OF

TIMOTHY REDDY

APOORVA IYENGAR

GREG CRAWFORD

AND

PRIYA KISHNANI

FOR

SPLICE SWITCHING OLIGONUCLEOTIDES TO RESTORE PHKG2

EXPRESSION IN GLYCOGEN STORAGE DISEASE IX

SPLICE SWITCHING OLIGONUCLEOTIDES TO RESTORE PHKG2 EXPRESSION IN GLYCOGEN STORAGE DISEASE IX

Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/415,774 filed 13 October 2022, which is incorporated by reference herein in its entirety for all purposes.

Incorporation by Reference of Sequence Listing Provided Electronically

[0002] The Sequence Listing submitted on 13 October 2023 is as an .xml file is incorporated by reference herein in its entirety. The electronic file is 51 kilobytes in size and titled “23- 2094-WO Sequence Listing"’.

Federal Funding Legend

[0003] This invention was made with Government support under Federal Grant No. RM1- HG011123 and R21-HG010747 awarded by the National Human Genome Research Institute (NIH/NHRGI). The Federal Government has certain rights to this invention.

Background

[0004] Systematically measuring the inheritance of splicing defects and mRNA abundance across rare disease trios and identifying their underlying pathogenic variants will meaningfully improve diagnosis of rare disease. RNA analysis can circumvent the challenges of non-coding variant interpretation by directly detecting the functional effects of non-coding variants. That improvement is primarily due to detection of aberrant splicing in mRNA. More broadly, 10% all known pathogenic variants in rare disease involve mRNA splicing. Unlike for other noncoding variant mechanisms, mRNA splicing can cause major changes in reading frame and therefore amino acid sequence. In many cases, altered reading frame introduces a premature termination codon. If that codon is at least 50-55 bp upstream of the final exon junction, the nonsense-mediated decay (NMD) surveillance mechanism ty pically degrades the transcript to prevent expression of truncated protein. The reduced transcript abundance causes loss-of- function. and NMD is therefore commonly implicated in both coding and non-coding pathogenic variants that cause rare disease. Furthermore, although trio DNA sequencing is used to improve rare disease diagnosis by adding segregation and de novo mutational analysis, previous diagnostic RNA-seq studies have not systematically included trio studies to analyze splicing or NMD in both parents and proband. [0005] Utilizing the approaches described in this disclosure will provide foundational knowledge regarding inheritance of splicing and NMD and how to leverage those data in a diagnostic setting. Advancing methods to detect and functionally interrogate non-coding pathogenic variants will establish new types of evidence that support a more inclusive variant classification framework. These are critical areas for expansion and improvement of genetic testing, and would facilitate numerous aspects of patient care. Early therapeutic intervention, setting expectations for likely disease course, and family planning often hinge on identifying the genetic variant(s) that cause disease. With up to 10% of Americans living with a rare disease, this proposal represents a substantial contribution to improving public health.

[0006] As set forth in the Examples, a new model of a rare genetic disease, specifically Glycogen Storage Disease (GSD) IX was created using precise genome editing in HEK293T cells. To do so, a single nucleotide splice variant that causes frameshift in PHKG2 was introduced that produced single cell-derived clones that are genetically identical other than at this putative pathogenic variant. These cells were assayed for enzymatic activity using identical conditions to those used on patient tissue to diagnose GSD IX in the clinic. These generated cells provide a novel and ideal model to study other pathogenic variants in GSD IX and can be used to test therapeutic options for GSD and other disease resulting from splicing errors.

[0007] Glycogen Storage Diseases (GSDs) are a family of rare inherited conditions affecting storage and processing of glycogen, which is a form of carbohydrate energy storage in cells, particularly in liver and muscle. A major cause of GSD type IX is a mutation in the PHKG2 gene that causes a loss of PHKG2 function and a subsequent deficiency in glycogen metabolism. GSD IX y2, also known as GSD IXc or PHKG2 -related phosphorylase kinase (PhK) deficiency is an autosomal recessive disease caused by pathogenic loss-of-function variants on both alleles of PHKG2, which encodes the catalytic subunit of PhK. Because this is the most critical subunit for PhK function, GSD IX y2 is the most severe of the GSD IX subtypes. Patients present with fasting hypoglycemia and ketosis, hepatomegaly, growth delay, elevated aspartate aminotransferase (AST)/alanine transaminase (ALT) liver enzymes, and hyperlipidemia. Eventually, >95% of patients with GSD IX y2 progress to liver fibrosis and/or cirrhosis. (Herbert M, et al., Phosphorylase Kinase Deficiency. 2011 May 31 [Updated 2018 Nov 1], In: GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993- 2023. Fernandes SA. et al., Mol Genet Metab., 2020 Nov; 131 :299-305).

[0008] GSD IX y2 is clinically diagnosed using a combination of biochemical and genetic testing. Typically, patients will have <10% PhK activity compared to the healthy population. Genetic testing usually covers the protein-coding regions of genes comprising the PhK heterotetramer (PHKAI, PHKA2, PH.KB, PHKG2, PHKG2). In many cases, however, patients who have typical symptoms and biochemical presentations of GSD IX do not ever receive a complete genetic diagnosis (pathogenic variants in trans on a single gene) due to limitations with this ty pe of genetic testing. These patients often struggle to receive high-quality clinical care and insurance coverage due to the lack of a genetic diagnosis, and may not qualify for gene-targeted clinical trials.

[0009] The present disclosure identifies a previously unknown genetic cause of GSD IX in patients wherein the patients have a defect in PHKG2 splicing, which causes disease. As shown herein, a new' cause of GSD IX is provided using genome editing to create the genetic variant in cells from an individual with no evidence of a GSD IX, and subsequently showing that the modified cells have the expected defects in glycogen processing. In particular, novel splice switching oligonucleotides were generated and identified. These SSOs were able to correct RNA splicing and facilitate proper gene expression, thereby demonstrating that diseases caused by splicing defects are a particularly good target for a splice switching oligonucleotides (SSOs) therapeutics. Thus, the present disclosure provides a novel and ideal model to study other pathogenic variants, and can be further used to test other SSO therapeutic options.

