WO2025166253A1 - Compositions and methods for treating usher syndrome - Google Patents

Abstract

As described below, the disclosure features compositions and methods for treating the deleterious effects of mutations resulting in Usher Syndrome. For example, antisense oligonucleotides targeting USH2A resulting in exon skipping and correcting splicing are used to treat individuals suffering from Usher Syndrome, type 2A and/or its symptoms. Compositions and kits containing antisense oligonucleotides of the disclosure are also provided.

Description

COMPOSITIONS AND METHODS FOR TREATING USHER SYNDROME

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/549,313, filed February 2, 2024, which is incorporated herein by reference in its entirety for all purposes.

BACKGROUND

Usher syndrome (USH) is the most common inherited form of combined deaf-blindness.

Mutations in USH2A account for half of all USH cases. However, the size of USH2A (~15.6Kb) limits gene therapy options since it exceeds the capacity of single or even dual vectors. As such, the large size of USH2A presents challeges for treatments. There is no cure for USH syndrome. Although mechanical devices such as hearing aids and cochlear implants can alleviate auditory deficiencies, there are few if any biological treatments for auditory or visual dysfunction in Usher patients.

Therefore, compositions and methods for treating USH2A and its symptoms, z.e., hearing and vision loss, are needed.

SUMMARY

As described below, the present disclosure provides antisense oligonucleotides (AONs or ASOs, used interchangeably herein) for the treatment of diseases and disorders associated with the deleterious effects of Usher Syndrome, Type 2 A (USH2A).

One aspect of the disclosure provides an antisense oligonucleotide (ASO) that includes 8-40 nucleotides or modified nucleotides, wherein the oligonucleotide is at least 80% complementary to a nucleic acid sequence in an USH2A allele comprising a mutation associated with aberrant splicing. In an embodiment of the antisense oligonucleotide described here, the oligonucleotide comprises a modified linkage selected from the group consisting of methylphosphonate, phosphodiester, phosphorodithioate, phosphorothioate, and phosphotriester linkages. Some embodiments of the described antisense oligonucleotide comprise a modified linkage that is a phosphorothioate linkage. In additional embodiments, the oligonucleotide comprises at least one modified sugar moity. For example, the modified sugar moiety is a 2'-O-methyl, a 2'-methoxyethoxy, a 2'-O-methoxyethyl, a 2'- dimethylaminooxyethoxy, a 2'-dimethylaminoethoxyethoxy, a 2'-fluoro, or a 2'-acetamide modification group. In one embodiment, the modified sugar moiety can be on every sugar moity of the antisense oligonucleotide. Other embodiments of the antisense oligonucleotides described here provide oligonucleotides having a modified nucleobase, where the modified nucleobase can comprise, for example, a locked nucleic acid (LNA) nucleobase. Additional embodiments of the disclosure provide antisense oligonucleotides that are a morpholino, a thiomorpholino, or a peptide nucleic acid. In further embodiments, the antisense oligonucleotides of the disclosure target a nucleic acid sequence having an USH2A mutation, where the mutation is in USH2A exon 6, exon 19, exon 20, or combinations thereof. In some embodiments, the mutation is a c.4338_4339del (p. Cysl447Gln/s^,Ter29) mutation or a C.949OA (p.Arg371=) mutation. Various embodiments provide for antisense oligonucleotides that comprise, consist essentially of, or consist of a nucleic acid sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of any one of those listed in TABLE 4, TABLE 5, and TABLE 6. Other embodiments are directed to antisense oligonucleotides that comprise, consist essentially of, or consist of a nucleic acid sequence selected from the group consisting of any one of those listed in TABLE 4, TABLE 5, and TABLE 6. In some embodiments, the ASOs described here increase USH2A expression or activity. For example, USH2A expression or activity is increased in the eye and/or in the ear of a subject when antisense oligonucleotides of the disclosure are used. Further embodiments of the disclosure provide for antisense oligonucleotides where the oligonucleotide comprises DNA residues, RNA residues, modified DNA or RNA residues, or combinations of any of these.

Another aspect of the disclosure provides a set of antisense oligonucleotides comprising two or more of the antisense oligonucleotides described here, for example, in TABLE 4, TABLE 5, and TABLE 6.

In a further aspect, a cell (or an isolated cell) comprising any one or more antisense oligonucleotides described here, for example, in TABLE 4, TABLE 5, and TABLE 6, and a mutation associated with Usher Syndrome, type 2A, is provided. Some embodiments are directed to the isolated cell comprising a c.4338_4339del (p. Cysl447Gln/s^,Ter29) mutation and/or a c.949C>A (p.Arg371=) mutation. In other embodiments, the cell is of an inner ear or a retina. Further embodiments provide for cells derived from a subject having or suspected of having Usher Syndrome, type 2A.

Various aspects of the disclosure are directed a pharmaceutical composition comprising an effective amount of the antisense oligonucleotides described here or the set of antisense oligonucleotides described here, and a pharmaceutically acceptable excipient.

One aspect disclosed here is to a method of restoring wild-type splicing of an USH2A allele comprising a mutation associated with Usher Syndrome, type 2A in a cell. In an embodiment desribed here, the method comprises contacting the cell with an effective amount of the antisense oligonucleotides disclosed here, thereby restoring wild-type splicing. Another embodiment of such a method provides a method where the cell is heterozygous or homozygous for the mutation associated with Usher Syndrome, type 2A. In other embodiments of the method, the mutation is in a splice acceptor site, a splice donor site, a splicing regulatory element, or combinations thereof. Further embodiments provide for a splicing regulatory element that is an exonic splicing enhancer (ESE). In various embodiments of the method described here, the mutation is a c.4338_4339del (p. Cysl447Gln/s^,Ter29) mutation or a c.949C>A (p.Arg371=) mutation.

In further aspects described here, a method of treating Usher Syndrome, type 2A in a subject is provided. Some embodiments directed to such methods comprise administering to the subject an effective amount of an antisense oligonucleotide disclosed here (e.g., TABLE 4, TABLE 5, and TABLE 6). In additional embodiments, the subject comprises a mutation associated with Usher Syndrome, type 2A. Other embodiments are directed to the Usher Syndrome, type 2A where the mutation is a c.4338_4339del (p. Cysl447Gln s^,Ter29) mutation or a C.949OA (p.Arg371=) mutation. In other embodiments, such methods of treating Usher Syndrome, type 2A in a subject are provided where the subject is heterozygous or homozygous for the mutation.

One aspect of the disclosure provides a method for treating a subject suffering from Usher Syndrome, type 2A, wherein the method comprises administering to the subject one or more of the described antisense oligonucleotides that bind to a target sequence selected from the group consisting of those listed in TABLE 1, TABLE 2, and TABLE 3. In some embodiments, such methods treat a subject having a homozygous or a heterozygous mutation associated with Usher Syndrome, type 2A.

Additional aspects of the disclosure provide a kit comprising the antisense oligonucleotide described here (e.g., TABLE 4, TABLE 5, and TABLE 6) or a set of such antisense oligonucleotides and directions for administering the antisense oligonucleotide to a subject.

Compositions and articles defined by the disclosure were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By a “nucleobase oligomer” is meant a compound that includes a chain of at least eight nucleobases joined together by linkage groups. Included in this definition are natural and non-natural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic acids. Numerous nucleobases and linkage groups may be employed in the nucleobase oligomers of the disclosure, including those described herein.

A “target sequence” comprises the nucleotide sequence of any of the sequences below or has at least about 85% (e.g., 90%, 95%, 97%, 98%, 99%, 100%) identity to such sequence. For example, a target sequence as used herein can include mutations in exons 6, 19, and 20 of the USH2a gene, such as an out-of-frame mutation (c.4338_4339del (p.Cysl447Gln s^,Ter29) and USH2A c.949C>A (p.Arg317=) mutation. Exemplary target sequences are as follows (Legend: underline=AG / GT, acceptor and donor sites; italics= mutation site):

TABLE 1: EXON 19 TARGET SEQUENCES

TABLE 2: EXON 20 TARGET SEQUENCES

TABLE 3: EXON 6 TARGET SEQUENCES

By “antisense oligonucleotide” or “ASO” or “AON”, used interchangeably, is meant a nucleobase oligomer, where the ASO is at least partially complementary to a target sequence, such as an USH2A target sequence. In some embodiments, the ASO is complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases of a target sequence. In some embodiments, the

ASO comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleobases that fail to form a complementary pair with the target sequence. The antisense oligonucleotide can contain modified bases, a modified backbone, or any other modification described herein or known in the art. Provided here are tables of antisense oligonucleotides directed against USH2A, where the ASOs are 25 basepairs (bp) in length with a melting temperature (Tm) of greater than 48°C (>48°C). Such ASOs are useful for targeting USH2A mutations or variants present in a human genome.

“Antisense oligonucleotide set” means a number of oligonucleotides that can be used, for example, alone or in a therapeutic composition. A set of anti-sense oligonucleotide set could comprise at least 2 or more ASOs (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000); or 2500 or fewer ASOs (e.g.,

2250, 1950, 1900, 1850, 1800, 1750, 1700, 1650, 1600, 1550, 1450, 1400, 1350, 1300, 1250, 1200, 1150, 950, 850, 750, 650, 550, 450, 350, 275, 225, 175, 150, 125, 95, 85, 75, 65, 55, 45, 35, 25, 19, 17, 15, 13, 11, 9, 7, 5, 3).

