784
GAAGAT
94564470
94564489
27217
27236
20
−0.0043718

GATGAA

GCGCTC

CA

32
4117
0.01708284
TGGCTG
94564465
94564483
27223
27241
19
−0.0009468

AAGATG

ATGAAG

C

33
4118
0.02096426
TCTCTG
94564461
94564479
27227
27245
19
0.00293462

GCTGAA

GATGAT

G

34
4080
0.01739695
CGTCTCT
94564459
94564475
27231
27247
17
−0.0006327

GGCTGA

AGAT

35
4136
0.02244261
TTGCCC
94564451
94564470
27236
27255
20
0.00441297

CGCGTC

TCTGGC

TG

36
4102
0.02139803
CACCGT
94564443
94564460
27246
27263
18
0.00336839

CTTTGCC

CCGCG

37
4146
0.0177441
ATAGCG
94564437
94564457
27249
27269
21
−0.0002855

CACCGT

CTTTGCC

CC

38
4081
0.01717889
GGCATA
94564434
94564450
27256
27272
17
−0.0008508

GCGCAC

CGTCT

39
4103
0.01417047
CAGGGC
94564431
94564448
27258
27275
18
−0.0038592

ATAGCG

CACCGT

40
4119
0.01682693
GAGCAC
94564426
94564444
27262
27280
19
−0.0012027

AGGGCA

TAGCGC

A

41
4082
0.00630405
AGAGGG
94564421
94564437
27269
27285
17
−0.0117256

AGCACA

GGGCA

42
4120
0.00571713
TGGGAG
94564417
94564435
27271
27289
19
−0.0123125

AGGGAG

CACAGG

G

43
4147
0.00235213
TAGGGT
94564407
94564427
27279
27299
21
−0.0156775

GCCCTG

GGAGAG

GGA

44
4148
0.01718938
TATCCA
94564398
94564418
27288
27308
21
−0.0008403

CTGTAG

GGTGCC

CTG

45
4083
0.00387113
TCTTCTA
94564393
94564409
27297
27313
17
−0.0141585

TCCACT

GTAG

46
4084
0.00368482
AGTGTC
94564389
94564405
27301
27317
17
−0.0143448

TTCTATC

CACT

47
4104
0.00409522
CAGAGT
94564386
94564403
27303
27320
18
−0.0139344

GTCTTCT

ATCCA

48
4137
0.00445977
GTTGGC
94564377
94564396
27310
27329
20
−0.0135699

ATACAG

AGTGTC

TT

49
4121
0.00750785
CCACGT
94564373
94564391
27315
27333
19
−0.0105218

TGGCAT

ACAGAG

T

50
4105
0.00641796
AAGTCC
94564369
94564386
27320
27337
18
−0.0116117

ACGTTG

GCATAC

51
4106
0.00402175
AAGAAG
94564366
94564383
27323
27340
18
−0.0140079

TCCACG

TTGGCA

52
4122
0.00421162
GCTTGA
94564361
94564379
27327
27345
19
−0.013818

AGAAGT

CCACGT

T

53
4107
0.00378806
AAGAGC
94564357
94564374
27332
27349
18
−0.0142416

TTGAAG

AAGTCC

54
4108
0.00324747
CGGAAG
94564354
94564371
27335
27352
18
−0.0147822

AGCTTG

AAGAAG

55
4085
0.00334551
AACACG
94564350
94564366
27340
27356
17
−0.0146841

GAAGAG

CTTGA

56
4123
0.01473282
CTTACA
94564345
94564363
27343
27361
19
−0.0032968

ACACGG

AAGAGC

T

57
4109
0.02021009
CTCCCTT
94564341
94564358
27348
27365
18
0.00218045

ACAACA

CGGAA

58
4149
0.01481722
CCAAAC
94564333
94564353
27353
27373
21
−0.0032124

CCCTCC

CTTACA

ACA

59
4086
0.01443598
CAGCCA
94564330
94564346
27360
27376
17
−0.0035937

AACCCC

TCCCT

60
4087
0.02024966
AGCAGC
94564328
94564344
27362
27378
17
0.00222002

CAAACC

CCTCC

61
4088
0.0372636
CGAGCA
94564326
94564342
27364
27380
17
0.01923396

GCCAAA

CCCCT

62
4110
0.07618036
TGGCGA
94564323
94564340
27366
27383
18
0.05815072

GCAGCC

AAACCC

63
4138
0.17283501
TGCAAT
94564317
94564336
27370
27389
20
0.15480537

TGGCGA

GCAGCC

AA

64
4597
0.10930086
AATTGG
94564320
94564336
27370
27386
17
0.09127122

CGAGCA

GCCAA

65
4598
0.09078839
CAATTG
94564319
94564336
27370
27387
18
0.07275875

GCGAGC

AGCCAA

66
4599
0.16039823
GCAATT
94564318
94564336
27370
27388
19
0.14236859

GGCGAG

CAGCCA

A

67
4600
0.16117871
TTGCAA
94564316
94564336
27370
27390
21
0.14314907

TTGGCG

AGCAGC

CAA

68
4601
0.11209091
CAATTG
94564319
94564335
27371
27387
17
0.09406127

GCGAGC

AGCCA

69
4602
0.23648176
GCAATT
94564318
94564335
27371
27388
18
0.21845211

GGCGAG

CAGCCA

70
4603
0.20595156
TGCAAT
94564317
94564335
27371
27389
19
0.18792192

TGGCGA

GCAGCC

A

71
4604
0.17100969
TTGCAA
94564316
94564335
27371
27390
20
0.15298005

TTGGCG

AGCAGC

CA

72
4605
0.14927085
CTTGCA
94564315
94564335
27371
27391
21
0.13124121

ATTGGC

GAGCAG

CCA

73
4606
0.26777524
GCAATT
94564318
94564334
27372
27388
17
0.2497456

GGCGAG

CAGCC

74
4607
0.29621478
TGCAAT
94564317
94564334
27372
27389
18
0.27818514

TGGCGA

GCAGCC

75
4608
0.31043846
TTGCAA
94564316
94564334
27372
27390
19
0.29240882

TTGGCG

AGCAGC

C

76
4609
0.26478391
CTTGCA
94564315
94564334
27372
27391
20
0.24675427

ATTGGC

GAGCAG

CC

77
4610
0.25010219
CCTTGC
94564314
94564334
27372
27392
21
0.23207255

AATTGG

CGAGCA

GCC

78
4611
0.26743515
TGCAAT
94564317
94564333
27373
27389
17
0.24940551

TGGCGA

GCAGC

79
4612
0.20968878
TTGCAA
94564316
94564333
27373
27390
18
0.19165914

TTGGCG

AGCAGC

80
4613
0.24661075
CTTGCA
94564315
94564333
27373
27391
19
0.22858111

ATTGGC

GAGCAG

C

81
4614
0.23289843
CCTTGC
94564314
94564333
27373
27392
20
0.21486879

AATTGG

CGAGCA

GC

82
4615
0.29501713
ACCTTG
94564313
94564333
27373
27393
21
0.27698749

CAATTG

GCGAGC

AGC

83
4616
0.27962315
TTGCAA
94564316
94564332
27374
27390
17
0.26159351

TTGGCG

AGCAG

84
4617
0.22421363
CTTGCA
94564315
94564332
27374
27391
18
0.20618399

ATTGGC

GAGCAG

85
4618
0.26986428
CCTTGC
94564314
94564332
27374
27392
19
0.25183464

AATTGG

CGAGCA

G

86
4619
0.29570147
ACCTTG
94564313
94564332
27374
27393
20
0.27767183

CAATTG

GCGAGC

AG

87
4620
0.26279915
CACCTT
94564312
94564332
27374
27394
21
0.24476951

GCAATT

GGCGAG

CAG

88
4089
0.17943073
CTTGCA
94564315
94564331
27375
27391
17
0.16140109

ATTGGC

GAGCA

89
4621
0.26260696
CCTTGC
94564314
94564331
27375
27392
18
0.24457732

AATTGG

CGAGCA

90
4622
0.31982099
ACCTTG
94564313
94564331
27375
27393
19
0.30179135

CAATTG

GCGAGC

A

91
4623
0.2558288
CACCTT
94564312
94564331
27375
27394
20
0.23779916

GCAATT

GGCGAG

CA

92
4624
0.23800896
TCACCTT
94564311
94564331
27375
27395
21
0.21997932

GCAATT

GGCGAG

CA

93
4625
0.25760784
CCTTGC
94564314
94564330
27376
27392
17
0.2395782

AATTGG

CGAGC

94
4626
0.29734234
ACCTTG
94564313
94564330
27376
27393
18
0.2793127

CAATTG

GCGAGC

95
4627
0.26139422
CACCTT
94564312
94564330
27376
27394
19
0.24336458

GCAATT

GGCGAG

C

96
4628
0.18097064
TCACCTT
94564311
94564330
27376
27395
20
0.162941

GCAATT

GGCGAG

C

97
4629
0.27847245
ATCACC
94564310
94564330
27376
27396
21
0.26044281

TTGCAA

TTGGCG

AGC

98
4090
0.26236346
ACCTTG
94564313
94564329
27377
27393
17
0.24433382

CAATTG

GCGAG

99
4630
0.31917424
CACCTT
94564312
94564329
27377
27394
18
0.3011446

GCAATT

GGCGAG

100
4631
0.76759466
TCACCTT
94564311
94564329
27377
27395
19
0.74956501

GCAATT

GGCGAG

101
4632
0.81860163
ATCACC
94564310
94564329
27377
27396
20
0.80057199

TTGCAA

TTGGCG

AG

102
4633
0.89239232
AATCAC
94564309
94564329
27377
27397
21
0.87436268

CTTGCA

ATTGGC

GAG

103
4634
0.84651316
CACCTT
94564312
94564328
27378
27394
17
0.82848352

GCAATT

GGCGA

104
4635
0.8390091
TCACCTT
94564311
94564328
27378
27395
18
0.82097946

GCAATT

GGCGA

105
4636
0.87739626
ATCACC
94564310
94564328
27378
27396
19
0.85936662

TTGCAA

TTGGCG

A

106
4637
0.87346315
AATCAC
94564309
94564328
27378
27397
20
0.85543351

CTTGCA

ATTGGC

GA

107
4638
0.90143132
GAATCA
94564308
94564328
27378
27398
21
0.88340168

CCTTGC

AATTGG

CGA

108
4124
0.44721392
AATCAC
94564309
94564327
27379
27397
19
0.42918427

CTTGCA

ATTGGC

G

109
4639
0.79968337
TCACCTT
94564311
94564327
27379
27395
17
0.78165373

GCAATT

GGCG

110
4640
0.80763727
ATCACC
94564310
94564327
27379
27396
18
0.78960763

TTGCAA

TTGGCG

111
4641
0.87411122
GAATCA
94564308
94564327
27379
27398
20
0.85608158

CCTTGC

AATTGG

CG

112
4642
0.80500233
GGAATC
94564307
94564327
27379
27399
21
0.78697268

ACCTTG

CAATTG

GCG

113
4643
0.88269558
ATCACC
94564310
94564326
27380
27396
17
0.86466594

TTGCAA

TTGGC

114
4644
0.87044459
AATCAC
94564309
94564326
27380
27397
18
0.85241495

CTTGCA

ATTGGC

115
4645
0.73199713
GAATCA
94564308
94564326
27380
27398
19
0.71396749

CCTTGC

AATTGG

C

116
4646
0.68348265
GGAATC
94564307
94564326
27380
27399
20
0.66545301

ACCTTG

CAATTG

GC

117
4647
0.82294769
AGGAAT
94564306
94564326
27380
27400
21
0.80491805

CACCTT

GCAATT

GGC

118
4648
0.84365284
AATCAC
94564309
94564325
27381
27397
17
0.8256232

CTTGCA

ATTGG

119
4649
0.78266251
GAATCA
94564308
94564325
27381
27398
18
0.76463287

CCTTGC

AATTGG

120
4650
0.67659075
GGAATC
94564307
94564325
27381
27399
19
0.65856111

ACCTTG

CAATTG

G

121
4651
0.67533495
AGGAAT
94564306
94564325
27381
27400
20
0.65730531

CACCTT

GCAATT

GG

122
4652
0.70200627
CAGGAA
94564305
94564325
27381
27401
21
0.68397663

TCACCTT

GCAATT

GG

123
4653
0.7782903
GAATCA
94564308
94564324
27382
27398
17
0.76026066

CCTTGC

AATTG

124
4654
0.78731012
GGAATC
94564307
94564324
27382
27399
18
0.76928048

ACCTTG

CAATTG

125
4655
0.78132802
AGGAAT
94564306
94564324
27382
27400
19
0.76329838

CACCTT

GCAATT

G

126
4656
0.3388734
CAGGAA
94564305
94564324
27382
27401
20
0.32084376

TCACCTT

GCAATT

G

127
4657
0.25626616
CCAGGA
94564304
94564324
27382
27402
21
0.23823652

ATCACC

TTGCAA

TTG

128
4091
0.60563805
GGAATC
94564307
94564323
27383
27399
17
0.58760841

ACCTTG

CAATT

129
4658
0.88473952
AGGAAT
94564306
94564323
27383
27400
18
0.86670988

CACCTT

GCAATT

130
4659
0.88226254
CAGGAA
94564305
94564323
27383
27401
19
0.8642329

TCACCTT

GCAATT

131
4660
0.85095103
CCAGGA
94564304
94564323
27383
27402
20
0.83292139

ATCACC

TTGCAA

TT

132
4661
0.83219493
CCCAGG
94564303
94564323
27383
27403
21
0.81416529

AATCAC

CTTGCA