Summary

[0010] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0011] The present disclosure provides, in part, three different novel splice switching oligonucleotides (SSOs) that comprise, consist of, or consist essentially of 2'-O-methyl RNA oligos with a phosphorothioate backbone and that bind to PHKG2 RNA and restore correct splicing of the gene (FIG. 1). The SSOs are RNA molecules with specific chemical modifications to increase their stability in cells and to reduce immune reactions. As shown herein, it is demonstrated that the SSOs correct the expression of PHK.G2 by delivering them to cells wdth the splicing variant. RT-PCR shows that those cells had increased abundance of the correctly spliced PHKG2 gene and decreased product of the incorrectly spliced and nonfunctional form of PHKG2. [0012] Accordingly, one aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed oAK176) and comprising the sequence mC*mU*mC*mA*mC*mC*mU*mC*mU*mU*mU*mG*mA*mG*mC*mC*mA*mA*mU *mC*mU*mA*mU*mA (SEQ ID NO: 1)

[0013] wherein the * indicates a phosphorothioate backbone, and the "m" indicates 2'-O- methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%. at least 90%, or at least 95% identity.

[0014] Another aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed oAK17) and comprising the sequence mC*mC*mU*mA*mG*mA*mG*mC*mA*mG*mU*mC*mA*mU*mC*mU*mC*mA*mC *mC*mU*mC*mU*mU (SEQ ID NO:2)

[0015] wherein the * indicates a phosphorothioate backbone, and the "m" indicates 2'-O- methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%, at least 90%, or at least 95% identity.

[0016] Another aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed oAKl 8) and comprising the sequence mG*mU*mA*mU*mU*mA*mC*mC*mU*mC*mU*mG*mG*mA*mG*mU*mC*mA*mG *mA*mC*mU*mG*mU*mC (SEQ ID NO:3)

[0017] wherein the * indicates a phosphorothioate backbone, and the "m" indicates 2'-O- methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%, at least 90%, or at least 95% identity.

[0018] Another aspect of the present disclosure provides a pharmaceutical composition comprising an SSO as in any of the preceding claims and a pharmaceutically acceptable buffer, excipient and/or carrier.

[0019] Another aspect of the present disclosure provides for methods of making the compositions as provided herein.

[0020] Another aspect of the present disclosure provides for methods of preventing and/or treating a glycogen storage disease IX (GSD IX) in a subject, the method comprising, consisting of, or consisting essentially of administering the subject a therapeutically effective amount of a composition and/or pharmaceutical composition thereof as provided herein to the subject such that the GSD IX is prevented and/or treated in the subject. [0021] In yet a further aspect, a kit for treating GSD IX is provided. The kit comprises, consists essentially of, or consists of at least one splice-switching oligonucleotide (SSO) described herein. In some aspects, the kit further comprises a second GSD IX therapy.

[0022] Another aspect of the present disclosure provides a method of generating a cell model of aberrant RNA splicing, the method comprising, identifying a pathogenic sequence of interest, utilizing a guide RNA and single stranded oligonucleotides via CRISPR/Cas9 gene editing, creating a model cell line with the pathogenic sequence of interest.

[0023] In an additional aspect, the method of generating a cell model provides a model of GSD IX, where in sequence of interest comprises a GSD IX mutation or non-coding sequence variant.

[0024] In a further aspect, the generated cell model is utilized to characterize a pathogenic sequence of interest. The pathogenic sequence of interest is associated with a frameshift, loss- of-function intolerance, patient symptoms in the Human Phenotype Ontology database. The generated cell model can identify an RNA splicing defect or presence of a pathogenic noncoding variant.

[0025] In an additional aspect of the invention, a method of utilizing the cell model identifies SSOs that restore aberrant gene splicing. The identified SSOs can be utilized to treat or mitigate RNA splicing defects in a patient in need thereof.

[0026] Another aspect of the present disclosure provides all that is described and illustrated herein.

Brief Description of the Drawings

[0027] The accompanying Figures and Examples are provided by way of illustration and not by way of limitation. The foregoing aspects and other features of the disclosure are explained in the following description, taken in connection with the accompanying example figures (also “FIG.”) relating to one or more embodiments, in which:

[0028] FIG. 1 provides three splice switching oligonucleotides (SSO) that bind RNA and restore gene splicing. The three SSOs consist essentially of 2'-O-methyl RNA oligos with a phosphorothioate backbone and have the ability to bind to PHK.G2 RNA.

[0029] FIG. 2 is a schematic of a GSD IX y2 family pedigree of two siblings with GSD IX y2 and no known familial history' of GSD. Patient A presented at 9 months of age with growth delay, hypoglycemia, hepatomegaly, elevated AST/ALT, Urine Hex4. Patient B presented at 1 month of age with delayed growth, hypoglycemia, and hepatomegaly. Both siblings have developed liver fibrosis, with Patient B progressing rapidly. [0030] FIG. 3 is data showing the results of short read RNA-seq of huffy coat from a pair of GSD IX y2 siblings as identified by the inventors. Patients A. B, and representative healthy control. Aberrant splicing to a previously unknown 76 bp pseudoexon is observed in both siblings. The pseudoexon donor site contains a rare mT>G variant that creates a cryptic splice donor (TT>TG). This variant has not previously been identified to be pathogenic or associated with GSD IX.

[0031] FIG. 4 is data of reverse transcription followed by PCR of the region surrounding c.556+1069T>G. Wild-type HEK293T cells express the expected canonical isoform product size of 304bp. Mutant cells express a reduced quantify of canonical isoform, and express an additional isoform consistent with a 76 bp pseudoexon insertion (380 bp).

[0032] FIG. 5 is a RT-qPCR analysis of PHKG2 expression. c.556+1069T>G reduces expression of PHKG2 in mutant HEK293T cells. Primers span exons 1-2 and are expected to amplify’ both the canonical and the pseudoexon-containing isoform.

[0033] FIG. 6 is a western blot analysis of PHKG2 protein content. 20 pg total protein from wild-type and homozygous c.556+1069T>G HEK293T cells was probed with anti-PHKG2 antibody and visualized via chemiluminescence. Mutant cells express a band of the expected size at a lower level than wild-type cells.

