WO2024196804A1 - Factor viii splice-modulating antisense oligonucleotides and methods of use - Google Patents

Abstract

Provided are splice-modulating antisense oligonucleotides (ASOs) that target a terminal stem loop structure at the 3' end of intron 15 (TSL-3-15) of a Factor VIII (F8) pre-mRNA. Also provided are compositions comprising the splice-modulating ASOs. In some embodiments, the compositions are formulated for administration to a subject. Methods of treating Hemophilia A (HA) in a subject in need thereof are also provided. In certain embodiments, the subject exhibits aberrant splicing of Factor VIII (F8) exon 16, and the methods comprise administering to the subject a therapeutically effective amount of a composition of the present disclosure.

Description

FACTOR VIII SPLICE-MODULATING ANTISENSE OLIGONUCLEOTIDES AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/453,036, filed March 17, 2023, which application is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under contract R35GM130361 and contract R01 GM095850 awarded by the National Institutes of Health. The Government has certain rights in the invention.

INTRODUCTION

Noncoding sequences (introns) interrupt protein coding information (exons) in most human genes. Conserved sequences known as splice sites (ss) demarcate exon-intron boundaries (1 ). Messenger RNA (mRNA) biogenesis requires intron removal from precursor transcripts and exon ligation (2, 3). The spliceosome assembles on each intron to catalyze the splicing reaction. This process involves the ordered assembly of five uracil-rich small nuclear ribonucleoprotein particles (U snRNPs) and hundreds of protein factors (4)(5). Exon definition is an initial spliceosome assembly step where splice site recognition occurs (6). In this early complex, U1 snRNP recognizes the 5'ss while the 3'ss and polypyrimidine tract bind U2 snRNP auxiliary factor (U2AF) (7-10). Some exonic sequences function as splicing enhancers or silencers, regulating this critical step of gene expression (11-16).

Aberrant splicing contributes to the etiology of many inherited diseases (17). Diseasecausing mutations often ablate splice sites sequences and exonic splicing enhancers. These lesions cripple the gene by producing a faulty message (18-20). Antisense oligonucleotides (ASOs) are a promising therapeutic modality for rescuing aberrant splicing. Their facile design exploits the chemical language of nucleic acid base pairing interactions. Yet, many problems including toxicity, delivery and stability hindered clinical translation (21 ). Solutions to these challenges exist, as multiple ASOs recently earned FDA approval(22). Notable examples include the splice-modulating drugs Nusinersen and Milasen. 15 years in development, Nusinersin was the first FDA approved cure for spinal muscular atrophy (23-25). By contrast, Milasen is a patientspecific ASO for treatment of Batten's disease, developed in only 16 months (26).

The F8 gene encodes a protease required for activation of the coagulation cascade. F8 deficiency causes Hemophilia A (HA), a potentially lethal inherited bleeding disorder. In some cases, aberrant splicing of the F8 pre-m RNA contributes to HA etiology (27-30). SUMMARY

Provided are splice-modulating antisense oligonucleotides (ASOs) that target a terminal stem loop structure at the 3’ end of intron 15 (TSL-3-15) of a Factor VIII (F8) pre-mRNA. Also provided are compositions comprising the splice-modulating ASOs. In some embodiments, the compositions are formulated for administration to a subject. Methods of treating Hemophilia A (HA) in a subject in need thereof are also provided. In certain embodiments, the subject exhibits aberrant splicing of Factor VIII (F8) exon 16, and the methods comprise administering to the subject a therapeutically effective amount of a composition of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : In vivo splicing assays reveal a highly fragile exon susceptible to mutation- induced aberrant splicing. (A) HA-causing mutations were tested for their impact on splicing across the F8 locus. Their positions are denoted by a black bar and their identifying number in the Human Gene Mutation Database (HGMD). (B) Each test exon was cloned into the HBB minigene to create a heterologous splicing reporter. (C) A representative agarose gel showing the various HA-causing mutations that can induce aberrant splicing of F8 exon 16, one of the most fragile exons discovered from the present splicing assays. (D) Percent-spliced-in (PSI) plot quantifying the impact HA-causing mutations have on F8 exon 16 splicing. Each mutation’s predicted impact on regulatory elements involved in splicing are annotated. Statistical significance between comparisons are denoted by asterisks that represent P-values with the following range of significance: ns = P > 0.05, * = P < 0.05, ** = P < 0.01 , *** = P < 0.001 , **** = P < 0.0001.

FIG. 2: ASO interference mapping (AIM) reveals splice-modulating ASOs for F8 exon 16 and a highly splicing-sensitive mutation in exon 16. (A) A mock schematic of AIM. Each ASO used in the present AIM experiments is 18 nucleotides in length, and are designed using ribose sugars that are heavily modified. The 2’-OH is modified to contain a methoxyethyl group (2’-MOE, highlighted), and the phosphate backbone is modified to a phosphorothioate backbone (highlighted). Each 18-mer ASO is contiguous by design, tiling across exon 16 and its flanking introns with no overlaps between each ASO. (B) Proof-of-concept demonstrating how the present ASOs and controls are expected to work in the AIM experiments. As shown in the annotative matrix above a representative agarose gel, the first two controls consist of the present 5’ss blocker ASO (positive control) and the present non-targeting ASO (negative control) being cotransfected with the WT exon 16 splicing reporter to demonstrate that the designed ASOs can modulate splicing. The last two controls consist of the WT exon 16 and A333G mutant splicing reporters without ASOs co-transfected to illustrate the typical splicing ratios expected to perhaps be seen from their splicing. Expected mRNA isoforms including or excluding the test exon are also annotated to the left of the agarose gel (C) and (D) show the AIM analysis of WT exon 16 and the A333G mutant of exon 16, respectively. AIM results are quantified using the PSI ratio. Each ASOs ability to significantly modulate splicing is annotated by color and corresponding effect (e.g., enhance or suppress splicing). Statistical significance between comparisons are denoted by asterisks that represent P-values with the following range of significance: ns = P > 0.05, * = P < 0.05, ** = P < 0.01 , *** = P < 0.001 , **** = P < 0.0001 .

FIG. 3: SHAPE probing identifies a native RNA structure (TSL-3-15) that is uniquely positioned at the 3’ss of F8 exon 16. (A) A normalized SHAPE reactivity vs. arc diagram plot comparing WT exon 16 and the A333G mutant. The plot uses SHAPE data that is plotted on the top portion of the plot, where each bar indicates the normalized SHAPE reactivity for each nucleotide position. The bottom portion of the plot depicts RNA structure predictions using the normalized SHAPE reactivity as a folding constraint. Each arc represents a base pairing interaction between the respective nucleotides involved within the sequence to form a given RNA structure. RNA structures unique to the WT or A333G mutant are depicted by their respective annotations. RNA structures that are shared and found in both sequence contexts are annotated black. A schematic model of exon 16 and its flanking introns are shown at the bottom of the plot to illustrate relative positions of SHAPE data. TSL-3-15 is specifically annotated to illustrate its position. (B) SHAPE-driven secondary structure prediction of TSL-3-15 depicted in its two dimensional structure. Cis- regulatory elements, core splicing signals, and ASOs are also annotated within the structure. Black represents core splicing signals such as the branchpoint motif, the poly-Y tract, and the consensus 3’ and 5’ss dinucleotides (these are explicitly indicated by arrows). Red-orange represents the bioinformatically predicted hnRNPAI binding sites. Each specific ASO and their target sequence within TSL-3-15 is annotated by a distinct color and sequence complementarity, respectively. All SHAPE probing data generated were done in vitro using the SHAPE reagent 2A3, and all subsequent data analysis was performed in RNA Framework.