TABLE 4: ANTISENSE OLIGONUCLEOTIDES TARGETING USH2A EXON 19

TABLE 5: ANTISENSE OLIGONUCLEOTIDES TARGETING EXON 20

TABLE 6: ANTISENSE OLIGONUCLEOTIDES TARGETING USH2A EXON 6

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. “Complementary” as used here refers to nucleotide bases linked by a hydrogen bond on opposite stranes of DNA or doublestranded RNA. For example, adenine and thymine in DNA (uracil in RNA) are complementary nucleobases that pair through the formation of hydrogen bonds, and guanine is a complementary base of cytosine.

By “polypeptide” as used here, is meant a functional protein or a functional fragment thereof, which is represented by one or more of the amino acid sequences described here. A “functional fragment” is used here to mean a portion of a polypeptide or protein that has at least some activity (z.e., 50% or greater (e.g., 60%, 70%, 80%, 90%, 100%); 100% or less (e.g., 95%, 85%, 75%, 65%, 55%; or 50%-100% (e.g., 51%-99%; 52%-98%; 53%-97%; 54%-96%; 55%-95%; 56%-94%; 57%- 93%; 58%-92%; 59%-91%; 60%-90%; 61%-89%; 62%-88%; 63%-87%; 64%-86%; 65%-85%; 66%- 84%; 67%-83%; 68%-82%; 69%-81%; 70%-80%; 71%-79%; 72%-78%; 73%-77%; 74%-76%) of a wildtype or full length protein. Functional fragments can have conservative substitutions of sequences disclosed here.

By “promoter” is meant a polynucleotide sufficient to direct transcription. In some embodiments, a promoter directs expression in the inner ear, e.g., in a hair cell of the inner ear. In some embodiments, the promoter is a heterologous promoter, for example, any one of the following: CMV promoter, a CBA promoter, a CASI promoter, a PGK promoter, a EFl promoter, an alpha9 nicotinic receptor promoter, a prestin promoter, a KCNQ4 promoter, a Myo7a promoter, a Myo6 promoter, a Gfil promoter, a Vglut3 promoter, Atohl and U7 promoter.

By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.

By “agent” is meant a peptide, nucleic acid molecule, or small compound. In one embodiment, the agent is an inhibitory nucleic acid molecule, such as an antisense oligonucleotide.

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In one embodiment, the disease affects hearing and vision and is associated with USH2A mutations or variants in a mammalian genome.

By “alteration” is meant a change (increase or decrease) in the sequence, expression levels, or activity of a gene or polypeptide as detected by standard art known methods including but not limited to those described here. As used here, an alteration includes a 10% change or greater (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100) in expression levels, 100% change or less (e.g., 95, 85, 75, 65, 55, 45, 35, 25, 15, 5) in expression levels, or a 5%-100% change (e.g., 6%-99%; 7%-98%; 8%-97%; 9%-96%; 10%- 95%; l l%-94%; 12%-93%) in expression levels.

By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence, or amount of the analyte to be detected. For example, a mutation in an USH2A polynucleotide or protein is detected.

By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an EUISA), biotin, digoxigenin, or haptens.

By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include Usher Syndrome, e.g., USH2A, or any genetic disorder associated with a mutation in the USH2A gene, not limited to exons 6, 19, and 20, where the genetic disorder results in a decrease in or loss of hearing and/or vision in an individual.

By “exon skipping” as used here is meant as the exclusion of an aberrant exon along adjacent exons when single exon skipping does not restore proper reading frame. Exon skipping involves inducing, producing, or increasing production within a cell of a mature mRNA that does not contain a particular exon that would normally be present in the mature mRNA without exon skipping. In some embodiments, “aberrant exon” refers to the presence of at least one mutation in an exon, e.g., exon 6, exon 19, exon 20, or combinations thereof (e.g., exons 19 and 20), in the USH2A mRNA. By “aberrant exon” or “aberrant USH2A exon” is meant the presence of at least one mutation in an exon of USH2A. such as but not limited to exon 6, exon 19, and exon 20.

By “effective amount” is meant the amount of an agent of the disclosure required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present disclosure for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount of an antisense oligonucleotide is the amount required to block abnormal splicing in a cell expressing an USH2A polynucleotide comprising a mutation, where the antisense oligonucleotide stably hybridizes to the target sequence in the RNA molecule under physiologically acceptable conditions.

The disclosure provides a number of targets that are useful for the development of highly specific agents or drugs to treat a disease or disorder characterized by the methods delineated here. In addition, the methods of the disclosure provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the disclosure provide a route for analyzing virtually any number of agents or compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains at least 10% or greater (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the entire length of the reference nucleic acid molecule or polypeptide; 99% or less (e.g., 95%, 85%, 75%, 65%,

55%, 45%, 35%, 25%, 15%, 5%); or 5%-99% (e.g., 6%-98%; 7%-97%; 8%-96%; 9%-95%; 10%-

94%; 11%-93%; 12%-92%; 13%-91%; 14%-90%; 15%-89%; 16%-88%; 17%-87%; 18%-86%; 19%-

85%; 20%-84%; 21%-83%; 22%-82%; 23%-81%; 24%-80%; 25%-79%; 26%-78%; 27%-77%; 28%- 76%; 29%-75%; 30%-74%; 31%-73%; 32%-72%; 33%-71%; 34%-70%; 35%-69%; 36%-68%; 37%- 67%; 38%-66%; 39%-65%; 40%-64%; 41%-63%; 42%-62%; 43%-61%; 44%-60%; 45%-59%; 46%-

58%; 47%-57%; 48%-56%; 49%-55%; 50%-54%; 51%-53%. A fragment can contain 10 or more nucleotides or amino acids (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000); 1500 or fewer nucleotides or amino acids (e.g., 1050, 950, 850, 750, 650, 550, 450, 350, 250, 150, 95, 85, 75, 65, 55, 45, 35, 25, 15).

“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. The antisense oligonucleotides of the disclosure in some embodiments, are “substantially complementary”, where there are some mismatches in the antisense oligonucleotide sequence that still allow for hybridization and functionality, z.e., inducing exon skipping or correction.

By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein. In one embodiment, an antisense RNA comprises at least about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or more nucleobases complementary to a target sequence. In one embodiment, the oligonucleotide comprises 18-22 nucleobases.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this disclosure is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the disclosure is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is free from the proteins and naturally-occurring organic molecules with which it is naturally associated by at least 60% or greater, by weight, (e.g., 70%, 80%, 90%, 100%); 100% or less, by weight, (e.g., 99%, 97%, 95%, 85%, 75%, 65%, 55%); or 50%-100% (e.g., 51%-99%; 52%-98%; 53%-97%; 54%-96%; 55%-95%; 56%-94%; 57%-93%; 58%-92%; 59%-91%; 60%-90%; 61%-89%; 62%-88%; 63%-87%; 64%-86%; 65%-85%; 66%-84%; 67%-83%; 68%-82%; 69%-81%; 70%-80%; 71%-79%; 72%-78%; 73%-77%; 74%-76%). The preparation can be at least 80%, 85%, 90%, 95%, 97%, 99%, or 100%, by weight, a polypeptide of the disclosure. An isolated polypeptide of the disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By “mutation” is meant an alteration in a sequence of a polypeptide or polynucleotide relative to a reference sequence. Exemplary mutations described herein are in the sequence of a Ush2a polypeptide or polynucleotide. In some embodiments, the mutation is c.4338_4339delCT (p.Cysl447Gln s^,Ter29). In other embodiments, the mutation is NM_206933.2 C.949OA (p.Arg371=).

As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition, such as Usher Syndrome, Usher Syndrome, Type 2, or Usher Syndrome, Type 2A.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition or agent. In one embodiment, a reference refers to the expression of an USH2A polynucleotide or polypeptide in an untreated control cell expressing an USH2A polynucleotide or polypeptide comprising a mutation associated with USH2A. In another embodiment, a reference refers to the expression of an USH2A polynucleotide or polypeptide in a cell expressing a wild-type USH2A polynucleotide or polypeptide.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 10, 15 or 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, and about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 5, 10, 15, 20, or 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, and about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By “specifically binds” is meant a polynucleotide that recognizes and binds a complementary sequence of the disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes other polynucleotide sequences. An antisense oligonucleotide can specifically bind its perfect complement or a sequence to which it is not perfectly complementary. In one embodiment, the antisense oligonucleotide specifically binds a polynucleotide that comprises 1, 2, 3, 4, 5 or more bases that are not perfectly complementary to the anti-sense oligonucleotide.

Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant to pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. U. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, or at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In a further embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, or less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, at least about 42° C, or at least about 68° C. In one embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a further embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Uaboratory Manual, Cold Spring Harbor Uaboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity or more (e.g., 60%, 70%, 80%, 90%); 100% or less (e.g., 99%, 97%, 95%, 85%, 75%, 65%, 55%) to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Such a sequence can be at least 50%-100% (e.g., 51 %-99%; 52%-98%; 53%-97%; 54%-96%; 55%-95%; 56%-94%; 57%-93%; 58%-92%; 59%-91%; 60%-90%; 61%-89%; 62%-88%; 63%-87%; 64%-86%; 65%-85%; 66%-84%; 67%-83%; 68%-82%; 69%-81%; 70%-80%; 71%-79%; 72%-78%; 73%-77%; 74%-76%) identical at the amino acid level or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BUAST, BESTFIT, GAP, or PIEEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.

Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^-3 and e ¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith, such as, Usher Syndrome (e.g., USH2A). It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

By “Ush2A polypeptide” is meant an amino acid sequence or fragment thereof having at least about 85% amino acid sequence identity to the sequence provided at NCBI Accession No. NCBI Ref: NP_996816.3, which is reproduced at FIGs. 10A-10B. In some embodiments, the sequence comprises one or more alterations relative to a reference sequence. In some embodiments, the alterations are a c.4338_4339del (p. Cysl447Gln/s^,Ter29) mutation or a c.949C>A (p.Arg371=) mutation. In embodiments, the Ush2A polypeptide binds harmonin and functions in hearing.

