WO2016054075A1

WO2016054075A1 - U2af65 variants and uses thereof

Info

Publication number: WO2016054075A1
Application number: PCT/US2015/052993
Authority: WO
Inventors: Clara L. KIELKOPF; Anant A. AGRAWAL
Original assignee: University of Rochester
Current assignee: University of Rochester
Priority date: 2014-09-29
Filing date: 2015-09-29
Publication date: 2016-04-07
Anticipated expiration: 2017-03-29

Abstract

Provided herein are compositions and methods for decreasing defective splicing of an mRNA transcript or treating disorder associated with defective mRNA splicing.

Description

U2AF VARIANTS AND USES THEREOF

This application claims the benefit of U.S. Provisional Application No. 62/056,934, filed on September 29, 2014 and U.S. Provisional Application No. 62/064,051, filed on October 15, 2014, which are incorporated herein by reference in their entirety. This invention was made with government support under grant number R01 GM070503 awarded by the National Institutes of Health, and DMR-0936384 awarded by the National Science Foundation. The government has certain rights in this invention.

I. BACKGROUND

1. Approximately 15% of the documented disease-causing point mutations disrupt consensus splice site elements in pre-mRNAs, including a polypyrimidine (Py) tract between a branch point sequence (BPS) and an AG dinucleotide at the junction of the 3' splice site. For example, disease-causing mutations in Py tracts have been documented in ~3,000 genes in the Human Gene Mutation Database, and an estimated 20% of these mutations affect regulatory splice site signals. One of the earliest reports of a splice site mutation as a major cause of inherited human disease was for β-thalassemia, for which splice site mutations in the human β-globin gene (HBB) are found in ~ 14% of patients, causing symptoms of mild to severe anemia.

2. With the emergence of high-throughput sequencing technologies, splice site mutations in specific transcripts have been identified as common contributors to neuromuscular disorders, metabolic disorders, cancers, leukemias, deafness, and blindness, among other disorders. Retinitis pigmentosa, the most prevalent form of inherited blindness in adults, represents one such disease that is primarily the consequence of mutations in splice sites of vision-relevant transcripts or splicing factors responsible for their recognition. Neurofibromatosis type I, a disease characterized by tumors of nerve tissue, is an inherited disorder in which nearly 30%> of the documented mutations disrupt neurofibromin 1 (NF1) splice sites.

3. The essential splicing factor U2AF⁶⁵ recognizes a polypyrimidine (Py) tract that is often mutated in these diseases. These mutations result in defective binding of U2AF⁶⁵ and reduced splicing of mRNA transcripts. In other cases, distal point mutations in the pre-mRNA can indirectly reduce U2AF⁶⁵ binding and splicing of the gene transcript via other protein-RNA effectors. What are needed are new therapeutics and treatment regimens that can rescue the aberrant splicing resulting from a mutated Py tract as well as methods of identifying said therapeutics.

II. SUMMARY

4. In one aspect, disclosed herein are U2AF⁶⁵ splice factor variants comprising one or more amino acid substitution at a contact residue of SEQ ID NO: 1 or a corresponding residue of U2AF⁶⁵ for a pre-mRNA Py tract splice site, wherein the variant increases splicing at the target splice site.

5. Also disclosed herein are methods of treating a disorder associated with an aberrantly spliced mRNA in a subject, comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF⁶⁵ variant comprises a substitution of at a contact residue for a pre-mRNA Py tract splice site.

6. Further provided herein is a method of decreasing defective splicing of an mRNA comprising contacting the mRNA with a polypeptide comprising a mutant U2AF⁶⁵. In the method, the mRNA has a mutation associated with defective splicing, and the variant U2AF⁶⁵ comprises at least one amino acid substitution and has an increases splicing of the target splice site and/or has increased binding affinity for a pyrimidine tract upstream of a 3 ^' splice site of the mRNA as compared to wild-type U2AF⁶⁵.

7. Also provided here are methods of engineering a splicing factor variant comprising mutating one of the nine nucleotide residues of a pre-mRNA splice site polypyrimidine (Py) tract of a gene to generate dU, dC, dG, and dA Py tract mutants; co-cystalizing the mutated Py tract mutants comprising a deoxy-ribose oligonucleotide backbone with a U2AF65 splice factor variant wherein the U2AF65 variant is a deletion mutant of the amino acids corresponding to residues 238-257 of SEQ ID NO: 1 ; comparing the binding affinity of the U2AF65 variant to the Py tract mutants; identifying contact residues of U2AF65 for a Py tract mutant; selecting amino acid substitutions at contact residues of U2AF65 that increase the binding affinity to a mutated residue of the Py tract and/or increase splicing at the target splice site, and substituting the native amino acid for an identified amino acid.

8. Additionally, disclosed herein are methods of engineering a splicing factor variant comprising co-cystalizing Py tract variants with a U2AF65 splice factor variant of SEQ ID NO: 1 ; comparing the binding affinity of the U2AF65 variant to the Py tract variants; identifying contact residues of U2AF65 for a Py tract variant; selecting amino acid substitutions at contact residues of U2AF65 that increase the binding affinity to a mutated residue of the Py tract and/or increase splicing at the target splice site, and substituting the native amino acid for an identified amino acid.

III. BRIEF DESCRIPTION OF THE DRAWINGS

9. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

10. Figure 1 shows fluorescence anisotropy changes during titration of the indicated fluorescein-labeled Py tract RNAs with U2AF⁶⁵ protein. The average data points and standard deviation of three independent experiments is given. Solid lines represent nonlinear fits of the data.

1 1. Figure 2 shows crystal structures of dU2AF⁶⁵ bound to Py tracts. Figures 2A-C show representative electron density showing the oligonucleotide binding register of the structures (A) dU2AF⁶⁵ bound to 5 ^' dUdUdUdU(BrdU)dAdU, (B) dU2AF65 bound to 5 ^'- dUdUdUdU(BrdU)dGdU, and (Q dU2AF65-D231 V bound to 5 ^'-dUdUdUdU(BrdU)dUdU. Both copies in the crystallographic asymmetric unit are shown. The purine nucleotides or synthetic D231V site are shown on the left. The 5-BrdU nucleotides are shown on the right. Kicked 2|F₀|-|F_C| electron density maps contoured at 1σ. Anomalous difference Fouriers are contoured at 4σ for the 5-BrdU of the purine -bound structures, for which data were collected near the bromine edge (0.92 A wavelength). Figure 2D shows the superposition of asymmetric units shows close match of overall conformations among the indicated dU2AF65 crystal structures.

12. Figure 3 shows data for a U2AF⁶⁵-D23 IV variant that improves splicing of X- linked retinitis pigmentosa causing RP2(U>A) mutant splice site in human cells. Figure 3A is an immunoblot showing equivalent expression of U2AF⁶⁵ and U2AF⁶⁵-D231V. Figure 3B and 3C show the results of a co-transfection experiment showing proportionate variation of the exon inclusion with the amount of U2AF⁶⁵ variant with either wild-type RP2 (B) or RP2(U4>A) (C) minigenes. The indicated amount of U2AF⁶⁵ or U2AF⁶⁵-D231V in pCMV6-XL5 (0, 0.5, or 1 μg) was mixed with 0.25 μg of the RP2 minigene vector. A bar graph of three independent biological replicates is shown. Figure 3C shows quantitative real-time PCR results using a Applied

Biosystems 7900HT Sequence Detection System. Reactions and their corresponding negative controls were repeated in triplicate using RNA purified from two biological samples. The relative levels of transcript expression of different isoforms of the same gene were quantified by using the second-derivative (2-AACt) maximum method. For RP2 amplification, the forward primer (5 '- CGAGCTGTACAAGTCCGGCC-3 ) (SEQ ID NO: 50) was used with specific reverse primers for either the exon-included or exon-skipped transcripts (respectively 5 ^'- GCAATAACAGGACCTTTGTTCAG-3 ^' (SEQ ID NO: 54) in the exon 3/exon 4 junction or 5 - TTTCAGATACAAACATCTTTGTTCAG-3 ^' (SEQ ID NO: 55) in the exon 3/exon 5 junction) for 40 cycles (94 15 s - 60 60 s). The qRT-PCR primers for GAPDH were 5 ^'- TGCACCACCAACTGCTTAGC (forward) (SEQ ID NO: 56) and 5 ^'-

GGCATGGACTGTGGTCATGAG (reverse) (SEQ ID NO: 53). Figure 3D shows results from quantitative real-time reverse transcription PCR experiments for the bicistronic construct described in Figure 4 A. ***, /?<0.003.

13. Figure 4 shows that a U2AF⁶⁵-D231V variant improves splicing of X-linked retinitis pigmentosa causing RP2(U>A) mutant splice site in human cells. Figure 4A shows the

experimental scheme. A bicistronic vector comprising either wild-type RP2 or mutated RP2(U>A) minigenes (Py tract sequences inset) and either wild-type U2AF⁶⁵ or the U2AF⁶⁵-D23 IV variant is transfected in HEK293T cells. Normally, exon 4 is included in the spliced RP2 transcript (410 bp). The U>A mutation in the 5 ' region of the Py tract causes exon 4 skipping (324 bp) and hence retinitis pigmentosa in patients. Figure 4B shows representative RT-PCR of mRNA isolated from the transfected cells. Products were separated by 2% agarose gel electrophoresis. Figure 4C is a bar graph of the average percent of the exon-included band relative to total amplified product and standard deviations from five independent RT-PCR experiments.

14. Figure 5 shows, via site-specific photo-crosslinking, that U2AF⁶⁵ directly contacts the 5 ^' and 3 ^' regions of the RP2 Py tract. Purified U2AF⁶⁵ (residues 141-342, 20 μΜ) was mixed with RP2 Py tract RNA oligonucleotide (24 μΜ) with a 4-thio-U photo-label at either the fourth nucleotide (i.e. in the 5 ^' region), as shown in Figure 5A or the tenth nucleotide (i.e. in the 3 ^' region) of the RNA site as shown in Figure 5B. The binding buffer comprised 100 mM NaCl/10 mM HEPES pH 6.8 and the total volume of each reaction was 10

Complexes were incubated on ice for 20 min then transferred a petri dish on ice, covered with plastic wrap, and exposed to 366 nm light from a Spectroline ENF-240C; specs-115V, 60Hz, 0.2 Amp hand-held lamp for the indicated time period. Samples subsequently were analyzed by 12.5% SDS-PAGE and Coomassie blue-stained. Controls include the corresponding R As synthesized without photo-label and protein without the addition of R A.

15. Figure 6 shows a comparison of nucleotides bound to a U2AF⁶⁵ sweet spot for Py tract substitutions. Figure 6 A is a schematic diagram of U2AF⁶⁵ recognizing the Py tract splice site signal in the context of accessory proteins (SFl, U2AF³⁵) and flanking pre-mRNA sequences (BPS, AG). Figures 6B shows the domains of the full length U2AF⁶⁵ protein. The boundaries of the Py tract-recognition domain (U2AF⁶⁵ 1,2, residues 141-342) and a shortened construct that is amenable to crystallization (dU2AF⁶⁵l,2, residues 148-237 and 258-336) are delineated by double-headed arrows. Figure 6C shows a representative crystal structure of U2AF⁶⁵1,2 bound to 9-U oligonucleotide at 1.6 A resolution. Figure 6D provides schematic views of penultimate bound nucleotides of dU2AF⁶⁵ structures. Crystallographic statistics are given in Table 14 and electron density is given in Figure 2.