FIG. 4: A combination of ASOs targeting TSL-3-15 can additively enhance splicing of a highly splicing-sensitive mutation by increasing 3’ss accessibility. (A) A representative agarose gel depicting the results from the in vivo splicing assays testing duo and trio ASO combinations’ ability to modulate splicing. Each splicing assay condition is annotated as shown in the matrix above the gel. Expected mRNA isoforms including or excluding the test exon are also annotated to the left of the agarose gel. (B) A plot quantifying the results from (A) using the PSI ratio. Each ASOs ability to significantly modulate splicing is annotated by color and corresponding effect (e.g., enhance or suppress splicing). Statistical significance between comparisons are denoted by asterisks that represent P-values with the following range of significance: ns = P> 0.05, * = P < 0.05, ** = P< 0.01 , *** = P< 0.001 , **** = P< 0.0001 . (C) An overlay plot comparing normalized 2A3 reactivities between two distinct SHAPE probing conditions used to probe the A333G mutant. One SHAPE condition probes A333G with ASOs present (annotated light blue), and the other condition probes A333G without ASOs present (annotated light red). Admixing of colors where this is indistinguishable overlap represents similar SHAPE reactivity values between the two probing conditions at that nucleotide position. The nucleotide positions where the ASOs bind, in addition to important splicing signals, are annotated in the plot.

FIG. 5: hnRNPAI cooperates with TSL-3-15 to amplify inhibitory effects at the 3’ss of F8 exon 16. (A) Representative Western blot and agarose gel depicting results from the hnRNPAI - ASO competition assay. Each condition tested in the assay is annotated as shown in the matrix above the gel. Epitopes targeted by specific antibodies in the Western blots are indicated to the left of their respective blots. Expected mRNA isoforms including or excluding the test exon are also annotated to the left of the agarose gel. (B) A plot quantifying the results from (A) using the PSI ratio. Co-transfection of the WT exon 16 splicing reporter with either the empty expression vector (PCG) or the hnRNPAI expression vector is indicated, respectively. (C) Representative Western blot and agarose gel electrophoresis depicting results from the SRSF6 splicing assay. Each condition tested in the assay is annotated as shown in the matrix above the gel. Epitopes targeted by specific antibodies in the Western blots are indicated to the left of their respective blots. Expected mRNA isoforms including or excluding the test exon are also annotated to the left of the agarose gel. (D) A plot quantifying the results from (C) using the PSI ratio. Cotransfection of the WT exon 16 splicing reporter with either the empty expression vector (PCG) or the hnRNPAI expression vector is indicated, respectively. Statistical significance between comparisons are denoted by asterisks that represent P-values with the following range of significance: ns = P > 0.05, * = P < 0.05, ** = P< 0.01 , *** = P< 0.001 , **** = P < 0.0001 .

FIG. 6: A combination of ASOs targeting TSL-3-15 can reverse aberrant splicing for a broad array of Hemophilia A associated variants of exon 16 by increasing 3’ss accessibility and blocking hnRNPAI binding. (A) A UCSC Genome Browser screenshot depicting the F8 exon 16 locus and the positions of HA-causing mutations tested in this study. The 3’ and 5’ splice sites are annotated in addition to TSL-3-15. Successful ASOs targeting TSL-3-15 are depicted using the same color scheme as previously shown in Fig. 3B. (B) A normalized SHAPE reactivity vs. arc diagram plot comparing WT exon 16 to multiple HA-causing mutations that induce aberrant splicing of exon 16. The plot uses SHAPE data for each sequence context (i.e., WT or MT) that is plotted on the top portion of the plot, where each bar indicates the normalized SHAPE reactivity at a nucleotide position for a given sequence context. The bottom portion of the plot depicts RNA structure predictions for each sequence context using their respective normalized SHAPE reactivity as a folding constraint. Each arc represents a base pairing interaction between the respective nucleotides involved within the sequence to form a given RNA structure. RNA structures unique to the WT or a specific MT are depicted by their respective color annotations. RNA structures that are shared and found in both WT or MT sequence contexts are annotated black. A schematic model of exon 16 and its flanking introns are shown at the bottom of the plot to illustrate relative positions of SHAPE data. TSL-3-15 is specifically annotated in yellow to illustrate its position. (C) A representative agarose gel depicting the results from the in vivo splicing assays testing the trio ASO combinations’ ability to reverse aberrant splicing of exon 16 induced by other HA-causing mutations. Each splicing assay condition included in this specific assay is annotated as shown in the matrix above the gel. Expected mRNA isoforms including or excluding the test exon are also annotated to the left of the agarose gel. (D) A plot quantifying the results from (C) using the PSI ratio. Each sequence context tested (WT or MT) is annotated by a distinct color. Statistical significance between comparisons are denoted by asterisks that represent P-values with the following range of significance: ns = P > 0.05, * = P < 0.05, ** = P < 0.01 , *** = P < 0.001 , **** = P < 0.0001 .

FIG. 7: The loss of a critical ESE in F8 exon 16 presumably amplifies the inhibitory nature of TSL-3-15 to alter exon definition and splicing fidelity. (A) A schematic depicting WT exon 16 (shown in light gray) in the splicing reporter context from in this study. In the WT context, a functional ESE recruits a positive splicing factor that controls the structure-function mechanism comprising TSL-3-15 and hnRNPAI at the 3’ss of exon 16. Doing so appears regulates the inhibitory effects of TSL-3-15 and hnRNPAI cooperation, increasing the accessibility of the 3’ss to the splicing machinery. (B) A schematic depicting the loss of the ESE in exon 16 due to the A333G mutation. Losing the ESE diminishes the ability to regulate TSL-3-15 and hnRNPAI cooperation at the 3’ss exon 16, leading to decreased accessibility of the 3’ss. (C) A schematic depicting the trio ASO combinations’ ability to reverse A333G-induced aberrant splicing of exon 16 by destabilizing TSL-3-15, and preventing the recruitment of hnRNPAI to the 3’ss. Collectively, the data-supported model indicates that the trio ASOs block the recruitment of a negative splicing factor and to increase the accessibility of the 3’ss to the splicing machinery. TSL-3-15 is annotated by a simplified depiction of an RNA secondary structure at the 3’ss of exon 16. RBPs binding to TSL-3-15 and this region such as hnRNPAI and U2AF are respectively annotated. The predicted ESE is annotated within exon 16, and its binding partner, presumably an RBP like SR proteins that are known to enhance splicing, is depicted as well. The loss of the ESE by the A333G mutation is annotated within exon 16.

FIG. 8: mRNA isoform levels quantified by a two-step end-labeled RT-PCR assay and capillary electrophoresis indicating that the majority of HA-causing mutations failed to induce exon skipping in a heterologous reporter context.

FIG. 9: mRNA isoform levels quantified by a two-step end-labeled RT-PCR assay and capillary electrophoresis indicating the effects of mutations in exon 7, 11 , 16 and 18 on exon inclusion.

FIG. 10: SHAPE probing data of HA mutants of exon 16.

FIG. 11 : In vivo splicing assay data for a duo combination of ASOs.

FIG. 12: Data demonstrating that a single ASO such as 91 -108 is capable of increasing the SHAPE activities for nucleotides comprising the poly-Y tract. DETAILED DESCRIPTION

Before the ASOs and methods of the present disclosure are described in greater detail, it is to be understood that the ASOs and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the ASOs and methods will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the ASOs and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the ASOs and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the ASOs and methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the ASOs and methods belong. Although any ASOs and methods similar or equivalent to those described herein can also be used in the practice or testing of the ASOs and methods, representative illustrative ASOs and methods are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present ASOs and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the ASOs and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the ASOs and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present ASOs and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

SPLICE-MODULATING ANTISENSE OLIGONUCLEOTIDES

Aspects of the present disclosure include splice-modulating antisense oligonucleotides (ASOs). In certain embodiments, the ASOs target a terminal stem loop structure at the 3’ end of intron 15 (TSL-3-15) of a Factor VIII (F8) pre-mRNA. Such ASOs are based on the inventors’ surprising discovery of the TSL-3-15 via RNA chemical probing, and of ASOs that target the TSL- 3-15 and rescue aberrant splicing of Factor VIII exon 16 resulting from point mutations in the gene encoding Factor VIII. Details regarding the ASOs of the present disclosure will now be provided.

In certain embodiments, a splice-modulating ASO of the present disclosure hybridizes to the F8 pre-mRNA at all or a portion of positions 1 -18, 37-54, 55-72, 73-90, 91 -108, or 469-486. For example, such an ASO may hybridize to 10 or more, 1 1 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17, or each of the contiguous nucleotides at positions 1 -18, 37- 54, 55-72, 73-90, 91 -108, or 469-486.