By “Ush2A polynucleotide” is meant a nucleic acid sequence encoding an Ush2A polypeptide. An exemplary Ush2A polynucleotide sequence is provided at FIGs 11A-11G.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0. 1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B show the generation of 2-O-Methyl RNA ASOs targeting exons 19 and 20 of USH2A. FIG. 1A is a schematic of the dual exon skipping strategy. The ASO exon-skipping strategy developed can be used for patients with pathogenic variants in USH2A exon 19 or 20, a region that overlaps more than 45 known mutations, including the out of frame c.4338_4339delCT (p.Cysl447Gln s^,Ter29) mutation, an USH2A founder mutation and a major cause of Usher Syndrome in French-Canadians. FIG. IB provides a table of the generated human ASOs that target the acceptor site of USH2A exons 19 and 20. To restore the reading frame and the synthesis of the usherin protein, ASOs were designed to be 25 basepairs (bp) in length with a 2-O-Methyl RNA modification. A functional group, z.e., phosphorothioate (PTO) bonds were added to the original sequence to obtain ASOs resistant to nuclease degradation. ASOs were purified by HPLC and produced by the Microsynth company based in Switzerland (microsynth.com/home-ch.html).

FIGs. 2A-2B demonstrate modeling of exons 19-20 skipping in humans. In-frame deletion of USH2A exon 19 and 20 in humans was performed and resulted in expression of a slightly shorter yet functional usherin protein (USH2A) encoded by USH2A gene (105 amino acids out of 5202 amino acids). FIG. 2A presents a schematic diagram of usherin, USH2A, protein domains. The linear usherin protein structure, from left to right, includes: a signal peptide, a Laminin G-Like domain (Lam GL), a Laminin N-Terminal domain (Lam NT), a series of Laminin EGF-Like domains (EGF Lam 1- 10), a series of Fibronectin III domains (FN3 1-4), a couple Laminin G domains (Lam G), a series of Fibronectin III domains (FN3 5-18; FN3 19-34), a transmembrane domain (TM), and a PDZ -binding motif (PDZ), where a horizontal bar depicts the region encoded by USH2A exons 16 to 25 spanning FN3 1-4 and the first Lam G domain, which encompasses the region encoded by exons 19 to 20. Exon 19-20 skipping leads to excision of a full and single fibronectin 3 domain. FIG. 2B provides an in silico assessment of the region encoded by exons 16-21 (Swissmodel) confirming the full excision of one fibronectin 3 domain (pseudo-USH2A^419-20; right side) and limited modification of the nearby fibronectin domains compared to the Wild-Type protein (USH2A^FL; left side).

FIGs. 3A-3B show ASO tiling the cryptic acceptor site to mediate USH2A exon 19 and/or exon 20 skipping in human retinoblastoma cells (WERI-RB1). FIG. 3A demonstrates via RT-PCR that USH2A exon 19 or exon 19+20 skipping was mediated by the ASOs designed for USH2A exon 19. FIG. 3B provides RT-PCR results showing that ASOs designed for USH2A exon 20 mediate USH2A exon 20 skipping. FIGs. 3C-3D show ASOs tiling the cryptic donor site to mediate USH2a exon 19 and/or 20 skipping in WERI-RB1 cells. FIG. 3C illustrates ASOs designed for USH2A exon 19 that mediate USH2A exon 19 or exon 19+20 skipping. FIG. 3D shows via RT-PCR ASOs designed for USH2A exon 20 that mediate USH2A exon skipping.

FIG. 4A presents dual exon skipping (USH2A exon 19+20) using ASO cocktails (e.g., ASO 1+4; ASO 1+5; ASO 1+6; ASO 2+4; ASO 2+5; ASO 2+6; ASO 3+4; ASO 3+5; ASO 3+6) in WERI- RB1 cells. FIG. 4B illustrates a cocktail of ASOs that efficiently leads to dual exon skipping (USH2A Exon 19 + 20).

FIG. 5 shows that cocktails of ASOs (three or all ASOs combined) efficiently lead to dual exon skipping (USH2A Exon 19 + 20) in WERI-RB1 cells.

FIG. 6 demonstrates that controls or control conditions do not lead to USH2A exon skipping. Scrambled ASO, untreated cells, and mock cells are not able to lead to USH2A exon 19 or 20 skipping, validating the specificity of the ASOs described here.

FIGs. 7A-7D illustrate ASOs leading to dual exon 19+20 skipping in control and hUSH2A c.4338-4339delCT mature retinal organoids (ROs), modeling of dual USH2A exon skipping in humans. FIG. 7A exemplifies how a blood sample was collected from a USH2A c.4338-4339delCT patient and whole exome sequencing was performed to determine the presence of other pathogenic mutations. Patient derived induced pluripotent stem cell (iPSC) lines as well as CRISPR/Cas9 corrected isogenic cell lines were generated. These cell lines are for generating retinal and inner ear organoids and validating the ASOs to specifically target the inner ear and the retina. FIG. 7B shows different maturation stages of retinal organoids (RO) at Day 60 (D60) and Day 90 (D90). FIG. 7C provides the results of RT-PCR performed on retinal organoids at D60 showing the high expression of the full length USH2A gene. FIG. 7D presents RT-PCR products that showed exon 19, 20 or 19+20 exons skipping after transfection of ASOs on both wild-type and hUSH2A c.4338-4339delCT ROs at 240 days (D240) in culture. Samples were treated with ASOs for one month. Each RT-PCR result corresponds to only one RO. Concentrations of ASOs used were lOpM for a single transfection or 5pM of each ASO for dual transfections.

FIG. 7E presents ASOs leading to dual exon 19+20 skippin in control and hUSH2Ac.4338- 4339delCT mature inner ear organoids (IEOS). RT-PCR products showed dual exons 19+20 skipping after transfection of ASOs on both wild-type and hUSH2A c.4338-4339delCT IEOs at 92 days (D92) in culture. Samples were treated with ASOs for 15 days. Each RT-PCR result corresponds to only one IEO. Concentrations of the ASOs used were lOpM for a single transfection or 5pM of each ASO for dual transfections.

FIGs. 8A-8E show the generation of zebrafish models for the human exon 20 c.4338_4339del mutation (exon 19 in zebrafish) and dual exon skip. FIG. 8 A shows two zebrafish lines generated in view of the prior validation that zebrafish can be used as a USH2A animal model. Fish model 1 has an out-of-frame indel in exon 18-19 (equivalent to human exons 19-20), which mimics the human mutation and displays severe retinal and auditory phenotypes. Fish model 2 has an in-frame deletion of exons 18-19 in zebrafish (equivalent to human exons 19-20), which mimics the dual exon skipping strategy. Retinal, balance, and auditory function will be assessed as well as immunolocalization of the Ush2a protein. FIG. 8B illustrates indel models generated to mimic the human mutation, USH2A^lvb3 containing an 8 bp insertion and USH2A^lvb4 with one substitution and 2 bp deletion, both resulting in frame shift and truncation. FIG. 8C demonstrates the loss of optokinetic reflex in the mutant USH2A^lvb3 as compared to wild type (WT). FIG. 8D presents a schematic of the generation of the dual exon-skip strategy. FIG. 8E shows the PCR results using a template prepared from uninjected control embryos (lane 1, Control). Lanes 2 and 3 are from CRISPR mix 1 (E18 -1666f + E19+5833r gRNA) and mix 2 (E18 -1666f + E19+1578r gRNA), respectively. Mix 1 resulted in validated skips and a 250 bp amplicon.

FIGs. 9A-9D illustrate ASO development for USH2A c.949C>A of exon 6. FIG. 9A shows the design and screening results of ASOs 1-20 (US001-US020) of TABLE 6 targeting c.949C>A, with exonic splicing enhancer (ESE) prediction results shown below the USH2A exon 6 sequence. RT-PCR results of ASO initial screening (top panel) and fine-tuning screening (bottom panel) are presented in FIG. 9B. FIG. 9C provides RT-qPCR analysis of the rescue of normal splicing by initial (top panel: ASOs 1-9) and fine-tuning (bottom panel: ASOs 10-20) screenings. FIG. 9D provides RT- qPCR results demonstrating the correction of mis-splicing in a patient’s induced pluripotent stem cells (iPSCs). RT-qPCR results of experiments conducted by treating iPSCs with translation inhibitor cycloheximide (CHX) and negative control (top panel) and ASOs 5-7 and 15-20 (bottom panel) from the initial and fine-tuning screenings, which demonstrated correction of mis-splicing in patient iPSCs.

FIGs. 10A-10B provide the human Usherin isoform B precursor protein sequence (NCBI Ref: NP_996816.3; Uniprot Ref: 075445) used as the basis for antisense oligonucleotide design.

FIGs. 11A-11G provide the humin Usherin (USH2A), transcript variant 2, mRNA (NCBI Ref: NM_206933.4; Ensembl Ref: ENST00000307340.8, USH2A-201) of the coding sequence used as the basis for antisense oligonucleotide design. The genomic human Usherin sequence on chromosome 1, where the gene includes both introns and exons (NCBI Ref: NB_009497.2) is also used as the basis for antisense oligonucleotide design.

DETAILED DESCRIPTION OF THE DISCLOSURE

The disclosure features antisense oligonucleotides (ASOs) that block abnormal or aberrant splicing of an USH2A gene comprising a mutation associated with an Usher Syndrome, type 2A (USH2A)-related disorder, and methods of using such antisense oligonucleotides to correct the deleterious effects of genetic hearing and vision loss in those individuals suffering from USH2A having mutations in their mammalian genome.