16. Figure 7 shows the structure of U2AF⁶⁵-D23 IV bound to the dU tract viewed at the penultimate nucleotide site.

17. Figure 8 shows the proposed method for selection of "tailored" U2AF⁶⁵ variants.

Figure 8 A shows the reporter minigene comprising the regulated and flanking exons and introns between RFP and YFP coding regions will be expressed in a stable HEK293T cell line. The exon- included splice-form produces both red and yellow fluorescence, whereas the exon-skipped (defective) splice-form frameshift introduces a STOP codon prior to YFP and hence primarily produces red fluorescence. The transiently-transfected U2AF⁶⁵ variant will be encoded on a BFP- vector. Figure 8B shows outcomes of cell sorting (URMC Flow Core). The ratio of yellow ("correct splicing":red (minigene expression) fluorescence will be plotted vs. blue fluorescence (U2AF⁶⁵ transfection) intensity for each cell count. Shaded ovals represent cell counts and least squares fits (expect linear for empty BFP vector and nonlinear for U2AF⁶⁵-constructs) are represented by dashed grey lines. U2AF⁶⁵ variants with the maximum increase relative to wild- type U2AF⁶⁵ will be selected for confirmation by immunoblot, qRT-PCR, and further rounds of optimization. The indicated controls will serve as a basis for comparison and for gating procedures. "X", stop codon; red star, defective splice site mutation. IV. DETAILED DESCRIPTION

18. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, 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.

A. Definitions

19. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

20. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. 21. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

22. "Optional" or "optionally" means that the subsequently described event or

circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

23. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

B. Compositions

24. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular U2AF⁶⁵ variant is disclosed and discussed and a number of modifications that can be made to a number of molecules including the U2AF⁶⁵ variant are discussed, specifically contemplated is each and every combination and permutation of U2AF⁶⁵ and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. 1. U2AF variants

25. Disease-causing mutations in a polypyrimidine (Py) consensus sequence preceding the 3 ^' splice site have been documented in approximately 3,000 genes in the Human Gene Mutation Database. The Py tract splice site signal is recognized by U2AF⁶⁵ (Figure 6), which in turn recruits core spliceosome components to a consensus "AG" dinucleotide at a proximal 3 ^' splice site. Mutations in the Py tract or elsewhere in the genome can result in decreased binding of U2AF⁶⁵, which results in defective splicing of an mRNA transcript.

26. The Py tract splice site signals of the major class of introns are recognized by the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor, 65 kDa (U2AF⁶⁵) (Fig. 6A), which acts in a complex with Splicing Factor 1 (SF1) and small (35 kDa) U2AF (U2AF³⁵) subunits that recognize the upstream BPS and consensus AG dinucleotide at the 3' splice site junction, respectively. The U2AF⁶⁵-SF1-U2AF³⁵ complex in turn stabilizes the association of core spliceosome components with the pre-mRNA. U2AF⁶⁵ has been shown to bind the SF3M55 subunit of the U2 snRNP, which ultimately displaces SF1, whereas SF1 interacts with the Ul snRNP at the 5' splice site and appears to be dispensable for the splicing of most human transcripts. The U2AF35 small subunit is an accessory factor to U2AF⁶⁵, required for splicing a subset of introns with degenerate Py tracts and conserved AG consensus. The central U2AF⁶⁵ subunit is required for splicing of most of the major U2 class of introns.

27. The two central U2AF⁶⁵ RNA recognition motifs of U2AF⁶⁵, RRM1 and RRM2, recognize the Py tract splice site signals (Fig. 6 A and B). Considering its central role in spliceosome recruitment, engineering U2AF⁶⁵ variants for improved affinity at specific Py tracts offers a potential approach to increase the use of an adjacent 3' splice site.

28. As used herein, U2AF⁶⁵ is a pre-mRNA splicing factor that guides splice site choice by recognizing a Py tract consensus sequence preceding the 3 ^' splice site. Examples of amino acid sequences for human U2AF⁶⁵ are provided herein as SEQ ID NO: 1 (isoform B) and SEQ ID NO: 3 (isoform A). An example of a nucleic acid sequence encoding SEQ ID NO: 1 is provided herein as SEQ ID NO: 2 and an example of a nucleic acid sequence encoding SEQ ID NO: 3 is provided herein as SEQ ID NO: 4. Fragments of U2AF⁶⁵ that have increased binding affinity for the Py tract, increase splicing at the target splice site and/or decrease defective splicing are also provided. These examples are not meant to limiting as other U2AF⁶⁵ sequences and fragments thereof from other species are available to those of skill in the art. 29. In one aspect, disclosed herein are U2AF variants comprising a modification in the nucleotide or amino acid sequence of U2AF⁶⁵ that changes the ability of U2AF⁶⁵ to bind the Py tract and/or increases splicing at the target splice site. It is understood that the polypeptides comprising a variant U2AF⁶⁵ set forth herein are non-naturally occurring polypeptides comprising a mutation introduced by genetic manipulation. The mutation can be, for example, a substitution of one or more amino acids, a deletion of one or more amino acids, or an insertion of one or more amino acids. In one aspect, disclosed herein are U2AF⁶⁵ splice factor variants comprising one or more amino acid substitutions at a contact residue of SEQ ID NO: 1 or a corresponding residue of U2AF⁶⁵ for a pre-mRNA Py tract splice site (including, but not limited to, those disclosed in Table 5), wherein the variant increases splicing at the target splice site and/or has increased binding affinity for the Py tract. The disclosed U2AF⁶⁵ splice factor variants can comprise a substitution at one or more contact residues of U2AF⁶⁵ as set forth in SEQ ID NO : 1, including but not limited to 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, and/or 339.

30. The mutation in U2AF⁶⁵ can be a conservative or a non-conservative substitution. For example, conservative substitutions can be made according to the following table:

31. Table 1. Amino Acid Substitutions.

Original Residue Exemplary Substitutions Original Residue Exemplary Substitutions

Ala Ser, Gly, Cys

Arg Lys, Gin, Met, He Leu lie, Val, Met

Asn Gin, His, Glu, Asp Lys Arg, Gin, Met, He

Asp Glu, Asn, Gin Met Leu, He, Val

Cys Ser, Met, Thr Phe Met, Leu, Tyr, Trp, His

Gin Asn, Lys, Glu, Asp Ser Thr, Met, Cys

Glu Asp, Asn, Gin Thr Ser, Met, Val

Gly Pro, Ala Trp Tyr, Phe

His Gin, Asn Tyr Trp, Phe, His

lie Leu, Val, Met Val He, Leu, Met

32. Additional conservative mutations can include a substitution of one polar amino acid residue (e.g., Serine, Threonine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate) for another polar residue or the substitution of a non-polar amino acid residue (e.g., Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, or Tryptophan). Other conservative substitutions include substituting an uncharged amino acid (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Serine, Cysteine, Threonine, Methionine, Asparagine, or Glutamine) for another uncharged amino acid. It is further recognized that more conservative substitutions can be made beyond simple polarity of the amino acid including, but not limited to substitution of one amino acid with an aromatic group (e.g.,

Phenylalanine, Tyrosine, or Tryptophan) for another; the substitution of a negatively charged 5 amino acid (e.g., Aspartate or Glutamate) for another; or the substitution of a positively charged amino acid (e.g., Lysine, Arginine, or Histidine) for another.

33. Accordingly, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 145 of SEQ ID NO: 1 or at a residue corresponding to residue 145 of SEQ ID NO: 1, wherein the substitution at residue 145 is the substitution of a

10 Arginine for another polar residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine,

Lysine, Histidine, Aspartate, Glutamate, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 145 is a R145Q substitution.

34. Also disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 147 of SEQ ID NO: 1 or at a residue corresponding to residue 147 of SEQ ID

15 NO: 1, wherein the substitution at residue 147 is the substitution of a Glutamine for nonaromatic or positively charged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, Aspartate, or Glutamate). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 147 is a Q147S, Q147T, Q147N, Q147A, Q147V, Q147I, Q147L, Q147M, Q147D, or Q147E substitution.

20 35. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the

substituted amino acid of U2AF⁶⁵ is at residue 150 of SEQ ID NO: 1 or at a residue corresponding to residue 150 of SEQ ID NO: 1, wherein the substitution at residue 150 is the substitution of a Arginine for a polar uncharged residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue

25 150 is a R150S, R150T, R150N, R150Q, or R150Y substitution.

36. In one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 155 of SEQ ID NO: 1 or at a residue corresponding to residue 155 of SEQ ID NO: 1, wherein the substitution at residue 155 is the substitution of a Asparagine for another polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, or Glutamine). For

30 example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 155 is a N155T, N155S, or N155Q substitution. 37. Further, in one aspect, disclosed herein are U2AF65 variants , wherein the substituted amino acid of U2AF65 is at residue 196 of SEQ ID NO: 1 or at a residue corresponding to residue 196 of SEQ ID NO: 1, wherein the substitution at residue 196 is the substitution of a Asparagine for uncharged nonaromatic residue or positively charged residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are U2AF65 variants wherein the substitution at residue 196 is a N196T, N196S, N196R, N196A, N196V, or N196Q substitution.

38. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 197 of SEQ ID NO: 1 or at a residue corresponding to residue 197 of SEQ ID NO: 1, wherein the substitution at residue 197 is the substitution of a Phenylalanine for another nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, or Tryptophan), more specifically a nonaromatic nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 197 is a F197V, F197I, or F197L substitution.

39. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 199 of SEQ ID NO: 1 or at a residue corresponding to residue 199 of SEQ ID NO: 1, wherein the substitution at residue 199 is the substitution of a

Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 199 is a F199Y or F199W

substitution.

40. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 215 of SEQ ID NO: 1 or at a residue corresponding to residue 215 of SEQ ID NO: 1, wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 215 is a D215N substitution.

41. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 227 of SEQ ID NO: 1 or at a residue corresponding to residue 227 of SEQ ID NO: 1, wherein the substitution at residue 227 is the substitution of a Arginine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Aspartate,

Glutamate, Lysine, Histidine, or Glutamine), more specifically a polar uncharged nonaromatic residue ((i.e., Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 227 is a R227Q substitution.

42. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 229 of SEQ ID NO: 1 or at a residue corresponding to residue 229 of SEQ ID NO: 1, wherein the substitution at residue 229 is the substitution of a Proline for another nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline), more specifically a nonpolar nonaromatic residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 229 is a P229G substitution.

43. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 23 Oof SEQ ID NO: 1 or at a residue corresponding to residue

230 of SEQ ID NO: 1, wherein the substitution at residue 230 is the substitution of a Histidine for a non-negatively charged amino acid residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Tyrosine, Threonine, Cysteine,

Asparagine, Glutamine, Lysine, or Arginine. For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 230 is a H230F, H230Y, H230I, H230L, H230Q, or H230R.

44. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 231 of SEQ ID NO: 1 or at a residue corresponding to residue

231 of SEQ ID NO: 1 , wherein the substitution at residue 231 is the substitution of a Aspartate for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine,

Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged amino acid residue with a small R group (e.g., Glycine, Alanine, Valine, Serine, Threonine, Cysteine, or Asparagine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 231 is a D231V, D231T, D231N, or D231S substitution.

45. Additionally, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 252 of SEQ ID NO: 1 or at a residue corresponding to residue 252 of SEQ ID NO: 1 , wherein the substitution at residue 252 is the substitution of a Threonine for another uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Tyrosine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue ((i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF variants wherein the substitution at residue 252 is a T252N, T252G, or T252P substitution.

46. Also, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 253 of SEQ ID NO: 1 or at a residue corresponding to residue 253 of SEQ ID NO: 1, wherein the substitution at residue 253 is the substitution of a Valine for another nonpolar residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue ((i.e., Glycine, Alanine,

Methionine, Leucine, Isoleucine, or Proline). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 253 is a V253G, V253S or V253P substitution.

47. Further, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of

U2AF⁶⁵ is at residue 254 of SEQ ID NO: 1 or at a residue corresponding to residue 254 of SEQ ID NO: 1, wherein the substitution at residue 254 is the substitution of a Valine for another uncharged residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine,

Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 254 is a V254G, V254I, V254A, V254I, V254P, V254T, or V254S substitution.

48. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 256 of SEQ ID NO: 1 or at a residue corresponding to residue 256 of SEQ ID NO: 1, wherein the substitution at residue 256 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 256 is a D256N, D256E, or D256K substitution.

49. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 260 of SEQ ID NO: 1 or at a residue corresponding to residue 260 of SEQ ID NO: 1, wherein the substitution at residue 260 is the substitution of a Lysine for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF variants wherein the substitution at residue 260 is a K260T, K260L, K260I, or K260Q substitution.

50. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 262 of SEQ ID NO: 1 or at a residue corresponding to residue

5 262 of SEQ ID NO: 1, wherein the substitution at residue 262 is the substitution of a

Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 262 is a F262Y substitution.

51. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 268 of SEQ ID NO: 1 or at a residue corresponding to residue

10 268 of SEQ ID NO: 1, wherein the substitution at residue 268 is the substitution of an Asparagine for another nonaromatic residue (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Arginine, Histidine, or Glutamine), more specifically wherein the nonaromatic residue is not positively charged (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine,

15 Aspartate, Glutamate, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 268 is a N268S, N268D, N268G, N268A, or N268T substitution.

52. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 272 of SEQ ID NO: 1 or at a residue corresponding to residue 272 of SEQ ID NO: 1, wherein the substitution at residue 272 is the substitution of a 0 Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine,

Glutamate, Lysine, Histidine, Arginine, or Glutamine), more specifically wherein the residue is a polar uncharged nonaromatic residue (i.e., Serine, Threonine, Cysteine, Asparagine, or

Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 272 is a D272N substitution.

5 53. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 287 of SEQ ID NO: 1 or at a residue corresponding to residue 287 of SEQ ID NO: 1, wherein the substitution at residue 287 is the substitution of a Alanine for another nonpolar residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline, Methionine,

Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue (i.e., Glycine,

30 Valine, Leucine, Isoleucine, Proline, or Methionine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 287 is an A287V or A287L substitution. 54. Also, in one aspect, disclosed herein are U2AF variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 289 of SEQ ID NO: 1 or at a residue corresponding to residue 289 of SEQ ID NO: 1, wherein the substitution at residue 289 is the substitution of a Asparagine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 289 is a N289R or N289Q substitution.

55. Additionally, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 292 of SEQ ID NO: 1 or at a residue corresponding to residue 292 of SEQ ID NO: 1 , wherein the substitution at residue 292 is the substitution of a Lysine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Asparagine, Histidine, Arginine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 292 is a K292Q substitution.

56. Also disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 293 of SEQ ID NO: 1 or at a residue corresponding to residue 293 of SEQ ID NO: 1, wherein the substitution at residue 293 is the substitution of an Aspartate for an uncharged residue (i.e., Phenylalanine, Tyrosine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically a nonaromatic uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 293 is a D293N, D293F, D293S, D293G, D293A, D293I, D293L, D293T, or D293V substitution.

57. Further, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 296 of SEQ ID NO: 1 or at a residue corresponding to residue 296 of SEQ ID NO: 1, wherein the substitution at residue 296 is the substitution of a Threonine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine,

Aspartate, or Glutamate), or more specifically, another polar uncharged residue (i.e., Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 296 is a T296N or T296Q substitution.

58. Additionally, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 298 of SEQ ID NO: 1 or at a residue corresponding to residue 298 of SEQ ID NO: 1, wherein the substitution at residue 298 is the substitution of a Leucine for a polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 298 is a L298G, L298S, L298N, or L298T substitution.

59. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 300 of SEQ ID NO: 1 or at a residue corresponding to residue 300 of SEQ ID NO: 1, wherein the substitution at residue 300 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 300 is a K300R substitution.

60. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 328 of SEQ ID NO: 1 or at a residue corresponding to residue 328 of SEQ ID NO: 1, wherein the substitution at residue 328 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 328 is a K328R substitution.

61. Also disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 329 of SEQ ID NO: 1 or at a residue corresponding to residue 329 of SEQ ID NO: 1, wherein the substitution at residue 329 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine,

Aspartate, or Glutamate), or more specifically, another charged residue (i.e., Arginine, Histidine, Aspartate, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 329 is a K329D substitution.

62. Further disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of

U2AF⁶⁵ is at residue 330 of SEQ ID NO: 1 or at a residue corresponding to residue 330 of SEQ ID NO: 1, wherein the substitution at residue 330 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine,

Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 330 is a L330V, L330A, or L330T substitution. 63. Additionally, in one aspect, disclosed herein are U2AF variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 331 of SEQ ID NO: 1 or at a residue corresponding to residue 331 of SEQ ID NO: 1 , wherein the substitution at residue 331 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine,

5 Alanine, Valine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 331 is a L331S, L331N, L331T, L331A, L331G, or L331Q substitution.

64. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 333 of SEQ ID NO: 1 or at a residue corresponding to residue

10 333 of SEQ ID NO: 1, wherein the substitution at residue 333 is the substitution of a Glutamine for another polar residue (i.e., Serine, Threonine, Cysteine, Asparagine, Lysine, Tyrosine, Histidine, Arginine, Glutamate, or Aspartate). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 333 is a Q333E, Q333N, Q333R, Q333S, or Q333T substitution.

65. Additionally disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid 15 of U2AF⁶⁵ is at residue 335 of SEQ ID NO: 1 or at a residue corresponding to residue 335 of SEQ

ID NO: 1 , wherein the substitution at residue 335 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or

Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue

335 is an A335S, A335T, or A335G substitution.

20 66. Further, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 336 of SEQ ID NO: 1 or at a residue corresponding to residue

336 of SEQ ID NO: 1, wherein the substitution at residue 336 is the substitution of a Serine for a nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue

25 336 is a S336P or S336G substitution.

67. Also, in one aspect, disclosed herein are U2AF⁶⁵ variants , wherein the substituted amino acid of U2AF⁶⁵ is at residue 339 of SEQ ID NO: 1 or at a residue corresponding to residue 339 of SEQ ID NO: 1, wherein the substitution at residue 339 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or

30 Glutamine). For example, disclosed herein are U2AF⁶⁵ variants wherein the substitution at residue 339 is an A339E, A339S, or A339T substitution. 68. As noted above, the disclosed U2AF splice factor variants can comprise one or more substitutions, such as, for example, two, three, four, five, six, seven, eight, nine, or ten

substitutions. In one aspect, disclosed herein are U2AF⁶⁵ splice factor variants comprising two or more substitutions (including, but not limited to, those disclosed in Table 5). It is understood that the two or more substitutions can comprise any combination of the substituted residues disclosed herein, including but not limited to residues 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, and/or 339 as set forth in SEQ ID NO: 1. For example, disclosed herein are

U2AF⁶⁵ splice factor variants comprising substitutions at residues 289 and 254; 256 and 260; 215 and 252; 253 and 287; or 292 and 272.

69. In one aspect, disclosed herein are U2AF⁶⁵ splice factor variants wherein the variant comprises any of the substitutions disclosed herein at residues 289 and 254 of SEQ ID NO: 1 or at a residue corresponding to residue 289 and 254 of SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising substitutions at residues 289 and 254, wherein the substitution at residue 289 is a substitution of an Asparagine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Histidine, Arginine, or Glutamine) and the substitution at residue 254 is substitution of a Valine for another uncharged nonaromatic residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein are U2AF⁶⁵ variants wherein the substitutions are N289Q and V254T.

70. Also disclosed, in one aspect are U2AF⁶⁵ splice factor variants wherein the variant comprises any of the substitutions disclosed herein at residues 256 and 260 of SEQ ID NO: 1 or at a residue corresponding to residues 256 and 260 of SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising substitutions at residues 256 and 260 wherein the substitution at residue 256 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine) and the substation at residues 260 is a substation of a Lysine for an uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein are U2AF⁶⁵ variants wherein the substitutions are D256K and K260T, D256K and K260L, D256K and K260I, D256N and K260Q, D256E and K260T, D256E and K260L, or D256E and K260I. 71. Also disclosed, are U2AF splice factor variants wherein the variant comprises any of the substitutions disclosed herein at residues 215 and 252 of SEQ ID NO: 1 or at a residue corresponding to residue 215 and 252 of SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising substitutions at residues 215 and 252, wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine) and wherein the substation at residue 252 is the substitution of a Threonine for another uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein are U2AF⁶⁵ variants wherein the substitutions are D215N and T252G or D215N and T252P.

72. Also disclosed, in one aspect are U2AF⁶⁵ splice factor variants wherein the variant comprises any of the substitutions disclosed herein at residues 253 and 287 of SEQ ID NO: 1 or at a residue corresponding to residue 253 and 287 of SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising at residues 253 and 287; wherein the substitution at residue 253 is the substitution of a Valine for another nonpolar nonaromatic residue ((i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, or Proline); and wherein the substation at residues 287 is a substitution of a Alanine for another nonpolar residue (i.e., Glycine, Valine, Leucine,

Isoleucine, Proline, or Methionine). In one aspect, disclosed herein are U2AF⁶⁵ variants wherein the substitutions are V253G and A287A, V253G and A287V, V253G and A287L, V253S and A287A, V253S and A287V, or V253S and A287L.

73. Additionally, disclosed herein are U2AF⁶⁵ splice factor variants wherein the variant comprises any of the substitutions disclosed herein at residues 292 and 272 of SEQ ID NO: 1 or at a residue corresponding to residue 292 and 272 of SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising at residues 292 and 272; wherein the substitution at residue 272 is the substitution of a Aspartate for a polar uncharged nonaromatic residue (i.e., Serine, Threonine, Cysteine, Asparagine, or Glutamine); and wherein the substitution at residue 292 is the substitution of a Lysine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Asparagine, Histidine, Arginine, or Glutamine). In one aspect, disclosed herein are U2AF⁶⁵ variants wherein the substitutions are K292Q and D272N.

74. In one aspect, the U2AF65 splice factor variants can be comprised in a polypeptide.

Thus, in one aspect, disclosed herein are polypeptides comprising a variantU2AF⁶⁵, wherein the variant comprises at least one amino acid substitution and wherein the variant increases splicing of the target splice cite and/or has an increased binding affinity for a pyrimidine tract upstream of a 3 ' splice site of the mR A as compared to wild-type U2AF⁶⁵. Optimally, the polypeptide is an isolated or a purified polypeptide. By isolated polypeptide or purified polypeptide is meant a polypeptide that is substantially free from the materials with which the polypeptide is normally associated in nature or in culture.

75. The polypeptides of the invention can be obtained by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide. When producing the polypeptide recombinantly, the nucleic acid encoding the polypeptide can include a signal sequence at the beginning of the coding sequence of U2AF⁶⁵. In addition, a polypeptide can be obtained by cleaving full length polypeptides. When the polypeptide is a fragment of a larger polypeptide, the isolated polypeptide is shorter than and excludes the full-length polypeptide of which it is a fragment. Reference to a fragment of a mutant U2AF⁶⁵, as used herein, means the fragment includes the mutation.

76. Also provided herein are nucleic acids encoding the polypeptides set forth herein.

Thus, in one aspect disclosed herein are nucleic acids encoding the U2AF⁶⁵ variants disclosed herein. As used herein, the term "nucleic acid" refers to single or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids. The nucleic acid may represent a coding strand or its complement, or any combination thereof. Vectors comprising the nucleic acids disclosed herein are also provided. These vectors include, but are not limited to, plasmids and viral vectors. Viral vectors include lentiviral vectors, adeno-associated viral vectors, herpes vectors and adenoviral vectors, to name a few.

77. It is understood and herein contemplated that the U2AF⁶⁵ variants disclosed herein are useful in the treatment of disorders associated with aberrant splicing of mRNA (including, but not limited to retinitis pigmentosa, β-thalassemia, or neurofribormatosis). In one aspect, disclosed herein are methods of decreasing defective splicing of an mRNA comprising contacting the mRNA with any of the U2AF⁶⁵ variants disclosed herein. Also disclosed are methods of treating a disorder associated with an aberrantly spliced mRNA in a subject, comprising administering to the subject any of the U2AF⁶⁵ variants disclosed herein. In one aspect, it is understood that the U2AF⁶⁵ variant can be administered to the subject as a polypeptide or polynucleotide. For example, in one aspect, disclosed herein are methods of treating a disorder associated with an aberrantly spliced mR A in a subject (including, but not limited to retinitis pigmentosa, β- thalassemia, or neurofribormatosis), comprising administering to the subject any of the U2AF⁶⁵ variants disclosed herein, wherein the U2AF⁶⁵ variant comprises a substitution of at residue 231 of SEQ ID NO: 1 or at corresponding residue of U2AF⁶⁵.