According to some embodiments, a splice-modulating ASO of the present disclosure hybridizes to the F8 pre-mRNA at all or a portion of positions 55-72, 73-90, or 91-108. In some instances, such an ASO hybridizes to 10 or more, 1 1 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17, or each of the contiguous nucleotides at positions 55-72, 73- 90, or 91 -108. ASOs are typically small (-15-30 nucleotides), synthetic, single-stranded nucleic acid polymers. In some instances, the ASOs comprise nucleotide modifications. Such modifications may impart useful properties, e.g. increase the biological stability of the ASOs (e.g. nuclease resistance), enhance target binding, increase tissue uptake and/or increase the physical stability of the duplex formed between the ASOs and target nucleic acids, etc..

In certain embodiments, the ASO induces steric block of a target sequence, and in such a way that it does not induce target cleavage via RNase H recruitment. For example, an ASO may comprise a chemistry which does not support RNase H cleavage (e.g., does not generate consecutive runs of DNA or DNA-like bases). For example, an ASO may comprise a “mixmer” pattern in which the ASO may comprise two or more different nucleic acid chemistries, but runs of more than 2 or 3 DNA or DNA-like bases (which would support RNase H-mediated cleavage) are avoided.

In certain embodiments, the ASO of the present disclosure may comprise DNA, RNA, and/or nucleotide analogues. The nucleotide analogues may be peptide nucleic acid (PNA), FANA, DANA, LNA, and other branched nucleic acids (ENA, cEt), phosphorodiamidate morpholino oligomer (PMO), and/or tricyclo DNA.

According to some embodiments, the ASO comprises an abasic site, i.e., the absence of a purine (adenine and guanine) or a pyrimidine (thymine, uracil and cytosine) nucleobase.

In certain embodiments, the ASO comprises a 3' to 5' phosphodiester (PO) linkage as naturally found in DNA or RNA. The ASO may comprise a modified internucleoside linkage, e.g. a phosphotriester linkage, a phosphorothioate (PS) linkage, a boranophosphate linkage, a phosphorodiamidate linkage, a phosphoamidate linkage, and/or a thiopho sphoramidate linkage. The modified internucleoside linkage may be other modifications known in the art.

According to some embodiments, the ASO comprises one or more asymmetric centers and thus give rise to enantiomers, diasteromers, and other stereoisomeric configurations, e.g. R, S. For example, stereochemistry may be constrained at one or more modified internucleoside linkages. For example, the oligonucleotide may comprise repeated left-left-right (or SSR) chiral PS centers.

In some instances, the ASO comprises a sugar moiety as found in naturally occurring RNA (e.g., a ribofuranosyl) or a sugar moiety as found in naturally occurring DNA (e.g., a deoxyribofuranosyl). The ASO may comprise a modified sugar moiety, i.e. a substituted sugar moiety or a sugar surrogate. Substituted sugar moieties include furanosyls comprising substituents at the 2'-position , the 3'-position, the 5 '-position and/or the 4'-position. A substituted sugar moiety may be a bicyclic sugar moiety (BNA). Sugar surrogates include morpholino, cyclohexeynl and cyclohexitol.

The modified sugar moiety may comprise a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-O- propyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'O-DMAEOE), or 2'0-N- methylacetoamido (2'0-NMA) modification or a locked or bridged ribose conformation (e.g., LNA, cEt or ENA). The modified sugar moiety may comprise other modifications known in the art.

According to some embodiments, the ASO comprises a terminal modification at its 5' and/or 3' end, such as a vinyl phosphonate, and/or inverted terminal bases.

In certain embodiments, the ASO comprises a nucleobase as found in naturally occurring RNA and DNA (i.e. adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), inosine (I), and 5-methyl C). The oligonucleotide may comprise a modified nucleobase, e.g. 5- hyrdoxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The inclusion of 5'methylcytosine may enhance base pairing by modifying the hydrophobic nature of the oligonucleotide.

In some instances, the ASO comprises a single type of nucleic acid chemistry (e.g. full PS -MOE, or full PMO) or combinations of different nucleic acid chemistries.

For example, each of the sugar moieties in the ASO may comprise a 2'-O-methoxyethyl (2'MOE) modification and each of the internucleoside linkages may be a phosphorothioate (i.e. a fully PS-MOE oligonucleotide). PS modifications are known to result in resistance to a broad spectrum of nucleases and increase protein binding, which also improves tissue uptake. 2'MOE modifications are known to enable enhanced binding affinity to the target mRNA with minimal toxicity and reduce plasma protein binding.

According to some embodiments, the ASO comprises a combination of PO and PS internucleoside linkages. This may facilitate fine tuning of the pharmacokinetics of the oligonucleotide.

In certain embodiments, the ASO is produced using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. Alternatively, An ASO may be produced biologically using an expression vector into which the oligonucleotide is sub-cloned in an antisense orientation, e.g., RNA transcribed from the inserted oligonucleotide will be of an antisense orientation to the target nucleic acid of interest.

COMPOSITIONS

Aspects of the present disclosure further include compositions comprising any of the ASOs of the present disclosure.

In certain embodiments, a composition of the present disclosure includes the ASO present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCI₂, KOI, MgSO₄), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl- 3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes an ASO of the present disclosure, and a pharmaceutically acceptable carrier.

The ASO can be incorporated into a variety of formulations for therapeutic administration. More particularly, the ASO can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

Formulations of the ASOs for administration to an individual (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

In pharmaceutical dosage forms, the ASOs can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers/excipients are merely examples and are in no way limiting.

For oral preparations, the ASOs can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The ASOs can be formulated for parenteral (e.g., intravenous, subcutaneous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, etc.) administration. In certain embodiments, the ASOs are formulated for injection by dissolving, suspending or emulsifying the ASOs in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

Pharmaceutical compositions that include the ASOs may be prepared by mixing the ASOs having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N- methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

An aqueous formulation of the ASOs may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the ASO against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

In some embodiments, the pharmaceutical composition includes the ASO, and one or more of the above-identified components (e.g. , a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).

METHODS OF USE

Aspects of the present disclosure further include methods of treating Hemophilia A (HA) in a subject in need thereof, e.g., a subject who exhibits aberrant splicing of Factor VIII (F8) exon 16. In certain embodiments, the methods comprise administering to the subject a therapeutically effective amount of a composition of the present disclosure, e.g., a composition formulated for administration to a subject comprising any of the ASOs or desired combinations thereof of the present disclosure.

The ASOs of the present disclosure may be administered via any suitable route of administration. In some instances, an ASO or combination thereof of the present disclosure is administered to the subject via parenteral administration. Non-limiting examples of parenteral routes of administration that find use in practicing the methods of the present disclosure include intravenous (IV) infusion and subcutaneous (SC) injection.

The ASOs of the present disclosure may be administered in a composition in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of HA, as compared to a control. With respect to HA, in some embodiments, the therapeutically effective amount is sufficient to reduce the clotting time of the subject, which in some instances may be assessed via a Prothrombin time (PT) test. An effective amount can be administered in one or more administrations.

As described above, aspects of the present disclosure include methods for treating HA in the subject. By treatment is meant at least an amelioration of one or more symptoms associated with the HA of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the HA being treated. As such, treatment also includes situations where the HA, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the HA, or at least the symptoms that characterize the HA.

KITS

Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of treating Hemophilia A (HA) in a subject in need thereof.

Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the ASOs or desirable combinations thereof of the present disclosure (e.g., present in a composition formulated for administration to a subject, such as any of the compositions of the present disclosure), and instructions for administering the ASO or combination thereof to the subject. As will be appreciated, the kits of the present disclosure may include any of the ASOs having any of the features described above in the section relating to the Splice-Modulating Antisense Oligonucleotides of the present disclosure, which are not reiterated herein for purposes of brevity.

The kits of the present disclosure may include a quantity of the ASO or combination of ASOs, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of an ASO or combination of ASOs of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the ASO or combination of ASOs calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the ASO or combination of ASOs employed, the effect to be achieved, and the pharmacodynamics associated with the ASO or combination of ASOs, in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the ASO or combination of ASOs.

The instructions (e.g., instructions for use (I FU)) included in the kits may be 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 (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable 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, the means for obtaining the instructions is recorded on a suitable substrate.