[0034] FIG. 7 illustrates impaired cellular function under hypoglycemic conditions. 200,000 mutant and wild-type HEK293T cells were grown with 0.5 g/L glucose and 55 mg/L Na pyruvate. Cells were counted after 40 hours to determine doubling time.

[0035] FIG. 8 shows a clinical test for phosphorylase kinase enzyme activity. Lysed mutant cells show a 10-fold decrease in phosphorylase kinase activity in vitro compared to wild-type, a similar level of change that w ould be seen in cases v. controls in human blood cells and liver biopsies. This assay was completed at the Duke University Medical Center Glycogen Storage Disease Laboratory.

[0036] FIG. 9 is an illustration of splice switching oligonucleotides (SSO) tested to target pseudoexon caused by c.556+1069T>G. SR splicing factor binding sites (medium gray) as predicted by ESEfinder 3.0 indicate putative exonic splicing enhancers. 3 hand-designed SSOs (dark gray) to target splice acceptor, splice donor, and putative exonic splicing enhancer. SSOs tiled across entire region (light gray) for a screen.

[0037] FIG. 10 is RT-PCR data illustrating SSOs cause dose-dependent splice-switching activity in a mutant HEK293T cell line. Cells were transfected with SSOs and RNA was collected 24 hours post-transfection. RT-PCR was performed and product was run on an agarose gel. Expected canonical isoform product size 304 bp; expected pseudoexon isoform product size 380 bp.

[0038] FIG. 11 is a mode of RT-qPCR assay designed for high-throughput quantitative screening of isoform expression changes after SSO treatment. This Taqman-style assay detects splice junctions unique to each isoform.

[0039] FIG. 12 is data demonstrating 2'-OMe SSOs induce splice switching. The three hand- designed 2’-OMe SSOs and a scrambled control with the same length and chemical modifications were transfected into biologically independent mutant HEK293T cell lines at 200 nM. SS03 had the highest efficiency, both for increasing canonical isoform expression and decreasing pseudoexon isoform expression.

[0040] FIG. 13 is data showing that 2 -MOE SSOs induce splice switching. The three hand- designed SSOs were synthesized with the 2’-M0E modification, which has previously advanced to FDA approval. These SSOs and a scrambled control with the same length and chemical modifications were transfected mutant HEK293T cell lines at 200 nM. SS03 had the highest splice-switching efficiency, and consistently improved canonical isoform expression in each biological replicate.

[0041] FIG. 14 is data showing combinations of SSOs induce splice-switching activity. The three hand-designed 2’-M0E SSOs were transfected into mutant HEK293T cells in a doseresponse curve using all possible combinations of SSOs. Fold change after treatment was plotted by total SSO concentration, e.g., 600 nM SSO1+2+3 = 200 nM SS01 + 200 nM SS02 + 200 nM SS03. The combinations had overall similar effects to an equal amount of a single SSO.

[0042] FIG. 15 is a screen of 2’-MOE SSOs tiled across the pseudoexon. 2'-M0E oligos were designed every 5 bp tiled across the pseudoexon from splice acceptor to splice donor and transfected into 3 biological replicate mutant HEK293T cells. RT-qPCR was performed 24 hours post-transfection. All oligos showed consistent reduction in pseudoexon isoform expression and were most effective at the splice acceptor and near predicted SR protein binding sites. oAKI109 showed the most substantial improvement in canonical isoform expression.

Detailed Description

[0043] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0044] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0045] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.

[0046] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0047] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

[0048] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0049] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%. it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0050] As used herein, "treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability' of developing a disease, disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder or condition. The term "effective amount" or '“therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0051] As used herein, the term "administering" an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0052] The term “biological sample” as used herein includes, but is not limited to, a sample containing tissues, cells, and/or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus, and tears. In one embodiment, the biological sample is a biopsy (such as a tumor biopsy). A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party ⁷ (e g., received from an intermediary ⁷ , such as a healthcare provider or lab technician).

[0053] The term "disease" as used herein includes, but is not limited to, any abnormal condition and/or disorder of a structure or a function that affects a part of an organism. It may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as cancer, autoimmune diseases and the like. The preferred disease to be treated by the oligonucleotides, compositions and methods herein are diseases that results from or are associated with a glycogen storage disease (GSD), for example, GSD IX.

[0054] "Contacting" as used herein, e.g., as in "contacting a sample" refers to contacting a sample directly or indirectly in vitro, ex vivo, or in vivo (i. e. , within a subj ect as defined herein). Contacting a sample may include addition of a compound to a sample, or administration to a subject. Contacting encompasses administration to a solution, cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture.

[0055] As used herein the term "reducing" or '‘repressing’’ are used interchangeably and refer to a decrease by a statistically significant amount. For example, in one embodiment, reducing refers to either partially or completely inhibiting an activity or decreasing or lowering an activity. In one embodiment, "reducing" means a decrease by at least 10% compared to a reference level, for example a decrease by at least about 15%. or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or up to and including a 100% decrease compared to a reference sample, or any decrease between about 10-100% compared to a reference level.

[0056] As used herein, the term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0057] "Nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. [0058] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. [0059] As used herein, the term “naked oligonucleotide'’ refers to the lack of an additional delivery vehicle. A “naked oligonucleotide” may or may not contain chemical modifications. [0060] As used herein, the term "altering the splicing of a pre-mRNA" refers to altering the splicing of a cellular pre-mRNA target resulting in an altered ratio of spliced products. Such an alteration of splicing can be detected by a variety ⁷ of techniques well known to one of skill in the art. For example, RT-PCR can be used on total cellular RNA to detect the ratio of splice products in the presence and the absence of an SSO.

[0061] As used herein, the term "variant" refers to a gene that is not normally expressed and may result in a truncated protein or reduced protein expression. Such variants may include but are not limited to RNA splicing defect, frameshift mutations, loss-of-function, non-coding, intolerance, patient symptoms in the Human Phenotype Ontology database, or those that contain a likely pathogenic coding variant in trans with the NMD or aberrant splicing in the proband.