The disclosure is based, at least in part, on the discovery of antisense oligonucleotides that restore or correct wild-type splicing of a USH2A gene having target mutations in exon 6, exon 19, exon 20 or any combinations threeof, of the USH2A gene, or that result in a functional transcript or polypeptide encoded by USH2A. The ASOs are designed to selectively block USH2A mutations and allow the production of a non-mutated, healthy pseudo-USH2A protein, z.e., usherin. In some embodiments, ASOs are based on an exon skipping strategy for individuals with pathogenic variants in exon 19 or 20, a region that overlaps over 45 known mutations, including the USH2A mutation NM_206933.4 c.4338_4339del (p. Cysl447Gln s^,Ter29) compared to wild-type USH2A (NG_009497.2), which is an USH2A founder mutation and major cause of Usher Syndrome in French-Canadians. Other embodiments are directed to ASOs designed to target USH2A variants of Usher Syndrome patients, such as those in Australia, France, and the Netherlands, including NM_206933.2 c.949C>A (p.Arg371=), compared to wild-type USH2A (NG_009497.2). This variant substitutes a cytosine at position 949 with an adenine in exon 6, where the splice site is in the Uaminin type IV domain, and a recurrent pathogenic variant that results in the truncation of exon 6 through activation of a cryptic splice donor site resulting in a synonymous pathogenic variant that does not change its amino acid in the coded protein (p.R317R). The disclosure provides exon skipping oligonucleotides and that can block USH2A mutations and those that splice correct c.949C>A, thus restoring normal gene function. These oligonucleotides can be used to treat Usher Syndrome, particularly Usher Syndrome, type 2A.

Usher Syndrome: USH2A-related Disorders

Usher syndrome (USH) is an autosomal recessive disorder affecting approximately 4-17 per 100,000 people and accounts for about 50% of all hereditary deaf-blindness cases. Usher syndrome, type 2A (USH2A)-related disorders are a result of genetic mutations in the USH2A gene, which plays a critical role in the development of the inner ear and light-sensitive tissue of the eyes, such as the retina. It is a genetic condition that is characterized by moderate to severe hearing loss from birth and progressive vision loss that begins in adolescence or adulthood.

The disclosure provides one or more antisense oligonucleotides (ASOs) (e.g., a set of antisense oligonucleotides) targeting USH2A exons 6, 19, and 20, and methods of using the antisense oligonucleotides for the treatment of USH2A-related diseases or disorders or symptoms thereof. The ASOs described here are designed based on the USH2A gene (USH2A gene, chromosome 1 [Homo sapiens] NCBI Ref Sequence: NG_009497.2), usherin protein encoded by USH2A (Usherin isoform B precursor [Homo sapiens] : NCBI Ref Sequence: NP_996816.3; Uniprot: 075445; FIGs. 10A-10B), and transcript (Usherin (USH2A), transcript variant 2 [Homo sapiens]: NCBI Reference Sequence: NM_206933.4; Ensembl: ENST00000307340.8 USH2A-201; FIGs. 11A-11G), all of which are incorporated here by reference in their entirety.

Antisense Oligonucleotides

In some embodiments, antisense oligonucleotides (ASOs) are provided that restore wild-type splicing of an USH2A gene having a mutation that leads to aberrant splicing of the gene. The ASO can comprise of an oligonucleotide sequence of 5 or more nucleotides (e.g., 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51); 50 or fewer nucleotides (e.g., 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4); or 5-40 nucleotides (e.g., 6- 39, 7-38, 8-37, 9-36, 10-35, 11-34, 12-33, 13-32, 14-31, 15-30, 16-29, 17-28, 18-27, 19-26, 20-25, 21- 24, 22-23), where the nucleotides are linked and/or modified. The antisense oligonucleotides of the disclosure comprises DNA residues, RNA residues, modified DNA or RNA residues, or combinations of any of these. In further embodiments, the ASOs comprise an oligonucleotide that is at least 80% (e.g., 82, 84, 86, 88, 90, 92, 94, 96, 98, 100) complementary to a nucleic acid sequence in an USH2A allele with a mutation associated with aberrant splicing. Some embodiments are directed to antisense oligonucleotides comprising a mutation in USH2A exon 6, exon 19, exon 20, or combinations thereof. For example, the mutation in USH2A can be c.4338_4339del (p. Cysl447Gln s^,Ter29) or NM_206933.2 C.949OA (p.Arg371=) in USH2A exon 20 and exon 6, respectively. In some embodiments, the genetic disorder present in an affected individual having mutations a region spanning USH2A exons 19 and 20 can be ameliorated if exon 19 and/or exon 20 is deleted or skipped allowing restoration of the normal reading frame. Other embodiments where the genetic disorder present in an affected individual having mutations in USH2A exon 6, which results in a truncation of exon 6 through activation of a cryptic splice donor site can be ameliorated if exon 6 and/or exon 7 is deleted or skipped allowing restoration of the normal reading frame. Exon skipping, or splice modulation in general, can be treated by antisense oligonucleotide therapy. Exon skipping is achieved using antisense oligonucleotides, which have the ability to hybridize to a sense target sequence, modulating splicing patterns of the target molecule. Antisense-mediated exon skipping has been shown to be useful for the restoration of a normal reading frame in genes impaired by mutations that alter undesirable gene splicing. This approach can be effective when the skipped exon is not critical for protein function, or when a non-productive exon is brought into play by a patient mutation.

Further embodiments of the disclosure include antisense oligonucleotides that comprise, consist essentially of, or consist of a nucleic acid sequence having at least 80% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, 100%) to a nucleotide sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 6, or modified antisense oligonucleotides thereof. Other embodiments provide for antisense oligonucleotides comprising, consisting essentially of, or consisting of a nucleic acid sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 6, or modified antisense oligonucleotides thereof. In some embodiments, the antisense oligonucleotides of the disclosure (e.g., TABLE 4, TABLE 5, and TABLE 6 or modified antisense oligonucleotides thereof), where the ASO increases USH2A expression or activity. Further aspects provide for antisense oligonucleotides that increase USH2A expression or activity in the eye, in the ear, or both. For example, USH2A expression or activity can increase in the retinal cells of the eye, in cells of the inner ear, or both. In some embodiments a set of antisense oligonucleotides can be used, where the set comprises two or more of the antisense oligonucleotides described here.

In one embodiment, an oligonucleotide or nucleobase oligomer of the disclosure comprises 2'- modified oligonucleotides where some or all intemucleotide linkages are modified to phosphorothioates or phosphodiester (PO). The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No. WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 Al).

As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred nucleobase oligomers useful in this disclosure include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone are also considered to be nucleobase oligomers.

Nucleobase oligomers that have modified oligonucleotide backbones or modified linkages include, for example, methylphosphonates, phosphodiesters, phosphotriesters, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

Nucleobase oligomers (e.g., ASOs) having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. In some embodiments, the antisense oligonucleotide described here is a morpholino, thiomorpholino, or a peptide nucleic acid. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

In other nucleobase oligomers, both the sugar and the intemucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with a complementary target sequence. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in “Peptide Nucleic Acids: Protocols and Applications” Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

In particular embodiments of the disclosure, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular -CH2-NH-O-CH2-, -CH2- N(CH3)-O-CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -O-N(CH3)-CH2-CH2-. In other embodiments, the oligonucleotides have morpholino backbone stmctures described in U.S. Pat. No. 5,034,506.

Nucleobase oligomers can also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2' position: -OH; F-; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, nucleobase oligomers include O[(CH2)_nO]_nCH3, O(CH2)_nOCH3, O(CH2)nNH2, O(CH2)_nCH3, O(CH₂)_nONH₂, and O(CH2)_nON[(CH2)_nCH3)]2, where n and m are from 1 to about 10. Other nucleobase oligomers include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Other embodiments described here include modifications such as 2'-O-methyl and 2'- methoxyethoxy ^'-O-C bC tOC fe, also known as 2'-O-(2 -methoxyethyl) or 2'-MOE). Another modification is 2'-dimethylaminooxyethoxy (i.e., O(CH2)2ON(CH3)2), also known as 2'-DMAOE). Other modifications include 2'-dimethylaminoethoxyethoxy, 2'-aminopropoxy (2'- OCH2CH2CH2NH2), 2'-fluoro (2'-F), and a 2'-acetamide. Some embodiments provide antisense oligonucleotides comprising a modified sugar moiety having a modification on every sugar moiety. Similar modifications can also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Nucleobase oligomers can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;

5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

Nucleobase oligomers can also include nucleobase modifications (or modified nucleobase) or substitutions. 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 other synthetic and natural nucleobases, such as methyl cytosine, 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5- propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5- halo (e.g., 5-bromo), 5 -trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8 -azaguanine and 8 -azaadenine; 7-deazaguanine and 7-deazaadenine; and 3- deazaguanine and 3 -deazaadenine. Other embodiments include a modified nucleobase comprising a locked nucleic acid (LNA) nucleobase. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebien, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the disclosure. These include 5-substituted pyrimidines, 6- azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. 5 -methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6° C-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2'-O-methoxyethyl or 2'-O-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.

Another modification of a nucleobase oligomer of the disclosure involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553- 6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 4: 1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 10: 1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 36:3651- 3654, 1995; Shea et al., Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference. The present disclosure also includes nucleobase oligomers that are chimeric compounds. “Chimeric” nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region or target sequence.