METHODS

The present disclosure also provides ASO interference mapping (AIM) methods of identifying a cis region that affects splicing of an exon of interest. In some embodiments, the methods comprise testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay. The splicing reporter assay reports splicing of the exon of interest, and an ASO that affects splicing of the exon of interest identifies the target sequence of the ASO as a sequence comprising a cis region that affects splicing of the exon of interest.

Also provided are methods of identifying an ASO that affects splicing of an exon of interest. In some embodiments, the methods comprise testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay. The splicing reporter assay reports splicing of the exon of interest, and wherein an ASO that affects splicing of the exon of interest identifies the ASO as an ASO that affects splicing of an exon of interest. According to some embodiments, the splicing reporter assay comprises co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement, such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

In some instances, the splicing reporter assay comprises co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement (e.g., a tube, well, or the like), such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

In certain embodiments, the exon of interest and its flanking introns or portions thereof are the wild-type exon of interest and its flanking introns or portions thereof. In other embodiments, the exon of interest and its flanking introns or portions thereof comprise a mutation. The mutation

Also provided are ASOs that target a cis region identified according to the methods above, wherein the ASO modulates splicing of the exon of interest in a desired manner. Also provided are ASOs identified as affecting splicing of an exon of interest according to the methods above, wherein the ASO modulates splicing of the exon of interest in a desired manner. Compositions comprising such ASOs, including compositions formulated for administration to a subject in need thereof or also provided, as are methods comprising administering such compositions to an individual in need thereof, e.g., where the ASO rescues a defect in splicing of the exon of interest in the subject.

For purposes of completeness, the present disclosure is further defined in the following numbered clauses.

1 . A splice-modulating antisense oligonucleotide (ASO) that targets a terminal stem loop structure at the 3’ end of intron 15 (TSL-3-15) of a Factor VIII (F8) pre-mRNA.

2. The splice-modulating ASO of clause 1 , wherein the splice-modulating ASO hybridizes to the F8 pre-mRNA at all or a portion of positions 1 -18, 37-54, 55-72, 73-90, 91-108, or 469- 486.

3. The splice-modulating ASO of clause 2, wherein the splice-modulating ASO hybridizes to the F8 pre-mRNA at all or a portion of positions 55-72, 73-90, or 91 -108.

4. The splice-modulating ASO of any one of clauses 1 to 3, comprising one or more linkages that confer resistance to nuclease degradation.

5. The splice-modulating ASO of clause 4, wherein the one or more linkages that confer resistance to nuclease degradation comprise one or more phosphorothioate (PS) linkages.

6. The splice-modulating ASO of any one of clauses 1 to 5, comprising one or more sugar modifications that confer resistance to nuclease degradation.

7. The splice-modulating ASO of clause 6, wherein the one or more sugar modifications comprise 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), or any combination thereof.

8. The splice-modulating ASO of any one of clauses 1 to 7, stably associated with one or more moieties that enhance cellular uptake of the splice-modulating ASO.

9. The splice-modulating ASO of clause 8, wherein the one or more moieties comprise a cell-penetrating peptide, octaguanidine dendrimers, or a combination thereof.

10. A composition comprising the splice-modulating ASO of any one of clauses 1 to 9.

11 . The composition of clause 10, comprising one, two, or each of: a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 55-72; a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 73-90; and a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 91 -108.

12. The composition of clause 10 or clause 1 1 formulated for administration to a subject.

13. The composition of clause 12 formulated for parenteral administration to the subject.

14. The composition of clause 13 formulated for intravenous administration to the subject. 15. A method of treating Hemophilia A (HA) in a subject in need thereof, wherein the subject exhibits aberrant splicing of Factor VIII (F8) exon 16, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of clauses 12 to 14.

16. The method according to clause 15, wherein the genome of the subject comprises a Factor VIII (F8) gene mutation selected from the group consisting of: G351 A, C348T, C321 A, C321T, C179T, A333G, and any combination thereof.

17. The method according to clause 15 or 16, wherein the administering is by parenteral administration.

18. The method according to clause 17, wherein the administering is by intravenous administration.

19. An ASO interference mapping (AIM) method of identifying a cis region that affects splicing of an exon of interest, the method comprising: testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay, wherein the splicing reporter assay reports splicing of the exon of interest, and wherein an ASO that affects splicing of the exon of interest identifies the target sequence of the ASO as a sequence comprising a cis region that affects splicing of the exon of interest.

20. The method according to clause 19, wherein the splicing reporter assay comprises: co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement, such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

21 . The method according to clause 19 or clause 20, wherein the exon of interest and its flanking introns or portions thereof are the wild-type exon of interest and its flanking introns or portions thereof.

22. The method according to clause 19 or clause 21 , wherein the exon of interest and its flanking introns or portions thereof comprise a mutation.

23. The method according to clause 22, wherein the mutation is a disease mutation.

24. A method of identifying an ASO that affects splicing of an exon of interest, the method comprising: testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay, wherein the splicing reporter assay reports splicing of the exon of interest, and wherein an ASO that affects splicing of the exon of interest identifies the ASO as an ASO that affects splicing of an exon of interest.

25. The method according to clause 24, wherein the splicing reporter assay comprises: co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement, such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

26. The method according to clause 24 or clause 25, wherein the exon of interest and its flanking introns or portions thereof are the wild-type exon of interest and its flanking introns or portions thereof.

27. The method according to clause 24 or clause 25, wherein the exon of interest and its flanking introns or portions thereof comprise a mutation.

28. The method according to clause 27, wherein the mutation is a disease mutation.

29. An ASO that targets a cis region identified according to the method of any one of clauses 19 to 23, wherein the ASO modulates splicing of the exon of interest in a desired manner.

30. An ASO identified as affecting splicing of an exon of interest according to the method of any one of clauses 24 to 28, wherein the ASO modulates splicing of the exon of interest in a desired manner.

31 . A composition comprising the splice-modulating ASO of clause 29 or clause 30.

32. The composition of clause 31 formulated for administration to a subject.

33. The composition of clause 32 formulated for parenteral administration to the subject.

34. The composition of clause 33 formulated for intravenous administration to the subject.

35. A method of rescuing a splicing defect in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of clauses 31 to 34, wherein the ASO rescues a defect in splicing of the exon of interest in the subject.

The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL

Example 1 - Multiple F8 exons are Susceptible to Aberrant Splicing in the Presence of HA-causing Mutations

We previously described inherited disease-causing mutations with the potential to alter the landscape of ESEs or ESSs ((18)). Among all candidate genes, F8 had the highest density (mutations per exon) and total number of putative splicing-sensitive point mutations ((17)). In this study, we investigated the impact of a wide array of HA-causing mutations on the splicing of eleven F8 exons (Fig. 1 A). To determine whether or not these HA-causing mutations can induce aberrant splicing, we analyzed 97 distinct mutations by generating heterologous splicing reporters where the wild type or mutant sequences for each F8 exon of interest is cloned into the HBB minigene (Fig. 1 B). Following transient transfections of each reporter into HEK293T cells, mRNA isoform levels are quantified by a two-step end-labeled RT-PCR assay and capillary electrophoresis. Among the eleven F8 exons tested, we found that the majority of HA-causing mutations failed to induce exon skipping in this heterologous reporter context (Fig. 8). By contrast, mutations in exon 7, 11 , 16, and 18 had striking effects on exon inclusion, indicating that these exons may be susceptible to aberrant splicing (Fig. 1 C; Fig 9). For example, Fig. 1 C shows splicing assays for 16 HA-causing variants of exon 16. In comparison to the WT exon 16 splicing reporter which is efficiently spliced (Lane 3), we found that the G351 A, C348T, C321A, C321 T, C179T, A333G mutations significantly reduced exon 16 inclusion (Fig. 1C, compare lane 3 to lanes 5 ,7, 10, 11 , 14 and 18; Fig. 1 D).

To investigate mechanisms contributing to aberrant splicing, we focused on F8 exon 16 which had the greatest proportion of splicing-sensitive mutations identified. Exon 16 encodes a large portion of the A3 domain which is required for efficient blood clotting (32). Clinical data repositories indicate that the coding sequence for the A3 domain is frequently disturbed by missense mutations that are linked to HA (33, 34). Our exon 16 splicing assays support the notion that aberrant splicing is a contributing etiology of HA as a result of skipping important coding sequences from F8 mRNA. Particularly, we observe that there are multiple HA-causing point mutations that can readily impact inclusion of exon 16, indicating that exon 16 is fragile and susceptible to aberrant splicing. Additionally, we identify that the A333G mutation induces the highest degree of exon 16 skipping, seeing a significant 5.23-fold decrease in its splicing relative to the WT.