[0062] As used herein, the term "complementary" is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an oligonucleotide and a DNA or RNA containing the target sequence. It is understood in the art that the sequence of an oligonucleotide need not be 100% complementary to that of its target. For example, for an SSO there is a sufficient degree of complementarity when, under conditions which permit splicing, binding to the target will occur and non-specific binding w ill be substantially avoided. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch, or hairpin structure). The SSOs of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, a SSO in which 18 of 20 nucleobases of the SSO are complementary to a target region, and would therefore specifically hybridize, w ould represent 90 percent complementarity. In this example, the remaining noncompl ementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of a SSO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol.. 1990, 215:403-410; Zhang et al., Genome Res., 1997. 7:649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2:482-489).

[0063] As used here, a protein or nucleic acid has at least a specified percentage of sequence homology ⁷ with a given SEQ ID NO, if the protein or nucleic acid in question has the same amino acid residues or bases, in the same sequence, in at least the specified percentage of residues or bases of the identified SEQ ID NO. In making nucleic acids with at least a given degree of sequence homology to a specified coding sequence, one skilled in the art, with the aid of a computer, could readily generate all nucleic acid sequences that would encode a given protein sequence. In making proteins with at least a given degree of sequence homology to specified protein sequence, one skilled in the art, guided by a knowledge of the physicochemical properties of amino acids, the position of a given residue within a protein, the known effects of certain amino acids on the conformation of proteins, and with the aid of a computer, could readily select certain amino acid substitutions at certain residue positions that would, with reasonable predictability, preserve the functional properties of the protein.

[0064] As used herein, the term “cell model’ ⁷ includes a cell line purposely modified to contain a gene variant or nucleotide sequence resulting in aberrant gene splicing. In one embodiment, the cell model is of Glycogen Storage Disease (GSD) IX. The cell model can be created using precise genome editing such as CRISPR/Cas9 in HEK293T cells. A nucleotide splice variant that causes frameshift in PHKG2 was introduced that produced single cell-derived clones that are genetically identical other than at this putative pathogenic variant. Such cell models can be created for other suspected gene variants of disease.

[0065] As used herein, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e., living organism, such as a patient). In some embodiments, the subject is suffering from a glycogen storage disease. In preferred embodiments, the subject is suffering from GSD IX.

[0066] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. A. Compositions

[0067] The present disclosure provides, in part, three different novel splice switching oligonucleotides (SSOs) that comprise, consist of, or consist essentially of 2'-O-methyl RNA oligos with a phosphorothioate backbone and have the ability to bind to PHKG2 RNA and restore correct splicing of the gene. The SSOs are RNA molecules with specific chemical modifications to increase their stability in cells and to reduce immune reactions. As shown herein, the inventors have demonstrated that the SSOs correct the expression of PHKG2 by delivering them to cells with the splicing variant, and then used RT-PCR to show that those cells had increased abundance of the correctly spliced PHKG2 gene and decreased product of the incorrectly spliced and non-functional form of PHKG2.

[0068] Accordingly, one aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed oAK176) and comprising the sequence mC*mU*mC*mA*mC*mC*mU*mC*mU*mU*mU*mG*mA*mG*mC*mC*mA*mA*mU *mC*mU*mA*mU*mA (SEQ ID NO: 1)

[0069] wherein the * indicates a phosphorothioate backbone, and the "m" indicates 2'-O- methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%, at least 90%, or at least 95% identity ⁷ .

[0070] Another aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed oAK17) and comprising the sequence mC*mC*mU*mA*mG*mA*mG*mC*mA*mG*mU*mC*mA*mU*mC*mU*mC*mA*mC *mC*mU*mC*mU*mU (SEQ ID NO:2)

[0071] wherein the * indicates a phosphorothioate backbone, and the "m" indicates 2'-O- methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%, at least 90%, or at least 95% identity ⁷ .

[0072] Another aspect of the present disclosure provides a splice switching oligonucleotide (SSO) comprising, consisting of, or consisting essentially of a 2'-O-methyl RNA oligo with a phosphorothioate backbone (termed 0AKI8) and comprising the sequence mG*mU*mA*mU*mU*mA*mC*mC*mU*mC*mU*mG*mG*mA*mG*mU*mC*mA*mG *mA*mC*mU*mG*mU*mC (SEQ ID NO:3)

[0073] wherein the *indi cates a phosphorothioate backbone, and the "m" indicates 2'-O-methyl RNA oligos, or any fragment or variant thereof, or a sequence with at least 85%, at least 90%, or at least 95% identity. [0074] A number of chemical modifications increase binding affinity, stability and delivery of oligonucleotides to cells and tissues and can be used in the practice of the current invention. Oligonucleotides containing 2'-0-Me phosphorothioate backbones have been used to correct aberrant splicing of modified luciferase pre-mRNA (Kotula et al., Nucleic Acid Ther, 2012, 22: 187-95) and by others to correct splicing of USH1C and rescue hearing and vestibular function (Lentz et al., Nat Med, 2013, 19:345-50). Additional studies have shown efficacy and, in some cases, superior efficacy of oligonucleotides containing other types of chemically modified backbones. For example, correction of splicing of modified enhanced green fluorescent protein pre-mRNA by oligonucleotides containing morpholino, peptide nucleic acid, locked nucleic acid and 2 -O-(2-methoxy ethyl) modified backbones (Sazani et al., Nucleic Acids Res. 2001, 29:3965-3974; Roberts et al., Mol Ther, 2006, 14:471-475; Sazani et al., NatBiotechnol, 2002, 20: 1228-1233; Veedu et al., Chem Biodivers, 2010, 7:536-542, the contents of which are incorporated by reference in their entireties), decrease of STAT3 and antitumor activity ⁷ in lymphoma and lung cancer as well as in patients in a Phase I dose escalation study by oligonucleotides containing 2'-O-(2-methoxyethyl) modified backbones (Hong et al., Sci Transl Med, 2015, 7:314ral85) and reduction of methyl-CpG-binding protein 2 (MeCP2) to rescue MECP2 duplication syndrome and correct MECP2 levels in cells from MECP2 duplication patients by oligonucleotides containing 2'-O-(2-methoxyethyl) modified backbones (Sztainberg et al., Nature, 2015, 528:123-126).