Chimeric nucleobase oligomers of the disclosure can be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

The nucleobase oligomers used in accordance with this disclosure can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art cab additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

The nucleobase oligomers of the disclosure can also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, enteral or parenteral administration (e.g., intravenous infusion, subcutaneous injection, intramuscular injection, intravitreal injection, intrathecal injection), oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference. The nucleobase oligomers of the disclosure encompass any pharmaceutically- or physiologically- acceptable salts, esters, or salts of such esters, or any other compound that, upon administration to an animal, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the disclosure, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the agent compound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., J. Pharma Sci., 66: 1-19, 1977). The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present disclosure. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the disclosure. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2- phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane- 1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-l,5-disulfonic acid, 2- or 3- phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds can also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides and other nucleobase oligomers, suitable pharmaceutically acceptable salts include (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (iv) salts formed from elemental anions such as chlorine, bromine, and iodine.

The present disclosure also includes pharmaceutical compositions and formulations that include the nucleobase oligomers or antisense oligonucleotides of the disclosure. The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral, enteral or parenteral (e.g., intraarterial, intracranial , intramuscular, intraperitoneal, intrathecal, intravenous, intraventricular, intravitreal, or subcutaneous via infusion or injection), oral, rectal, topical or other formulations.

Methods

In some embodiments, the disclosure provides a method of restoring wild-type splicing of an USH2A allele comprising a mutation associated with Usher Syndrome, type 2A in a cell. Such methods comprise: contacting the cell with an effective amount of the antisense oligonucleotides of the disclosure, thereby restoring wild-type splicing in the cell. In some aspects, the cell is in vitro or in vivo and/or the cell is heterozygous or homozygous for the mutation. The method associated with Usher Syndrome, type 2A is located in a splice acceptor site, a splice donor site, or a splicing regulatory element, such as an exonic splicing enhancer (ESE). Some embodiments provide a method of restoring wild-type splicing of an USH2A allele comprising at least one USH2A mutation. In one aspect, the USH2A mutation is in exon 6, exon 19, exon 20, or combinations thereof. Other aspects described here provide for an USH2A mutation that is a c.4338_4339del (p. Cysl447Gln s^,Ter29) or a C.949OA (p.Arg371=) mutation.

The disclosure provides methods of treating a disease and/or disorder or symptoms thereof associated with Usher Syndrome, for example, Usher Syndrome, type 2A. The USH2A-related disease or disorder can be treated by administering a therapeutically effective amount of an antisense oligonucleotide or a pharmaceutical composition comprising such an antisense oligonucleotide targeting an USH2A target sequence (for example, TABLEs 1-3) herein to a subject (e.g., a mammal such as a human) having or suspected of having an USH2A-related disease or disorder. Additional methods for treating a subject suffering from Usher Syndrome, type 2A described here, include those that comprise administering to the subject one or more antisense oligonucleotides described here that binds to a target sequence selected from the group consisting of those listed in TABLE 1, TABLE 2, and TABLE 3. Thus, one embodiment is a method of treating a subject suffering from or suspected of having a disease or disorder or symptom thereof associated with aberrant splicing in, for example, an USH2A gene. Such methods of treating described here include administering to the subject or mammal a therapeutic and/or effective amount of an agent described here sufficient to treat the disease or disorder or symptom thereof, under conditions such that the USH2A-related disease or disorder, or its symptoms, is treated. In some embodiments for treating an USH2A-related disease or disorder, the agent is an antisense oligonucleotide comprising an oligonucleotide of linked nucleotides or modified nucleotides. Embodiments of the disclosure can include antisense oligonucleotides comprising an oligonucleotide of 8-40 linked nucleotides or modified nucleotides. In further embodiments, the oligonucleotide is at least 80% complementary to a nucleic acid sequence in an USH2A allele having a mutation associated with aberrant splicing. Some embodiments provide for antisense oligonucleotides selected from TABLEs 4-6 or modified nucleotides. Methods using antisense oligonucleotides described here comprise administering to a subject suffering from, susceptible to an USH2A-related disease or disorder, or having symptoms thereof, in need of treatment, where the subject comprises or is suspected of having a mutation associated with Usher Syndrome, type 2A. In some aspects, the mutation is a located in USH2A exon 6, exon 19, exon 20, or any combinations thereof. Other aspects are directed to an USH2A-related disease or disorder in a subject, where the subject is heterozygous or homozygous for the mutation resulting in the USH2A- related disease or disorder. In further aspects of the disclosure, the subject treated for a USH2A- related disease or disorder has a c.4338_4339del (p. Cysl447Gln s^,Ter29) mutation and/or a C.949OA (p.Arg371=) mutation.

In some embodiments, the methods described here include administering to the subject (including a subject identified as in need of such treatment) an effective amount or a therapeutic amount of an antisense oligonucleotide described here, or a composition comprising such antisense oligonucleotide(s). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g, measurable by a test or diagnostic method).

The therapeutic methods of the disclosure (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the antisense oligonucleotides herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for an USH2A-related disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The compounds herein may be also used in the treatment of any other disorders in which aberrant splicing is implicated.

In one embodiment, the disclosure provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (marker) (e.g., a protein whose expression or activity is disrupted by aberrant splicing) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with aberrant splicing, in which the subject has been administered a therapeutic amount of an antisense oligonucleotide herein sufficient to treat the disease or symptoms thereof. The level, length or activity of the marker determined in the method can be compared to the level, length or activity of the marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status. In certain embodiments, a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In other embodiments, a pre-treatment level of marker in the subject is determined prior to starting treatment according to this disclosure; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Additional embodiments are directed to the use of an antisense oligonucleotide or set of antisense oligonucleotides (e.g., TABLEs 4-6) to restore wild-type splicing of an USH2A allele comprising a mutation associated with Usher Syndrome, type 2A in a cell, to treat an USH2A -related disease or disorder, or symptoms thereof, or to block aberrant splicing in a subject suffering from an USH2A -related disease or disorder by binding such antisense oligonucleotides to a USH2A target sequence, for example, TABLEs 1-3. Such antisense oligonucleotides described here, for example, the ASOs of TABLEs 4-6, can be used in the manufacture of a medicament for the treatment of an USH2A -related disease or disorder, such as Usher Syndrome, type 2A.

Therapy

Therapy can be provided wherever therapy associated with a genetic disorder is performed: at home, the doctor's office, a clinic, a hospital’s outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy’s effects closely and make any adjustments that are needed. The duration of the therapy depends on the kind of genetic disorder being treated, the age and condition of the patient, the type of the patient’s disease, and how the patient’s body responds to the treatment.

An antisense oligonucleotide or antisense nucleobase oligomer of the disclosure can be administered within a pharmaceutically-acceptable or physiologically acceptable carrier (e.g., diluent, excipient), in unit dosage form. Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from an USH2A-related disease or disorder, or symptoms thereof. Administration can begin before the patient is symptomatic. Any appropriate route of administration can be employed, for example, administration can be enteral, parenteral, intraarterial, intracapsular, intracistemal, intracranial, intramuscular, intranasal, intraorbital, intraperitoneal, intrathecal, intratympanic, intravenous, intraventricular, intravitreal, ophthalmic, subcutaneous, topical (e.g., aerosol, ointment, drops), or oral administration. For example, therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for antisense oligonucleotides include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, nanoparticles, nanolipids, and liposomes. Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or be oily solutions for administration in the form of eye, ear, or nasal drops, or as a gel.

The formulations can be administered to human patients or subjects in therapeutically effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. Some embodiments provide a dosage of an antisense oligonucleotide of the disclosure that depends on such variables as the type and extent of the USH2A- related disorder, the overall health status of the particular subject or patient, the formulation of the agent (e.g., antisense oligonucleotide), excipients, and its route of administration.

For any of the methods of application described above, an antisense oligonucleotide of the disclosure can be administered intravenously or is applied to the site of the needed intervention (e.g., by injection, by topical eye or ear drops).

Isolated Cells, Organoids, and Pharmaceutical Compositions

In some embodiments of the disclosure, an isolated cell or an organoid (e.g., inner ear, retinal) comprises an antisense oligonucleotide described here (e.g., TABLES 4-6) and a mutation associated with Usher Syndrome. Another embodiment provides an isolated cell or an organoid comprising an antisense oligonucleotide described here (e.g., TABLES 4-6) and an USH2A mutation. In some aspects, the USH2A mutation is a mutation found in exon 6, exon 19, exon 20, or combinations thereof. Other aspects provide for such isolated cells or organoids described here, where the mutation is a c.4338_4339del (p. Cysl447Gln s^,Ter29) mutation and/or a c.949C>A (p.Arg371=) mutation. In some embodiments, the isolated cell is a pluripotent stem cell. Other embodiments are directed to the isolated cell, where the cell is of the inner ear or of the retina. Additional embodiments provide for an organoid, where the organoid is an inner ear organoid or a retinal organoid. In further embodiments, an organoid or an embroyoid body of the disclosure can contain cells comprising an antisense oligonucleotide described here and a mutation associated with Usher Syndrome, type 2A, where the organoid or the embroyoid body are multi-cellular structures that contain organ-specific or embryonic cell types, such as those of the ear (e.g., inner ear) or the eye (e.g., retina).

Various embodiments described here are directed to a pharmecutical composition comprising an effective amount of an antisense oligonucleotide of TABLES 4-6, a set of antisense oligonucleotides of TABLES 4-6, or an isolated cell comprising an antisense oligonucleotide of TABLES 4-6, and a pharmaceutically acceptable or physiologically acceptable carrier (e.g., excipient, diluent).