Example 2 - Modulation of F8 exon 16 splicing by Antisense Oligonucleotides (ASOs)

To identify ASOs that can modulate splicing of F8 exon 16, we performed ASO interference mapping (AIM). In these AIM experiments, we designed and tested non-overlapping, phosphorothioate substituted, 2’-methoxyethyl modified 18-mer ASOs that span exon 16 and its flanking introns in a splicing reporter context (Fig. 2A). We also designed ASO controls demonstrating that the chemistry works as expected to modulate splicing (Fig. 2B). In all AIM experiments, we include an ASO that has no sequence specificity for exon 16, serving as our non-targeting (NT) control (Fig. 2B, lane 1 ), and an ASO “blocker” that specifically targets the 5’ss of exon 16 to directly inhibit its splicing (Fig. 2B, lane 2). By tiling ASOs across exon 16 and its flanking introns, we hypothesized that AIM can effectively delineate regions with c/s-regulatory functions that can be exploited to enhance the splicing of exon 16 and its HA variants.

We first performed AIM on exon 16 WT to identify any c/s-regulatory elements that may be required for its splicing. Each individual ASO is co-transfected with the WT exon 16 splicing reporter into each well of a 96-well tissue culture plate culturing HEK293T cells. Effectively, each well corresponds to an ASO that targets a specific position of exon 16 or its flanking introns. AIM analysis on WT exon 16 indicates that all ASOs targeting the exon, except for a few, strongly inhibited its splicing (Fig. 2C). By contrast, individual ASOs targeting the flanking introns had little impact on exon 16 splicing relative to our controls. A couple of ASOs that target the intronic region downstream of the 5’ss, 469-486 and 505-522, are indicated to be statistically significant in enhancing splicing relative to the WT control with no ASO. 469-486 results in a 1.1891 -fold increase in splicing (P-value = 0.0106), whereas 505-522 results in a 1.1879-fold increase in splicing (P-value = 0.0114). ASOs that target the flanking intronic sequences upstream of the 3’ss appear to modulate splicing positively or negatively in a subtle, non-statistically significant manner.

To determine if any ASOs could rescue splicing-sensitive mutants of exon 16, we then performed AIM using the A333G mutation as a model. Compellingly, we discovered multiple ASOs that enhanced splicing of the A333G mutant (Fig. 2D). Co-transfection of ASOs 1 -18, 37- 54, 55-72, 91 -108, or 469-486 with A333G resulted in a statistically significant increase in exon 16 inclusion relative to its control with no ASOs co-transfected. Of note, these ASOs target the flanking introns of the A333G mutant of exon 16. ASOs 1 -18, 37-54, 55-72, and 91 -108 target regions upstream and adjacent to the 3'ss, with 37-54 exhibiting the largest rescue effect by increasing splicing by 2.015-folds (P-value <0.0001 ). ASO 469-486 targets a region downstream of the 5’ss, rescuing splicing by 1 .696-folds (P-value = 0.0007). Taken together, our AIM data indicates that targeting regions upstream or downstream of F8 exon 16 with ASOs may rescue splicing of splicing-sensitive mutants by perturbing the influence of inhibitory elements found in the flanking introns.

Example 3 - SHAPE-MaP-seg Reveals an Inhibitory RNA Structure (TSL-3-15) at the 3’ss of F8 Exons

Several studies predict that disease-causing mutations can alter RNA structures and their biological functions. To determine how splicing-sensitive mutants may influence the formation of RNA structures in F8 exon 16, we performed selective 2’-hydroxyl acylation analyzed by primer extension and mutational profiling coupled to high-throughput sequencing (SHAPE-MaP-seq) on in vitro transcribed RNA. All in vitro RNA corresponding to WT exon 16 or its HA mutants contain the same sequence context as we tested in our splicing reporter assays. For all chemical probing experiments performed in this study, 2-aminopyridine-3-carboxylic acid imidazolide (2A3) was selected as our SHAPE reagent due to its moderate improvement in accurately producing data- driven folding predictions, compared to other SHAPE reagents conventionally used in chemical probing experiments (35).

We interrogated the RNA structure profiles for several splicing-sensitive variants of F8 exon 16 that we discovered, comparing each of their 2A3 reactivities and SHAPE-driven folding predictions to WT exon 16. With a particular focus on the A333G mutation, SHAPE probing of this mutant shows that it primarily induces local rearrangements to RNA structures nearby, as well as in creating some long-range base-pairing interactions that involve the flanking introns (Fig. 3A). Surprisingly, when comparing the entire structure profile to the WT, most of the RNA structures detected in the A333G mutant appear unchanged. Analyzing the SHAPE probing data of other HA mutants of exon 16 also revealed similar findings where these mutations primarily induced changes to 2A3 reactivities proximal to their positions, and that the majority of 2A3 reactivities between the WT and mutants also remain unchanged (Fig. 10). Taken together, these SHAPE-MaP-Seq experiments suggest that HA-causing mutations in exon 16 will generally cause changes to local RNA structures.

Intriguingly, when analyzing all of our WT and mutant SHAPE-driven structure predictions, we discovered a “Y-shaped” RNA structure that forms at the 3’ss of F8 exon 16 (Fig. 3B). Our SHAPE-driven structure predictions indicate that an upstream region of F8 intron 15 base-pairs with the branchpoint and poly-Y tract, potentially occluding their accessibility to splicing factor 1 (SF1 ) and U2AF. When cross-referencing our SHAPE and AIM data together, we discover that ASOs that individually improved splicing of the A333G mutant hybridize to this structure. These ASOs include 55-72 and 91 -108, which had a statistically significant improvement on A333G splicing, and 73-90 which improved splicing of the A333G mutant in a non-significant manner (A333GNO ASO, PSI = 14.87%, Std Dev. = 0.028; A333GASO 73-90, PSI = 20.80%, Std Dev. = 0.033). Our analysis indicates that ASOs capable of enhancing exon 16 splicing and its HA mutants like A333G may be doing so by destabilizing this 3’ss structure, which we will refer to as TSL-3-15 (terminal stem loop at the 3’ end of intron 15).

To determine if TSL-3-15 may contain any functional binding sites for RBPs, we used RBPmap to identify RBP consensus motifs within the structure (36). In this analysis, we used an input sequence that contained the exact intron 15 sequence that flanks exon 16 in all of our splicing reporter contexts. We included a high stringency constraint to match known RBP motifs within our input sequence, as well as a conservation filter to selectively identify motifs that best match sequences from the human and mouse genomes. Intriguingly, RBPmap recognized two hnRNPAI binding motifs within TSL-3-15 (Fig. 3B, binding sites are highlighted in dark red bubbles with white text). The first predicted motif is found at nucleotide positions 84-90 (UUAGGGA), and the second predicted motif is found at nucleotide positions 99-105 (CUAAGGA). We term these predicted hnRNPAI binding sites as ISS-15-1 and ISS-15-2, respectively. Based on published research, these predicted binding sites contain a motif that contains or highly resembles the hnRNPAI consensus motif, UAGG (37, 38). These predicted hnRNPAI binding sites are positioned within the three-way junction of TSL-3-15. Intriguingly, ASOs 73-90 and 91 -108 directly binds ISS-15-1 and ISS-15-2, respectively. These ASOs, when used individually, improved splicing of the A333G mutant. Together, we hypothesize that there is a structure-function relationship where TSL-3-15 weakens the accessibility of the 3’ss to U2AF due to structural constraints, in addition to recruiting a pair of hnRNPAI proteins that cooperates with this structure to amplify inhibitory effects at the 3’ss. As a whole, our data analysis suggests that co-transfecting more than one ASO targeting TSL-3-15 may further destabilize this structure to attenuate 3’ss accessibility and thereby restore proper splicing of exon 16.