[0075] It will be obvious to one skilled in the art that additional oligomer chemistries can be used to practice the invention including phosphorodiamidate-linked morpholino oligomers (PMO) or locked nucleic acid (LNA) oligomers.

[0076] The SSOs of the present disclosure can be made through the well-known technique of solid phase synthesis. Any other means for such synthesis known in the art can additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. Suitable SSOs can also be ordered from suitable oligomer companies, for example Bio Basic (biobasic.com) which can provide SSOs containing chemically modified 2'-0-Me phosphorothioate backbones.

[0077] The bases of the SSO can be the conventional cytosine, guanine, adenine and uracil or thymidine bases. Alternatively, modified bases can be used. Of particular interest are modified bases that increase binding affinity ⁷ . One non-limiting example of preferred modified bases are the so-called G-clamp or 9-(aminoethoxy)phenoxazine nucleotides, cytosine analogues that form 4 hydrogen bonds with guanosine. (Flanagan et al., 1999, Proc. Natl. Acad. Sci. 96:3513; Holmes, 2003, Nucleic Acids Res. 31:2759). Specific examples of other bases include, but are not limited to, 5 -methylcytosine (MeC), isocytosine, pseudoisocytosine. 5-(l-propynyl)- cytosine, 5-bromouracil, 5-(l-propynyl)-uracil, 5-propyny-6,5-methylthiazoleuracil, 6- aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7- propyne-7-deazaguanine and 2-chloro-6-aminopurine.

[0078] Compositions comprising the splice-switching oligonucleotides are also provided. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art (Amon, R. (Ed.) Synthetic Vaccines 1 :83-92, CRC Press, Inc., Boca Raton, Fla., 1987). They include liquid media suitable for use as vehicles to introduce the splice-switching oligonucleotides into a subject.

[0079] SSOs can be used for in vivo delivery. SSOs that have been formulated using phosphorothioate backbones and 2' modifications that are such as those described in the Examples below can be delivered as naked oligos. To reach the liver, as is needed for GSD IX y2, they can be delivered subcutaneously, intravenously, or intraperitoneally, and are taken up by cells via endocytosis/pinocytosis.

[0080] In some embodiments, the SSO's are stored in a lyophilized product until use. In other embodiments, SSOs can be resuspended in nuclease free water and stored.

[0081] Cells can be transfected w ith SSOs in Opti-MEM medium, and cells can be cultured in medium known in the art, for example. Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 100IU penicillin, 100 pg/mL streptomycin, 110 mg/L Na pyruvate, 42 mg/L L-glutamine, and 4.5 g/L or 0.5 g/L D-glucose.

B. Pharmaceutical Compositions

[0082] In another aspect, the present disclosure provides compositions comprising one or more of the SSOs as described herein and an appropriate carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.

[0083] When used to treat or prevent a disease or disorder (e.g., a glycogen storage disease such as GSD IX), the SSOs (also referred to herein as compounds) described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and/or disorders and/or the symptoms associated with such diseases and/or disorders (e.g., a glycogen storage disease such as GSD IX). Suitable examples of such additional therapeutic agents may include special diets, enzyme replacement therapy, and the like. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.

[0084] SSOs that have been formulated using phosphorothioate backbones and 2' modifications that are such as those described herein can be delivered as naked oligos. To reach the liver, as is needed for GSD IX y2, they can be delivered subcutaneously, intravenously, or intraperitoneally, and are taken up by cells via endocytosis/pinocytosis.

[0085] Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.

[0086] The compounds may be formulated in the pharmaceutical composition per se. or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

[0087] Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[0088] For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

[0089] Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0090] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0091] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate): lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.

[0092] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.

[0093] Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

[0094] For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. [0095] For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.

[0096] For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).

[0097] Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

[0098] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0099] The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

[0100] The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.

[0101] Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity' and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.

[0102] Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity' of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophy lactic effect. For example, the compounds may be administered once per week, several times per week (e g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

C. Methods

[0103] Methods of preventing and/or treating a subject suffering from a disease are also contemplated. The method comprises, consists of, or consists essentially of administering to the subject a therapeutically effective amount of a splice-switching oligonucleotide described herein or a composition comprising the SSO such that the disease is treated. In preferred embodiments, the disease comprises a glycogen storage disease. In a preferred embodiment, the glycogen storage disease comprises GSD IX.

[0104] In some embodiments, the method of prevention and/or treatment further comprises administering to the subject a second therapy. The combination of the SSOs and the second therapy results in an increase in the efficacy of the treatment of the disease than the second therapy administered alone.

D. Kits

[0105] The present disclosure further provides kits comprising the SSOs provided herein and for carrying out the subject methods as provided herein. For example, in one embodiment, a subject kit may comprise, consist of, or consist essentially of one or more SSOs or pharmaceutical compositions thereof. In other embodiments, a kit may further include other components. Such components may be provided individually or in combinations, and may provide in any suitable container such as a vial, a bottle, or a tube. Examples of such components include, but are not limited to, one or more additional reagents, such as one or more dilution buffers; one or more reconstitution solutions; one or more wash buffers; one or more storage buffers, one or more control reagents and the like, Components (e.g, reagents) may also be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). Suitable buffers include, but are not limited to, phosphate buffered saline, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.

[0106] In addition to above-mentioned components, a subject kit can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (z.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data fde present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

E. Sequences

[0107] mC*mU*mC*mA*mC*mC*mU*mC*mU*mU*mU*mG*mA*mG*mC*mC*mA*m A*mU*mC*mU*mA*mU*mA (SEQ ID NO: 1)

[0108] mC*mC*mU*mA*mG*mA*mG*mC*mA*mG*mU*mC*mA*mU*mC*mU*mC*m

A*mC*mC*mU*mC*mU*mU (SEQ ID NO:2)

[0109] mG*mU*mA*mU*mU*mA*mC*mC*mU*mC*mU*mG*mG*mA*mG*mU*mC*m A*mG*mA*mC*mU*mG*mU*mC (SEQ ID NO:3)

For SEQ ID NO: 1 - SEQ ID NO:3, indicate a phosphorothioate backbone, and the "m" indicates 2'-O-methyl RNA oligos.