ATT

133
4662
0.88970276
AGGAAT
94564306
94564322
27384
27400
17
0.87167312

CACCTT

GCAAT

134
4663
0.87956906
CAGGAA
94564305
94564322
27384
27401
18
0.86153942

TCACCTT

GCAAT

135
4664
0.81659418
CCAGGA
94564304
94564322
27384
27402
19
0.79856454

ATCACC

TTGCAA

T

136
4665
0.85952746
CCCAGG
94564303
94564322
27384
27403
20
0.84149781

AATCAC

CTTGCA

AT

137
4666
0.69318589
CCCCAG
94564302
94564322
27384
27404
21
0.67515625

GAATCA

CCTTGC

AAT

138
4125
0.29460087
CCCAGG
94564303
94564321
27385
27403
19
0.27657123

AATCAC

CTTGCA

A

139
4667
0.36645782
CAGGAA
94564305
94564321
27385
27401
17
0.34842818

TCACCTT

GCAA

140
4668
0.83743902
CCAGGA
94564304
94564321
27385
27402
18
0.81940938

ATCACC

TTGCAA

141
4669
0.29444226
CCCCAG
94564302
94564321
27385
27404
20
0.27641262

GAATCA

CCTTGC

AA

142
4670
0.23897641
ACCCCA
94564301
94564321
27385
27405
21
0.22094677

GGAATC

ACCTTG

CAA

143
4671
0.22377272
CCAGGA
94564304
94564320
27386
27402
17
0.20574308

ATCACC

TTGCA

144
4672
0.27703321
CCCAGG
94564303
94564320
27386
27403
18
0.25900356

AATCAC

CTTGCA

145
4673
0.22181682
CCCCAG
94564302
94564320
27386
27404
19
0.20378717

GAATCA

CCTTGC

A

146
4674
0.73692266
ACCCCA
94564301
94564320
27386
27405
20
0.71889302

GGAATC

ACCTTG

CA

147
4675
0.16174868
TACCCC
94564300
94564320
27386
27406
21
0.14371904

AGGAAT

CACCTT

GCA

148
4676
0.2452912
CCCAGG
94564303
94564319
27387
27403
17
0.22726156

AATCAC

CTTGC

149
4677
0.23007754
CCCCAG
94564302
94564319
27387
27404
18
0.2120479

GAATCA

CCTTGC

150
4678
0.20199157
ACCCCA
94564301
94564319
27387
27405
19
0.18396193

GGAATC

ACCTTG

C

151
4679
0.22664884
TACCCC
94564300
94564319
27387
27406
20
0.2086192

AGGAAT

CACCTT

GC

152
4680
0.24065276
CTACCC
94564299
94564319
27387
27407
21
0.22262312

CAGGAA

TCACCTT

GC

153
4681
0.31432345
CCCCAG
94564302
94564318
27388
27404
17
0.29629381

GAATCA

CCTTG

154
4682
0.27533803
ACCCCA
94564301
94564318
27388
27405
18
0.25730839

GGAATC

ACCTTG

155
4683
0.35359545
TACCCC
94564300
94564318
27388
27406
19
0.33556581

AGGAAT

CACCTT

G

156
4684
0.29786175
CTACCC
94564299
94564318
27388
27407
20
0.27983211

CAGGAA

TCACCTT

G

157
4685
0.84163308
GCTACC
94564298
94564318
27388
27408
21
0.82360344

CCAGGA

ATCACC

TTG

158
4686
0.28817154
ACCCCA
94564301
94564317
27389
27405
17
0.2701419

GGAATC

ACCTT

159
4687
0.25414838
TACCCC
94564300
94564317
27389
27406
18
0.23611874

AGGAAT

CACCTT

160
4689
0.87305965
GCTACC
94564298
94564317
27389
27408
20
0.85503

CCAGGA

ATCACC

TT

161
4690
0.82648716
TGCTAC
94564297
94564317
27389
27409
21
0.80845752

CCCAGG

AATCAC

CTT

162
4111
0.14924213
CTACCC
94564299
94564316
27390
27407
18
0.13121249

CAGGAA

TCACCT

163
4691
0.19736827
TACCCC
94564300
94564316
27390
27406
17
0.17933863

AGGAAT

CACCT

164
4692
0.3686295
GCTACC
94564298
94564316
27390
27408
19
0.35059986

CCAGGA

ATCACC

T

165
4693
0.79136767
TGCTAC
94564297
94564316
27390
27409
20
0.77333803

CCCAGG

AATCAC

CT

166
4694
0.82715435
CTGCTA
94564296
94564316
27390
27410
21
0.80912471

CCCCAG

GAATCA

CCT

167
4695
0.19457674
CTACCC
94564299
94564315
27391
27407
17
0.1765471

CAGGAA

TCACC

168
4696
0.81253152
GCTACC
94564298
94564315
27391
27408
18
0.79450188

CCAGGA

ATCACC

169
4697
0.77605781
TGCTAC
94564297
94564315
27391
27409
19
0.75802817

CCCAGG

AATCAC

C

170
4698
0.8033507
CTGCTA
94564296
94564315
27391
27410
20
0.78532106

CCCCAG

GAATCA

CC

171
4699
0.76580739
TCTGCT
94564295
94564315
27391
27411
21
0.74777775

ACCCCA

GGAATC

ACC

172
4700
0.20463344
GCTACC
94564298
94564314
27392
27408
17
0.1866038

CCAGGA

ATCAC

173
4701
0.19263715
TGCTAC
94564297
94564314
27392
27409
18
0.17460751

CCCAGG

AATCAC

174
4702
0.25031864
CTGCTA
94564296
94564314
27392
27410
19
0.232289

CCCCAG

GAATCA

C

175
4703
0.22951121
TCTGCT
94564295
94564314
27392
27411
20
0.21148157

ACCCCA

GGAATC

AC

176
4704
0.1954459
CTCTGCT
94564294
94564314
27392
27412
21
0.17741626

ACCCCA

GGAATC

AC

177
4092
0.13500456
TGCTAC
94564297
94564313
27393
27409
17
0.11697492

CCCAGG

AATCA

178
4705
0.16096575
CTGCTA
94564296
94564313
27393
27410
18
0.1429361

CCCCAG

GAATCA

179
4706
0.158593
TCTGCT
94564295
94564313
27393
27411
19
0.14056336

ACCCCA

GGAATC

A

180
4707
0.13411114
CTCTGCT
94564294
94564313
27393
27412
20
0.1160815

ACCCCA

GGAATC

A

181
4708
0.20781816
GCTCTG
94564293
94564313
27393
27413
21
0.18978852

CTACCC

CAGGAA

TCA

182
4709
0.0784893
CTGCTA
94564296
94564312
27394
27410
17
0.06045966

CCCCAG

GAATC

183
4710
0.0891908
TCTGCT
94564295
94564312
27394
27411
18
0.07116116

ACCCCA

GGAATC

184
4711
0.05290537
CTCTGCT
94564294
94564312
27394
27412
19
0.03487573

ACCCCA

GGAATC

185
4712
0.15401065
GCTCTG
94564293
94564312
27394
27413
20
0.13598101

CTACCC

CAGGAA

TC

186
4713
0.09604376
GGCTCT
94564292
94564312
27394
27414
21
0.07801412

GCTACC

CCAGGA

ATC

187
4714
0.13741142
TCTGCT
94564295
94564311
27395
27411
17
0.11938178

ACCCCA

GGAAT

188
4715
0.1047728
CTCTGCT
94564294
94564311
27395
27412
18
0.08674316

ACCCCA

GGAAT

189
4716
0.23153099
GCTCTG
94564293
94564311
27395
27413
19
0.21350135

CTACCC

CAGGAA

T

190
4717
0.27661374
GGCTCT
94564292
94564311
27395
27414
20
0.2585841

GCTACC

CCAGGA

AT

191
4139
0.15666069
AGGCTC
94564291
94564310
27396
27415
20
0.13863105

TGCTAC

CCCAGG

AA

192
4718
0.13584046
CTCTGCT
94564294
94564310
27396
27412
17
0.11781081

ACCCCA

GGAA

193
4719
0.48672796
GCTCTG
94564293
94564310
27396
27413
18
0.46869832

CTACCC

CAGGAA

194
4720
0.37749689
GGCTCT
94564292
94564310
27396
27414
19
0.35946725

GCTACC

CCAGGA

A

195
4721
0.50288272
GCTCTG
94564293
94564309
27397
27413
17
0.48485308

CTACCC

CAGGA

196
4722
0.43230889
GGCTCT
94564292
94564309
27397
27414
18
0.41427924

GCTACC

CCAGGA

197
4723
0.19564733
GGCTCT
94564292
94564308
27398
27414
17
0.17761769

GCTACC

CCAGG

198
4126
0.04292774
CGTGAG
94564287
94564305
27401
27419
19
0.02489809

GCTCTG

CTACCC

C

199
4112
0.00596452
AATTCG
94564283
94564300
27406
27423
18
−0.0120651

TGAGGC

TCTGCT

200
4127
0.01072732
GGTCAA
94564279
94564297
27409
27427
19
−0.0073023

TTCGTG

AGGCTC

T

201
4093
0.01129358
CAAGGT
94564276
94564292
27414
27430
17
−0.0067361

CAATTC

GTGAG

202
4150
0.00813254
CCTCCC
94564270
94564290
27416
27436
21
−0.0098971

CAAGGT

CAATTC

GTG

203
4151
0.01433631
GGCTCA
94564261
94564281
27425
27445
21
−0.0036933

CGCCCT

CCCCAA

GGT

204
4094
0.02260101
CAGGCT
94564259
94564275
27431
27447
17
0.00457137

CACGCC

CTCCC

205
4113
0.01461124
CACCAG
94564256
94564273
27433
27450
18
−0.0034184

GCTCAC

GCCCTC

206
4140
0.02414921
CCAGAA
94564250
94564269
27437
27456
20
0.00611957

CACCAG

GCTCAC

GC

TABLE 4

Start
Stop

on
on

SEQ

Start

SEQ
SEQ

ID
DG

Chr1
End Chrl
ID
ID

NO:
ID
PSI
Sequence
[hg19/b37]
[hg19/b37]
NO: 1
NO: 1
length
dPSI

2
4128
0.00852007
ATACCT
94564626
94564645
27061
27080
20
-0.0095096

TGTGTT

ACATGG

CG

3
4073
0.02900731
GGGAAT
94564622
94564638
27068
27084
17
0.01097767

ACCTTG

TGTTA

4
4141
0.07391646
AGAACC
94564615
94564635
27071
27091
21
0.05588682

TGGGAA

TACCTT

GTG

5
4114
0.01283934
CTAACC
94564606
94564624
27082
27100
19
-0.0051903

CACAGA

ACCTGG

G

6
4129
0.01265609
CCACGT
94564599
94564618
27088
27107
20
-0.0053736

CCTAAC

CCACAG

AA

7
4130
0.01522623
GAAAGA
94564590
94564609
27097
27116
20
-0.0028034

CACCCA

CGTCCT

AA

8
4095
0.00990721
TAGGAA
94564587
94564604
27102
27119
18
-0.0081224

AGACAC

CCACGT

9
4074
0.02108604
GGTAGG
94564585
94564601
27105
27121
17
0.0030564

AAAGAC

ACCCA

10
4115
0.02587134
CCCTGT
94564579
94564597
27109
27127
19
0.0078417

GGTAGG

AAAGAC

A

11
4096
0.01078192
CTGCCC
94564576
94564593
27113
27130
18
-0.0072477

TGTGGT

AGGAAA

12
4075
0.01630967
AACTGC
94564574
94564590
27116
27132
17
-0.00172

CCTGTG

GTAGG

13
4076
0.01604054
GAAACT
94564572
94564588
27118
27134
17
-0.0019891

GCCCTG

TGGTA

14
4097
0.00826475
CTAGAA
94564569
94564586
27120
27137
18
-0.0097649

ACTGCC

CTGTGG

15
4131
0.01220225
GGCAAC
94564562
94564581
27125
27144
20
-0.0058274

ACTAGA

AACTGC

CC

16
4142
0.01869085
GGAGAA
94564554
94564574
27132
27152
21
0.00066121

GAGGCA

ACACTA

GAA

17
4098
0.0129716
CAGGGA
94564551
94564568
27138
27155
18
-0.005058

GAAGAG

GCAACA

18
4077
0.01036923
ACTGCA
94564547
94564563
27143
27159
17
-0.0076604

GGGAGA

AGAGG

19
4132
0.01542554
GAGCGA
94564541
94564560
27146
27165
20
-0.0026041

ACTGCA

GGGAGA

AG

20
4133
0.0144133
TCCATG
94564536
94564555
27151
27170
20
-0.0036163

AGCGAA

CTGCAG

GG

21
4134
0.01992633
GGGACT
94564531
94564550
27156
27175
20
0.00189669

CCATGA

GCGAAC

TG

22
4078
0.01516713
TCCGGG
94564528
94564544
27162
27178
17
-0.0028625

ACTCCA

TGAGC

23
4143
0.01312488
AGCGCC
94564519
94564539
27167
27187
21
-0.0049048

AGGTCC

GGGACT

CCA

24
4144
0.01627758
GTCCTTC
94564512
94564532
27174
27194
21
-0.0017521

AGCGCC

AGGTCC

GG

25
4145
0.01750626
CAGGCG
94564504
94564524
27182
27202
21
-0.0005234

ATGTCC

TTCAGC

GCC

26
4116
0.01152383
CCTCGC
94564496
94564514
27192
27210
19
-0.0065058

TGCAGG

CGATGT

C

27
4099
0.03132164
GGAGGG
94564490
94564507
27199
27216
18
0.013292