Example 4 - Combinations of ASOs additively enhance exon 16 splicing in the A333G Mutant By Destabilizing TSL-3-15

To test the hypothesis that a combination of ASOs can additively enhance splicing of the A333G mutant even further compared to a single ASO when targeting TSL-3-15, we performed additional in vivo splicing assays where A333G is co-transfected with ASOs 55-72, 73-90 and 91 -108. All three ASOs directly hybridize to the partner strand that envelopes the branchpoint and poly-Y tract, in addition to the predicted hnRNPAI -dependent silencers ISS-15-1 and ISS- 15-2. Remarkably, we discover that this trio ASO combination had a striking effect on exon 16 splicing empirically (Fig. 4A). Compared to the A333G mutant control without any ASOs cotransfected (Fig. 4A, lane 4), in addition to a duo combination that worked best from a preliminary screen (Fig. 4A, lane 5; Fig. 1 1 ), we observed that the trio combination had the strongest additive effect on exon 16 splicing (Fig. 4A, lane 6). Quantitatively, relative to the A333G control with no ASOs (PSI = 19.12%), the trio ASO combination of ASOs 55-72, 73-90, and 91 -108 enhanced splicing of A333G the most significantly (PSI = 77.78%, P-value <0.0001 ), increasing the inclusion of exon 16 by 4.068-folds (Fig. 4B). Most notably as well, this trio ASO combination restores splicing of the A333G mutant to levels that are not significantly different from WT exon 16.

To test the hypothesis that the trio ASO combinations targeting TSL-3-15 increases the accessibility of the poly-Y tract, we performed additional SHAPE-MaP-seq experiments to determine if the ASOs are destabilizing base-pairing interactions that comprise the structure. We adapted the same SHAPE-MaP-seq approach previously described with the exception that prior to SHAPE probing, the in vitro RNA template was unfolded and then re-folded with the ASOs present. In order to draw comparisons between the probing condition with ASOs present and the control condition without ASOs, we calculated the averaged SHAPE reactivity for each nucleotide from the respective datasets and plotted them on the same axis. Differences between each dataset’s SHAPE reactivities are represented by their distinct color annotation, whereas predominant admixing of colors represent minimal differences. Accordingly, the results from this experiment show that there is an increased shift in SHAPE reactivities for nucleotides that surround or comprise the poly-Y tract in the condition with ASOs 55-72 and 73-90 present, compared to the minus ASO condition (Fig. 4C). Additionally, a single ASO such as 91 -108 is also capable of increasing the SHAPE activities for nucleotides comprising the poly-Y tract (Fig. 12). These results indicate that our ASOs enhance exon 16 splicing by destabilizing TSL-3-15 to increase the accessibility of the 3’ss. Moreover, the data supports our structure-function hypothesis and illuminates a mechanism of action for our ASOs where the reversal of aberrant splicing is achieved by destabilizing TSL-3-15 to increase the accessibility of the poly-Y tract to U2AF. Collectively, these findings invite the exciting hypothesis that simply targeting TSL-3-15 with ASOs can potentially rescue any splicing-sensitive mutants of exon 16.

Example 5 - TSL-3-15 Cooperates with hnRNPAI to Desensitize F8 Exon 16 Definition

We believe our SHAPE probing and combinatorial ASO experiments point towards a structure-function mechanism for FS exon 16 where TSL-3-15 occludes 3’ss accessibility, and in recruiting hnRNPAI to repress splicing, as indicated by RBPmap’s prediction of two putative ISSs. To test the hypothesis that promising ASO combinations are acting to enhance splicing by also blocking hnRNPAI binding to ISS-15-1 and ISS-15-2 within TSL-3-15, we performed what we describe as an hnRNPAI -ASO competition assay. To elaborate, we repeated the same combinatorial ASO procedure but with two additional conditions added to the experimental design. Essentially, ASOs and a splicing reporter were co-transfected with either an empty expression vector, or an hnRNPAI expression vector. Conditions with the hnRNPAI expression vector should lead to the overexpression of hnRNPAI , which hypothetically will inhibit splicing of exon 16. If splicing inhibition directed by hnRNPAI is attenuated with ASOs that target the predicted silencers ISS-15-1 and ISS-15-2, this would therefore indicate that hnRNPAI indeed interacts with TSL-3-15. We chose to test whether a duo or trio combination of ASOs would be more effective in attenuating splicing by targeting the silencers in TSL-3-15. In either a duo or trio combination, we ensure the inclusion of ASO 55-72 as it directly binds to the partner strand that base-pairs with the poly-Y tract, and in either combination, we include or exclude a given ASO that independently or simultaneously targets ISS-15-1 or ISS-15-2. We also note that as these hnRNPAI sites are found in a RNA structure that is formed across all F8 exon 16 contexts SHAPE probed in this study, we reasoned that using WT exon 16 would sufficiently conclude whether ASOs targeting TSL-3-15 can attenuate hnRNPAI -dependent inhibition of splicing.

We performed the hnRNPAI -ASO competition assay and were able to demonstrate that overexpression of hnRNPAI can strongly inhibit splicing of WT exon 16 (compare lane 4 to lane 1 ) (Fig. 5A). In comparison to the condition where WT exon 16 was co-transfected with the hnRNPAI expression vector but not with any ASO combinations present (lane 4), we can empirically observe that both a duo or trio ASO combination can attenuate the inhibitory effects of hnRNPAI on exon 16 splicing (compare lane 4 to lane 5 and 6). It is compelling to observe that ASO 55-72 and 73-90 is sufficient to attenuate hnRNPAI -directed inhibition of splicing (lane 5). In this case, only ISS-15-1 is sterically masked by ASO 73-90. More strikingly in the trio ASO combination when 55-72 relieves the poly-Y tract and both ISS-15-1 and ISS-15-2 are respectively blocked by 73-90 and 91 -108, we see the strongest rescue effect where splicing is considerably improved in the presence of hnRNPAI overexpression (lane 6). This assay evidently demonstrates that hnRNPAI binds to the predicted ISSs in the structure, as its inhibitory effects are clearly reduced when ASOs bind to their respective binding sites within TSL-3-15.

By coincidence, we also identified the UAGG motif within the sequence of ASO 91 -108 (5’- AGGTCCTTAGGGTTTACA -3’), inviting the hypothesis that ASO 91 -108 may attenuate exon 16 splicing in our hnRNPAI -ASO competition assay by directly binding to hnRNPAI . A piece of evidence that appears to support this hypothesis is that, compared to the duo combination without ASO 91 -108, only when this ASO is included in a trio combination can we observe the strongest rescue effect on A333G splicing (Fig. 5A, compare lanes 5 and 6). To test this hypothesis, we repeated the hnRNPAI -ASO competition assay using a different heterologous HBB splicing reporter that ASO 91 -108 has no specificity for. This splicing reporter contains SRSF6 exon 6, a reporter that we previously used in a prior work (39). We know that, when hnRNPAI is overexpressed, splicing of SRSF6 exon 6 will be suppressed. The SRSF6 exon 6 splicing reporter is co-transfected with either the empty expression vector, the hnRNPAI expression vector, or both the hnRNPAI expression vector and ASO 91 -108. We observe that, relative to the empty expression vector co-transfected with SRSF6 exon 6 (lane 1 ), co-transfection of this splicing reporter with the hnRNPAI expression vector inhibited splicing as expected (compare lane 1 to lanes 2 and 3) (Fig. 5C). Importantly, in comparing the hnRNPAI co-transfection condition without ASO 91 -108 added (Fig. 5C, lane 2) to the condition with the ASO present (Fig. 5C, lane 3), we do not see any significant changes in SRSF6 exon 6 splicing (Fig 5D). We conclude from these results that ASO 91 -108 does not attenuate hnRNPAI -directed inhibition of splicing by binding to the splicing factor itself. Instead, this experiment supports our hypothesis that the trio ASO combinations are indeed blocking bonafide hnRNPAI -dependent silencers found within TSL-3-15.