[0110] Another aspect of the present disclosure provides all that is described and illustrated herein. The following Examples are provided by way of illustration and not by way of limitation.

Examples

Example 1: Identifying Rare Disease Induced by RNA Splicing Defects

[01 1 1] A systematic study of NMD across rare disease trios is described in the present disclosure. Identifying segregation of NMD is hampered by identification of which transcripts originate from which allele (phasing), as each read may not span all heterozygous variants across the transcript. A novel statistical was used to jointly model allele-specific expression (ASE) and phasing that is more accurate than previously published methods. The model will be extended to identify patterns of ASE and gene expression outliers that reflect Mendelian inheritance patterns in trios. This will substantially improve detection of NMD and establishes anew type of evidence supporting non-coding variant pathogenicity.

[0112] The cohort of undiagnosed patients includes rare and ultra-rare (i.e., one known case) diseases. Identify ing the genes or variants underlying their conditions leads to the discovery of new diseases and new causes of known diseases. Identifying new pathogenic splice variants in particular leads to new precision therapies by leveraging existing platforms that have successfully manipulated splicing in clinical trials. In addition, as with many rare disease studies, this also provides insight into the basic biological function of the causal genes. [0113] Genetic diagnosis of rare disease remains a major gap, particularly for non-coding and splice-altering variants. Today, those variants are ty pically labeled as having uncertain significance, leaving the patient in limbo. Identifying and interrogating the function of noncoding pathogenic variants on a large scale will lay the groundwork to guide future classification of non-coding variants. In addition, rare variants discovered to be either pathogenic or benign by this study can be added to OMIM, ClinVar, and other databases used to inform genetic diagnosis and construct genetic testing. Identifying the genetic variant(s) underlying a rare disease is important for early detection, early intervention, family planning, and development of precision therapies. However, with tens to hundreds of variants present in each gene, it is often challenging to identify which of these variants are pathogenic, especially for non-coding variants. The disclosed studies provide for identifying non-coding causes of disease by testing variant identification and functional interrogation techniques in a case where the genomic search space for pathogenic non-coding variants is limited to a single gene.

[0114] Identification of a novel cause of GSD IX. Tw o siblings with GSD IX y2 and no prior family history of the disease were identified (FIG. 2). The siblings had a typical clinical presentation and <20% phosphorylase kinase activity in liver biopsies, but whole-genome sequencing did not yield a complete genetic diagnosis: both had a likely pathogenic variant at c.96-HG>A (Bali D.S., et al., Moi Genet Metab. 2014 Mar,l l l(3):309-313); however, their second pathogenic allele was unknown even after whole-genome sequencing.

[0115] Candidate variant identification. It was hypothesized that the siblings were compound heterozygous at PHKG2 and the missing variant was non-coding, and therefore performed RNA-seq on huffy ⁷ coat from both siblings and healthy controls (FIG. 3). This identified a 76 bp pseudoexon ~ 1 kb downstream of exon 6 of PHKG2, likely caused by a rare variant carried by both siblings (c.556+ 1069T>G) creating a cryptic GGT splice donor. This pseudoexon disrupts the kinase domain of PHKG2, its insertion leading to frameshift and a premature termination codon. Based on RNA-seq of the full quad, C.556+ 1069T>G and c.96-11G>A and their effects on splicing are detected in different parents, indicating that the variants are present on different alleles in the affected siblings.

[0116] Short-read RNA-seq of patient samples. Frozen buffy coat samples from patients and non-GSD IX controls were thawed and RNA was prepared through Trizol-chloroform extraction. 1 ug of RNA w as used to generate RNA-seq libraries using the TruSeq Stranded mRNA Library Prep kit. 50bp paired end libraries were sequenced on aNovaseq 6000 S-prime flow cell. Reads were aligned to the hgl9 genome build using STAR 2. 7 in two-pass mode with WASP filtering to minimize reference allele bias. BAM alignment files were visualized in the Integrative Genomics Viewer browser and were inspected manually for alterations in splicing.

Example 2: Developing a New Isogenic Model of Rare Disease - GSD IX

[0117] To establish functional evidence to assess pathogenicity of C.556+ 1069T>G, a set of isogenic HEK.293T cell lines were created. These cells provide a new model of GSD IX y2 and have identical genetic background other than at the variant of interest. CRISPR/Cas9 gene editing was utilized with a guide RNA cutting at the variant site and single-stranded oligodeoxynucleotides (ssODNs) providing homologous template to induce the variant of interest in the cell lines. Single-cell derived clones were then isolated and screened via allelespecific PCR and Sanger sequencing to confirm homozygous for either the wild-type or mutant allele, with no other mutations in the region.

[0118] CRISPR editing. A gRNA was designed to cut 0-1 bp 3' of the variant site. gRNA oligonucleotides were annealed and phosphorylated in T4 Ligation buffer (NEB) and T4 Polynucleotide Kinase (NEB). pX330 SpCas9-gRNA vector (Addgene #42230) was digested 15 mins at 37 °C with BbsI and purified. The gRNA duplex was ligated into the digested pX330 vector using T4 ligase (NEB) for 10 mins at room temperature. 4 pL ligated product was transformed into Endura electrocompetent cells (1800V, 10 pF, 600Q. 1 mm cuvette) and recovered in Lucigen Recovery Media for 1 hr at 37 °C and 225 rpm. Plasmid was purified through the Pure Yield Plasmid Miniprep System (Promega) and gRNA insertion validated through Sanger Sequencing (GeneWiz). Two custom single stranded oligo DNA nucleotides (ssODN) homology -directed repair templates corresponding to the desired T>G edit (one symmetric, one asymmetric) were designed using the Horizon HDR Donor Designer and manufactured by IDT.