CCTCGC

TGCAGG

28
4100
0.04411962
GCTCCA
94564484
94564501
27205
27222
18
0.02608998

GGAGGG

CCTCGC

29
4079
0.02378016
GCGCTC
94564482
94564498
27208
27224
17
0.00575051

CAGGAG

GGCCT

30
4101
0.01407391
TGAAGC
94564478
94564495
27211
27228
18
-0.0039557

GCTCCA

GGAGGG

31
4135
0.0122176
GAAGAT
94564470
94564489
27217
27236
20
-0.005812

GATGAA

GCGCTC

CA

32
4117
0.00913255
TGGCTG
94564465
94564483
27223
27241
19
-0.0088971

AAGATG

ATGAAG

C

33
4118
0.01154571
TCTCTG
94564461
94564479
27227
27245
19
-0.0064839

GCTGAA

GATGAT

G

34
4080
0.01103206
CGTCTCT
94564459
94564475
27231
27247
17
-0.0069976

GGCTGA

AGAT

35
4136
0.01414565
TTGCCC
94564451
94564470
27236
27255
20
-0.003884

CGCGTC

TCTGGC

TG

36
4102
0.01511915
CACCGT
94564443
94564460
27246
27263
18
-0.0029105

CTTTGCC

CCGCG

37
4146
0.01070549
ATAGCG
94564437
94564457
27249
27269
21
-0.0073241

CACCGT

CTTTGCC

CC

38
4081
0.01051709
GGCATA
94564434
94564450
27256
27272
17
-0.0075125

GCGCAC

CGTCT

39
4103
0.01277919
CAGGGC
94564431
94564448
27258
27275
18
-0.0052504

ATAGCG

CACCGT

40
4119
0.01240376
GAGCAC
94564426
94564444
27262
27280
19
-0.0056259

AGGGCA

TAGCGC

A

41
4082
0.01090273
AGAGGG
94564421
94564437
27269
27285
17
-0.0071269

AGCACA

GGGCA

42
4120
0.01957139
TGGGAG
94564417
94564435
27271
27289
19
0.00154175

AGGGAG

CACAGG

G

43
4147
0.00065793
TAGGGT
94564407
94564427
27279
27299
21
-0.0173717

GCCCTG

GGAGAG

GGA

44
4148
0.00875718
TATCCA
94564398
94564418
27288
27308
21
-0.0092725

CTGTAG

GGTGCC

CTG

45
4083
0.01195793
TCTTCTA
94564393
94564409
27297
27313
17
-0.0060717

TCCACT

GTAG

46
4084
0.00608376
AGTGTC
94564389
94564405
27301
27317
17
-0.0119459

TTCTATC

CACT

47
4104
0.00557296
CAGAGT
94564386
94564403
27303
27320
18
-0.0124567

GTCTTCT

ATCCA

48
4137
0.01727846
GTTGGC
94564377
94564396
27310
27329
20
-0.0007512

ATACAG

AGTGTC

TT

49
4121
0.00523364
CCACGT
94564373
94564391
27315
27333
19
-0.012796

TGGCAT

ACAGAG

T

50
4105
0.0132362
AAGTCC
94564369
94564386
27320
27337
18
-0.0047934

ACGTTG

GCATAC

51
4106
0.01811265
AAGAAG
94564366
94564383
27323
27340
18
8.3006E-05

TCCACG

TTGGCA

52
4122
0.00735466
GCTTGA
94564361
94564379
27327
27345
19
-0.010675

AGAAGT

CCACGT

T

53
4107
0.00854169
AAGAGC
94564357
94564374
27332
27349
18
-0.009488

TTGAAG

AAGTCC

54
4108
0.00238904
CGGAAG
94564354
94564371
27335
27352
18
-0.0156406

AGCTTG

AAGAAG

55
4085
0.00493693
AACACG
94564350
94564366
27340
27356
17
-0.0130927

GAAGAG

CTTGA

56
4123
0.00374432
CTTACA
94564345
94564363
27343
27361
19
-0.0142853

ACACGG

AAGAGC

T

57
4109
0.01006963
CTCCCTT
94564341
94564358
27348
27365
18
-0.00796

ACAACA

CGGAA

58
4149
0.01178247
CCAAAC
94564333
94564353
27353
27373
21
-0.0062472

CCCTCC

CTTACA

ACA

59
4086
0.00939203
CAGCCA
94564330
94564346
27360
27376
17
-0.0086376

AACCCC

TCCCT

60
4087
0.03079641
AGCAGC
94564328
94564344
27362
27378
17
0.01276677

CAAACC

CCTCC

61
4088
0.29179785
CGAGCA
94564326
94564342
27364
27380
17
0.27376821

GCCAAA

CCCCT

62
4110
0.08947937
TGGCGA
94564323
94564340
27366
27383
18
0.07144973

GCAGCC

AAACCC

63
4138
0.22120365
TGCAAT
94564317
94564336
27370
27389
20
0.20317401

TGGCGA

GCAGCC

AA

64
4597
0.47513581
AATTGG
94564320
94564336
27370
27386
17
0.45710617

CGAGCA

GCCAA

65
4598
0.72634299
CAATTG
94564319
94564336
27370
27387
18
0.70831335

GCGAGC

AGCCAA

66
4599
0.51076267
GCAATT
94564318
94564336
27370
27388
19
0.49273303

GGCGAG

CAGCCA

A

67
4600
0.23376829
TTGCAA
94564316
94564336
27370
27390
21
0.21573864

TTGGCG

AGCAGC

CAA

68
4601
0.74320192
CAATTG
94564319
94564335
27371
27387
17
0.72517227

GCGAGC

AGCCA

69
4602
0.59473771
GCAATT
94564318
94564335
27371
27388
18
0.57670806

GGCGAG

CAGCCA

70
4603
0.66762071
TGCAAT
94564317
94564335
27371
27389
19
0.64959107

TGGCGA

GCAGCC

A

71
4604
0.58471501
TTGCAA
94564316
94564335
27371
27390
20
0.56668537

TTGGCG

AGCAGC

CA

72
4605
0.65609249
CTTGCA
94564315
94564335
27371
27391
21
0.63806285

ATTGGC

GAGCAG

CCA

73
4606
0.72313482
GCAATT
94564318
94564334
27372
27388
17
0.70510518

GGCGAG

CAGCC

74
4607
0.8716546
TGCAAT
94564317
94564334
27372
27389
18
0.85362496

TGGCGA

GCAGCC

75
4608
0.74564326
TTGCAA
94564316
94564334
27372
27390
19
0.72761362

TTGGCG

AGCAGC

C

76
4609
0.78299129
CTTGCA
94564315
94564334
27372
27391
20
0.76496165

ATTGGC

GAGCAG

CC

77
4610
0.67006409
CCTTGC
94564314
94564334
27372
27392
21
0.65203445

AATTGG

CGAGCA

GCC

78
4611
0.85497825
TGCAAT
94564317
94564333
27373
27389
17
0.83694861

TGGCGA

GCAGC

79
4612
0.52063801
TTGCAA
94564316
94564333
27373
27390
18
0.50260837

TTGGCG

AGCAGC

80
4613
0.68203054
CTTGCA
94564315
94564333
27373
27391
19
0.6640009

ATTGGC

GAGCAG

C

81
4614
0.37065258
CCTTGC
94564314
94564333
27373
27392
20
0.35262294

AATTGG

CGAGCA

GC

82
4615
0.4217697
ACCTTG
94564313
94564333
27373
27393
21
0.40374006

CAATTG

GCGAGC

AGC

83
4616
0.71775973
TTGCAA
94564316
94564332
27374
27390
17
0.69973009

TTGGCG

AGCAG

84
4617
0.7403724
CTTGCA
94564315
94564332
27374
27391
18
0.72234275

ATTGGC

GAGCAG

85
4618
0.55691816
CCTTGC
94564314
94564332
27374
27392
19
0.53888852

AATTGG

CGAGCA

G

86
4619
0.81497515
ACCTTG
94564313
94564332
27374
27393
20
0.79694551

CAATTG

GCGAGC

AG

87
4620
0.72321098
CACCTT
94564312
94564332
27374
27394
21
0.70518134

GCAATT

GGCGAG

CAG

88
4089
0.82127394
CTTGCA
94564315
94564331
27375
27391
17
0.8032443

ATTGGC

GAGCA

89
4621
0.88664722
CCTTGC
94564314
94564331
27375
27392
18
0.86861758

AATTGG

CGAGCA

90
4622
0.87451707
ACCTTG
94564313
94564331
27375
27393
19
0.85648742

CAATTG

GCGAGC

A

91
4623
0.89267292
CACCTT
94564312
94564331
27375
27394
20
0.87464328

GCAATT

GGCGAG

CA

92
4624
0.56133913
TCACCTT
94564311
94564331
27375
27395
21
0.54330949

GCAATT

GGCGAG

CA

93
4625
0.73532055
CCTTGC
94564314
94564330
27376
27392
17
0.71729091

AATTGG

CGAGC

94
4626
0.82730273
ACCTTG
94564313
94564330
27376
27393
18
0.80927309

CAATTG

GCGAGC

95
4627
0.8159207
CACCTT
94564312
94564330
27376
27394
19
0.79789106

GCAATT

GGCGAG

C

96
4628
0.59808349
TCACCTT
94564311
94564330
27376
27395
20
0.58005385

GCAATT

GGCGAG

C

97
4629
0.67216645
ATCACC
94564310
94564330
27376
27396
21
0.65413681

TTGCAA

TTGGCG

AGC

98
4090
0.88361284
ACCTTG
94564313
94564329
27377
27393
17
0.8655832

CAATTG

GCGAG

99
4630
0.86571736
CACCTT
94564312
94564329
27377
27394
18
0.84768772

GCAATT

GGCGAG

100
4631
0.92856185
TCACCTT
94564311
94564329
27377
27395
19
0.91053221

GCAATT

GGCGAG

101
4632
0.88361444
ATCACC
94564310
94564329
27377
27396
20
0.8655848

TTGCAA

TTGGCG

AG

102
4633
0.92078171
AATCAC
94564309
94564329
27377
27397
21
0.90275207

CTTGCA

ATTGGC

GAG

103
4634
0.92540904
CACCTT
94564312
94564328
27378
27394
17
0.9073794

GCAATT

GGCGA

104
4635
0.8837001
TCACCTT
94564311
94564328
27378
27395
18
0.86567046

GCAATT

GGCGA

105
4636
0.84273478
ATCACC
94564310
94564328
27378
27396
19
0.82470514

TTGCAA

TTGGCG

A

106
4637
0.90290584
AATCAC
94564309
94564328
27378
27397
20
0.8848762

CTTGCA

ATTGGC

GA

107
4638
0.77352068
GAATCA
94564308
94564328
27378
27398
21
0.75549104

CCTTGC

AATTGG

CGA

108
4124
0.87866651
AATCAC
94564309
94564327
27379
27397
19
0.86063687

CTTGCA

ATTGGC

G

109
4639
0.91849987
TCACCTT
94564311
94564327
27379
27395
17
0.90047023

GCAATT

GGCG

110
4640
0.79921991
ATCACC
94564310
94564327
27379
27396
18
0.78119027

TTGCAA

TTGGCG

111
4641
0.84375916
GAATCA
94564308
94564327
27379
27398
20
0.82572952

CCTTGC

AATTGG

CG

112
4642
0.89609416
GGAATC
94564307
94564327
27379
27399
21
0.87806452

ACCTTG

CAATTG

GCG

113
4643
0.9454494
ATCACC
94564310
94564326
27380
27396
17
0.92741976

TTGCAA

TTGGC

114
4644
0.92651139
AATCAC
94564309
94564326
27380
27397
18
0.90848175

CTTGCA

ATTGGC

115
4645
0.85076613
GAATCA
94564308
94564326
27380
27398
19
0.83273649

CCTTGC

AATTGG

C

116
4646
0.8129502
GGAATC
94564307
94564326
27380
27399
20
0.79492056

ACCTTG

CAATTG

GC

117
4647
0.79016891
AGGAAT
94564306
94564326
27380
27400
21
0.77213927

CACCTT

GCAATT

GGC

118
4648
0.90098533
AATCAC
94564309
94564325
27381
27397
17
0.88295569

CTTGCA

ATTGG

119
4649
0.72815081
GAATCA
94564308
94564325
27381
27398
18
0.71012116

CCTTGC

AATTGG

120
4650
0.64728201
GGAATC
94564307
94564325
27381
27399
19
0.62925237

ACCTTG

CAATTG

G

121
4651
0.76330538
AGGAAT
94564306
94564325
27381
27400
20
0.74527574

CACCTT

GCAATT

GG

122
4652
0.62727959
CAGGAA
94564305
94564325
27381
27401
21
0.60924995

TCACCTT

GCAATT

GG

123
4653
0.78546741
GAATCA
94564308
94564324
27382
27398
17
0.76743777

CCTTGC

AATTG

124
4654
0.8267452
GGAATC
94564307
94564324
27382
27399
18
0.80871556

ACCTTG

CAATTG

125
4655
0.82641003
AGGAAT
94564306
94564324
27382
27400
19
0.80838039

CACCTT

GCAATT

G

126
4656
0.7584858
CAGGAA
94564305
94564324
27382
27401
20
0.74045616

TCACCTT

GCAATT

G

127
4657
0.70433919
CCAGGA
94564304
94564324
27382
27402
21
0.68630955

ATCACC

TTGCAA

TTG

128
4091
0.96455353
GGAATC
94564307
94564323
27383
27399
17
0.94652389

ACCTTG

CAATT

129
4658
0.89000659
AGGAAT
94564306
94564323
27383
27400
18
0.87197695

CACCTT