Test Compounds and Extracts

In one aspect, the disclosure provides methods of screening for agents that have the ability to inhibit aberrant splicing. In various embodiments, a test compound can be an antisense oligonucleotide that targets an USH2A splice site (splice donor site or splice acceptor site) or a splice enhancer site. Additionally, other agents can be screened for the desired activity.

In general, agents, such as small molecule compounds, are known in the art or are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the disclosure. Compounds used in screens can include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.

Numerous methods are also available for generating random or directed synthesis (e.g., semisynthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. For example, a library of 8,000 novel small molecules is available, which was created using combinatorial methods of Diversity-Oriented Synthesis (DOS) (Comer et al, Proc Natl Acad Sci U S A 108, 6751 (Apr 26, 2011 ; Lowe et al, J Org Chem 77, 7187 (Sep 7, 2012); Marcaurelle et al, J Am Chem Soc 132, 16962 (Dec 1, 2010)) — to investigate chemical compounds not represented in traditional pharmaceutical libraries (Schreiber, S. L. (2000). Science 287, 1964-1969; Schreiber et al, Nat Biotechnol 28, 904 (Sep, 2010), each of which is herein incorporated by reference in their entirety). Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Set. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91: 11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261: 1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. M. Mi' i' . 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

Libraries of compounds can be presented in solution (e.g., Houghten, Biotechniques 13:412- 421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89: 1865-1869, 1992) or on phage (Scotl and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et a/. Proc. Natl. Acad. Sci. 87:6378- 6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.

When a crude extract is identified as containing a compound of interest, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that achieves a desired biological effect. Methods of fractionation and purification of such heterogenous extracts are known in the art. Small molecules of the disclosure can have a molecular weight below 2,000 daltons, between 300 and 1,000 daltons, or between 400 and 700 daltons. In some embodiments, these small molecules are organic molecules.

Kits

The disclosure provides, in some embodiments, kits for the treatment or prevention of a genetic disease associated with Usher Syndrome, such as USH2A-related disease or disorder, or symptoms thereof. In one embodiment, the kit includes a pharmaceutical pack comprising an effective amount of an antisense oligonucleotide described here that targets a region of USH2A having a mutation resulting in aberrant splicing. For example, the antisense oligonucleotide targets a sequence of exon 6, exon 19, or exon 20 (see, e.g., TABLES 1-3). Additional embodiments are directed to antisense oligonucleotides that target a splice acceptor site, a splice donor site, or an exonic splicing enhancer. In some embodiments, the kit comprises a pharmaceutical composition comprising any one or more of the described antisense oligonucleotides or a cell comprising any one or more of the described antisense oligonucleotides, and a pharmaceutically acceptable or physiologically acceptable carrier, where the pharmaceutical composition is present in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition or pharmaceutical composition of the disclosure; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired compositions of the disclosure or combinations thereof are provided together with instructions for administering them to a subject having a genetic disorder associated with Usher Syndrome, the instructions will generally include information about the use of the agent (e.g., antisense oligonucleotide, pharmaceutical composition, cell(s)) for the treatment or prevention of the genetic disorder. In other embodiments, the instructions include at least one of the following: description of the oligonucleotide(s); dosage schedule and administration for treatment of a genetic disorder or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Eaboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the disclosure, and, as such, may be considered in making and practicing the disclosure. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

EXAMPLES

Example 1: Design of 2-O-Methyl RNA ASOs Targeting USH2A Exon 19 and Exon 20

An exon skipping strategy was developed that can be used for patients with pathogenic variants USH2A exon 19 or exon 20, which overlaps more than 45 known mutations including the USH2A variant, designated c.4338_4339delCT. For example, a dual exon skipping strategy was the basis for designing antisense oligonucleotides (ASOs) targeting USH2A exon 19 and exon 20. See, FIGs. 1A-1B. This variant resulted is a frameshift of Cysteine at position 1447 to Glutamine, where protein translation was terminated at position 29 (Cysl447Gln s^,Ter29), which resulted in defective splicing having an out-of-frame mutation in exon 20. The use of the antisense oligonucleotides of the disclosure (see, FIG. IB) restored the normal reading frame and the synthesis of the usherin protein. The ASOs were purified by HPLC and produced by the Microsynth company based in Switzerland (microsynth.com/home-ch.html). Using such ASOs would similarly be useful for treating patients having any mutations in this region as identified, for example, in the Deafness variant database (deafhessvariationdatabase.org) .

A series of ASOs targeting human USH2A exons 19 and 20 located at different regions in and around these exons were designed using Genomnis, eSkip-Finder, and ESE3.0Finder software. For example, regions that included exonic splicing enhancer sequence (ESE) and the acceptor site were targeted. Splicing factors were analysed online using ESE3.0Finder (default threshold: SF2/ASF= 1.956, SF2/ASF IgM-BRCAl =1.867, SC35=2.383, SRp40=2.67, SRp55=2.676), eSkip- Finder (parameters calculated for 0.1 pM of 2'0Me introduced in cultured cells (rhabdomyosarcoma cells)) and Genomnis (Human Splicing Finder Professional) websites. ASOs sequences were designed around the acceptor site region for USH2A Exon 19 and Exon 20. ASOs were designed with 25bp, Tm>48°C and GC% between 30 and 60% (FIG. IB). ASOs sequences were synthetized by MicroSynth (Switzerland) company at 0.2 pM and IpM. ASOs were resuspended in ultrapure water at a stock of lOOpM and stored at -20 °C. Example 2: Validation of Exon Skipping Strategy

In silico analysis of the effect of USH2A exon 19 and exon 20 skipping validated the hypothesis that in-frame deletion of human USH2A exon 19 and exon 20 would result in a shorter yet still functional USH2A protein (USH2A^A19-2°; usherin) (105 amino acids of 5202 amino acids) as compared to wild type protein (USH2A^WT). The in silico 3D modeling was built using Benchling AlphaFold beta feature, and the structural files were analyzed by ChimeraX-1.2.5. FIG. 2A depicts the deletion of USH2A exon 19 and 20 in humans, which resulted in a functional usherin protein (105 amino acids out of 5202 amino acids) lacking a single fibronectin 3 domain. FIG. 2B demonstrates the in silico assessment of the region encoded by exons 16-21, which confirmed the complete removal of one fibronectin 3 domain (pseudo-USH2A^A19-2°) compared to the wild-type protein (USH2A FL or USH2A^WT). The in silico modeling of the effect of USH2A exon 19 and 20 skipping (USH2A^A19-2°) protein was compared to the wild type (WT) protein (USH2A^WT). This script allowed for the specific selection of the region of USH2A^WT protein encoded by the exon 16 to 21 (from 1054 to 1543 AA), responsible for the synthesis of four complete fibronectin type III domains (FN3-1 to FN3-4) and one complete Laminin-G domain; and the region of USH2A^A19-2° protein encoded by the exon 16 to 21 lacking exons 19 and 20 (from 1054 to 1438 AA), responsible for the synthesis of only three of the four complete fibronectin type III domains (FN3-1 to FN3-4, lacking the FN3-3 domain) and one complete Laminin-G domain compared to USH2A^WT protein. The structural file (mmCIF (PDBx)) was download and ChimeraX-1.2.5 software was used to analyze the 3D structure and align the sequences of the USH2A^WT and USH2A^e

Example 3: Treatment of Retinoblastoma Cells with ASOs

Human Retinoblastoma cells (WERI-RB1) were ordered from ATCC (HTB-169). WERI- RB1 cells were cultured in 25cm² flask with RPMI-1640 (ATCC, ref 30-2001) media containing 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate supplemented with 10% FBS (Fetal Bovine Serum - ThermoFisher Scientific, ref #16140089) at 37°C and 5% CO2. Every 3-5 days, cells were centrifuged (1200rpm - Imin), the supernatant was removed and replaced by warm and fresh supplemented media. Depending on the confluency, a dilution of 1/4 to 1/6 was applied. For ASOs transfection, cells were seeded into a 24- well plate at LOxlO⁵ (ImL/well). After 24 hours in culture, ASOs were applied on WERI-RB1 cells by transfection using lipofectamine 2000 (Thermofisher Scientific - ref #11668027). Concentrations between 10 and 200nM of ASOs were used. A control, 2-O-Methyl RNA ASO (Spinraza® - Nusinersen, sequence: 5’-UCACUUUCAUAAUGCUGG-3’) with phophorothioate (PTO) bonds was used to validate the potential skipping action of the ASOs developed. For each condition (except the untreated condition), 2pL of lipofectamine 2000 were used. A mixture of ASOs and lipofectamine 2000 diluted in OPTI-MEM (Gibco, ref #31985062) was made and incubated for 15 min at room temperature (RT) before being added onto cells. After 48 hours of expression at 37°C and 5% CO2, cells were harvested. Cells were collected into a 1.5mL RNAse-free tube and centrifuged for 3min at 12000 rpm. The supernatant was removed, and the pellet was rapidly snap frozen and stored at -80°C until further analysis. At least 3 experiments were performed for each condition.

Antisense oligonucleotides were transfected into retinoblastoma cells (WERI-RB1) and determined their capacity to induce single or dual exon skipping using RT-PCR. The ASOs 1-6 from FIG. IB were used at concentrations of 200 nM, 100 nM, 500 nM, 25 nM, and 10 nM (See, FIGs. 3A- 3B, from left to right, respectively). At least three experiments were performed for each condition. The ASOs were designed for USH2A exon 19 induced skipping of USH2A exon 19 or both USH2A exons 19 and 20 (FIG. 3 A) and those designed for USH2A exon 20 mediated USH2A exon 20 skipping alone.