Table 1. Guide RNA and Homologous Template Sequences for CRISPR Editing [0119] To determine whether C.556+ 1069T>G induced the 76 bp pseudoexon observed in the GSD IX patients, RT-PCR (Reverse Transcription followed by PCR) was performed on the mRNA region surrounding the pseudoexon in the HEK293T cell clones. HEK293T cells were cultured in DMEM high glucose, L-glutamine, 110 mg/L sodium pyruvate (Gibco cat. no. 11995073) supplemented with penicillin/streptomycin. Cells were grown to 70% confluent in a 24-well culture plate and transfected using 400 ng pX330, 1 pL 10 pM ssODN, 1.5 pL Lipofectamine 3000. and 1 pL P3000 reagent. 48 hours after transfection, cells were plated in a 96-well plate for clonal isolation through limiting dilution. After 10-14 days, colonies were screened for edits through allele-specific PCR, genotype confirmed through Sanger sequencing (GeneWiz), and positive clones expanded.

[0120] It was observed that the mutant cells expressed an isoform of PHKG2 including a 76bp insertion that was not present in wild-type cells (FIG. 4). Through Sanger sequencing, it was confirmed that the 76 bp pseudoexon seen in this assay was identical to the pseudoexon observed in the patient RNA-seq. It was observed through a Taqman qPCR assay (ThermoFisher Scientific, assay Hs04963859 ml) and Western blot (Proteintech. Cat no. 15109-1- AP) that the mutant cell lines had significantly reduced mRNA and protein expression (FIG. 5 - FIG. 6)

Table 2. RT-PCR Primers.

[0121] Mutant cells displayed impaired doubling time under low-glucose conditions (FIG. 7), indicating impaired glycogenolysis, or ability to metabolize stored glycogen. PhK enzyme activity was analyzed from frozen HEK293T cell pellets at the Glycogen Storage Disease Laboratory at Duke University Medical Center using standard spectrophotometric methods. Enzyme activity was measured indirectly by measuring the amount of glucose or phosphate released using glucose reagent (Infinity. Cat. TR15421) or phosphate reagent (Inorganic Phosphorous, Cat. TR30026) from ThermoScientific (Fisher Diagnostics, Middletown, VA, USA). Enzyme activity was expressed as pmol/min/mg protein).

[0122] Most significantly, mutant cells showed a 90% reduction on PhK enzyme activity in a clinical diagnostic test at the Duke Biochemical Labs (FIG. 8), which is consistent with the level of reduction observed in patients compared to healthy controls. Therefore, this cell line model is ideal to test candidate GSD IX y2 therapies. Furthermore, the methods outlined above can be utilized to generate additional cell models to study other disease resulting from splicing defects.

Example 3; Splice-Switching Oligonucleotides (SSOs) and Restoration of Gene Splicing [0123] SSOs are RNA molecules with specific chemical modifications to increase their stability in cells, increase their specificity to their target, and to reduce immune reactions. As shown herein, it is demonstrated that the SSOs correct the expression of PHKG2 by delivering them to cells with the splicing variant, and then used RT-PCR to show that those cells had increased abundance of the correctly spliced PHKG2 mRNA and decreased product of the incorrectly spliced and non-functional form of PHKG2.

[0124] A major goal of rare disease research is to identify new treatments that prolong life and quality of life for patients. Despite recent advances in gene therapy and gene editing, 90% of rare diseases do not have an FDA-approved treatment. Most therapies currently being developed focus on resolving defects caused by protein-coding pathogenic variants. Despite that focus, splicing, which is regulated by non-coding variants, is responsible for 10% of human disease (Stenson PD, et al., Human Gene Mutation Database (HGMD), Hum Mutat. 2003 21 :577-81). Rare diseases caused by splicing defects are a particularly good target for a class of therapeutics known as splice switching oligonucleotides (SSOs). This is a type of antisense oligonucleotide that base-pairs w ith a pre-mRNA molecule in order to inhibit RNA-RNA base pairing events or splicing factor binding events that are critical for the aberrant splicing event to occur. Successfully blocking those interactions would block the aberrant splicing and restore the normal mRNA sequence, reading frame, and amino acid sequence of the protein. Other SSOs have advanced to clinical trials, including the FDA-approved Spinraza (Corey DR. Nusinersen, an antisense oligonucleotide drug for spinal muscular atrophy. (Cheng L et al., Identification of spinal circuits involved in touch-evoked dynamic mechanical pain. Nat Neurosci, 2017 Jun; 20:804-814) and the Therapeutic Milasen (Kim J et al., Patient- Customized Oligonucleotide Therapy for a Rare Genetic Disease, N Engl J Med.. 2019 Oct; 381: 1644-1652).

[0125] Chemical modifications tested. The formulation of standard antisense RNA oligonucleotides decreases mRNA abundance of the target gene via RNase H recruitment, which would be contrary’ to the ultimate goal of increasing abundance of functional protein. For that reason, SSOs must be chemically modified to be resistant to RNase H. There are several modifications that can be used to that end, as well as to improve SSO stability’, binding affinity to the target pre-mRNA, and increase cellular uptake. All SSOs were synthesized byintegrated DNA Technologies.

[0126] Backbone modification: In all SSOs discussed in this application, phosphorothioate bonds were used in place of phosphodi ester bonds, replacing a non-bridging oxygen atom with a sulfur atom. This modification increases stability in vivo and improves SSO retention by reducing renal clearance (Havens MA et al., Splice-switching antisense oligonucleotides as therapeutic drugs, Nucleic Acids Res.. 2016 Aug; 44:6549-6563).

[0127] 2' modifications: Modifying the 2' position causes the SSO to be resistant to RNase H. The most widely used modifications are 2'-O-methyl (2'-OMe) and 2'-O-methoxyethyl (2M0E), with 2'-M0E modified oligos showing the most promise in vivo and in clinical trials. Applicants have tested both of these modifications, as the 2'-OMe modification is more readily taken up by in vitro cell culture models.

[0128] SSO sequence design. Three SSOs to inhibit pseudoexon inclusion were designed (FIG. 10) to block the splice donor site, splice acceptor site, and a predicted exonic splicing enhancer. That was done using ESEfinder 3.0 (Cartegni L et al.. ESEfinder: A web resource to identify exonic splicing enhancers, Nucleic Acids Res., 2003 Jul; 31(13):3568-3571), which predicts serine/arginine (SR) rich protein binding to the input sequence. SR proteins are highly conserved and integral to pre-mRNA splicing, making this a good target for SSOs. To further optimize SSO sequence, we chose oligo lengths 24-25 nucleotides reducing off-target effects inherent to shorter sequences approaching the lower bound of 12 nucleotides, and preventing decreases in cellular uptake that occur at the upper bound of >25 nucleotides. SSOs were also designed to avoid self-complementarity and G-quartet structures, which are known to reduce efficiency. These SSOs were tested with both the 2'-0Me and the 2'-M0E modifications.