GCAATT

130
4659
0.74886526
CAGGAA
94564305
94564323
27383
27401
19
0.73083562

TCACCTT

GCAATT

131
4660
0.8928542
CCAGGA
94564304
94564323
27383
27402
20
0.87482456

ATCACC

TTGCAA

TT

132
4661
0.8040571
CCCAGG
94564303
94564323
27383
27403
21
0.78602745

AATCAC

CTTGCA

ATT

133
4662
0.88681006
AGGAAT
94564306
94564322
27384
27400
17
0.86878042

CACCTT

GCAAT

134
4663
0.80587159
CAGGAA
94564305
94564322
27384
27401
18
0.78784195

TCACCTT

GCAAT

135
4664
0.7487059
CCAGGA
94564304
94564322
27384
27402
19
0.73067626

ATCACC

TTGCAA

T

136
4665
0.85609438
CCCAGG
94564303
94564322
27384
27403
20
0.83806474

AATCAC

CTTGCA

AT

137
4666
0.64796081
CCCCAG
94564302
94564322
27384
27404
21
0.62993117

GAATCA

CCTTGC

AAT

138
4125
0.91268401
CCCAGG
94564303
94564321
27385
27403
19
0.89465437

AATCAC

CTTGCA

A

139
4667
0.82019394
CAGGAA
94564305
94564321
27385
27401
17
0.8021643

TCACCTT

GCAA

140
4668
0.78970497
CCAGGA
94564304
94564321
27385
27402
18
0.77167533

ATCACC

TTGCAA

141
4669
0.80707813
CCCCAG
94564302
94564321
27385
27404
20
0.78904849

GAATCA

CCTTGC

AA

142
4670
0.61545569
ACCCCA
94564301
94564321
27385
27405
21
0.59742605

GGAATC

ACCTTG

CAA

143
4671
0.80883562
CCAGGA
94564304
94564320
27386
27402
17
0.79080598

ATCACC

TTGCA

144
4672
0.83456855
CCCAGG
94564303
94564320
27386
27403
18
0.81653891

AATCAC

CTTGCA

145
4673
0.69793978
CCCCAG
94564302
94564320
27386
27404
19
0.67991014

GAATCA

CCTTGC

A

146
4674
0.63673921
ACCCCA
94564301
94564320
27386
27405
20
0.61870957

GGAATC

ACCTTG

CA

147
4675
0.64104813
TACCCC
94564300
94564320
27386
27406
21
0.62301849

AGGAAT

CACCTT

GCA

148
4676
0.87014332
CCCAGG
94564303
94564319
27387
27403
17
0.85211368

AATCAC

CTTGC

149
4677
0.77803887
CCCCAG
94564302
94564319
27387
27404
18
0.76000923

GAATCA

CCTTGC

150
4678
0.84159721
ACCCCA
94564301
94564319
27387
27405
19
0.82356757

GGAATC

ACCTTG

C

151
4679
0.81830134
TACCCC
94564300
94564319
27387
27406
20
0.8002717

AGGAAT

CACCTT

GC

152
4680
0.87797865
CTACCC
94564299
94564319
27387
27407
21
0.85994901

CAGGAA

TCACCTT

GC

153
4681
0.86670248
CCCCAG
94564302
94564318
27388
27404
17
0.84867284

GAATCA

CCTTG

154
4682
0.87625691
ACCCCA
94564301
94564318
27388
27405
18
0.85822727

GGAATC

ACCTTG

155
4683
0.84275371
TACCCC
94564300
94564318
27388
27406
19
0.82472406

AGGAAT

CACCTT

G

156
4684
0.84487036
CTACCC
94564299
94564318
27388
27407
20
0.82684072

CAGGAA

TCACCTT

G

157
4685
0.70957679
GCTACC
94564298
94564318
27388
27408
21
0.69154715

CCAGGA

ATCACC

TTG

158
4686
0.84873383
ACCCCA
94564301
94564317
27389
27405
17
0.83070419

GGAATC

ACCTT

159
4687
0.81850076
TACCCC
94564300
94564317
27389
27406
18
0.80047112

AGGAAT

CACCTT

207
4688
0.85763794
CTACCC
94564299
94564317
27389
27407
19
0.8396083

CAGGAA

TCACCTT

160
4689
0.77144079
GCTACC
94564298
94564317
27389
27408
20
0.75341115

CCAGGA

ATCACC

TT

161
4690
0.80045646
TGCTAC
94564297
94564317
27389
27409
21
0.78242682

CCCAGG

AATCAC

CTT

162
4111
0.3795993
CTACCC
94564299
94564316
27390
27407
18
0.36156966

CAGGAA

TCACCT

163
4691
0.82615894
TACCCC
94564300
94564316
27390
27406
17
0.80812929

AGGAAT

CACCT

164
4692
0.83877867
GCTACC
94564298
94564316
27390
27408
19
0.82074903

CCAGGA

ATCACC

T

165
4693
0.84312158
TGCTAC
94564297
94564316
27390
27409
20
0.82509194

CCCAGG

AATCAC

CT

166
4694
0.75358321
CTGCTA
94564296
94564316
27390
27410
21
0.73555356

CCCCAG

GAATCA

CCT

167
4695
0.71573819
CTACCC
94564299
94564315
27391
27407
17
0.69770855

CAGGAA

TCACC

168
4696
0.775299
GCTACC
94564298
94564315
27391
27408
18
0.75726936

CCAGGA

ATCACC

169
4697
0.78009723
TGCTAC
94564297
94564315
27391
27409
19
0.76206759

CCCAGG

AATCAC

C

170
4698
0.67240676
CTGCTA
94564296
94564315
27391
27410
20
0.65437712

CCCCAG

GAATCA

CC

171
4699
0.73032379
TCTGCT
94564295
94564315
27391
27411
21
0.71229414

ACCCCA

GGAATC

ACC

172
4700
0.61028686
GCTACC
94564298
94564314
27392
27408
17
0.59225721

CCAGGA

ATCAC

173
4701
0.69254508
TGCTAC
94564297
94564314
27392
27409
18
0.67451543

CCCAGG

AATCAC

174
4702
0.70030276
CTGCTA
94564296
94564314
27392
27410
19
0.68227312

CCCCAG

GAATCA

C

175
4703
0.55123289
TCTGCT
94564295
94564314
27392
27411
20
0.53320325

ACCCCA

GGAATC

AC

176
4704
0.44734228
CTCTGCT
94564294
94564314
27392
27412
21
0.42931264

ACCCCA

GGAATC

AC

177
4092
0.78761999
TGCTAC
94564297
94564313
27393
27409
17
0.76959035

CCCAGG

AATCA

178
4705
0.83351676
CTGCTA
94564296
94564313
27393
27410
18
0.81548712

CCCCAG

GAATCA

179
4706
0.61126527
TCTGCT
94564295
94564313
27393
27411
19
0.59323563

ACCCCA

GGAATC

A

180
4707
0.34441052
CTCTGCT
94564294
94564313
27393
27412
20
0.32638087

ACCCCA

GGAATC

A

181
4708
0.57416296
GCTCTG
94564293
94564313
27393
27413
21
0.55613332

CTACCC

CAGGAA

TCA

182
4709
0.20688401
CTGCTA
94564296
94564312
27394
27410
17
0.18885437

CCCCAG

GAATC

183
4710
0.37699084
TCTGCT
94564295
94564312
27394
27411
18
0.3589612

ACCCCA

GGAATC

184
4711
0.16262582
CTCTGCT
94564294
94564312
27394
27412
19
0.14459618

ACCCCA

GGAATC

185
4712
0.39432372
GCTCTG
94564293
94564312
27394
27413
20
0.37629408

CTACCC

CAGGAA

TC

186
4713
0.30527196
GGCTCT
94564292
94564312
27394
27414
21
0.28724232

GCTACC

CCAGGA

ATC

187
4714
0.66369416
TCTGCT
94564295
94564311
27395
27411
17
0.64566452

ACCCCA

GGAAT

188
4715
0.49201464
CTCTGCT
94564294
94564311
27395
27412
18
0.473985

ACCCCA

GGAAT

189
4716
0.65363111
GCTCTG
94564293
94564311
27395
27413
19
0.63560147

CTACCC

CAGGAA

T

190
4717
0.70829044
GGCTCT
94564292
94564311
27395
27414
20
0.6902608

GCTACC

CCAGGA

AT

191
4139
0.33884001
AGGCTC
94564291
94564310
27396
27415
20
0.32081037

TGCTAC

CCCAGG

AA

192
4718
0.46989482
CTCTGCT
94564294
94564310
27396
27412
17
0.45186518

ACCCCA

GGAA

193
4719
0.51069562
GCTCTG
94564293
94564310
27396
27413
18
0.49266597

CTACCC

CAGGAA

194
4720
0.39270541
GGCTCT
94564292
94564310
27396
27414
19
0.37467577

GCTACC

CCAGGA

A

195
4721
0.38953287
GCTCTG
94564293
94564309
27397
27413
17
0.37150323

CTACCC

CAGGA

196
4722
0.27990987
GGCTCT
94564292
94564309
27397
27414
18
0.26188022

GCTACC

CCAGGA

197
4723
0.0791666
GGCTCT
94564292
94564308
27398
27414
17
0.06113696

GCTACC

CCAGG

198
4126
0.01690878
CGTGAG
94564287
94564305
27401
27419
19
-0.0011209

GCTCTG

CTACCC

C

199
4112
0.0039981
AATTCG
94564283
94564300
27406
27423
18
-0.0140315

TGAGGC

TCTGCT

200
4127
0
GGTCAA
94564279
94564297
27409
27427
19
-0.0180296

TTCGTG

AGGCTC

T

201
4093
0.00230947
CAAGGT
94564276
94564292
27414
27430
17
-0.0157202

CAATTC

GTGAG

202
4150
0.00677073
CCTCCC
94564270
94564290
27416
27436
21
-0.0112589

CAAGGT

CAATTC

GTG

203
4151
0.00776482
GGCTCA
94564261
94564281
27425
27445
21
-0.0102648

CGCCCT

CCCCAA

GGT

204
4094
0.01458947
CAGGCT
94564259
94564275
27431
27447
17
-0.0034402

CACGCC

CTCCC

205
4113
0.01159775
CACCAG
94564256
94564273
27433
27450
18
-0.0064319

GCTCAC

GCCCTC

206
4140
0.01532544
CCAGAA
94564250
94564269
27437
27456
20
-0.0027042

CACCAG

GCTCAC

GC

Example 2 the Splicing of ABCA4 is Disrupted in the c.4773+3A>G Variant and can be Partially Rescued Through the Use of Antisense Oligonucleotides

To confirm exon 33 skipping in the chr1: 94487399:T:C [hg19/b37] (c.4773+3A>G) variant, wild type and variant containing minigenes were constructed containing exons 32-34 and the corresponding introns, 32 and 33 (FIG. 2A). Minigenes were then transfected into HEK293T and ARPE19 cells to examine the effect of the c.4773+3A>G variant on splicing. As seen in FIG. 2B, wildtype minigenes showed both exon 33 inclusion, represented by the upper band, and exclusion. c.4773+3A>G mutants, however, showed little exon 33 inclusion indicating the chr1: 94487399:T:C [hg9/b37] mutation induces exon 33 skipping.

To examine the ability of antisense oligonucleotides to promote exon 33 inclusion in the c.4773+3A>G variant the minigenes above were co-transfected with antisense oligonucleotides having sequences set forth in SEQ ID NOs: 208-315 (see Table 5). Antisense oligonucleotides were tiled along exon 33 and intron 33 Antisense oligonucleotides were cotransfected with the mutant minigene containing the c.4773+3A>G variant in HEK293T cells. RT-PCR was conducted to analyze the effect on the splicing of the minigene. Samples were measured by capillary electrophoresis. These results were quantified and are set forth in Table 5. Observing Table 5 it is clear that targeting the intronic regions surrounding exon 33 induces exon 33 inclusion in c.4773+3A>G variant minigenes (high percent spliced in/correctly (PSI) and change in PSI as compared to mutant PSI (dPSI). These observations also suggest antisense oligonucleotides targeting certain regions or “hotspots” in intron 33 (positions 104314-104336 in SEQ ID NO: 1; chr1: 94487370-94487392), e.g., those complementary to a nucleobase sequence in SEQ ID NOs: 260-287, may be particularly useful in the treatment of retinal disease associated with exon 33 skipping (e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease caused by the c.4773+3A>G mutation).