Cocktails of two and three ASOs, as well as all six ASOs of FIG. IB, at the aforementioned concentrations were used and demonstrated efficient dual exon skipping of USH2A exons 19 and 20. See, FIGs. 4 - 5. RT-PCR demonstrates that co-transfection of ASOs designed for USH2A exons 19 and 20 mediate USH2A dual exon skipping using all six ASOs (FIG. 5: Left panel) and different combinations of three ASOs (FIG. 5: Center and Right panels). The skipping efficiency appears to be concentration dependent. Whereas, under control conditions, no USH2A exon skipping was observed. The controls were scrambled ASO, untreated cells (WERI-RB1), and mock cells (see, FIG. 6).

To analyze the human USH2A transcripts from untreated and ASOs-treated WERI-RB1 cells, total RNA was isolated combining Trizol/chloroform extraction protocol with PureLink RNA mini kit (Thermofisher Scientific, ref #12183018A). Total RNA (2 pg) was used for RT-PCR one-Step using SuperScript™ IV One-Step RT-PCR System (ThermoFisher Scientific, ref #12594100). Primer sets used amplified the region of human USH2A exon 17 to exon 24 (Forward 5’- CAAAGACTAAGTCCACCTAAGATGC-3 ’, Reverse 5 -GCAAAGACAATCAAACCTTCAGG- 3’). The PCR program used was adapted from the recommended protocol from the company: (1) reverse transcription: 50°C - lOmin, (2) Reverse transcriptase inactivation/initial denaturation: 98°C - 2min, (3) amplification: 98°C - 2min, 90°C - lOsec, 59°C - lOsec, 72°C - Imin, (4) Final extension: 72°C - 5min, 35 cycles. Between 20pL to 40pL of PCR products were analyzed on 3% agarose gel. HyperLadder™ 50bp (Bioline, ref # BIO-33054) were used to estimate the weight of each band. Potential bands were cautiously extracted from the agarose gel, purified with Monarch® DNA Gel Extraction kit (New England Biolab, ref #T1020L) and then sent to sequencing for validation (Azenta company).

Example 4: Assessment of ASOs in Retinal (RO) and Inner Ear (IEO) Organoids

Whole exome sequencing was performed on a blood sample from an USH2A patient having USH2A c.4338_4339delCT to determine whether other pathogenic mutations were present. Patient- derived iPSC lines were previously generated as were CRISPR/Cas9 corrected isogenic cell lines, which were used to generate retinal organoids (ROs) and inner ear organoids (lEOs). These organoids are used to validate that the ASOs specifically target the retina and inner ear. See, FIG. 7A. Day 60 and Day 90 retinal organoids were generated. See, FIG. 7B. RT-PCR analysis performed on Day 60 ROs showed high expression of the full length USH2A gene, containing both USH2A exons 19 and 20. See, FIG. 7C. Retinal organoids were generated following the protocol described in Fligor et al., (Sci Rep. 8: 14520, 2018, 2020, doi: 10. 1038/s41598-018-32871-8; Methods Cell Biol. 159:279- 302, 2020, doi: 10.1016/bs.mcb.2020.02.005). While inner ear organoids were generated following the protocol described by Koehler et al. (Nat Biotechnol.35 5 3AS 9, 2017, doi: 10.1038/nbt.3840) and Doda et al. (Development. 150, 2023, doi: 10.1242/dev.201865).

Example 5: Zebrafish models

In order to validate the functionality of the USH2A protein lacking only one fibronectin type- 3 domain (dual exon skipping), two zebrafish models are used. One of the zebrafish models having the jjSH2A⁴³³⁸-^4339delCT mutation (exon 19 in zebrafish), was generated by In Vivo Biosystems company. In parallel, the USH2A⁴⁸¹⁸'¹⁹ zebrafish identified as Fl in FIG. 8A, has an out-of-frame indel in exon 18-19 (equivalent to human exon 19-20), will also undergo ASO treatment F2 has an in-frame deletion of exon 18-19, which mimics the dual exon skipping strategy. See, FIG. 8A. These models were confirmed by genotype, propagated through two generations, and maintained as homozygous viable adults. Two zebrafish models, USH2A^lvb3 and USH2A^lvb4 (FIG. 8B), mimic human USH2A c.4338_4339delCT mutation and exhibit an out-of-frame indel in exons 18-19 (equivalent to human USH2A exons 19-20), which introduced premature termination codons in USH2A exon 19 of zebrafish, resulted in impaired photoreceptor function and survival. Immunostaining performed on homozygous USH2A^lvb3 mutant larvae at 6 days post-fertilization confirmed the loss of Usherin localization from the peri ciliary region of photoreceptors. Immunostaining performed at 6dpf on USH2A^lvb3 and wild type zebrafish, demonstrated that USH2A protein, usherin, was not detected in the mutant retina (results not shown). Acetylated tubulin was used to visualize the periciliary region of photoreceptors. Visual function showed loss ofor reduction of optokinetic reflex (OKR) in USH2A ^lvb3 mutants (FIG. 8C). The dual exon skip models were used to determine if, in contrast to in-frame deletion of zebrafish exons 18 and 19, the dual exon skipping leads to production of a functional protein while preserving vision and hearing. DNA samples from embryos injected with the CRISPR/Cas9 cocktail targeting intronic regions flanking exons 18 and 19 indicated successful excision of this region (see, FIGs. 8D-8E). In FIG. 8D, the USH2A⁴⁸¹⁸'¹⁹ mimics the dual exon skipping strategy with an in-frame deletion of exons 18-19 in zebrafish, where the forward primer (F Primer), upstream CRISPR cut site [e 166f] are positioned upstream of exon 18, and the downstream CRISPR cut 2 site [E 19+ 1578r], reverse primer (R Primer 2), Downstream CRISPR cut 1 site [E19+5833r], and reverse primer 1 (R Primer 1) are positioned, respectively, downstream of exon 19. FIG. 8E shows validation of the dual exon 18-19 skipping via PCR (Control: uninjected controls; Mixl: E18 -1666f + E19+5833r gRNA; Mix2: E18 -1666f + E19+1578r gRNA). The zebrafish models will be further assessed for vision (OKR), hearing (acoustical startle reflex), and balance (swimming behaviors) to determine their phenotypes. Retinal and inner ear histology will also be performed to assess development, structure, and cell death.

Example 6: ASO Development for USH2A c.949C>A

Antisense oligonucleotides 1-20 (US001-US020) of TABLE 6 targeting USH2A C.949OA were designed and screened (see, FIG. 9A). Genomic DNA was collected from an USH2A C.949OA patient and a splicing reporter minigene construct was generated, as well as patient fibroblasts and iPSC lines. RT-PCR and qPCR assays were used to quantify normal and mis-spliced products and confirmed the truncation of exon 6 in USH2A c.949C>A cells. Nine ASOs were designed and synthesized (see, TABLE 6, ASOs 1-9) having 22 nucleotides (nt) each. These ASOs were used to tile the cryptic donor site and screened using the splicing reporter minigene assay in order to identify those that rescued proper splicing. ASOs 1-7 reduced usage of the cryptic donor, although ASOs 1-6 also caused exon 6 skipping (see, FIG. 9B, upper panel).

Further analysis of the surrounding exonic splic enhancer (ESE) led to the design of additional ASOs 10-20, which were 15-20 nt long. ASOs 10-20 reduced usage of the cryptic donor (see, FIG. 9B, lower panel), and of these, several were found to increase normal splicing (see, FIG. 9C). Experiments in patient iPSCs validated several of these leads, z.e., ASOs 5, 19 (see, FIG. 9D, lower panel), which decreased mis-splicing in patient iPSCs.

ASOs were screened using a minigene assay and several ASOs that correct the mis-splicing of USH2A exon 6 were identified. These will be tested in patient iPSC-derived retinal and inner ear organoids as well as control cell lines (Sox2-GFP or isogenic controls) as described in Example 4. ASOs will be administered via gymnosis to confirm rescue of normal USH2A splicing patterns (via RT-PCR, qPCR, RNAseq) and usherin protein levels (via immunohistochemistry and western blot). HEK293 cells were cotransfected for 48 hours (plasmid Ipg or 0.5 pg/ ASO 200 nM per well) in a 12- well plate and then analysed after RT-PCR. The ASOs blocked cryptic donor sites reduced mis- splicing and increased USH2A normal splicing.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Embodiment 1. An antisense oligonucleotide (ASO) comprising 8-40 nucleotides or modified nucleotides, wherein the oligonucleotide is at least 80% complementary to a nucleic acid sequence in an USH2A allele comprising a mutation associated with aberrant splicing. Embodiment 2. The ASO of embodiment 1, wherein the oligonucleotide comprises a modified linkage selected from the group consisting of methylphosphonate, phosphodiester, phosphorodithioate, phosphorothioate, and phosphotriester linkages.

Embodiment 3. The ASO of embodiment 2, wherein the modified linkage is a phosphorothioate linkage.

Embodiment 4. The ASO of any one of embodiments 1-3 wherein the oligonucleotide comprises at least one modified sugar moiety.

Embodiment 5. The ASO of embodiment 4, wherein the modified sugar moiety is a 2'-O- methyl, a 2'-methoxyethoxy, a 2'-O-methoxyethyl, a 2'-dimethylaminooxyethoxy, a 2'- dimethylaminoethoxyethoxy, a 2'-fluoro, or a 2'-acetamide modification group.

Embodiment 6. The ASO of any one of embodiments 4-5, wherein the modified sugar moity comprises a modification on every sugar moiety.

Embodiment 7. The ASO of any one of embodiments 1-6, wherein the oligonucleotide comprises a modified nucleobase.