2. Homology/identity

78. It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of homology to specific known sequences. For example SEQ ID NO: 2 sets forth a particular sequence of an U2AF⁶⁵ and SEQ ID NO: 1 sets forth a particular sequence of the protein encoded by SEQ ID NO: 2, an U2AF⁶⁵ protein. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

79. Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman A dv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

80. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci.

USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment.

81. It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology/identity to specific known sequences. For example, SEQ ID NO: 1 sets forth a particular sequence of U2AF⁶⁵. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80%> or 85% or 90%> or 95% homology to the stated sequence. Those of skill in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

82. Another way of calculating homology can be performed by published algorithms.

Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman A dv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

83. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

84. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology to a particular sequence wherein the variants are conservative mutations.

85. As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 1 is set forth in SEQ ID NO:2. It is understood that for this mutation all of the nucleic acid sequences that encode this particular derivative of the U2AF⁶⁵ splicing factor are also disclosed including for example sequences which set forth a degenerate nucleic acid sequences that encode the particular polypeptide set forth in SEQ ID NO:l . It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular U2AF65 from which that protein arises is also known and herein disclosed and described.

C. Methods of Treating a Disorder

86. As used herein, a polypyrimidine or pyrimidine tract (Py tract) is a region of mRNA that promotes the assembly of the spliceosome, the protein complex specialized for carrying out RNA splicing. This tract is rich with pyrimidine (Py) nucleotides, primarily uracil, and is located about 5-40 base pairs before the 3 ^' end of the intron to be spliced. Nine nucleotides within the Py tract are generally recognized by the RNA Recognition Motif 1 (RRMl) and the RNA Recognition Motif 2 (RRM2) domains of U2AF⁶⁵ (See Figure 2B), which recognizes, for example, and not to be limiting, the sequence 5 ^'-ULJUUUUUCC-3 ^' (Singh et al. "Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins," Science 268: 1173-1176 (1995)). The nine nucleotides within the Py tract can occur, for example, four nucleotides, five nucleotides, six nucleotides, seven nucleotides or more upstream of a 3 ^' splice site as shown in the non-limiting example below. N is G, A, U or C; Py is U or C; X is in integer, i.e. 0,1, 2, 3, 4, 5, 6, 7 etc.

3 ' splice site 5 ^' -PyPyPyPyN_xPyPyPyPyN_xC AG J, -3 '

87. As used throughout, reference to a variant U2AF⁶⁵ with an increased splicing of a target splice site (e.g., a Py tract 3' splice site) and/or increased binding affinity for a Py tract means that the mutant U2AF⁶⁵ has increased splicing of the target splice site and/or has increased binding affinity for a sequence of nine nucleotides that is recognized by the RRMl and the RRM2 domains of U2AF⁶⁵ and is upstream of a 3 ^' splice site. It is understood that these nine nucleotides can be consecutive nucleotides or interspersed with other nucleotides as long as the RRMl and the RRM2 domains of U2AF⁶⁵ recognize the nucleotide sequence. Based on the composition of the Py tract and available sequences that are recognized by the RRMl and the RRM2 domains of U2AF⁶⁵, one of skill in the art would know how to identify a Py tract that is upstream of a 3 ' end of the intron to be spliced.

88. For example, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased binding affinity for and/or increased splicing at a Py tract comprising uracil, cytosine, adenine or guanine in the first position or nucleotide of the Py tract. Further, the U2AF variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the second position or nucleotide of the Py tract. Also, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the third position or nucleotide of the Py tract. Further, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the fourth position or nucleotide of the Py tract. Also, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the fifth position or nucleotide of the Py tract. Also, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the sixth position or nucleotide of the Py tract. Further, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the seventh position or nucleotide of the Py tract. Also, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the eighth position or nucleotide of the Py tract. As used herein, the eighth position corresponds to the penultimate position of the Py tract. Further, the U2AF⁶⁵ variants (or polypeptides or nucleic acids encoding said variants) provided herein can have an increased splicing at the target splice site and/or binding affinity for a Py tract comprising uracil, cytosine, adenine or guanine in the ninth position or nucleotide of the Py tract.

89. Using known sequences for the Py tracts of m NAs comprising a mutation associated with defective splicing, one of skill in the art can use the methods provided herein to make mutant U2AF⁶⁵ polypeptides that have an increased affinity for uracil, cytosine, adenine or guanine at any one of the nine nucleotides of the Py tract. As set forth above, the mutation can be in the Py tract or elsewhere in the genome such that binding of U2AF⁶⁵ to the Py tract is decreased. These U2AF⁶⁵ variants (including the polypeptides or nucleic acids encoding said variants) can be used to correct aberrant splicing caused by a defective or mutant Py tract and to indirectly overcome other splicing defects by strengthening the use of a splice site that has been compromised by mutations in other regions of the genome. Thus, the methods provided herein can be used to improve the use of splice sites that are inactivated or weakened by mutations in the Py tract or elsewhere in the genome.

90. For example, as set forth in the Examples, a mutant U2AF⁶⁵ comprising an aspartic acid to valine substitution at position 231 (U2AF⁶⁵-D231V), can be used to decrease defective splicing of an mRNA comprising a uracil at the eighth nucleotide or position of the Py tract upstream of a 3 ^' splice site of an mRNA. Numerous diseases are associated with an mRNA comprising a mutation that results in decreased or defective binding of U2AF⁶⁵ to the Py tract. Thus, for example, if the disease is associated with an mRNA that has a uracil at the eighth position of the Py tract, U2AF⁶⁵-D23 IV can be used to decrease defective splicing of the mRNA associated with the disease. Non-limiting examples of these diseases include, but are not limited to, retinitis pigmentosa, β-thalassemia, neurofibromatosis, hepatocellular carcinoma, acute myeloid leukemia, ataxia telangiectasia, breast cancer, cystic fibrosis and xeroderma pigmentosum. Tables 2-4 provide representative splice site candidates for decreasing defective mRNA splicing with U2AF65 variants, for example, U2AF⁶⁵-D231V. In Tables 2-4 and 6-13, lowercase font indicates an intron; capital letters represent exons; | represents an intron-exon junction; bold/underlined nucleotides indicate a Py tract; and Freq is the prevalence among patients with the indicated disorder. Also provided are the Gene Name and NCBI Reference No. for each mRNA associated with the specified disease.

Table 2-He atocellular carcinoma.

Table 4- Representative inherited splice site mutations for personalized therapy.

In addition to U2AF -D23 IV, other variant U2AF proteins are provided that increase splicing at the target splice site and/or have an increased binding affinity for a uracil at the eighth position of the Py tract. For example, as shown in Table 5, aspartic acid at position 231 can be replaced with threonine, serine or asparagine. It is understood that an amino acid substitution can be made at position 231 or at a position corresponding to amino acid position 231 of SEQ ID NO: 1. For example, a substitution can be made in other U2AF⁶⁵ sequences, such as, for example, a U2AF⁶⁵ sequence from another species, such that, the substitution corresponds to position 231 of SEQ ID NO: 1. One of skill in the art would know how to align amino acid sequences in order to identify a position that corresponds to position 231 of SEQ ID NO: 1 or any other position in SEQ ID NO: 1, for example, the positions set forth in Table 5. In addition to mutants that increase splicing at the target splice site and/or have an increased affinity for a uracil at the eighth position of the Py tract, mutants that increase splicing at the target splice site and/or have an increased affinity for uracil at other positions are also provided. Similarly, mutants that increase splicing at the target splice site and/or have an increased affinity for cytosine, adenine or guanine at numerous positions within the Py tract are also provided (See Table 5). As used throughout, the numbering of the amino acid residues is based on the wild- type sequence for U2AF⁶⁵ set forth herein as SEQ ID NO: 1 or SEQ ID NO: 2.

s

91. Once a particular mRNA of interest is identified, one of skill in the art can determine the nucleotide sequence of the Py tract, in particular the nucleotide at a specific position of the Py tract, and use a mutant U2AF⁶⁵, for example, a mutant described in Table 5, to decrease defective mRNA splicing. Set forth below, in Tables 6-12, are additional examples of mRNAs that can be targeted using the methods and compositions set forth herein.

Table 6- Duchenne muscular dystrophy.

Table 11- Myelodysplastic syndrome (MDS) patients carrying somatic mutations in U2AF accessor subunit

92. In one aspect disclosed herein are methods of treating a disorder associated with an aberrantly spliced mRNA or decreasing defective splicing of an mRNA in a subject, comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF65 variant comprises a substitution of at a contact residue for a pre-mRNA Py tract splice site, and wherein administration of the U2AF⁶⁵ variant increases splicing at a target splice site. It is understood and herein disclosed that the substitution of the pre-mRNA Py tract causes defective binding of U2AF⁶⁵ and defective splicing. Thus, in one aspect, disclosed herein are methods of treating a disorder associated with an aberrantly spliced mRNA or decreasing defective splicing of an mRNA in a subject; wherein the substitution of the pre-mRNA Py tract causes defective binding of U2AF⁶⁵ and defective splicing, the method comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF65 variant comprises a substitution of at a contact residue for a pre-mRNA Py tract splice site, and wherein

administration of the U2AF⁶⁵ variant increases splicing at a target splice site.

93. It is understood and herein contemplated that any of the U2AF65 variants disclosed herein can be used in the treatment methods or methods of decreasing defective splicing disclosed herein. Accordingly, in one aspect, disclosed herein are methods of treating a disorder associated with an aberrantly spliced mRNA or decreasing defective splicing of an mRNA in a subject, comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF⁶⁵ splice factor variants comprise one or more amino acid substitutions at a contact residue of SEQ ID NO: 1 or a corresponding residue of U2AF⁶⁵ for a pre-mRNA Py tract splice site (including, but not limited to, those disclosed in Table 5), wherein the variant increases splicing at the target splice site and/or has increased binding affinity for the Py tract. As noted above, the disclosed U2AF⁶⁵ splice factor variants for use in the methods disclosed herein can comprise a substitution at one or more contact residues of U2AF⁶⁵ as set forth in SEQ ID NO : 1, including but not limited to 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, and/or 339.

94. Accordingly, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 145 of SEQ ID NO: 1 or at a residue corresponding to residue 145 of SEQ ID NO: 1, wherein the substitution at residue 145 is the substitution of a Arginine for another polar residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine, Lysine, Histidine, Aspartate, Glutamate, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 145 is a R145Q substitution.

95. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 147 of SEQ ID NO: 1 or at a residue corresponding to residue 147 of SEQ ID NO: 1, wherein the substitution at residue 147 is the substitution of a Glutamine for nonaromatic or positively charged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, Aspartate, or Glutamate). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 147 is a Q147S, Q147T, Q147N, Q147A, Q147V, Q147I, Q147L, Q147M, Q147D, or Q147E substitution.

96. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 150 of SEQ ID NO: 1 or at a residue corresponding to residue 150 of SEQ ID NO: 1, wherein the substitution at residue 150 is the substitution of a Arginine for a polar uncharged residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 150 is a R150S, R150T, R150N, R150Q, or R150Y substitution.

97. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 155 of SEQ ID NO: 1 or at a residue corresponding to residue 155 of SEQ ID NO: 1, wherein the substitution at residue 155 is the substitution of a Asparagine for another polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 155 is a N155T, N155S, or N155Q substitution.

98. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF65 is at residue 196 of SEQ ID NO: 1 or at a residue corresponding to residue 196 of SEQ ID NO: 1, wherein the substitution at residue 196 is the substitution of a Asparagine for uncharged nonaromatic residue or positively charged residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 196 is a N196T, N196S, N196R, N196A, N196V, or N196Q substitution.

99. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 197 of SEQ ID NO: 1 or at a residue corresponding to residue 197 of SEQ ID NO: 1, wherein the substitution at residue 197 is the substitution of a Phenylalanine for another nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, or Tryptophan), more specifically a nonaromatic nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 197 is a F197V, F197I, or F197L substitution.

100. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 199 of SEQ ID NO: 1 or at a residue corresponding to residue 199 of SEQ ID NO: 1, wherein the substitution at residue 199 is the substitution of a Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 199 is a F199Y or F199W substitution. 101. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 215 of SEQ ID NO: 1 or at a residue corresponding to residue 215 of SEQ ID NO: 1 , wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 215 is a D215N substitution.

102. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 227 of SEQ ID NO: 1 or at a residue corresponding to residue 227 of SEQ ID NO: 1 , wherein the substitution at residue 227 is the substitution of a Arginine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Aspartate, Glutamate, Lysine, Histidine, or Glutamine), more specifically a polar uncharged nonaromatic residue ((i.e., Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 227 is a R227Q substitution.

103. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 229 of SEQ ID NO: 1 or at a residue corresponding to residue 229 of SEQ ID NO: 1 , wherein the substitution at residue 229 is the substitution of a Proline for another nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline), more specifically a nonpolar nonaromatic residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 229 is a P229G substitution.

104. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 230of SEQ ID NO: 1 or at a residue corresponding to residue 230 of SEQ ID NO: 1, wherein the substitution at residue 230 is the substitution of a Histidine for a non-negatively charged amino acid residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamine, Lysine, or Arginine. For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 230 is a H230F, H230Y, H230I, H230L, H230Q, or H230R.

105. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 231 of SEQ ID NO: 1 or at a residue corresponding to residue 231 of SEQ ID NO: 1, wherein the substitution at residue 231 is the substitution of a Aspartate for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or

Glutamine), more specifically an uncharged amino acid residue with a small R group (e.g., Glycine, Alanine, Valine, Serine, Threonine, Cysteine, or Asparagine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 231 is a D231V, D231T, D231N, or D231S substitution.

106. Additionally, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 252 of SEQ ID NO: 1 or at a residue corresponding to residue 252 of SEQ ID NO: 1, wherein the substitution at residue 252 is the substitution of a Threonine for another uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Tyrosine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue ((i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 252 is a T252N, T252G, or T252P substitution. 107. Also, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 253 of SEQ ID NO: 1 or at a residue corresponding to residue 253 of SEQ ID NO: 1, wherein the substitution at residue 253 is the substitution of a Valine for another nonpolar residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue ((i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 253 is a V253G, V253S or V253P substitution.

108. Further, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 254 of SEQ ID NO: 1 or at a residue corresponding to residue 254 of SEQ ID NO: 1, wherein the substitution at residue 254 is the substitution of a Valine for another uncharged residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine,

Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 254 is a V254G, V254A, V254I, V254P, V254T, or V254S substitution.

109. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 256 of SEQ ID NO: 1 or at a residue corresponding to residue 256 of SEQ ID NO: 1, wherein the substitution at residue 256 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 256 is a D256N, D256E, or D256K substitution. 1 10. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 260 of SEQ ID NO: 1 or at a residue corresponding to residue 260 of SEQ ID NO: 1 , wherein the substitution at residue 260 is the substitution of a Lysine for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 260 is a K260T, K260L, K260I, or K260Q substitution.

1 1 1. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 262 of SEQ ID NO: 1 or at a residue corresponding to residue 262 of SEQ ID NO: 1 , wherein the substitution at residue 262 is the substitution of a Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 262 is a F262Y substitution.

1 12. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 268 of SEQ ID NO: 1 or at a residue corresponding to residue 268 of SEQ ID NO: 1 , wherein the substitution at residue 268 is the substitution of an Asparagine for another nonaromatic residue (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Arginine, Histidine, or Glutamine), more specifically wherein the nonaromatic residue is not positively charged (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Aspartate, Glutamate, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF variant wherein the substitution at residue 268 is a N268S, N268D, N268G, N268A, or N268T substitution.

113. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 272 of SEQ ID NO: 1 or at a residue corresponding to residue 272 of SEQ ID NO: 1, wherein the substitution at residue 272 is the substitution of a Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine), more specifically wherein the residue is a polar uncharged nonaromatic residue (i.e., Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 272 is a D272N substitution.

114. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 287 of SEQ ID NO: 1 or at a residue corresponding to residue 287 of SEQ ID NO: 1, wherein the substitution at residue 287 is the substitution of a Alanine for another nonpolar residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline, Methionine, Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline, or Methionine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 287 is an A287V or A287L substitution.

115. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 289 of SEQ ID NO: 1 or at a residue corresponding to residue 289 of SEQ ID NO: 1, wherein the substitution at residue 289 is the substitution of a Asparagine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 289 is a N289R or N289Q substitution. 116. Additionally, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 292 of SEQ ID NO: 1 or at a residue corresponding to residue 292 of SEQ ID NO: 1, wherein the substitution at residue 292 is the substitution of a Lysine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Asparagine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 292 is a K292Q substitution.

117. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 293 of SEQ ID NO: 1 or at a residue corresponding to residue 293 of SEQ ID NO: 1, wherein the substitution at residue 293 is the substitution of an Aspartate for an uncharged residue (i.e., Phenylalanine, Tyrosine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically a nonaromatic uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 293 is a D293N, D293F, D293S, D293G, D293A, D293I, D293L, D293T, or D293V substitution.

118. Further, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 296 of SEQ ID NO: 1 or at a residue corresponding to residue 296 of SEQ ID NO: 1, wherein the substitution at residue 296 is the substitution of a Threonine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another polar uncharged residue (i.e., Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 296 is a T296N or T296Q substitution. 119. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 298 of SEQ ID NO: 1 or at a residue corresponding to residue 298 of SEQ ID NO: 1, wherein the substitution at residue 298 is the substitution of a Leucine for a polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 298 is a L298G, L298S, L298N, or L298T substitution.

120. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 300 of SEQ ID NO: 1 or at a residue corresponding to residue 300 of SEQ ID NO: 1, wherein the substitution at residue 300 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 300 is a K300R substitution.

121. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 328 of SEQ ID NO: 1 or at a residue corresponding to residue 328 of SEQ ID NO: 1, wherein the substitution at residue 328 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 328 is a K328R substitution.

122. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 329 of SEQ ID NO: 1 or at a residue corresponding to residue 329 of SEQ ID NO: 1, wherein the substitution at residue 329 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another charged residue (i.e., Arginine, Histidine, Aspartate, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 329 is a K329D substitution.

123. Further disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 330 of SEQ ID NO: 1 or at a residue corresponding to residue 330 of SEQ ID NO: 1, wherein the substitution at residue 330 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 330 is a L330V, L330A, or L330T substitution.

124. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 331 of SEQ ID NO: 1 or at a residue corresponding to residue 331 of SEQ ID NO: 1, wherein the substitution at residue 331 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine,

Asparagine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 331 is a L331 S, L33 IN, L33 IT, L331 A, L331G, or L331Q substitution.

125. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 333 of SEQ ID NO: 1 or at a residue corresponding to residue 333 of SEQ ID NO: 1, wherein the substitution at residue 333 is the substitution of a Glutamine for another polar residue (i.e., Serine, Threonine, Cysteine,

Asparagine, Lysine, Tyrosine, Histidine, Arginine, Glutamate, or Aspartate). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 333 is a Q333E, Q333N, Q333R, Q333S, or Q333T substitution.

126. Additionally disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 335 of SEQ ID NO: 1 or at a residue corresponding to residue 335 of SEQ ID NO: 1, wherein the substitution at residue 335 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 335 is an A335S, A335T, or A335G substitution.

127. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 336 of SEQ ID NO: 1 or at a residue corresponding to residue 336 of SEQ ID NO: 1, wherein the substitution at residue 336 is the substitution of a Serine for a nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods comprising administering a U2AF⁶⁵ variant wherein the substitution at residue 336 is a S336P or S336G substitution.

128. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising

administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 339 of SEQ ID NO: 1 or at a residue corresponding to residue 339 of SEQ ID NO: 1, wherein the substitution at residue 339 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or Glutamine). For example, disclosed herein are methods comprising administering a U2AF variant wherein the substitution at residue 339 is an A339E, A339S, or A339T substitution.

129. The method of decreasing defective splicing of an mRNA includes contacting the mRNA with a polypeptide comprising a mutant U2AF⁶⁵, wherein the mRNA comprises a mutation associated with defective splicing and wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution. In the methods set forth herein, the mRNA can be in a cell or cell-free mRNA. The cell can be an in vitro, ex vivo or in vivo. As used throughout, the term mRNA includes any mRNA comprising a pyrimidine tract upstream of a 3 ' splice site, including unspliced precursor mRNA (pre-mRNA). The mutant U2AF⁶⁵ has an increased binding affinity for a pyrimidine tract upstream of a 3 ^' splice site of the mRNA as compared to wild-type U2AF⁶⁵. In the methods provided herein, the mutant U2AF⁶⁵ can comprise at least one amino acid substitution, wherein the amino acid substitution increases binding affinity for a pyrimidine tract upstream of a 3 ' splice site of the mRNA as compared to wild-type U2AF⁶⁵.

130. As used throughout, defective or aberrant splicing of an mRNA is splicing that results in defective or aberrant mRNA transcripts. Such mRNAs can be degraded or the proteins encoded by these defective transcripts can be truncated or can have missing domains. Defective splicing can be caused by mutations in the mRNA, for example, a mutation that creates a new splice site, a mutation that reduces the binding affinity of U2AF⁶⁵ for a Py tract, a mutation that weakens an existing splice site, a mutation that eliminates a splice site, a mutation that results in exon skipping, a mutation that results in intron inclusion, a mutation that activates a cryptic splice site, or a mutation that results in pseudoexon inclusion, to name a few. These mutations can occur at sites usually associated with splicing, for example, within a splice site, or within a Py tract upstream of a 3 ' splice site, or in other sites of the genome, upstream or downstream of a Py tract or a 3 ' splice site. As used throughout, a mutation in an mRNA can be, but is not limited to, an insertion of one or more nucleotides, a deletion of one or more nucleotides, substitution of one or more nucleotides or an inversion. These mutations optionally alter protein function, which may result in a disease state or a propensity to develop a disease state.

131. In the methods provided herein, a decrease in defective splicing can be about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percentage in between as compared to a control cell or a control value. In the methods provided herein, a control can be a defectively spliced mRNA that has been contacted with a wild-type U2AF⁶⁵. For example, and not to be limiting, if a 25% decrease in a defective transcript is observed after contacting the mR A with a polypeptide comprising a mutant U2AF⁶⁵, wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution and has an increased binding affinity for a pyrimidine tract upstream of a 3 ^' splice site of the mRNA, it is understood that the method decreases defective splicing. As used throughout, a decrease in defective splicing results in an increase in correctly spliced mRNA transcripts. Therefore, a decrease in defective splicing can also be measured by detecting an increase in correctly spliced mRNA transcripts. This increase can be an increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater as compared to a control cell or a control value. The amount of defective or correctly spliced transcripts can be determined by methods standard in the art, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), as described in the Examples.

132. In another example, a control can be a cell that comprises a defectively spliced mRNA and has been contacted with a wild-type U2AF⁶⁵. For example, and not to be limiting, if a 25% decrease in a defective transcript is observed after contacting the cell with a polypeptide comprising a mutant U2AF⁶⁵, wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution and has an increased binding affinity for a pyrimidine tract upstream of a 3 ' splice site of the mRNA, it is understood that the method decreases defective splicing.

133. Also, in the methods of treating a disease or disorder associated with defective splicing, the change in defective splicing can correlate with a reduction in one or more symptoms of the related disease state or a reduced risk for developing the disease state. Thus, the methods can slow the onset or progression of one or more symptoms of a disease state or can reduce the frequency or severity of one or more symptoms of a disease state.

134. As used throughout, an increase in binding affinity to a Py tract can be an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or greater as compared to a control, for example, the binding of wild-type U2AF⁶⁵ to the Py tract. The increase can also be a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or higher as compared to a control. Methods for measuring binding affinity are known in the art and provided in the Examples.