TABLE 5

SEQ

Start
End
Start on
Stop on

ID
DG

Chr1
Chr1
SEQ ID
SEQ ID

NO:
ID
PSI
Sequence
[hg19/b37]
[hg19/b37]
NO: 1
NO: 1
length
dPSI

208
2870
0
TAAAA
94487435
94487454
104252
104271
20
−0.0213675

ACCCA

ACAAG

TGCTT

209
2868
0
TTAAA
94487434
94487453
104253
104272
20
−0.0213675

AACCC

AACAA

GTGCT

210
2869
0
CTTAA
94487433
94487452
104254
104273
20
−0.0213675

AAACC

CAACA

AGTGC

211
2872
0
GCTTA
94487432
94487451
104255
104274
20
−0.0213675

AAAAC

CCAAC

AAGTG

212
2871
0
CGCTT
94487431
94487450
104256
104275
20
−0.0213675

AAAAA

CCCAA

CAAGT

213
2862
0.00052457
CCCCG
94487401
94487420
104286
104305
20
−0.020843

CTCAC

ATTCA

TGATC

214
2874
0.00080667
CACAC
94487397
94487416
104290
104309
20
−0.0205609

CCCGC

TCACA

TTCAT

215
2875
0.00474358
TGTTT
94487391
94487410
104296
104315
20
−0.0166239

ACACA

CCCCG

CTCAC

216
4284
0.04784349
TCTCC
94487382
94487402
104304
104324
21
0.02647596

AGTCT

GTTTA

CACAC

C

217
2876
0.08451165
CTCCA
94487383
94487402
104304
104323
20
0.06314412

GTCTG

TTTAC

ACACC

218
4290
0.02633169
CCAGT
94487385
94487402
104304
104321
18
0.00496416

CTGTTT

ACACA

CC

219
4359
0.0160642
CAGTC
94487386
94487402
104304
104320
17
−0.0053033

TGTTT

ACACA

CC

220
4320
0.04684946
ATCTC
94487381
94487401
104305
104325
21
0.02548193

CAGTC

TGTTT

ACACA

C

221
4304
0.03986191
TCTCC
94487382
94487401
104305
104324
20
0.01849438

AGTCT

GTTTA

CACAC

222
4317
0.05247774
CTCCA
94487383
94487401
104305
104323
19
0.03111022

GTCTG

TTTAC

ACAC

223
4288
0.02440678
TCCAG
94487384
94487401
104305
104322
18
0.00303925

TCTGTT

TACAC

AC

224
4345
0.0152116
CCAGT
94487385
94487401
104305
104321
17
−0.0061559

CTGTTT

ACACA

C

225
4338
0.02968089
AATCT
94487380
94487400
104306
104326
21
0.00831336

CCAGT

CTGTTT

ACACA

226
4297
0.02919964
ATCTC
94487381
94487400
104306
104325
20
0.00783211

CAGTC

TGTTT

ACACA

227
4295
0.02665574
TCTCC
94487382
94487400
104306
104324
19
0.00528821

AGTCT

GTTTA

CACA

228
4300
0.02227967
CTCCA
94487383
94487400
104306
104323
18
0.00091214

GTCTG

TTTAC

ACA

229
4307
0.01566261
TCCAG
94487384
94487400
104306
104322
17
−0.0057049

TCTGTT

TACAC

A

230
4348
0.02854314
AAATC
94487379
94487399
104307
104327
21
0.00717561

TCCAG

TCTGTT

TACAC

231
4331
0.01222792
AATCT
94487380
94487399
104307
104326
20
−0.0091396

CCAGT

CTGTTT

ACAC

232
4357
0.01851217
TCTCC
94487382
94487399
104307
104324
18
−0.0028554

AGTCT

GTTTA

CAC

233
4339
0.01564375
CTCCA
94487383
94487399
104307
104323
17
−0.0057238

GTCTG

TTTAC

AC

234
4347
0.01732577
CAAAT
94487378
94487398
104308
104328
21
−0.0040418

CTCCA

GTCTG

TTTAC

A

235
4319
0.02028748
AAATC
94487379
94487398
104308
104327
20
−0.00108

TCCAG

TCTGTT

TACA

236
4316
0.02157724
AATCT
94487380
94487398
104308
104326
19
0.00020972

CCAGT

CTGTTT

ACA

237
4308
0.01404085
ATCTC
94487381
94487398
104308
104325
18
−0.0073267

CAGTC

TGTTT

ACA

238
4299
0.01686652
TCTCC
94487382
94487398
104308
104324
17
−0.004501

AGTCT

GTTTA

CA

239
4318
0.02311438
TCAAA
94487377
94487397
104309
104329
21
0.00174686

TCTCC

AGTCT

GTTTA

C

240
2877
0.0159866
CAAAT
94487378
94487397
104309
104328
20
−0.0053809

CTCCA

GTCTG

TTTAC

241
4315
0.02033591
AAATC
94487379
94487397
104309
104327
19
−0.0010316

TCCAG

TCTGTT

TAC

242
4324
0.01464558
ATCTC
94487381
94487397
104309
104325
17
−0.0067219

CAGTC

TGTTT

AC

243
4311
0.0241704
CTCAA
94487376
94487396
104310
104330
21
0.00280287

ATCTC

CAGTC

TGTTT

A

244
2878
0.01586952
TCAAA
94487377
94487396
104310
104329
20
−0.005498

TCTCC

AGTCT

GTTTA

245
4334
0.01096985
CAAAT
94487378
94487396
104310
104328
19
−0.0103977

CTCCA

GTCTG

TTTA

246
4306
0.0082054
AAATC
94487379
94487396
104310
104327
18
−0.0131621

TCCAG

TCTGTT

TA

247
4336
0.00893915
AATCT
94487380
94487396
104310
104326
17
−0.0124284

CCAGT

CTGTTT

A

248
4332
0.01779842
CTCAA
94487376
94487395
104311
104330
20
−0.0035691

ATCTC

CAGTC

TGTTT

249
4314
0.02020412
TCAAA
94487377
94487395
104311
104329
19
−0.0011634

TCTCC

AGTCT

GTTT

250
4352
0.02273897
CAAAT
94487378
94487395
104311
104328
18
0.00137144

CTCCA

GTCTG

TTT

251
4303
0.01092555
AAATC
94487379
94487395
104311
104327
17
−0.010442

TCCAG

TCTGTT

T

252
4342
0.03608537
TACTC
94487374
94487394
104312
104332
21
0.01471785

AAATC

TCCAG

TCTGTT

253
4346
0.03163721
ACTCA
94487375
94487394
104312
104331
20
0.01026968

AATCT

CCAGT

CTGTT

254
4277
0.02538751
TCAAA
94487377
94487394
104312
104329
18
0.00401999

TCTCC

AGTCT

GTT

255
4341
0.0133478
CAAAT
94487378
94487394
104312
104328
17
−0.0080197

CTCCA

GTCTG

TT

256
4361
0.03839499
CTACT
94487373
94487393
104313
104333
21
0.01702747

CAAAT

CTCCA

GTCTG

T

257
4328
0.02221052
ACTCA
94487375
94487393
104313
104331
19
0.00084299

AATCT

CCAGT

CTGT

258
4358
0.01898736
CTCAA
94487376
94487393
104313
104330
18
−0.0023802

ATCTC

CAGTC

TGT

259
4343
0.01753224
TCAAA
94487377
94487393
104313
104329
17
−0.0038353

TCTCC

AGTCT

GT

260
4298
0.07456743
CCTAC
94487372
94487392
104314
104334
21
0.05319991

TCAAA

TCTCC

AGTCT

G

261
4289
0.05263352
TACTC
94487374
94487392
104314
104332
19
0.031266

AAATC

TCCAG

TCTG

262
4355
0.05632484
ACTCA
94487375
94487392
104314
104331
18
0.03495732

AATCT

CCAGT

CTG

263
4312
0.04068388
CTCAA
94487376
94487392
104314
104330
17
0.01931635

ATCTC

CAGTC

TG

264
4285
0.10321842
TCCTA
94487371
94487391
104315
104335
21
0.0818509

CTCAA

ATCTC

CAGTC

T

265
4329
0.06474209
CTACT
94487373
94487391
104315
104333
19
0.04337457

CAAAT

CTCCA

GTCT

266
4349
0.07991069
TACTC
94487374
94487391
104315
104332
18
0.05854316

AAATC

TCCAG

TCT

267
4282
0.05279718
ACTCA
94487375
94487391
104315
104331
17
0.03142965

AATCT

CCAGT

CT

268
4305
0.10192797
ATCCT
94487370
94487390
104316
104336
21
0.08056044

ACTCA

AATCT

CCAGT

C

269
2863
0.12769861
TCCTA
94487371
94487390
104316
104335
20
0.10633108

CTCAA

ATCTC

CAGTC

270
4340
0.10554271
CCTAC
94487372
94487390
104316
104334
19
0.08417518

TCAAA

TCTCC

AGTC

271
4309
0.07190236
CTACT
94487373
94487390
104316
104333
18
0.05053484

CAAAT

CTCCA

GTC

272
4322
0.06185338
TACTC
94487374
94487390
104316
104332
17
0.04048585

AAATC

TCCAG

TC

273
4354
0.09178354
AATCC
94487369
94487389
104317
104337
21
0.07041601

TACTC

AAATC

TCCAG

T

274
4286
0.07464417
ATCCT
94487370
94487389
104317
104336
20
0.05327664

ACTCA

AATCT

CCAGT

275
4323
0.05544928
TCCTA
94487371
94487389
104317
104335
19
0.03408175

CTCAA

ATCTC

CAGT

276
4313
0.0777456
CCTAC
94487372
94487389
104317
104334
18
0.05637807

TCAAA

TCTCC

AGT

277
4296
0.06060062
AAATC
94487368
94487388
104318
104338
21
0.0392331

CTACT

CAAAT

CTCCA

G

278
2867
0.11830793
AATCC
94487369
94487388
104318
104337
20
0.0969404

TACTC

AAATC

TCCAG

279
4294
0.05698576
ATCCT
94487370
94487388
104318
104336
19
0.03561823

ACTCA

AATCT

CCAG

280
4364
0.05505851
TCCTA
94487371
94487388
104318
104335
18
0.03369098

CTCAA

ATCTC

CAG

281
4350
0.06485799
CCTAC
94487372
94487388
104318
104334
17
0.04349046

TCAAA

TCTCC

AG

282
4287
0.04057979
AAAAT
94487367
94487387
104319
104339
21
0.01921226

CCTAC

TCAAA

TCTCC

A

283
4330
0.03754774
AAAAA
94487366
94487386
104320
104340
21
0.01618022

TCCTA

CTCAA

ATCTC

C

284
4326
0.03679981
AAAAT
94487367
94487386
104320
104339
20
0.01543229

CCTAC

TCAAA

TCTCC

285
4356
0.03101451
AAATC
94487368
94487386
104320
104338
19
0.00964698

CTACT

CAAAT

CTCC

286
4335
0.02140241
AATCC
94487369
94487386
104320
104337
18
3.4885E−05

TACTC

AAATC

TCC

287
4344
0.02608654
ATCCT
94487370
94487386
104320
104336
17
0.00471901

ACTCA

AATCT

CC

288
4337
0.01612763
AAAAA
94487366
94487385
104321
104340
20
−0.0052399

TCCTA

CTCAA

ATCTC

289
4283
0.01625135
AAAAT
94487367
94487385
104321
104339
19
−0.0051162

CCTAC

TCAAA

TCTC

290
4310
0.00703731
TCAAA
94487364
94487384
104322
104342
21
−0.0143302

AATCC

TACTC

AAATC

T

291
4360
0.01312168
AAAAA
94487366
94487384
104322
104340
19
−0.0082458

TCCTA

CTCAA

ATCT

292
4302
0.00716491
AAAAT
94487367
94487384
104322
104339
18
−0.0142026

CCTAC

TCAAA

TCT

293
4333
0.00594284
AAATC
94487368
94487384
104322
104338
17
−0.0154247

CTACT

CAAAT

CT

294
4327
0.00735476
CAAAA
94487365
94487383
104323
104341
19
−0.0140128

ATCCT

ACTCA

AATC

295
4293
0.0062991
AAAAT
94487367
94487383
104323
104339
17
−0.0150684

CCTAC

TCAAA

TC

296
4301
0.00766725
AGTCA
94487362
94487382
104324
104344
21
−0.0137003

AAAAT

CCTAC

TCAAA

T

297
2879
0.0306372
GTCAA
94487363
94487382
104324
104343
20
0.00926968

AAATC

CTACT

CAAAT

298
4325
0.00521359
TCAAA
94487364
94487382
104324
104342
19
−0.0161539

AATCC

TACTC

AAAT

299
4281
0.00556784
CAAAA
94487365
94487382
104324
104341
18
−0.0157997

ATCCT

ACTCA

AAT

300
4278
0.00674261
AGTCA
94487362
94487381
104325
104344
20
−0.0146249

AAAAT

CCTAC

TCAAA

301
4363
0.01433914
GTCAA
94487363
94487381
104325
104343
19
−0.0070284

AAATC

CTACT

CAAA

302
4321
0.0030924
CAAAA
94487365
94487381
104325
104341
17
−0.0182751

ATCCT

ACTCA

AA

303
2880
0.03800592
AAGTC
94487361
94487380
104326
104345
20
0.0166384

AAAAA

TCCTA

CTCAA

304
4353
0.00893723
AGTCA
94487362
94487380
104326
104344
19
−0.0124303

AAAAT

CCTAC

TCAA

305
4280
0.00531292
GTCAA
94487363
94487380
104326
104343
18
−0.0160546

AAATC

CTACT

CAA

306
4291
0.00374818
TCAAA
94487364
94487380
104326
104342
17
−0.0176193

AATCC

TACTC

AA

307
4279
0.00686827
AAGTC
94487361
94487379
104327
104345
19
−0.0144993

AAAAA

TCCTA

CTCA

308
4275
0.00534896
AGTCA
94487362
94487379
104327
104344
18
−0.0160186

AAAAT

CCTAC

TCA

309
4276
0.00592412
GTCAA
94487363
94487379
104327
104343
17
−0.0154434

AAATC

CTACT

CA

310
4351
0.00988739
AAGTC
94487361
94487378
104328
104345
18
−0.0114801

AAAAA

TCCTA

CTC

311
4292
0.00570931
AGTCA
94487362
94487378
104328
104344
17
−0.0156582

AAAAT

CCTAC

TC

312
4362
0.00618523
AAGTC
94487361
94487377
104329
104345
17
−0.0151823

AAAAA

TCCTA

CT

313
2881
0.0253028
TTAAG
94487355
94487374
104332
104351
20
0.00393528

CAAGT

CAAAA

ATCCT

314
2864
0.00584037
TCATT
94487342
94487361
104345
104364
20
−0.0155272

CATGG

TAGTT

AAGCA

315
2865
0.00560728
CTCAT
94487341
94487360
104346
104365
20
−0.0157602

TCATG

GTAGT

TAAGC

Example 3 the Splicing of ABCA4 is Disrupted in the c.5196+1137G>A Variant and can be Partially Rescued Through the Use of Antisense Oligonucleotides

To confirm partial intron 36 inclusion (i.e. pseudo exon inclusion) in the chr1: 94484001:C:T [hg19/b37] (c.5196+1137G>A) variant, wild type and variant containing minigenes were constructed containing exons 36-37 and the corresponding intron 36 (FIG. 3A). Minigenes were then transfected into HEK293T and ARPE19 cells to examine the effect of the c.5196+1137G>A variant on splicing. As seen in FIG. 3B, wildtype minigenes showed little to no intron 36 inclusion, represented by the upper band. c.5196+1137G>A mutants, however, showed no partial intron 36 inclusion (i.e. pseudo exon 36.1 inclusion) indicating the chr1:94484001:C:T [hg19/b37] mutation induces intron 36 inclusion.