Example 6 - Trio ASO Combination Targeting TSL-3-15 Additively Rescues Splicing of a Broad Array of HA-linked Variants of Exon 16

Our experiments indicate that the splicing fidelity of F8 exon 16 appears to be regulated in part, by an RNA structure that weakens the 3’ss and recruits hnRNPAI to this exon-intron junction (Fig 6A). Because this feature is targeted by ASOs that rescue splicing of the A333G mutant, we reasoned that targeting TSL-3-15 might rescue splicing of other splicing-sensitive mutants of exon 16. To test this hypothesis, we co-transfected the WT, G351 A, C348T, C321 A, C321 T, and C179T reporter with the NT ASO or the trio ASO combination. Similar to the A333G mutant, we can observe that co-transfecting ASOs 55-72, 73-90, and 91 -108 in combination can rescue the splicing of other splicing-sensitive HA-causing mutants of exon 16, including the WT context (Fig. 6C). Relative to the NT ASO condition for WT exon 16 (lane 1 ), we can observe that the trio combination can strengthen the splicing efficiency of WT exon 16 splicing in a nonsignificant manner (lane 2). In comparing the NT ASO condition for each exon 16 mutant (lanes 4, 7, 10, 13 and 16) to their condition with the trio combination co-transfected (lanes 5, 8, 1 1 , 14, and 17), we can see that aberrant splicing of these HA mutants were ameliorated with all three ASOs present. For each exon 16 splicing reporter co-transfected with the trio combination, relative to their respective NT ASO condition, this trio combination of ASOs significantly improved their splicing by simply targeting TSL-3-15 (Fig. 6D).

Materials and Methods

F8 Splicing Reporters

The sequences of wild-type (WT) F8 exons were amplified from human genomic DNA (Promega) using WT PCR primers. Following gel purification, PCR products were ligated into pACT7_SC14 (HBB minigene reporter from Lynch Lab) using homology-based cloning technology (In-Fusion HD Cloning kit from Takara Bio). Following sequence verification, each plasmid was used as a template for site-directed mutagenesis via overlap-extension PCR using mutagenesis primers. Mutant (MT) splicing reporter constructs were then sequence-validated using Sanger sequencing to confirm successful cloning and identity of splicing reporters. The designation for each F8 point mutation, and therefore each MT F8 exon presented in this study, is based on the nucleotide being mutated (e g., A>C), and its position within the sequence context tested (i.e., length of flanking introns included and size of exon tested).

Cell-based in vivo Splicing Assays

HEK293T cells (ATCC) were cultured in 6-well tissue culture plates (CytoOne, USA Scientific) using Dulbecco’s Modified Eagle Medium (Gibco, supplemented with 10% FBS) at 37°C, 5% CO2. The cells were transiently transfected at -60-80% confluency with 2.5ug of each F8 splicing reporter using Lipofectamine 2000 (Invitrogen). Total RNA was harvested from cells 24-hours post-transfection using the Direct-zol RNA Miniprep kits (Zymo Research). Each in vivo splicing assay was performed a minimum of three times.

AIM and Combinatorial ASO Experiments

2’-methoxyethyl (2’MOE) phosphorothioate substituted ASOs complementary to F8 exon 16 and flanking introns were designed from the reverse complement of the F8 sense sequence, creating non-overlapping 18-mers. F8 exon 16 ASOs were designed to contiguously tile across the exon and its flanking introns. ASOs were synthesized by Integrated DNA Technologies (IDT). Each ASO is designated by their complementary positions in the F8 exon 16 reporter. HEK293T cells (ATCC) were cultured in 96-well tissue culture plates (Perkin Elmer) as described above. Cells were transiently transfected with 250ng of WT or MT splicing reporter and 10pmol of each ASO using Lipofectamine 2000 (Invitrogen). 24-hours post transfection, cells were harvested and prepared for total RNA purification using the Quick-DNA/RNA Viral MagBead kit from Zymo Research and an Agilent Bravo NGS A liquid handler (31 ). Each experiment type (e.g., AIM or combinatorial ASO assays) was performed a minimum of three times. hnRNPAI overexpression and western blot analysis

HEK293T cells (ATCC) were cultured in 6-well tissue culture plates as described above. Cells were co-transfected with 1250ng of the WT splicing reporter, 1250ng of either an empty expression vector or a T7-tagged hnRNPAI expression vector, and 50pmol of ASO(s) as described above. Total RNA and protein were isolated 24-hours post transfection using a RSB lysis buffer (10mM Tris pH 7.0, 100mM NaCI, 5mM MgCI2, 0.5% NP40, 0.5% Triton X-100, and EDTA-free Protease Inhibitor Cocktail (Roche)). Following a 10 minute incubation on ice, the cell lysate was then centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatant was then collected and aliquoted for two separate applications. The first aliquot, comprising -90% of the cell lysate, was prepared for total RNA purification using the Direct-zol RNA Miniprep kits from Zymo Research. The remaining -10% of the cell lysate was then homogenized into a denaturing buffer solution containing 4X NuPAGE™ LDS Sample Buffer in preparation for polyacrylamide gel electrophoresis (Invitrogen™ NuPAGE™, 4 to 12%, Bis-Tris, 1.0-1.5 mm, Mini Protein Gels), and subsequent Western blots. For western blots, protein samples were transferred to a Immobilon NC membrane (Millipore) using a Genie Blotter (Idea Scientific). Membranes probed with anti-HSP90 (Santa Cruz Biotech) and anti-T7 (Novagen) monoclonal antibodies and visualized by HRP conjugated secondary antibodies and chemiluminescence (Pierce). These experiments were performed a minimum of three times.

Two-step RT-qPCR and Analysis of Splicing Reporter Assays

A minimum of 500 ng of purified total RNA was used as input for all first-strand cDNA synthesis using Multiscribe Reverse Transcriptase (Applied Biosystems). The resulting cDNA was then used as a template for endpoint PCR amplification using specific primers that detect our mRNA splicing reporter isoforms. The forward primer of the pair contains a 5’FAM modification. The resulting amplicons were then analyzed using agarose gel electrophoresis to empirically evaluate mRNA isoforms detected. The abundance of each 5’FAM labeled mRNA isoform is quantified using capillary electrophoresis and fragment analysis (UC Berkeley, DNA Sequencing Center). For fragment analysis, each sample is suspended in a formamide solution that contains a proper size standard for sizing detected fragments (GeneScan 1200 Liz, Applied Biosystems). Analysis was performed in PeakScanner (Thermofisher). Calculating Splicing Efficiency using Percent-Spliced-ln (PSI) Index Formula

Based on fragment analysis data collected, subsequent quantification of splicing efficiency is achieved by comparing relative fluorescence units (RFU) between 5’FAM labeled reporter isoforms that include or exclude an exon of interest. The RFU detected for each reporter isoform is then plugged into the following formula to calculate the PSI index, which reflects the splicing efficiency of an exon in either the WT or MT context:

Included Iso form RFU

PSI = - - -

Included Isoform RFU + Excluded Isoform RFU

The mean PSI for a given reporter context is then calculated using all its respective replicates for a corresponding experiment. Statistical significance in the differences between the mean PSI of the control group(s) vs the experimental group(s) is determined using analysis of variance (ANOVA), and Dunett’s post-hoc test. All statistical tests for PSI analysis were done in GraphPad Prism 9. Values are determined to be statistically significant if calculated the P-value is below an alpha value of < 0.05.

In vitro Transcribed RNA for F8 exon 16 WT and Its MTs

Templates for WT or MT F8 exon 16 pre-mRNA sequences corresponding to the reporter plasmid inserts were synthesized by Primerization, and transcribed in vitro using T7 RNA polymerase. RNA was purified by denaturing PAGE and eluted from gel slices overnight in 10 mM Tris pH 7.5, 480 mM sodium acetate, 1 mM EDTA, 0.1 % SDS. Following ethanol precipitation transcripts were resuspended in ddH2O and quantified by spectrophotometry.

In vitro SHAPE Probing of F8 Targets

F8 exon 16 in vitro transcribed pre-mRNA sequences were first denatured by incubating at 95°C for 3 minutes in 65mM Na-HEPES (pH 8.0). The denatured RNA was then allowed to slowly cool to room temperature (RT) for 15 minutes, after which MgCI2 was supplemented to 1 mM for a total volume of 15pL and incubated at RT for an additional 5 minutes. To chemically modify RNA, 2-aminopyridine-3-carboxylic acid imidazolide (2A3) was added to a final concentration of 100mM and incubated for 2 minutes at 37°C. The reaction was then quenched using dithiothreitol (DTT) to a final concentration of 500mM at RT for 10 minutes. Reactions which substituted 2A3 for anhydrous dimethyl sulfoxide (DMSO) were used as negative controls. All modified RNAs, including negative controls, were then purified using RNA Clean & Concentrator- 5 from Zymo Research.