135. Provided herein is a method of determining the nucleic acid sequence of the Py tract of an mRNA that is aberrantly spliced in the subject, and administering to the subject a polypeptide encoding a mutant U2AF⁶⁵, wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution and has an increased binding affinity for a Py tract upstream of a 3 ' splice site of the mRNA as compared to wild-type U2AF⁶⁵. The method can further comprise determining how the mutation in the mRNA affects the Py tract in order to select the appropriate mutant U2AF⁶⁵ that will correct aberrant splicing of the mRNA. The mutation that causes aberrant splicing can be, for example, a mutation that creates a new splice site, a mutation that reduces the binding affinity of U2AF⁶⁵ for a Py tract, a mutation that weakens an existing splice site, a mutation that eliminates a splice site, a mutation that results in exon skipping, a mutation that results in intron inclusion, a mutation that activates a cryptic splice site, or a mutation that results in pseudoexon inclusion, to name a few.

136. Once the sequence of a Py tract of an mRNA comprising a mutation associated with defective splicing is determined, one of skill in the art can use the methods provided herein to make mutant U2AF⁶⁵ polypeptides that have an increased affinity for uracil, cytosine, adenine or guanine at any one of the nine nucleotides of the Py tract. These mutant U2AF⁶⁵ polypeptides can be used to correct aberrant splicing caused by a defective or mutant Py tract and to indirectly overcome other splicing defects by strengthening the use of a splice site that has been

compromised by mutations in other regions of the genome of the subject. In addition to decreasing defective or aberrant splicing, the methods herein can be used to increase splicing of an mRNA, by strengthening or increasing the use of a splice site, even in the absence of a defect in the mRNA. For example, provided herein is a method of increasing the use of a splice site in an mRNA comprising contacting the mRNA with a polypeptide comprising a mutant U2AF⁶⁵, wherein the mRNA comprises a splice site that is underutilized as compared to other splice sites in the mRNA, and wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution and has an increased binding affinity for a pyrimidine tract upstream of a 3 ^' splice site of the mRNA as compared to wild-type U2AF⁶⁵.

137. Also provided is a method of treating a disorder associated with an mRNA that is aberrantly spliced in a subject, comprising administering to the subject with a disorder associated with the mRNA that is aberrantly spliced, a polypeptide encoding a mutant U2AF⁶⁵, wherein the mRNA comprises a mutation that is associated with aberrant splicing and wherein the mutant U2AF⁶⁵ comprises at least one amino acid substitution and has an increased binding affinity for a pyrimidine tract upstream of a 3 ^' splice site of the mRNA as compared to wild-type U2AF⁶⁵. Non- limiting examples of these disorders are provided above in Tables 2-4 and 6-12. Optionally, the method further comprises detecting the mR A mutation associated with aberrant splicing in the subject.

138. The methods provided herein can optionally further comprise administering to a subject or contacting a cell with a splice-site switching oligonucleotide (SSO) (See, for example, Kole et al. Nat. Rev. Drug Discover. 11 : 125 (2012); and Bauman et al. Oligonucleotides 19(1): 1- 13 (2009).

139. As noted above, the disclosed methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions, such as, for example, two, three, four, five, six, seven, eight, nine, or ten substitutions. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising two or more substitutions (including, but not limited to, those disclosed in Table 5). It is understood that the two or more substitutions can comprise any combination of the substituted residues disclosed herein, including but not limited to residues 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, and/or 339 as set forth in SEQ ID NO: 1. For example, disclosed herein are U2AF⁶⁵ splice factor variants comprising substitutions at residues 289 and 254; 256 and 260; 215 and 252; 253 and 287; or 292 and 272.

140. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the variant comprises any of the substitutions disclosed herein at residues 289 and 254 of SEQ ID NO: 1 or at a residue corresponding to residue 289 and 254 of SEQ ID NO: 1. For example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the U2AF⁶⁵ splice factor variants comprises substitutions at residues 289 and 254, wherein the substitution at residue 289 is a substitution of an Asparagine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Histidine, Arginine, or Glutamine) and the substitution at residue 254 is substitution of a Valine for another uncharged nonaromatic residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein are methods wherein the U2AF⁶⁵ substitutions are N289Q and V254T.

141. Also disclosed, in one aspect are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the variant comprises any of the substitutions disclosed herein at residues 256 and 260 of SEQ ID NO: 1 or at a residue corresponding to residues 256 and 260 of SEQ ID NO: 1. For example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the U2AF⁶⁵ splice factor variants comprises substitutions at residues 256 and 260 wherein the substitution at residue 256 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine) and the substation at residues 260 is a substation of a Lysine for an uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine,

Leucine, Methionine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein methods wherein the U2AF⁶⁵ substitutions are D256K and K260T, D256K and K260L, D256K and K260I, D256N and K260Q, D256E and K260T, D256E and K260L, or D256E and K260I.

142. Also disclosed, are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the variant comprises any of the substitutions disclosed herein at residues 215 and 252 of SEQ ID NO: 1 or at a residue corresponding to residue 215 and 252 of SEQ ID NO: 1. For example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the U2AF⁶⁵ splice factor variants comprises substitutions at residues 215 and 252, wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine) and wherein the substation at residue 252 is the substitution of a Threonine for another uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Cysteine, Asparagine, or Glutamine). In one aspect, disclosed herein are methods wherein the U2AF⁶⁵ substitutions are D215N and T252G or D215N and T252P.

143. Also disclosed, in one aspect methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the variant comprises any of the substitutions disclosed herein at residues 253 and 287 of SEQ ID NO: 1 or at a residue corresponding to residue 253 and 287 of SEQ ID NO: 1. For example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the U2AF⁶⁵ splice factor variants comprises substitutions at residues 253 and 287; wherein the substitution at residue 253 is the substitution of a Valine for another nonpolar nonaromatic residue ((i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, or Proline); and wherein the substation at residues 287 is a substitution of a Alanine for another nonpolar residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline, or Methionine). In one aspect, disclosed herein are methods wherein the U2AF⁶⁵ substitutions are V253G and

A287A, V253G and A287V, V253G and A287L, V253S and A287A, V253S and A287V, or V253S and A287L.

144. Additionally, disclosed herein methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the variant comprises any of the substitutions disclosed herein at residues 292 and 272 of SEQ ID NO: 1 or at a residue corresponding to residue 292 and 272 of SEQ ID NO: 1. For example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the U2AF⁶⁵ splice factor variants comprises substitutions at residues 292 and 272; wherein the substitution at residue 272 is the substitution of a Aspartate for a polar uncharged nonaromatic residue (i.e., Serine, Threonine, Cysteine,

Asparagine, or Glutamine); and wherein the substitution at residue 292 is the substitution of a Lysine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Asparagine, Histidine, Arginine, or Glutamine). In one aspect, disclosed herein are methods wherein the U2AF⁶⁵ substitutions are K292Q and D272N.

145. It is understood and herein contemplated that the relevance of a contact residue can vary based on the Py tract sequence. A residue that is a contact residue for an adenine at position 1 of the Py tract may not be a contact residue if position 1 is a guanine, cysteine, or uracil. Similarly, a contact residue at position 1 of the Py tract may not be a contact residue at positions 2, 3, 4, 5, 6, 7, 8, or 9 of the Py tract.

146. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 268, 293, 296, 298, and/or 300.

147. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 268 of SEQ ID NO: 1 or at a residue corresponding to residue 268 of SEQ ID NO: 1, wherein the substitution at residue 268 is the substitution of an Asparagine for another nonaromatic residue (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Arginine,

Histidine, or Glutamine), more specifically wherein the nonaromatic residue is not positively charged (i.e., Glycine, Alanine, Methionine, Valine, Leucine, Isoleucine, Proline, Serine,

Threonine, Cysteine, Aspartate, Glutamate, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 1 ; and wherein the U2AF⁶⁵ variant comprises a N268S, N268D, N268G, N268A, or N268T substitution.

148. Also, for example disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 293 of SEQ ID NO: 1 or at a residue corresponding to residue 293 of SEQ ID NO: 1, wherein the substitution at residue 293 is the substitution of an Aspartate for an uncharged residue (i.e., Phenylalanine, Tyrosine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically a nonaromatic uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are U2AF⁶⁵ methods wherein the substitution at residue 293 is a D293N, D293F, D293S, D293G, D293A, D293I, D293L, D293T, or D293V substitution. Also, for example disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 1; and wherein the U2AF⁶⁵ variant comprises a D293N substitution. Also, for example disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 1; and wherein the U2AF⁶⁵ variant comprises a D293N, D293F, D293S, D293G, D293A, D293I, D293L, D293T, or D293V substitution. Also, for example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 1; and wherein the U2AF⁶⁵ variant comprises a D293N or D293F substitution.

149. Further, for example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 296 of SEQ ID NO: 1 or at a residue corresponding to residue 296 of SEQ ID NO: 1, wherein the substitution at residue 296 is the substitution of a Threonine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another polar uncharged residue (i.e., Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 1; and wherein the substitution at residue 296 is a T296N or T296Q substitution.

150. Additionally, for example, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract comprising administering to a subject a U2AF variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 298 of SEQ ID NO: 1 or at a residue corresponding to residue 298 of SEQ ID NO: 1, wherein the substitution at residue 298 is the substitution of a Leucine for a polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, or 5 Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 1; and wherein the substitution at residue 298 is a L298G, L298S, L298N, or L298T substitution.

151. Also, in one aspect, disclosed herein are methods of treating a disorder associated 10 with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 300 of SEQ ID NO: 1 or at a residue corresponding to residue 300 of SEQ ID NO: 1, wherein the substitution at residue 300 is the substitution of a Lysine for

15 another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine,

Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 1 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 1; and wherein the substitution at residue 300 is

20 a K300R substitution.

152. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues

25 262, 328, 329, 330, 331, and/or 333.

153. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the

30 substituted amino acid of U2AF⁶⁵ is at residue 262 of SEQ ID NO: 1 or at a residue corresponding to residue 262 of SEQ ID NO: 1, wherein the substitution at residue 262 is the substitution of a Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre -mRNA Py tract; wherein the mutation is a cytosine or adenine at position 2; and wherein the substitution at residue 262 is a F262Y substitution.

154. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 328 of SEQ ID NO: 1 or at a residue corresponding to residue 328 of SEQ ID NO: 1, wherein the substitution at residue 328 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another positively charged residue (i.e., Arginine or Histidine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre- mRNA Py tract; wherein the mutation is an cytosine at position 2; and wherein the substitution at residue 328 is a K328R substitution.

155. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 329 of SEQ ID NO: 1 or at a residue corresponding to residue 329 of SEQ ID NO: 1, wherein the substitution at residue 329 is the substitution of a Lysine for another polar residue (Serine, Cysteine, Asparagine, Glutamine, Tyrosine, Lysine, Arginine, Histidine, Aspartate, or Glutamate), or more specifically, another charged residue (i.e., Arginine, Histidine, Aspartate, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 2; and wherein the substitution at residue 329 is a K329D substitution.

156. Further disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 330 of SEQ ID NO: 1 or at a residue corresponding to residue 330 of SEQ ID NO: 1, wherein the substitution at residue 330 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine,

5 Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 2; and wherein the substitution at residue 330 is a L330V, L330A, or L330T substitution.

10 157. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 331 of SEQ ID NO: 1 or at a residue corresponding

15 to residue 331 of SEQ ID NO: 1 , wherein the substitution at residue 331 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py

20 tract; wherein the mutation is a cytosine at position 2; and wherein the substitution at residue 331 is a L331S, L331N, L331T, L331A, or L331G substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 2; and wherein the substitution at residue 331 is a L331S, L331T, L331A, or L331G substitution.

25 158. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 333 of SEQ ID NO: 1 or at a residue corresponding to residue

30 333 of SEQ ID NO: 1, wherein the substitution at residue 333 is the substitution of a Glutamine for another polar residue (i.e., Serine, Threonine, Cysteine, Asparagine, Lysine, Tyrosine, Histidine, Arginine, Glutamate, or Aspartate). For example, For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 2 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 2; and wherein the substitution at residue 333 is a Q333R substitution.

159. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 331, 333, 335, 336, and/or 339.

160. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 331 of SEQ ID NO: 1 or at a residue corresponding to residue 331 of SEQ ID NO: 1 , wherein the substitution at residue 331 is the substitution of a Leucine for another uncharged residue (i.e., Tyrosine, Phenylalanine, Tryptophan, Glycine, Alanine, Valine,

Isoleucine, Proline, Methionine, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 3; and wherein the substitution at residue 331 is a L331Q substitution.

161. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 333 of SEQ ID NO: 1 or at a residue corresponding to residue 333 of SEQ ID NO: 1, wherein the substitution at residue 333 is the substitution of a Glutamine for another polar residue (i.e., Serine, Threonine, Cysteine, Asparagine, Lysine, Tyrosine, Histidine, Arginine, Glutamate, or Aspartate). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre- mRNA Py tract; wherein the mutation is a cytosine at position 3; and wherein the substitution at residue 333 is a Q333E substitution. For example, 3 disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre- mRNA Py tract; wherein the mutation is a guanine at position 3; and wherein the substitution at residue 333 is a Q333N, Q333S, or Q333T substitution.

162. Additionally disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 335 of SEQ ID NO: 1 or at a residue corresponding to residue 335 of SEQ ID NO: 1, wherein the substitution at residue 335 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or

Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 3; and wherein the substitution at residue 335 is an A335S, A335T, or A335G substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre- mRNA Py tract; wherein the mutation is a adenine at position 3; and wherein the substitution at residue 335 is an A335G substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre- mRNA Py tract; wherein the mutation is a guanine at position 3; and wherein the substitution at residue 335 is an A335S or A335G substitution.

163. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 336 of SEQ ID NO: 1 or at a residue corresponding to residue 336 of SEQ ID NO: 1, wherein the substitution at residue 336 is the substitution of a Serine for a nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 3; and wherein the substitution at residue 336 is a S336P or S336G substitution.

164. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 339 of SEQ ID NO: 1 or at a residue corresponding to residue 339 of SEQ ID NO: 1, wherein the substitution at residue 339 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 3 of the pre-mRNA Py tract;

wherein the mutation is a cytosine at position 3; and wherein the substitution at residue 339 is an A339S or A339T substitution.

165. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 254, 256, 260, 289, and/or 339.

166. Further, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 254 of SEQ ID NO: 1 or at a residue corresponding to residue 254 of SEQ ID NO: 1, wherein the substitution at residue 254 is the substitution of a Valine for another uncharged residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 4; and wherein the substitution at residue 254 is a V254T, or V254S substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 4; and wherein the substitution at residue 254 is a V254T substitution.

167. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 256 of SEQ ID NO: 1 or at a residue corresponding to residue 256 of SEQ ID NO: 1, wherein the substitution at residue 256 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 4; and wherein the substitution at residue 256 is a D256N or, D256E substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 4; and wherein the substitution at residue 256 is a D256K substitution.

168. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 260 of SEQ ID NO: 1 or at a residue corresponding to residue 260 of SEQ ID NO: 1, wherein the substitution at residue 260 is the substitution of a Lysine for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or

Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Valine, Leucine, Methionine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 4; and wherein the substitution at residue 260 is a K260T, K260L, K260I, or K260Q substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mR A Py tract; wherein the mutation is a adenine at position 4; and wherein the substitution at residue 260 is a K260Q substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 4; and wherein the substitution at residue 260 is a K260T, K260L, or K260I substitution

169. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 289 of SEQ ID NO: 1 or at a residue corresponding to residue 289 of SEQ ID NO: 1, wherein the substitution at residue 289 is the substitution of a Asparagine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Lysine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 4; and wherein the substitution at residue 289 is a N289R substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre- mRNA Py tract; wherein the mutation is a cytosine at position 4; and wherein the substitution at residue 289 is a N289Q substitution.

170. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 339 of SEQ ID NO: 1 or at a residue corresponding to residue 339 of SEQ ID NO: 1, wherein the substitution at residue 339 is the substitution of an Alanine for a polar uncharged residue (i.e., Serine, Threonine, Cysteine, Asparagine, Tyrosine, Glycine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 4 of the pre-mRNA Py tract; wherein the mutation is a guanine at position 4; and wherein the substitution at residue 339 is an A339E, substitution. 171. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mR A or method of decreasing defective splicing; wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 215, 252, 253, 254, and/or 287.

172. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 215 of SEQ ID NO: 1 or at a residue corresponding to residue 215 of SEQ ID NO: 1, wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 5; and wherein the substitution at residue 215 is a D215N substitution.

173. Additionally, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 252 of SEQ ID NO: 1 or at a residue corresponding to residue 252 of SEQ ID NO: 1, wherein the substitution at residue 252 is the substitution of a Threonine for another uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline,

Methionine, Serine, Tyrosine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue ((i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 5; and wherein the substitution at residue 252 is a T252N substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 5; and wherein the substitution at residue 252 is a T252G, or T252P substitution.

174. Also, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 253 of SEQ ID NO: 1 or at a residue corresponding to residue 253 of SEQ ID NO: 1, wherein the substitution at residue 253 is the substitution of a Valine for another nonpolar residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue ((i.e., Glycine, Alanine,

Methionine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 5; and wherein the substitution at residue 253 is a V253G or V253P substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 5; and wherein the substitution at residue V253G or V253S substitution.

175. Further, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 254 of SEQ ID NO: 1 or at a residue corresponding to residue 254 of SEQ ID NO: 1, wherein the substitution at residue 254 is the substitution of a Valine for another uncharged residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged nonaromatic residue (i.e., Glycine, Alanine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 5; and wherein the substitution at residue 254 is a V254G, V254A, V254P, V254T, or V254S substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 5; and wherein the substitution at residue 254 is a V254T or V254I substitution.

176. Further, in one aspect, disclosed herein are methods of treating a disorder

5 associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 287 of SEQ ID NO: 1 or at a residue corresponding to residue 287 of SEQ ID NO: 1 , wherein the substitution at residue 287 is the substitution of a

10 Alanine for another nonpolar residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline,

Methionine, Phenylalanine, or Tryptophan), more specifically a nonpolar nonaromatic residue (i.e., Glycine, Valine, Leucine, Isoleucine, Proline, or Methionine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 5 of the pre-mRNA Py tract; wherein the mutation is a adenine at position 5;

15 and wherein the substitution at residue 287 is an A287V or A287L substitution.

177. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues

20 155, 196, 272, and/or 292.

178. In one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino

25 acid of U2AF⁶⁵ is at residue 155 of SEQ ID NO: 1 or at a residue corresponding to residue 155 of SEQ ID NO: 1 , wherein the substitution at residue 155 is the substitution of a Asparagine for another polar uncharged residue (i.e., Serine, Tyrosine, Threonine, Cysteine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract; wherein the mutation is

30 an uracil at position 6; and wherein the substitution at residue 155 is a N155T or N155S. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract; wherein the mutation is an cytosine at position 6; and wherein the substitution at residue 155 is a N155Q substitution.

179. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mR A or method of decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 196 of SEQ ID NO: 1 or at a residue corresponding to residue 196 of SEQ ID NO: 1, wherein the substitution at residue 196 is the substitution of a Asparagine for uncharged nonaromatic residue or positively charged residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre- mRNA Py tract; wherein the mutation is an uracil at position 6; and wherein the substitution at residue 196 is a at residue 196 is a N196Q substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract; wherein the mutation is an cytosine at position 6; and wherein the substitution at residue 196 is a at residue 196 is a N196T, N196S, N196R, N196A, or N196V substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 6; and wherein the substitution at residue 196 is a N196T substitution.

180. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 272 of SEQ ID NO: 1 or at a residue corresponding to residue 272 of SEQ ID NO: 1, wherein the substitution at residue 272 is the substitution of a Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Histidine, Arginine, or Glutamine), more specifically wherein the residue is a polar uncharged nonaromatic residue (i.e., Serine, Threonine, Cysteine, Asparagine, or

Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 6; and wherein the substitution at residue 272 is a D272N substitution.

181. Additionally, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 292 of SEQ ID NO: 1 or at a residue corresponding to residue 292 of SEQ ID NO: 1, wherein the substitution at residue 292 is the substitution of a Lysine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Aspartate, Glutamate, Asparagine, Histidine, Arginine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 6 of the pre- mRNA Py tract; wherein the mutation is an adenine at position 6; and wherein the substitution at residue 292 is a K292Q substitution.

182. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 197, 199, 215, 227, 229, and/or 230.

183. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 197 of SEQ ID NO: 1 or at a residue corresponding to residue 197 of SEQ ID NO: 1, wherein the substitution at residue 197 is the substitution of a Phenylalanine for another nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, or Tryptophan), more specifically a nonaromatic nonpolar residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, or Proline). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre- mRNA Py tract; wherein the mutation is an adenine at position 7; and wherein the substitution at residue 197 is a F197V, F197I, or F197L substitution. 184. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 199 of SEQ ID NO: 1 or at a residue corresponding to residue 199 of SEQ ID NO: 1, wherein the substitution at residue 199 is the substitution of a

Phenylalanine for another aromatic residue (i.e., Tyrosine or Tryptophan). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract; wherein the mutation is an guanine at position 7; and wherein the substitution at residue 199 is a F199Y or F199W substitution.

185. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 215 of SEQ ID NO: 1 or at a residue corresponding to residue 215 of SEQ ID NO: 1, wherein the substitution at residue 215 is the substitution of an Aspartate for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Glutamate, Lysine, Arginine, Histidine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract; wherein the mutation is a cytosine at position 7; and wherein the substitution at residue 215 is a D215N substitution.

186. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 227 of SEQ ID NO: 1 or at a residue corresponding to residue 227 of SEQ ID NO: 1, wherein the substitution at residue 227 is the substitution of a Arginine for another polar residue (i.e., Serine, Tyrosine, Threonine, Cysteine, Asparagine, Aspartate,

Glutamate, Lysine, Histidine, or Glutamine), more specifically a polar uncharged nonaromatic residue ((i.e., Serine, Threonine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract; wherein the mutation is an cytosine at position 7; and wherein the substitution at residue 227 is a R227Q substitution.

187. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the 5 disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 229 of SEQ ID NO: 1 or at a residue corresponding to residue 229 of SEQ ID NO: 1, wherein the substitution at residue 229 is the substitution of a Proline for another nonpolar residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine,

10 Methionine, Leucine, Isoleucine, Proline), more specifically a nonpolar nonaromatic residue (i.e., Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract; wherein the mutation is an guanine or uracil at position 7; and wherein the substitution at residue 229 is a P229G substitution.

15 188. Also, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 23 Oof SEQ ID NO: 1 or at a residue corresponding to residue

20 230 of SEQ ID NO: 1, wherein the substitution at residue 230 is the substitution of a Histidine for a non-negatively charged amino acid residue (i.e., Phenylalanine, Tryptophan, Glycine, Alanine, Valine, Methionine, Leucine, Isoleucine, Proline, Serine, Tyrosine, Threonine, Cysteine,

Asparagine, Glutamine, Lysine, or Arginine. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7

25 of the pre-mRNA Py tract; wherein the mutation is an uracil at position 7; and wherein the

substitution at residue 230 is a H230F, H230Y, H230I, H230L, or H230Q substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 7 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 7; and wherein the substitution at residue 230 is a H230F, H230Y, H230I, or

30 H230L substitution. 189. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 147, 150, and/or 231.

190. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 147 of SEQ ID NO: 1 or at a residue corresponding to residue 147 of SEQ ID NO: 1, wherein the substitution at residue 147 is the substitution of a Glutamine for nonaromatic or positively charged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, Aspartate, or Glutamate). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract; wherein the mutation is an adenine at position 8; and wherein the substitution at residue 147 is a Q147S, Q147T, Q147N, Q147A, Q147V, Q147I, Q147L, Q147M, Q147D, or Q147E substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract; wherein the mutation is an guanine at position 8; and wherein the substitution at residue 147 is a Q147S, Q147T, Q147N, Q147A, Q147V, Q147I, or Q147L substitution.