To examine the ability of antisense oligonucleotides to promote intron 36 exclusion in the c.5196+1137G>A variant the minigenes above were co-transfected with antisense oligonucleotides having sequences set forth in SEQ ID NOs: 316-385 and 463-596 (see Table 6). Antisense oligonucleotides were tiled along intron 36. Antisense oligonucleotides were cotransfected with the mutant minigene containing the c.5196+1137G>A variant in HEK293T cells. RT-PCR was conducted to analyze the effect on the splicing of the minigene. Samples were measured by capillary electrophoresis. These results were quantified and are set forth in Table 6. Observing Table 6 it is clear that targeting intron 36 promotes intron 36 exclusion in c.5196+1137G>A variant minigenes (high percent spliced in/correctly (PSI) and change in PSI as compared to mutant PSI (dPSI). These observations suggest antisense oligonucleotides targeting this region or “hotspot” (positions 107659-107800 in SEQ ID NO: 1; chr1: 94483906-94484047), e.g., those complementary to a nucleobase sequence in SEQ ID NOs: 316-374 and 463-596, may be particularly useful in the treatment of retinal disease associated with intron 36 inclusion (e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease caused by the c.5196+1137G>A mutation).

TABLE 6

SEQ

Start
End
Start on
Stop on

ID
DG

Chr1
Chr1
SEQ ID
SEQ ID

NO:
ID
PSI
Sequence
[hg19/b37]
[hg19/b37]
NO: 1
NO: 1
length
dPSI

316
3892
0.92360722
TTTAGTT
94484028
94484047
107659
107678
20
0.06408227

GCTACT

GATAAT

C

317
3877
0.94385808
ATTTAG
94484027
94484046
107660
107679
20
0.08433313

TTGCTA

CTGATA

AT

318
3891
0.92758041
AATTTA
94484026
94484045
107661
107680
20
0.06805546

GTTGCT

ACTGAT

AA

319
3893
0.94758288
AATAAT
94484023
94484042
107664
107683
20
0.08805793

TTAGTT

GCTACT

GA

320
3883
0.98602336
AGAGAG
94484015
94484034
107672
107691
20
0.12649841

GAAATA

ATTTAG

TT

321
3910
0.98162144
GAGAGA
94484014
94484033
107673
107692
20
0.12209649

GGAAAT

AATTTA

GT

322
3902
0.97208233
GAAGAG
94484011
94484030
107676
107695
20
0.11255738

AGAGGA

AATAAT

TT

323
3846
0.98452774
AGAAGA
94484010
94484029
107677
107696
20
0.12500279

GAGAGG

AAATAA

TT

324
3899
0.97837164
ACAGAA
94484008
94484027
107679
107698
20
0.11884669

GAGAGA

GGAAAT

AA

325
3905
0.97124617
AGACAG
94484006
94484025
107681
107700
20
0.11172122

AAGAGA

GAGGAA

AT

326
3876
0.96455012
GTGTAG
94484002
94484021
107685
107704
20
0.10502517

ACAGAA

GAGAGA

GG

327
3881
0.96973536
TGTGTA
94484001
94484020
107686
107705
20
0.1102104

GACAGA

AGAGAG

AG

328
3867
0.97895873
CTTGTGT
94483999
94484018
107688
107707
20
0.11943377

AGACAG

AAGAGA

G

329
3865
0.98220319
TCCTTGT
94483997
94484016
107690
107709
20
0.12267824

GTAGAC

AGAAGA

G

330
3872
0.98844395
TTTCCTT
94483995
94484014
107692
107711
20
0.128919

GTGTAG

ACAGAA

G

331
3869
0.9720131
ATGAGT
94483988
94484007
107699
107718
20
0.11248815

GTTTCCT

TGTGTA

G

332
3873
0.96449385
TTATGA
94483986
94484005
107701
107720
20
0.1049689

GTGTTTC

CTTGTGT

333
3871
0.99006429
TGCATTT
94483981
94484000
107706
107725
20
0.13053934

ATGAGT

GTTTCCT

334
3870
0.98567671
CGTGCA
94483979
94483998
107708
107727
20
0.12615175

TTTATG

AGTGTT

TC

335
3882
0.96968677
CCGTGC
94483978
94483997
107709
107728
20
0.11016182

ATTTAT

GAGTGT

TT

336
3901
0.99148614
CCCCGT
94483976
94483995
107711
107730
20
0.13196119

GCATTT

ATGAGT

GT

337
3843
0.99443585
CCTCCC
94483973
94483992
107714
107733
20
0.1349109

CGTGCA

TTTATG

AG

338
3851
0.97596017
CTCCTCC
94483971
94483990
107716
107735
20
0.11643522

CCGTGC

ATTTAT

G

339
3907
0.96166339
CCTCCTC
94483970
94483989
107717
107736
20
0.10213844

CCCGTG

CATTTAT

340
3878
0.98624354
CTGACC
94483966
94483985
107721
107740
20
0.12671859

TCCTCCC

CGTGCA

T

341
3844
0.98651575
TTCTGA
94483964
94483983
107723
107742
20
0.1269908

CCTCCTC

CCCGTG

C

342
3847
0.99420524
GTTCTG
94483963
94483982
107724
107743
20
0.13468029

ACCTCC

TCCCCG

TG

343
3906
0.98854692
GGTTCT
94483962
94483981
107725
107744
20
0.12902197

GACCTC

CTCCCC

GT

344
3845
0.95483698
CAGGTT
94483960
94483979
107727
107746
20
0.09531203

CTGACC

TCCTCCC

C

345
3888
0.95721508
TCAGGT
94483959
94483978
107728
107747
20
0.09769013

TCTGAC

CTCCTCC

C

346
3890
0.96554722
TTCAGG
94483958
94483977
107729
107748
20
0.10602227

TTCTGA

CCTCCTC

C

347
3880
0.95891421
TTTCAG
94483957
94483976
107730
107749
20
0.09938926

GTTCTG

ACCTCC

TC

348
3884
0.94176661
AAAGGC
94483951
94483970
107736
107755
20
0.08224166

TTTCAG

GTTCTG

AC

349
3894
0.9538534
AAGAAA
94483948
94483967
107739
107758
20
0.09432845

GGCTTT

CAGGTT

CT

350
3849
0.96705708
CAAAGA
94483946
94483965
107741
107760
20
0.10753213

AAGGCT

TTCAGG

TT

351
3850
0.95722885
CCAAAG
94483945
94483964
107742
107761
20
0.0977039

AAAGGC

TTTCAG

GT

352
3889
0.95791095
TCCAAA
94483944
94483963
107743
107762
20
0.098386

GAAAGG

CTTTCA

GG

353
3911
0.9793179
TTATCC
94483941
94483960
107746
107765
20
0.11979295

AAAGAA

AGGCTT

TC

354
3895
0.98777759
TGCTCTT
94483936
94483955
107751
107770
20
0.12825264

ATCCAA

AGAAAG

G

355
3879
0.98118092
TGATGC
94483933
94483952
107754
107773
20
0.12165597

TCTTATC

CAAAGA

A

356
3868
0.97667072
GTTGAT
94483931
94483950
107756
107775
20
0.11714577

GCTCTT

ATCCAA

AG

357
3848
0.97718318
GCAGTT
94483928
94483947
107759
107778
20
0.11765823

GATGCT

CTTATCC

A

358
3842
0.98372299
TGCAGT
94483927
94483946
107760
107779
20
0.12419804

TGATGC

TCTTATC

C

359
3866
0.97507399
CTGCAG
94483926
94483945
107761
107780
20
0.11554904

TTGATG

CTCTTAT

C

360
3857
0.97729685
CCTGCA
94483925
94483944
107762
107781
20
0.1177719

GTTGAT

GCTCTT

AT

361
3864
0.98073104
ACCTGC
94483924
94483943
107763
107782
20
0.12120609

AGTTGA

TGCTCTT

A

362
3859
0.96952222
TACCTG
94483923
94483942
107764
107783
20
0.10999727

CAGTTG

ATGCTC

TT

363
3852
0.97693621
GTACCT
94483922
94483941
107765
107784
20
0.11741126

GCAGTT

GATGCT

CT

364
3856
0.96942457
GGTACC
94483921
94483940
107766
107785
20
0.10989962

TGCAGT

TGATGC

TC

365
3855
0.95768882
TGGTAC
94483920
94483939
107767
107786
20
0.09816386

CTGCAG

TTGATG

CT

366
3858
0.98094362
GTGGTA
94483919
94483938
107768
107787
20
0.12141866

CCTGCA

GTTGAT

GC

367
3861
0.97641827
TGTGGT
94483918
94483937
107769
107788
20
0.11689331

ACCTGC

AGTTGA

TG

368
3853
0.98023491
ATGTGG
94483917
94483936
107770
107789
20
0.12070996

TACCTG

CAGTTG

AT

369
3854
0.9297235
AATGTG
94483916
94483935
107771
107790
20
0.07019855

GTACCT

GCAGTT

GA

370
3860
0.97283359
CAATGT
94483915
94483934
107772
107791
20
0.11330864

GGTACC

TGCAGT

TG

371
3875
0.96553215
GCCAAT
94483913
94483932
107774
107793
20
0.1060072

GTGGTA

CCTGCA

GT

372
3862
0.97278364
AGGGCC
94483910
94483929
107777
107796
20
0.11325868

AATGTG

GTACCT

GC

373
3863
0.97702638
ACAGGG
94483908
94483927
107779
107798
20
0.11750143

CCAATG

TGGTAC

CT

374
3874
0.9517307
TCACAG
94483906
94483925
107781
107800
20
0.09220574

GGCCAA

TGTGGT

AC

375
3897
0.37628761
ATTAGC
94483899
94483918
107788
107807
20
−0.4832373

ATCACA

GGGCCA

AT

376
3903
0.17207263
TATTAG
94483898
94483917
107789
107808
20
−0.6874523

CATCAC

AGGGCC

AA

377
3900
0.21244089
TATATT
94483896
94483915
107791
107810
20
−0.6470841

AGCATC

ACAGGG

CC

378
3908
0.14872555
TTTATAT
94483894
94483913
107793
107812
20
−0.7107994

TAGCAT

CACAGG

G

379
3887
0.25938883
CCTTTTA
94483891
94483910
107796
107815
20
−0.6001361

TATTAG

CATCAC

A

380
3896
0.26261845
TCCTTTT
94483890
94483909
107797
107816
20
−0.5969065

ATATTA

GCATCA

C

381
3904
0.45104715
GCTCCTT
94483888
94483907
107799
107818
20
−0.4084778

TTATATT

AGCATC

382
3886
0.53710195
AGCTCC
94483887
94483906
107800
107819
20
−0.322423

TTTTATA

TTAGCA

T

383
3898
0.39262608
TAGCTC
94483886
94483905
107801
107820
20
−0.4668989

CTTTTAT

ATTAGC

A

384
3909
0.81437018
GGCCTA
94483882
94483901
107805
107824
20
−0.0451548

GCTCCTT

TTATATT

385
3885
0.78147426
CCGGTG
94483876
94483895
107811
107830
20
−0.0780507

GGCCTA

GCTCCTT

T

463
6033
0.99708567
TGAGTG
94483989
94484005
107701
107717
17
0.13756072

TTTCCTT

GTGT

464
6034
0.99700831
ATGAGT
94483988
94484005
107701
107718
18
0.13748336

GTTTCCT

TGTGT

465
6035
0.99528582
TATGAG
94483987
94484005
107701
107719
19
0.13576086

TGTTTCC

TTGTGT

466
6036
0.98994658
TTTATG
94483985
94484005
107701
107721
21
0.13042163

AGTGTT

TCCTTGT

GT

467
6037
0.99670278
ATGAGT
94483988
94484004
107702
107718
17
0.13717783

GTTTCCT

TGTG

468
6038
0.99552504
TATGAG
94483987
94484004
107702
107719
18
0.13600009

TGTTTCC

TTGTG

469
6039
0.99370127
TTATGA
94483986
94484004
107702
107720
19
0.13417632

GTGTTTC

CTTGTG

470
6040
0.99364496
TTTATG
94483985
94484004
107702
107721
20
0.13412001

AGTGTT

TCCTTGT

G

471
6041
0.99742833
ATTTAT
94483984
94484004
107702
107722
21
0.13790338

GAGTGT

TTCCTTG

TG

472
6042
0.99386028
TATGAG
94483987
94484003
107703
107719
17
0.13433532

TGTTTCC

TTGT

473
6043
0.9948824
TTATGA
94483986
94484003
107703
107720
18
0.13535745

GTGTTTC

CTTGT

474
6044
0.99560869
TTTATG
94483985
94484003
107703
107721
19
0.13608373

AGTGTT

TCCTTGT

475
6045
0.98836088
ATTTAT
94483984
94484003
107703
107722
20
0.12883593

GAGTGT

TTCCTTG

T

476
6046
0.99812564
CATTTAT
94483983
94484003
107703
107723
21
0.13860069

GAGTGT

TTCCTTG

T

477
6047
0.99661461
TTATGA
94483986
94484002
107704
107720
17
0.13708966

GTGTTTC

CTTG

478
6048
0.98365619
TTTATG
94483985
94484002
107704
107721
18
0.12413124

AGTGTT

TCCTTG

479
6049
0.99452638
ATTTAT
94483984
94484002
107704
107722
19
0.13500143

GAGTGT

TTCCTTG

480
6050
0.97742354
CATTTAT
94483983