[0129] Because sequence-dependent cellular uptake is not easily predictable, 17 2'-M0E SSOs tiled across the pseudoexon region were screened (FIG. 10).

Table 3. SSO Sequences and Modifications.

[0130] In Table 3, denotes a phosphorothioate bond, “mN” denotes N base with 2’-0Me modification, “i2MOErN” denotes internal N base with 2’-M0E modification, ”52MOErN" denotes 5’ N base with 2’-M0E modification, and “32MOErN” denotes 3’ N base with 2’- MOE modification. With the exception of SEQ ID NO: 1 - SEQ ID NO: 3, the sequences in Table 3 (SEQ ID NO:4 - SEQ ID NO:23) are RNA-like, but as a result of modifications, the skilled person in the art would recognize these sequences as neither necessarily DNA nor RNA. For example, in an aspect, SEQ ID NO:4 - SEQ ID NO:23 can be considered MOE RNA.

[0131] Each 2'OMe SSO in a single homozygous mutant cell line was initially tested to determine whether splice-switching at this specific pseudoexon locus could be induced. Each SSO was transfected in a dose-response curve, from 0 to 200 nM SSO, and RNA collected 24 hours later for RT-PCR (FIG. 9). SSO transfection of hand-designed SSOs was performed as follows. Homozygous mutant c.556+1069T>G HEK293T cells were plated to 70% confluent on a 24-well plate. SSOs were transfected at doses 0, 10, 25, 50, 100, and 200 nM using 1.5 pL Lipofectamine 3000 and 2 pL P3000 reagent, as per manufacturer's instructions. Cells were incubated for 24 hours after transfection and lysed for RT-PCR or qRT-PCR. Tiled SSOs: Identical to hand-designed SSOs, but performed in a 96-well plate with 0.3 pL Lipofectamine 3000, 0.2 pL P3000 reagent, and at 100 nM SSO. After performing RT-PCR and running the products on an agarose gel, it was observed that each of the 3 2'-0Me SSOs caused spliceswitching in a dose- dependent manner. (FIG. 9).

[0132] To more precisely quantify the splice-switching effects of each SSO, and to enable higher-throughput screening, we designed a probe-based qPCR assay to detect the canonical and pseudoexon-containing isoforms separately. This allowed us to better disentangle their individual quantities, as the RT-PCR assay would have different levels of efficiency at each differently -si zed isoform. Primer/probe mixes were synthesized at a primerl: primer: probe ratio of 2:2: 1 by Integrated DNA Technologies (PrimeTime qPCR probe assays). Cells were lysed and RT-qPCR was performed using the Cells-to-CT 2-step Taqman format kit (Invitrogen, AMI 728) as per manufacturer's instructions. The qPCR assay for the canonical isoform (qAKl) amplifies both the canonical and pseudoexon isoforms, but the probe will only detect the canonical isoform using a split design crossing the exon 6/7 boundary. The assay for the pseudoexon (qAKI2) amplifies only the pseudoexon isoform, and the probe detects only the pseudoexon isoform by crossing the exon 6/pseudoexon boundary (FIG. 11).

Table 4. Isoform Specific qPCR Assay Primer and Probe Design.

[0133] In Table 4, FAM denotes fluorescein amidite dye, 3IABkFQ denotes 3' Iowa Black quencher, ZEN denotes internal quencher proprietary to Integrated DNA Technologies.

[0134] Quantification of splice-switching activity by 2'-OMe and 2' -MOE designed SSOs is described as follows. Five biologically independent homozygous mutant cell lines were transfected with 200 nM of each of the three SSOs, using both the 2'-OMe (FIG. 12) and 2'- MOE (FIG. 13) formulations. Using both formulations, it was observed the largest effect size with SS03, with canonical isoform expression increasing by 2.2x using 2'-OMe SS03 and 1.8x using 2'-MOE SS03. Pseudoexon isoform expression decreased by ~2x using both formulations of SS03.

[0135] Next, combinations of 2 -MOE SSOs were tested. The efficacy of combinations of SSOs were tested to determine whether targeting multiple sites - the splice acceptor (SS01), an exonic splice enhancer (SS02), and the splice donor SS03 - would result in higher spliceswitching activity than any one of those loci alone.

[0136] Each 2'-M0E SSO was transfected alone, as well as every possible combination of SSOs, in a dose-response curve (FIG. 14). Clear synergistic effects were observed; however, these results may indicate that combining the most effective SSO (SS03) with SSO 1, SS02, or both SSO 1+SS02 may allow lower overall dosage for a given effect size. [0137] We tiled 2'-MOE SSOs every 5 base pairs across the genomic region containing the pseudoexon and performed a screen of all 17 compared to a scrambled control 2'-M0E SSO. We also reduced the SSO length to 22 bases to improve cellular intake. Each SSO was transfected at 100 nM concentration into 3 biological replicate homozygous mutant cells and collected RNA 24 hours post-transfection. To quantify the splice-switching effects, qAKIl and qAKI2 RT-qPCR assays were performed as described above (FIG. 11).

[0138] It was found that all SSOs induced a significant decrease in pseudoexon isoform (qAKI2 assay) expression of at least 50% compared to a scrambled SSO negative control (FIG. 15). oAKI103 induced the largest decrease, expressing ~ 10% pseudoexon isoform expression compared to the same cell lines transfected with a scrambled control oligo. There is a clear trend of SSOs improving in efficiency (with respect to pseudoexon expression) as they approach the 5' end of the pre-mRNA molecule. This is due to the higher presence of predicted SR protein binding sites at that locus.

[0139] One skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present disclosure described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present disclosure. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the present disclosure as defined by the scope of the claims. [0140] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.