Embodiment 8. The ASO of embodiment 7, wherein the modified nucleobase comprises a locked nucleic acid (LN A) nucleobase.

Embodiment 9. The ASO of any one of embodiments 1-8, wherein the ASO is a morpholino, thiomorpholino, or peptide nucleic acid.

Embodiment 10. The ASO of any one of embodiments 1-9, wherein the mutation is in USH2A exon 6, exon 19, exon 20, or combinations thereof.

Embodiment 11. The ASO of embodiment 10, wherein the mutation is c.4338_4339del (p. Cysl447Gln/s^,Ter29) or C.949OA (p.Arg371=).

Embodiment 12. The ASO of any of embodiments 1-11, wherein the ASO comprises, consists essentially of, or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 6.

Embodiment 13. The ASO of any of embodiments 1-12, wherein the ASO comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 6.

Embodiment 14. The ASO of any one of embodiments 1-13, wherein the ASO increases USH2A expression or activity.

Embodiment 15. The ASO of embodiment 14, wherein the ASO increases USH2A expression or activity in the eye.

Embodiment 16. The ASO of embodiment 14, wherein the ASO increases USH2A expression or activity in the ear.

Embodiment 17. The ASO of any one of embodiments 1-16, wherein said oligonucleotide comprises DNA residues, RNA residues, modified DNA or RNA residues, or combinations of any of these. Embodiment 18. A set of antisense oligonucleotides comprising two or more of the antisense oligonucleotides of any one of embodiments 1-17.

Embodiment 19. An isolated cell comprising an antisense oligonucleotide of any one of embodiments 1-17 and a mutation associated with Usher Syndrome, type 2A.

Embodiment 20. The isolated cell of embodiment 19, wherein the cell comprises a c.4338_4339del (p. Cysl447Gln s^,Ter29) mutation and/or a C.949OA (p.Arg371=) mutation.

Embodiment 21. The isolated cell of embodiment 19 or embodiment 20, wherein the cell is of an inner ear or a retina.

Embodiment 22. The isolated cell of any one of embodiments 19-21, wherein the cell is derived from a subject having or suspected of having Usher Syndrome, type 2A.

Embodiment 23. A pharmaceutical composition comprising an effective amount of the antisense oligonucleotides of any one of embodiments 1-17 or the set of antisense oligonucleotides of embodiment 18, and a pharmaceutically acceptable excipient.

Embodiment 24. A method of restoring wild-type splicing of an USH2A allele comprising a mutation associated with Usher Syndrome, type 2A in a cell, the method comprising: contacting the cell with an effective amount of the antisense oligonucleotides of any one of embodiments 1-17, thereby restoring wild-type splicing.

Embodiment 25. The method of embodiment 24, wherein the cell is heterozygous or homozygous for the mutation.

Embodiment 26. The method of embodiment 24 or 25, wherein the mutation is in a splice acceptor site, a splice donor site, or a splicing regulatory element.

Embodiment 27. The method of embodiment 26, wherein the splicing regulatory element is an exonic splicing enhancer (ESE).

Embodiment 28. The method of any one of embodiments 24-27, wherein the mutation is a c.4338_4339del (p. Cys l447Gln/sTcr29) or a c.949C>A (p.Arg371=) mutation.

Embodiment 29. A method of treating Usher Syndrome, type 2A in a subject, the method comprising: administering to the subject an effective amount of an antisense oligonucleotide of any one of embodiments 1-17.

Embodiment 30. The method of embodiment 29, wherein the subject comprises a mutation associated with Usher Syndrome, type 2A.

Embodiment 31. The method of embodiment 29 or 30, wherein the mutation is a c.4338_4339del (p. Cys l447Gln/sTcr29) or c.949C>A (p.Arg371=) mutation.

Embodiment 32. The method of any one of embodiments 29-31, wherein the subject is heterozygous or homozygous for the mutation.

Embodiment 33. A method for treating a subject suffering from Usher Syndrome, type 2A, wherein the method comprises administering to the subject one or more antisense oligonucleotides of embodiments 1-17 that binds to a target sequence selected from the group consisting of those listed in TABLE 1, TABLE 2, and TABLE 3.

Embodiment 34. The method of embodiment 33, wherein the subject comprises a homozygous or a heterozygous mutation associated with Usher Syndrome, type 2A. Embodiment 35. A kit comprising the antisense oligonucleotide of any one of embodiments

1-17 or a set of antisense oligonucleotides of embodiment 18, and directions for administering the antisense oligonucleotide to a subject.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS What is claimed is:

1. An antisense oligonucleotide (ASO) comprising 8-40 nucleotides or modified nucleotides, wherein the oligonucleotide is at least 80% complementary to a nucleic acid sequence in an USH2A allele comprising a mutation associated with aberrant splicing.

2. The ASO of claim 1, wherein the oligonucleotide comprises a modified linkage selected from the group consisting of methylphosphonate, phosphodiester, phosphorodithioate, phosphorothioate, and phosphotriester linkages.

3. The ASO of claim 2, wherein the modified linkage is a phosphorothioate linkage.

4. The ASO of any one of claims 1-3 wherein the oligonucleotide comprises at least one modified sugar moiety.

5. The ASO of claim 4, wherein the modified sugar moiety is a 2'-O-methyl, a 2'- methoxyethoxy, a 2'-O-methoxyethyl, a 2'-dimethylaminooxyethoxy, a 2'- dimethylaminoethoxyethoxy, a 2'-fluoro, or a 2'-acetamide modification group.

6. The ASO of any one of claims 4-5, wherein the modified sugar moity comprises a modification on every sugar moiety.

7. The ASO of any one of claims 1-6, wherein the oligonucleotide comprises a modified nucleobase.

8. The ASO of claim 7, wherein the modified nucleobase comprises a locked nucleic acid (LN A) nucleobase.

9. The ASO of any one of claims 1-8, wherein the ASO is a morpholino, thiomorpholino, or peptide nucleic acid.

10. The ASO of any one of claims 1-9, wherein the mutation is in USH2A exon 6, exon 19, exon 20, or combinations thereof.

11. The ASO of claim 10, wherein the mutation is c.4338_4339del (p. Cysl447Gln^Ter29) or C.949OA (p.Arg371=).

12. The ASO of any of claims 1-11, wherein the ASO comprises, consists essentially of, or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleotide sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 66.

13. The ASO of any of claims 1-12, wherein the ASO comprises, consists essentially of, or consists of a nucleic acid sequence selected from the group consisting of those listed in TABLE 4, TABLE 5, and TABLE 6.

14. The ASO of any one of claims 1-13, wherein the ASO increases USH2A expression or activity.

15. The ASO of claim 14, wherein the ASO increases USH2A expression or activity in the eye.

16. The ASO of claim 14, wherein the ASO increases USH2A expression or activity in the ear.

17. The ASO of any one of claims 1-16, wherein said oligonucleotide comprises DNA residues,

RNA residues, modified DNA or RNA residues, or combinations of any of these.

18. A set of antisense oligonucleotides comprising two or more of the antisense oligonucleotides of any one of claims 1-17.

19. An isolated cell comprising an antisense oligonucleotide of any one of claims 1-17 and a mutation associated with Usher Syndrome, type 2A.

20. The isolated cell of claim 19, wherein the cell comprises a c.4338_4339del (p. Cysl447Gln/s^,Ter29) or C.949OA (p.Arg371=) mutation.

21. The isolated cell of claim 19 or claim 20, wherein the cell is of an inner ear or a retina.

22. The isolated cell of any one of claims 19-21, wherein the cell is derived from a subject having or suspected of having Usher Syndrome, type 2A.

23. A pharmaceutical composition comprising an effective amount of the antisense oligonucleotides of any one of claims 1-17 or the set of antisense oligonucleotides of claim 18, and a pharmaceutically acceptable excipient.

24. A method of restoring wild-type splicing of an USH2A allele comprising a mutation associated with Usher Syndrome, type 2A in a cell, the method comprising: contacting the cell with an effective amount of the antisense oligonucleotides of any one of claims 1- 17, thereby restoring wild-type splicing.

25. The method of claim 24, wherein the cell is heterozygous or homozygous for the mutation.

26. The method of claim 24 or 25, wherein the mutation is in a splice acceptor site, a splice donor site, or a splicing regulatory element.

27. The method of claim 26, wherein the splicing regulatory element is an exonic splicing enhancer (ESE).

28. The method of any one of claims 24-27, wherein the mutation is a c.4338_4339del (p. Cysl447Gln/s^,Ter29) or a C.949OA (p.Arg371=) mutation.

29. A method of treating Usher Syndrome, type 2A in a subject, the method comprising: administering to the subject an effective amount of an antisense oligonucleotide of any one of claims 1-17.

30. The method of claim 29, wherein the subject comprises a mutation associated with Usher Syndrome, type 2A.

31. The method of claim 29 or 30, wherein the mutation is a c.4338_4339del (p. Cysl447Gln/s^,Ter29) or c.949C>A (p.Arg371=) mutation.

32. The method of any one of claims 29-31, wherein the subject is heterozygous or homozygous for the mutation.

33. A method for treating a subject suffering from Usher Syndrome, type 2A, wherein the method comprises administering to the subject one or more antisense oligonucleotides of claims 1-17 that binds to a target sequence selected from the group consisting of those listed in TABLE 1, TABLE 2, and TABLE 3.

34. The method of claim 33, wherein the subject comprises a homozygous or a heterozygous mutation associated with Usher Syndrome, type 2A.

35. A kit comprising the antisense oligonucleotide of any one of claims 1-17 or a set of antisense oligonucleotides of claim 18 and directions for administering the antisense oligonucleotide to a subject.