94484002
107704
107723
20
0.11789859

GAGTGT

TTCCTTG

481
6051
0.99790655
GCATTT
94483982
94484002
107704
107724
21
0.1383816

ATGAGT

GTTTCCT

TG

482
6052
0.99011281
TTTATG
94483985
94484001
107705
107721
17
0.13058786

AGTGTT

TCCTT

483
6053
0.99628751
ATTTAT
94483984
94484001
107705
107722
18
0.13676256

GAGTGT

TTCCTT

484
6054
0.99774963
CATTTAT
94483983
94484001
107705
107723
19
0.13822468

GAGTGT

TTCCTT

485
6055
0.99672063
GCATTT
94483982
94484001
107705
107724
20
0.13719568

ATGAGT

GTTTCCT

T

486
6056
0.99696414
TGCATTT
94483981
94484001
107705
107725
21
0.13743919

ATGAGT

GTTTCCT

T

487
6057
0.998537
ATTTAT
94483984
94484000
107706
107722
17
0.13901204

GAGTGT

TTCCT

488
6058
0.99733283
CATTTAT
94483983
94484000
107706
107723
18
0.13780788

GAGTGT

TTCCT

489
6059
0.99794292
GCATTT
94483982
94484000
107706
107724
19
0.13841796

ATGAGT

GTTTCCT

490
6060
0.99779486
GTGCAT
94483980
94484000
107706
107726
21
0.13826991

TTATGA

GTGTTTC

CT

491
6061
0.99868652
CATTTAT
94483983
94483999
107707
107723
17
0.13916157

GAGTGT

TTCC

492
6062
0.99832234
GCATTT
94483982
94483999
107707
107724
18
0.13879739

ATGAGT

GTTTCC

493
6063
0.99765297
TGCATTT
94483981
94483999
107707
107725
19
0.13812802

ATGAGT

GTTTCC

494
6064
0.98029775
GTGCAT
94483980
94483999
107707
107726
20
0.1207728

TTATGA

GTGTTTC

C

495
6065
0.99754751
CGTGCA
94483979
94483999
107707
107727
21
0.13802256

TTTATG

AGTGTT

TCC

496
6066
0.9946547
GCATTT
94483982
94483998
107708
107724
17
0.13512975

ATGAGT

GTTTC

497
6067
0.99593138
TGCATTT
94483981
94483998
107708
107725
18
0.13640642

ATGAGT

GTTTC

498
6068
0.9909698
GTGCAT
94483980
94483998
107708
107726
19
0.13144485

TTATGA

GTGTTTC

499
6069
0.99372888
CCGTGC
94483978
94483998
107708
107728
21
0.13420393

ATTTAT

GAGTGT

TTC

500
6070
0.99159647
TGCATTT
94483981
94483997
107709
107725
17
0.13207151

ATGAGT

GTTT

501
6071
0.99707014
GTGCAT
94483980
94483997
107709
107726
18
0.13754519

TTATGA

GTGTTT

502
6072
0.99356046
CGTGCA
94483979
94483997
107709
107727
19
0.13403551

TTTATG

AGTGTT

T

503
6073
0.99731285
CCCGTG
94483977
94483997
107709
107729
21
0.1377879

CATTTAT

GAGTGT

TT

504
6074
0.99667542
GTGCAT
94483980
94483996
107710
107726
17
0.13715047

TTATGA

GTGTT

505
6075
0.99654701
CGTGCA
94483979
94483996
107710
107727
18
0.13702206

TTTATG

AGTGTT

506
6076
0.99430514
CCGTGC
94483978
94483996
107710
107728
19
0.13478019

ATTTAT

GAGTGT

T

507
6077
0.99864031
CCCGTG
94483977
94483996
107710
107729
20
0.13911536

CATTTAT

GAGTGT

T

508
6078
0.99513775
CCCCGT
94483976
94483996
107710
107730
21
0.1356128

GCATTT

ATGAGT

GTT

509
6079
0.98996838
CGTGCA
94483979
94483995
107711
107727
17
0.13044343

TTTATG

AGTGT

510
6080
0.99932461
CCGTGC
94483978
94483995
107711
107728
18
0.13979966

ATTTAT

GAGTGT

511
6081
0.98981026
CCCGTG
94483977
94483995
107711
107729
19
0.13028531

CATTTAT

GAGTGT

512
6082
0.99093164
TCCCCG
94483975
94483995
107711
107731
21
0.13140669

TGCATTT

ATGAGT

GT

513
6083
0.99524727
CCGTGC
94483978
94483994
107712
107728
17
0.13572232

ATTTAT

GAGTG

514
6084
0.99255254
CCCGTG
94483977
94483994
107712
107729
18
0.13302759

CATTTAT

GAGTG

515
6085
0.99366018
CCCCGT
94483976
94483994
107712
107730
19
0.13413523

GCATTT

ATGAGT

G

516
6086
0.99911074
TCCCCG
94483975
94483994
107712
107731
20
0.13958579

TGCATTT

ATGAGT

G

517
6087
0.99968834
CTCCCC
94483974
94483994
107712
107732
21
0.14016339

GTGCAT

TTATGA

GTG

518
6088
1
CCCGTG
94483977
94483993
107713
107729
17
0.14047505

CATTTAT

GAGT

519
6089
0.9965087
CCCCGT
94483976
94483993
107713
107730
18
0.13698375

GCATTT

ATGAGT

520
6090
0.99896379
TCCCCG
94483975
94483993
107713
107731
19
0.13943884

TGCATTT

ATGAGT

521
6091
0.99920439
CTCCCC
94483974
94483993
107713
107732
20
0.13967944

GTGCAT

TTATGA

GT

522
6092
0.99359014
CCTCCC
94483973
94483993
107713
107733
21
0.13406519

CGTGCA

TTTATG

AGT

523
6093
1
CCCCGT
94483976
94483992
107714
107730
17
0.14047505

GCATTT

ATGAG

524
6094
0.99679413
TCCCCG
94483975
94483992
107714
107731
18
0.13726918

TGCATTT

ATGAG

525
6095
0.995319
CTCCCC
94483974
94483992
107714
107732
19
0.13579405

GTGCAT

TTATGA

G

526
6096
1
TCCTCCC
94483972
94483992
107714
107734
21
0.14047505

CGTGCA

TTTATG

AG

527
6097
0.98989575
TCCCCG
94483975
94483991
107715
107731
17
0.1303708

TGCATTT

ATGA

528
6098
0.99149171
CTCCCC
94483974
94483991
107715
107732
18
0.13196676

GTGCAT

TTATGA

529
6099
0.99354399
CCTCCC
94483973
94483991
107715
107733
19
0.13401904

CGTGCA

TTTATG

A

530
6100
0.99448301
TCCTCCC
94483972
94483991
107715
107734
20
0.13495806

CGTGCA

TTTATG

A

531
6101
0.99703138
CTCCTCC
94483971
94483991
107715
107735
21
0.13750643

CCGTGC

ATTTAT

GA

532
6102
0.99558543
CTCCCC
94483974
94483990
107716
107732
17
0.13606047

GTGCAT

TTATG

533
6103
0.99912813
CCTCCC
94483973
94483990
107716
107733
18
0.13960318

CGTGCA

TTTATG

534
6104
0.99498711
TCCTCCC
94483972
94483990
107716
107734
19
0.13546216

CGTGCA

TTTATG

535
6105
0.99606456
CCTCCTC
94483970
94483990
107716
107736
21
0.1365396

CCCGTG

CATTTAT

G

536
6106
0.99538394
CCTCCC
94483973
94483989
107717
107733
17
0.13585899

CGTGCA

TTTAT

537
6107
0.99116241
TCCTCCC
94483972
94483989
107717
107734
18
0.13163746

CGTGCA

TTTAT

538
6108
0.98809019
CTCCTCC
94483971
94483989
107717
107735
19
0.12856524

CCGTGC

ATTTAT

539
6109
0.99708577
ACCTCC
94483969
94483989
107717
107737
21
0.13756082

TCCCCG

TGCATTT

AT

540
6110
0.99257134
TCCTCCC
94483972
94483988
107718
107734
17
0.13304639

CGTGCA

TTTA

541
6111
0.9921426
CTCCTCC
94483971
94483988
107718
107735
18
0.13261765

CCGTGC

ATTTA

542
6112
0.99077156
CCTCCTC
94483970
94483988
107718
107736
19
0.13124661

CCCGTG

CATTTA

543
6113
0.92250391
ACCTCC
94483969
94483988
107718
107737
20
0.06297896

TCCCCG

TGCATTT

A

544
6114
0.99325004
GACCTC
94483968
94483988
107718
107738
21
0.13372509

CTCCCC

GTGCAT

TTA

545
6115
0.99636481
CTCCTCC
94483971
94483987
107719
107735
17
0.13683986

CCGTGC

ATTT

546
6116
0.99413994
CCTCCTC
94483970
94483987
107719
107736
18
0.13461499

CCCGTG

CATTT

547
6117
0.99570644
ACCTCC
94483969
94483987
107719
107737
19
0.13618149

TCCCCG

TGCATTT

548
6118
0.99405885
GACCTC
94483968
94483987
107719
107738
20
0.1345339

CTCCCC

GTGCAT

TT

549
6119
0.99754622
TGACCT
94483967
94483987
107719
107739
21
0.13802127

CCTCCC

CGTGCA

TTT

550
6120
0.97369837
CCTCCTC
94483970
94483986
107720
107736
17
0.11417342

CCCGTG

CATT

551
6121
0.95975907
ACCTCC
94483969
94483986
107720
107737
18
0.10023411

TCCCCG

TGCATT

552
6122
0.9985255
GACCTC
94483968
94483986
107720
107738
19
0.13900055

CTCCCC

GTGCAT

T

553
6123
0.9904905
TGACCT
94483967
94483986
107720
107739
20
0.13096555

CCTCCC

CGTGCA

TT

554
6124
0.99407828
CTGACC
94483966
94483986
107720
107740
21
0.13455333

TCCTCCC

CGTGCA

TT

555
6125
0.99485913
ACCTCC
94483969
94483985
107721
107737
17
0.13533418

TCCCCG

TGCAT

556
6126
0.99153982
GACCTC
94483968
94483985
107721
107738
18
0.13201487

CTCCCC

GTGCAT

557
6127
0.99438632
TGACCT
94483967
94483985
107721
107739
19
0.13486137

CCTCCC

CGTGCA

T

558
6129
0.99675885
GACCTC
94483968
94483984
107722
107738
17
0.13723389

CTCCCC

GTGCA

559
6130
0.99704147
TGACCT
94483967
94483984
107722
107739
18
0.13751652

CCTCCC

CGTGCA

560
6131
0.99707416
CTGACC
94483966
94483984
107722
107740
19
0.13754921

TCCTCCC

CGTGCA

561
6132
0.9970857
TCTGAC
94483965
94483984
107722
107741
20
0.13756075

CTCCTCC

CCGTGC

A

562
6133
0.99736692
TTCTGA
94483964
94483984
107722
107742
21
0.13784197

CCTCCTC

CCCGTG

CA

563
6134
0.9916746
TGACCT
94483967
94483983
107723
107739
17
0.13214965

CCTCCC

CGTGC

564
6135
0.99740995
CTGACC
94483966
94483983
107723
107740
18
0.137885

TCCTCCC

CGTGC

565
6136
1
TCTGAC
94483965
94483983
107723
107741
19
0.14047505

CTCCTCC

CCGTGC

566
6137
0.98683302
GTTCTG
94483963
94483983
107723
107743
21
0.12730807

ACCTCC

TCCCCG

TGC

567
6138
0.99762799
CTGACC
94483966
94483982
107724
107740
17
0.13810304

TCCTCCC

CGTG

568
6139
0.98803138
TCTGAC
94483965
94483982
107724
107741
18
0.12850643

CTCCTCC

CCGTG

569
6140
0.99322322
TTCTGA
94483964
94483982
107724
107742
19
0.13369827

CCTCCTC

CCCGTG

570
6141
0.99086404
GGTTCT
94483962
94483982
107724
107744
21
0.13133909

GACCTC

CTCCCC

GTG

571
6142
0.99460361
TCTGAC
94483965
94483981
107725
107741
17
0.13507865

CTCCTCC

CCGT

572
6143
0.9978076
TTCTGA
94483964
94483981
107725
107742
18
0.13828264

CCTCCTC

CCCGT

573
6144
0.99947537
GTTCTG
94483963
94483981
107725
107743
19
0.13995042

ACCTCC

TCCCCG

T

574
6145
0.99781033
AGGTTC
94483961
94483981
107725
107745
21
0.13828538

TGACCT

CCTCCC

CGT

575
6146
0.99578042
TTCTGA
94483964
94483980
107726
107742
17
0.13625547

CCTCCTC

CCCG

576
6147
0.99733058
GTTCTG
94483963
94483980
107726
107743
18
0.13780562

ACCTCC

TCCCCG

577
6148
1
GGTTCT
94483962
94483980
107726
107744
19
0.14047505

GACCTC

CTCCCC

G

578
6149
0.99758052
AGGTTC
94483961
94483980
107726
107745
20
0.13805557

TGACCT

CCTCCC

CG

579
6150
0.99711125
CAGGTT
94483960
94483980
107726
107746
21
0.1375863

CTGACC

TCCTCCC

CG

580
6151
0.99860493
GTTCTG
94483963
94483979
107727
107743
17
0.13907998

ACCTCC

TCCCC

581
6152
0.99723212
GGTTCT
94483962
94483979
107727
107744
18
0.13770717

GACCTC

CTCCCC

582
6153
0.99282364
AGGTTC
94483961
94483979
107727
107745
19
0.13329869

TGACCT

CCTCCC

C

583
6154
0.99716907
TCAGGT
94483959
94483979
107727
107747
21
0.13764412

TCTGAC

CTCCTCC

CC

584
6155
0.99847681
GGTTCT
94483962
94483978
107728
107744
17
0.13895186

GACCTC

CTCCC

585
6156
0.99567493
AGGTTC
94483961
94483978