SHAPE-MaP Library Assembly and Sequencing

Modified RNAs were fragmented to a median size of 200nt by incubation at 94°C for 1 minute using NEBNext® Magnesium RNA Fragmentation Kit and then purified using NEB’s recommended ethanol precipitation protocol. Purified RNA was then prepared for reverse transcription, incubating the RNA with 1 pL of 10mM dNTPs and 2pL of 20pM random hexamers at 70°C for 5 minutes, followed by immediate transfer to ice. Reverse transcription reactions were then supplemented with 4 L of 5X RT buffer (250mM Tris-HCI pH 8.3, 375mM KCI), 2pL of 0.1 M DTT, 1 L of 120 mM MnCI2, 10 U of SUPERase RNase Inhibitor, and 200 U of Superscript II Reverse Transcriptase (SSII) [ThermoFisher Scientific, cat. 18064014] to a final volume of 20 L. These reactions were then incubated at 25°C for 10 minutes to allow for partial primer extension, followed by incubation at 42°C for 3 hours to enable efficient extension. SSII was then heat- inactivated by incubation at 75°C for 20 minutes. Reverse transcription reactions were then supplemented with EDTA to a final concentration of 6mM to chelate Mn2+ ions and incubated at RT for 5 minutes. MgCI₂ was then added to a final concentration of 6mM for each reaction. Reverse transcription reactions were then used as input for NEBNext® Ultra™ II DNA library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), using NEBNext Multiplex Oligos for Illumina® (Unique Dual index UMI Adaptors DNA Set 1 , cat. E7395). Subsequent reactions were performed following manufacturer instructions.

SHAPE-MaP Data Analysis and RNA Structure Prediction

All the relevant data analysis steps were conducted using RNA Framework v2.6.9. Reads produced from Illumina libraries were pre-processed and mapped using the rf-map module (parameters: -b2 -mp “-no-mixed -no-discordant” -bs) ensuring only paired-end mates with expected mate orientation were considered with Bowtie2. The mutational signal was obtained using the rf-count module (parameters: -m -pp -nd -ni) enabling mutation counts of reads produced from properly paired mates. Mutational signal was normalized relative to an unmodified control using parameters (-sm 3 -nm 1 -mu 0.05), based on a scoring method from Siegfried et al., 2014 and further normalized using the 2-8% normalization approach provided by RNA Framework. Normalized reactivities were then supplied to RNAstructure to generate data-driven predicted structure models.

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Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1 . A splice-modulating antisense oligonucleotide (ASO) that targets a terminal stem loop structure at the 3’ end of intron 15 (TSL-3-15) of a Factor VIII (F8) pre-mRNA.

2. The splice-modulating ASO of claim 1 , wherein the splice-modulating ASO hybridizes to the F8 pre-mRNA at all or a portion of positions 1 -18, 37-54, 55-72, 73-90, 91 -108, or 469-486.

3. The splice-modulating ASO of claim 2, wherein the splice-modulating ASO hybridizes to the F8 pre-mRNA at all or a portion of positions 55-72, 73-90, or 91 -108.

4. The splice-modulating ASO of any one of claims 1 to 3, comprising one or more linkages that confer resistance to nuclease degradation.

5. The splice-modulating ASO of claim 4, wherein the one or more linkages that confer resistance to nuclease degradation comprise one or more phosphorothioate (PS) linkages.

6. The splice-modulating ASO of any one of claims 1 to 5, comprising one or more sugar modifications that confer resistance to nuclease degradation.

7. The splice-modulating ASO of claim 6, wherein the one or more sugar modifications comprise 2'-O-methyl (2'-OMe), 2'-0-methoxyethyl (2'-MOE), or any combination thereof.

8. The splice-modulating ASO of any one of claims 1 to 7, stably associated with one or more moieties that enhance cellular uptake of the splice-modulating ASO.

9. The splice-modulating ASO of claim 8, wherein the one or more moieties comprise a cell-penetrating peptide, octaguanidine dendrimers, or a combination thereof.

10. A composition comprising the splice-modulating ASO of any one of claims 1 to 9.

11 . The composition of claim 10, comprising one, two, or each of: a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 55-72; a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 73-90; and a splice-modulating ASO that hybridizes to the F8 pre-mRNA at all or a portion of positions 91 -108.

12. The composition of claim 10 or claim 11 formulated for administration to a subject.

13. The composition of claim 12 formulated for parenteral administration to the subject.

14. The composition of claim 13 formulated for intravenous administration to the subject.

15. The composition of claim 13 formulated for subcutaneous administration to the subject.

16. A method of treating Hemophilia A (HA) in a subject in need thereof, wherein the subject exhibits aberrant splicing of Factor VIII (F8) exon 16, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 12 to 14.

17. The method according to claim 16, wherein the genome of the subject comprises a Factor VIII (F8) gene mutation selected from the group consisting of: G351 A, C348T, C321 A, C321T, C179T, A333G, and any combination thereof.

18. The method according to claim 16 or 17, wherein the administering is by parenteral administration.

19. The method according to claim 18, wherein the administering is by intravenous administration.

20. A kit comprising the splice-modulating ASO of any one of claims 1 to 9 or the composition of any one of claims 12 to 14; and instructions for administering the ASO or composition to a subject having Hemophilia A (HA).

21 . The kit of claim 20, wherein the ASO or composition is present in one or more unit dosages.

22. The kit of claim 20, wherein the ASO or composition is present in two or more unit dosages.

23. An ASO interference mapping (AIM) method of identifying a cis region that affects splicing of an exon of interest, the method comprising: testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay, wherein the splicing reporter assay reports splicing of the exon of interest, and wherein an ASO that affects splicing of the exon of interest identifies the target sequence of the ASO as a sequence comprising a cis region that affects splicing of the exon of interest.

24. The method according to claim 23, wherein the splicing reporter assay comprises: co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement, such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

25. The method according to claim 23 or claim 24, wherein the exon of interest and its flanking introns or portions thereof are the wild-type exon of interest and its flanking introns or portions thereof.

26. The method according to claim 23 or claim 25, wherein the exon of interest and its flanking introns or portions thereof comprise a mutation.

27. The method according to claim 26, wherein the mutation is a disease mutation.

28. A method of identifying an ASO that affects splicing of an exon of interest, the method comprising: testing tiled or non-overlapping ASOs of known sequence that span the exon of interest and its flanking introns or portions thereof in a splicing reporter assay, wherein the splicing reporter assay reports splicing of the exon of interest, and wherein an ASO that affects splicing of the exon of interest identifies the ASO as an ASO that affects splicing of an exon of interest.

29. The method according to claim 28, wherein the splicing reporter assay comprises: co-transfecting an individual ASO and a splicing reporter comprising the exon of interest and its flanking introns or portions thereof into cells present in a confinement, such that each confinement corresponds to an ASO that targets a specific position of the exon of interest or its flanking introns or portions thereof; and qualitatively or quantitively assaying for splicing of the exon of interest.

30. The method according to claim 28 or claim 29, wherein the exon of interest and its flanking introns or portions thereof are the wild-type exon of interest and its flanking introns or portions thereof.

31 . The method according to claim 28 or claim 29, wherein the exon of interest and its flanking introns or portions thereof comprise a mutation.

32. The method according to claim 31 , wherein the mutation is a disease mutation.

33. An ASO that targets a cis region identified according to the method of any one of claims 23 to 27, wherein the ASO modulates splicing of the exon of interest in a desired manner.

34. An ASO identified as affecting splicing of an exon of interest according to the method of any one of claims 28 to 32, wherein the ASO modulates splicing of the exon of interest in a desired manner.

35. A composition comprising the splice-modulating ASO of claim 33 or claim 34.

36. The composition of claim 35 formulated for administration to a subject.

37. The composition of claim 36 formulated for parenteral administration to the subject.

38. The composition of claim 37 formulated for intravenous administration to the subject.

39. A method of rescuing a splicing defect in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 35 to 38, wherein the ASO rescues a defect in splicing of the exon of interest in the subject.