107728
107745
18
0.13614998

TGACCT

CCTCCC

586
6157
0.99506277
CAGGTT
94483960
94483978
107728
107746
19
0.13553782

CTGACC

TCCTCCC

587
6158
0.99636379
TTCAGG
94483958
94483978
107728
107748
21
0.13683884

TTCTGA

CCTCCTC

CC

588
6159
0.99109538
AGGTTC
94483961
94483977
107729
107745
17
0.13157043

TGACCT

CCTCC

589
6160
0.98907762
CAGGTT
94483960
94483977
107729
107746
18
0.12955267

CTGACC

TCCTCC

590
6161
0.98093795
TCAGGT
94483959
94483977
107729
107747
19
0.121413

TCTGAC

CTCCTCC

591
6162
0.99262906
CAGGTT
94483960
94483976
107730
107746
17
0.13310411

CTGACC

TCCTC

592
6163
0.99141297
TCAGGT
94483959
94483976
107730
107747
18
0.13188801

TCTGAC

CTCCTC

593
6164
0.95402775
TTCAGG
94483958
94483976
107730
107748
19
0.0945028

TTCTGA

CCTCCTC

594
6165
0.99038866
TCAGGT
94483959
94483975
107731
107747
17
0.1308637

TCTGAC

CTCCT

595
6166
0.98818288
TTCAGG
94483958
94483975
107731
107748
18
0.12865793

TTCTGA

CCTCCT

596
6167
0.96431084
TTCAGG
94483958
94483974
107732
107748
17
0.10478589

TTCTGA

CCTCC

Example 4 the Splicing of ABCA4 is Disrupted in the c.5714+5G>A Variant and can be Partially Rescued Through the Use of Antisense Oligonucleotides

To confirm exon 40 skipping in the chr1: 94476351:C:T [hg19/b37] (c.5714+5G>A) variant, wild type and variant containing minigenes were constructed containing exons 39-41 and the corresponding introns, 38, 39, 40 and 41 (FIG. 4A). Minigenes were then transfected into HEK293T cells to examine the effect of the c.5714+5G>A variant on splicing. As seen in FIG. 4B, wildtype minigenes showed only exon 40 inclusion, represented by the upper band. c.5714+5G>A mutants, however, showed mostly exon 40 exclusion, represented by the lower band, and some exon 40 inclusion indicating the chr1:94476351:C:T [hg19/b37] mutation induces exon 40 skipping.

To examine the ability of antisense oligonucleotides to promote exon 40 inclusion in the c.5714+5G>A variant the minigenes above were co-transfected with antisense oligonucleotides having sequences set forth in SEQ ID NOs: 386-449 (see Table 7). Antisense oligonucleotides were tiled along exon 40 and the surrounding introns. Antisense oligonucleotides were cotransfected with the mutant minigene containing the c.5714+5G>A variant in HEK293T cells. RT-PCR was conducted to analyze the effect on the splicing of the minigene. Samples were measured by capillary electrophoresis. These results were quantified and are set forth in Table 7. Observing Table 7 it is clear that targeting the intronic regions surrounding exon 7 or exon 7 induces exon 7 inclusion in c.5714+5G>A variant minigenes (high percent spliced in/correctly (PSI) and change in PSI as compared to mutant PSI (dPSI)). These observations suggest antisense oligonucleotides targeting these regions or “hotspots” (positions 115149-115205, 115357-115378 and 115384-115450 in SEQ ID NO: 1; chr1: 94476501-94476557, 94476328-94476349 and chr1: 94476256-94476322), e.g., those complementary to a nucleobase sequence in SEQ ID NOs: 390-394 for hotspot 1 and SEQ ID NOs: 438-449 for hotspot 2, may be particularly useful in the treatment of retinal disease associated with exon 40 skipping (e.g., retinitis pigmentosa, cone-rod dystrophy, or Stargardt disease caused by the c.5714+5G>A mutation).

TABLE 7

Start
Stop

on
on

SEQ

SEQ
SEQ

ID
DG

Start Chr1
End Chr1
ID
ID

NO:
ID
PSI
Sequence
[hg19/b37]
[hg19/b37]
NO: 1
NO: 1
length
dPSI

386
4245
0.16351047
ACCAGG
94476566
94476584
115122
115140
19
0.00013772

CCTTAT

GTGGGA

A

387
4255
0.15063859
ACTAGA
94476561
94476580
115126
115145
20
−0.0127342

CCAGGC

CTTATGT

G

388
4209
0.14451858
CCCACT
94476558
94476574
115132
115148
17
−0.0188542

AGACCA

GGCCT

389
4267
0.11805199
GCCACA
94476544
94476564
115142
115162
21
−0.0453208

GCACAG

GGCCCA

CTA

390
4268
0.17173042
AGACCT
94476537
94476557
115149
115169
21
0.00835767

GGCCAC

AGCACA

GGG

391
4246
0.16997976
GCTCAC
94476526
94476544
115162
115180
19
0.00660701

CCCACA

GACCTG

G

392
4269
0.21080501
GCCGCC
94476517
94476537
115169
115189
21
0.04743226

CCAGCT

CACCCC

ACA

393
4270
0.4678908
CCACTT
94476509
94476529
115177
115197
21
0.30451805

CAGCCG

CCCCAG

CTC

394
4271
0.18572087
AATTGA
94476501
94476521
115185
115205
21
0.02234812

GTCCAC

TTCAGC

CGC

395
4227
0.07507214
AACAGG
94476495
94476512
115194
115211
18
−0.0883006

AATTGA

GTCCAC

396
4247
0.04474524
CATCAA
94476491
94476509
115197
115215
19
−0.1186275

CAGGAA

TTGAGT

C

397
4210
0.03055414
GGCATC
94476489
94476505
115201
115217
17
−0.1328186

AACAGG

AATTG

398
4228
0.11932321
CTGGGC
94476486
94476503
115203
115220
18
−0.0440495

ATCAAC

AGGAAT

399
4248
0.14264007
CTCACC
94476481
94476499
115207
115225
19
−0.0207327

TGGGCA

TCAACA

G

400
4211
0.06308102
CTCCTC
94476478
94476494
115212
115228
17
−0.1002917

ACCTGG

GCATC

401
4212
0.08435033
GTGCTC
94476475
94476491
115215
115231
17
−0.0790224

CTCACC

TGGGC

402
4256
0.08861896
GCAGAG
94476470
94476489
115217
115236
20
−0.0747538

TGCTCCT

CACCTG

G

403
4249
0.06416822
GGATTT
94476464
94476482
115224
115242
19
−0.0992045

GCAGAG

TGCTCCT

404
4257
0.06926567
CCAGTG
94476454
94476473
115233
115252
20
−0.0941071

GAACGG

ATTTGC

AG

405
4213
0.01971552
GTCCCA
94476451
94476467
115239
115255
17
−0.1436572

GTGGAA

CGGAT

406
4214
0.0438786
AGGTCC
94476449
94476465
115241
115257
17
−0.1194942

CAGTGG

AACGG

407
4229
0.02575892
ATCAGG
94476446
94476463
115243
115260
18
−0.1376138

TCCCAG

TGGAAC

408
4230
0.13447573
TCCCAA
94476441
94476458
115248
115265
18
−0.028897

TCAGGT

CCCAGT

409
4231
0.05533741
TCTTCCC
94476438
94476455
115251
115268
18
−0.1080353

AATCAG

GTCCC

410
4272
0.01480694
ACAGGT
94476432
94476452
115254
115274
21
−0.1485658

TCTTCCC

AATCAG

GT

411
4258
0.07816009
GGCAAA
94476427
94476446
115260
115279
20
−0.0852127

CAGGTT

CTTCCC

AA

412
4232
0.14657467
CATGGC
94476424
94476441
115265
115282
18
−0.0167981

AAACAG

GTTCTT

413
4215
0.04375712
ACCATG
94476422
94476438
115268
115284
17
−0.1196156

GCAAAC

AGGTT

414
4216
0.02380441
CCACCA
94476420
94476436
115270
115286
17
−0.1395683

TGGCAA

ACAGG

415
4233
0.03588861
CCACCA
94476417
94476434
115272
115289
18
−0.1274841

CCATGG

CAAACA

416
4217
0.08374322
CCCTTCC
94476412
94476428
115278
115294
17
−0.0796295

ACCACC

ATGG

417
4234
0.10068959
CACCCC
94476409
94476426
115280
115297
18
−0.0626832

TTCCAC

CACCAT

418
4235
0.0860025
AGTACA
94476402
94476419
115287
115304
18
−0.0773703

CCACCC

CTTCCA

419
4259
0.03802999
GAGGAA
94476397
94476416
115290
115309
20
−0.1253428

GTACAC

CACCCC

TT

420
4250
0.11174784
GGTCAG
94476391
94476409
115297
115315
19
−0.0516249

GAGGAA

GTACAC

C

421
4218
0.10316017
CAGGGT
94476388
94476404
115302
115318
17
−0.0602126

CAGGAG

GAAGT

422
4219
0.21595241
CCAGCA
94476384
94476400
115306
115322
17
0.05257966

GGGTCA

GGAGG

423
4236
0.18745955
CTGGAC
94476379
94476396
115310
115327
18
0.0240868

CAGCAG

GGTCAG

424
4260
0
GTGGCG
94476373
94476392
115314
115333
20
−0.1633728

CTGGAC

CAGCAG

GG

425
4237
0.09913076
GAAGAA
94476367
94476384
115322
115339
18
−0.064242

GTGGCG

CTGGAC

426
4238
0.0837757
GAGGAA
94476364
94476381
115325
115342
18
−0.0795971

GAAGTG

GCGCTG

427
4261
0.09184707
ATTGGG
94476357
94476376
115330
115349
20
−0.0715257

AGAGGA

AGAAGT

GG

428
4220
0.13158774
CCATTG
94476355
94476371
115335
115351
17
31 0.031785

GGAGAG

GAAGA

429
4239
0.08859458
GTACCA
94476352
94476369
115337
115354
18
−0.0747782

TTGGGA

GAGGAA

430
4273
0.07765108
CATGGA
94476345
94476365
115341
115361
21
−0.0857217

TGTACC

ATTGGG

AGA

431
4251
0.04522755
GTGTGG
94476339
94476357
115349
115367
19
−0.1181452

CATGGA

TGTACC

A

432
4221
0.12038155
AGGGTG
94476336
94476352
115354
115370
17
−0.0429912

TGGCAT

GGATG

433
4252
0.18419996
GGCCCA
94476331
94476349
115357
115375
19
0.02082721

GGGTGT

GGCATG

G

434
4240
0.29185317
ACTGGC
94476328
94476345
115361
115378
18
0.12848042

CCAGGG

TGTGGC

435
4262
0.09500995
TGAGCT
94476318
94476337
115369
115388
20
−0.0683628

GCCCAC

TGGCCC

AG

436
4263
0.11642409
TGCCCT
94476313
94476332
115374
115393
20
−0.0469487

GAGCTG

CCCACT

GG

437
4264
0.06303642
CTGGAT
94476308
94476327
115379
115398
20
−0.1003363

GCCCTG

AGCTGC

CC

438
4222
0.28020735
TTCTGG
94476306
94476322
115384
115400
17
0.11683459

ATGCCC

TGAGC

439
4241
0.19171274
GTCCAG
94476300
94476317
115389
115406
18
0.02833999

TTCTGG

ATGCCC

440
4223
0.28203905
TAAGGT
94476296
94476312
115394
115410
17
0.1186663

CCAGTT

CTGGA

441
4242
0.18281706
GTATAA
94476293
94476310
115396
115413
18
0.01944431

GGTCCA

GTTCTG

442
4253
0.22976438
GTGGGT
94476289
94476307
115399
115417
19
0.06639163

ATAAGG

TCCAGT

T

443
4243
0.24376363
GAAATG
94476278
94476295
115411
115428
18
0.08039088

ACCATG

TGGGTA

444
4274
0.11565453
TGAGGA
94476270
94476290
115416
115436
21
−0.0477182

AAGAAA

TGACCA

TGT

445
4254
0.18343226
GCTCCT
94476265
94476283
115423
115441
19
0.02005951

GAGGAA

AGAAAT

G

446
4224
0.25878428
GGGCTC
94476263
94476279
115427
115443
17
0.09541153

CTGAGG

AAAGA

447
4244
0.19718093
GTGGGG
94476260
94476277
115429
115446
18
0.03380818

CTCCTG

AGGAAA

448
4225
0.22573324
GAGTGG
94476258
94476274
115432
115448
17
0.06236049

GGCTCC

TGAGG

449
4226
0.17536592
TGGAGT
94476256
94476272
115434
115450
17
0.01199317

GGGGCT

CCTGA

OTHER EMBODIMENTS

Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

	Number	Date	Country
Parent	PCT/CA2020/050954	Jul 2020	US
Child	17572321		US

OLIGONUCLEOTIDE THERAPY FOR STARGARDT DISEASE

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE

Provisional Applications (1)

Continuations (1)