191. Additionally, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 150 of SEQ ID NO: 1 or at a residue corresponding to residue 150 of SEQ ID NO: 1, wherein the substitution at residue 150 is the substitution of a Arginine for a polar uncharged residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract; wherein the mutation is an guanine at position 8; and wherein the substitution at residue 150 is a R150S, R150T, R150N, R150Q, or R150Y substitution.

192. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 231 of SEQ ID NO: 1 or at a residue corresponding to residue 231 of SEQ ID NO: 1 , wherein the substitution at residue 231 is the substitution of a Aspartate for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged amino acid residue with a small R group (e.g., Glycine, Alanine, Valine, Serine, Threonine, Cysteine, or Asparagine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre- mRNA Py tract; wherein the mutation is an guanine at position 8; and wherein the substitution at residue 231 is a D23 IN substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 8 of the pre- mRNA Py tract; wherein the mutation is an uracil at position 8; and wherein the substitution at residue 231 is a D231V, D231T, D231N, or D231S substitution.

193. Accordingly, disclosed herein, methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing; wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract comprising administering to a subject with the disorder a U2AF⁶⁵ variant; wherein the U2AF⁶⁵ variant comprises a substitution at residues 145, 147, and/or 231.

194. Accordingly, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 145 of SEQ ID NO: 1 or at a residue corresponding to residue 145 of SEQ ID NO: 1, wherein the substitution at residue 145 is the substitution of a Arginine for another polar residue (i.e., Serine, Threonine, Tyrosine, Cysteine, Asparagine, Lysine, Histidine, Aspartate, Glutamate, or Glutamine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract; wherein the mutation is an guanine at position 9; and wherein the substitution at residue 145 is a R145Q substitution.

195. Also disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 147 of SEQ ID NO: 1 or at a residue corresponding to residue 147 of SEQ ID NO: 1, wherein the substitution at residue 147 is the substitution of a Glutamine for nonaromatic or positively charged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Serine, Threonine, Cysteine, Asparagine, Aspartate, or Glutamate). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract; wherein the mutation is an uracil at position 9; and wherein the substitution at residue 147 is a Q147S, Q147T, Q147M, Q147E, Q147L, or Q147D substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre- mRNA Py tract; wherein the mutation is an adenine at position 9; and wherein the substitution at residue 147 is a Q147T, Q147N, Q147V, Q147I, or Q147L substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract; wherein the mutation is an guanine at position 9; and wherein the substitution at residue 147 is a Q147S, Q147T, Q147N, Q147V, Q147I, or Q147L substitution.

196. Further, in one aspect, disclosed herein are methods of treating a disorder associated with aberrantly spliced mRNA or method of decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mRNA Py tract comprising administering to a subject a U2AF⁶⁵ variant comprising one or more amino acid substitutions; wherein the substituted amino acid of U2AF⁶⁵ is at residue 231 of SEQ ID NO: 1 or at a residue corresponding to residue 231 of SEQ ID NO: 1 , wherein the substitution at residue 231 is the substitution of a Aspartate for an uncharged residue (i.e., Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine, Serine, Tyrosine, Threonine, Cysteine, Asparagine, or Glutamine), more specifically an uncharged amino acid residue with a small R group (e.g., Glycine, Alanine, Valine, Serine, Threonine, Cysteine, or Asparagine). For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre- mR A Py tract; wherein the mutation is an guanine at position 9; and wherein the substitution at residue 231 is a D231N or D231S substitution. For example, disclosed herein are methods of treating or decreasing defective splicing wherein the disorder results from a mutation at position 9 of the pre-mR A Py tract; wherein the mutation is an guanine at position 9; and wherein the substitution at residue 231 is a D23 IN substitution.

197. It is understood and herein contemplated that the methods disclosed herein can be used to treat any disorder associated with aberrantly spliced mRNA or decreased defective splicing. For example, the disorder can be pigmentosa, β-thalassemia, neurofribormatosis,

Duchenne muscular dystrophy, Spinal muscular atrophy, general cancers, acute myeloid leukemia, Hepatocellular carcinoma, Myelodysplasia syndrome (MDS), and hematological malignancies.

198. In the methods provided herein, the cell-free mRNA or the mRNA in a cell can be in vitro. In in vivo methods, the mRNA is present in a subject. As used throughout, by subject is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

1. Pharmaceutical carriers/Delivery of pharmaceutical products

199. As described above, the U2AF⁶⁵ comprising compositions can also be

administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

200. The compositions may be administered by injection (e.g., subcutaneous, intramuscular, intravenous, intra-arterial, intraperitoneal), by continuous intravenous infusion, cutaneously, dermally, transdermally, orally (e.g., tablet, pill, liquid medicine, edible film strip), by implanted osmotic pumps, by suppository, or by aerosol spray. Routes of administration include, but are not limited to, topical, intradermal, intrathecal, intralesional, intratumoral, intrabladder, intravaginal, intra-ocular, intrarectal, intrapulmonary, intracranial, intraventricular, intraspinal, dermal, subdermal, intra-articular, placement within cavities of the body, nasal inhalation, pulmonary inhalation, impression into skin, and electroporation.

201. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

202. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S.

Patent No. 3,610,795, which is incorporated by reference herein.

203. In an example in which a nucleic acid is employed, for example a nucleic acid encoding a polypeptide comprising a mutant U2AF⁶⁵, the nucleic acid can be delivered

intracellularly (for example by expression from a nucleic acid vector or by receptor-mediated mechanisms), or by an appropriate nucleic acid expression vector which is administered so that it becomes intracellular, for example by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (such as a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (for example Jo Hot et ah, Proc. Natl. Acad. Sci. USA 1991, 88: 1864-8). Nucleic acid carriers also include, polyethylene glycol (PEG), PEG-liposomes, branched carriers composed of histidine and lysine (HK polymers), chitosan-thiamine pyrophosphate carriers, surfactants, nanochitosan carriers, and D5W solution. The present disclosure includes all forms of nucleic acid delivery, including naked DNA, plasmid and viral delivery, integrated into the genome or not.

204. As mentioned above, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al, Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al, Mol. Cell. Biol. 6:2895, 1986). The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al, Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al, Blood 84: 1492-1500, 1994), lentiviral vectors (Naidini et al, Science 272:263-267, 1996), and pseudotyped retroviral vectors (Agrawal et al, Exper. Hematol. 24:738-747, 1996).

205. Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al,

Blood 87:472-478, 1996) to name a few examples. This invention can be used in conjunction with any of these or other commonly used gene transfer methods.

206. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al, Bioconjugate Chem., 4:3-9, (1993); Battelli, et al, Cancer Immunol.

Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al, Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as

"stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al, Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

207. The compositions, including antibodies, can be used therapeutically in

combination with a pharmaceutically acceptable carrier.

208. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA

1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the U2AF⁶⁵ variant, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

209. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

210. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

211. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously,

intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

212. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

213. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

214. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..

215. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as

hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. 216. These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, for example, sugars, sodium chloride, and the like can also be included. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

217. Solid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.

218. Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.

219. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They can contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above- mentioned excipients. 220. Liquid dosage forms for oral administration of the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

221. Besides such inert diluents, the composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.

222. Suspensions, in addition to the active compounds, can contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

b) Therapeutic Uses

223. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose

administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al, Antibodies in Human Diagnosis and Therapy, Haber et al, eds., Raven Press, New York (1977) pp. 365-389. 2. Nucleic Acid Delivery

224. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the U2AF⁶⁵-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE

(GIBCO-BPvL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

225. As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al, Proc. Natl. Acad. Sci. U.S.A. 85:4486, 1988; Miller et al, Mol. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a U2AF⁶⁵ variant. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al, Blood 84: 1492-1500, 1994), lentiviral vectors (Naidini et al, Science 272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al, Exper. Hematol. 24:738-747, 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al, Blood 87:472-478, 1996). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods. 226. Parenteral administration of the nucleic acid or vector, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. For additional discussion of suitable formulations and various routes of administration of therapeutic compounds, see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.

3. Delivery of the compositions to cells

227. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. A., et al, Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

a) Nucleic acid based delivery systems

228. Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

229. As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as a variant U2AF⁶⁵ disclosed herein into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Viral vectors are , for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

230. Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

231. It is understood and herein contemplated that the modification to achieve the U2AF⁶⁵ variant can be achieved by any means known in the art including techniques that manipulate genomic DNA, messenger and/or non-coding RNA and/or proteins including but not limited to endogenous or exognenous control elements (e.g., siRNA, shRNA, small molecule inhibitor, and antisense oligonucleotide) and mutations in or directly targeting the coding region of the gene, mRNA, or protein or a control element or mutation in a regulator region operably linked to the gene, mRNA, or protein. As such, the technologies or mechanisms that can be employed to modulate a gene of interest include but are not limited to technologies and reagents that target genomic DNA to result in an edited genome (e.g., homologous recombination to introduce a mutation such as a deletion into a gene, zinc finger nucleases, meganucleases, transcription activator-like effectors (e.g., TALENs), triplexes, mediators of epigenetic modification, and CRISPR and rAAV technologies).

b) Non-nucleic acid based systems

232. The disclosed U2AF⁶⁵ variants can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.

233. Thus, the U2AF⁶⁵ comprising compositions can comprise, in addition to the disclosed U2AF⁶⁵ variant or vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355.

Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

234. In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN,

LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ). 235. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al, Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al, Bioconjugate Chem., 4:3-9, (1993); Battelli, et al, Cancer Immunol.

Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al, Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al, Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104 : 179- 187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

236. Nucleic acids that are delivered to cells which are to be integrated into the host cell genome, typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can become integrated into the host genome. 237. Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote

homologous recombination are known to those of skill in the art.

c) In vivo/ex vivo

238. As described above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).

239. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteo liposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or

homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. In one aspect, cells are removed from a subject, U2AF⁶⁵ is administered to the cells, and transplanted into a subject. In autologous transfers the cells are transplanted back into the donating subject. In syngeneic transfers, cells are removed, treated with U2AF⁶⁵, and administered to a recipient subject.

4. Expression systems

240. The nucleic acids that are delivered to cells typically contain expression controlling systems. For example, the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. a) Viral Promoters and Enhancers

241. Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as:

polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al, Nature, 273: 113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HmdIII E restriction fragment (Greenway, P.J. et al, Gene 18: 355-360 (1982)). Of course, promoters from the host cell or related species also are useful herein.

242. Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3^* (Lusky, M.L., et al, Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al, Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

243. The promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

244. In certain embodiments the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTR.

245. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

246. Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mR A expression. These regions are transcribed as

polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early

polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.

b) Markers

247. The viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed. Preferred marker genes are the E. Coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

248. In some embodiments the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow independent of a supplemented media. Two examples are: CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

249. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1 : 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al, Mol. Cell. Biol. 5 : 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

250. It is understood and herein contemplated that engineering a splicing factor variant is useful for finding new therapies for aberrant mRNa splicing. Accordingly, in one aspect disclosed herein are methods of engineering a splicing factor variant comprising mutating one of the nine nucleotide residues of a pre-mRNA splice site polypyrimidine (Py) tract of a gene to generate dU, dC, dG, and dA Py tract mutants; co-cystalizing the mutated Py tract mutants comprising a deoxy-ribose oligonucleotide backbone with a U2AF⁶⁵ splice factor variant wherein the U2AF⁶⁵ variant is a deletion mutant of the amino acids corresponding to residues 238-257 of SEQ ID NO: 1 ; comparing the binding affinity of the U2AF⁶⁵ variant to the Py tract mutants; identifying contact residues of U2AF⁶⁵ for a Py tract mutant; selecting amino acid substitutions at contact residues of U2AF⁶⁵ that increase the binding affinity to a mutated residue of the Py tract, and substituting the native amino acid for an identified amino acid. In one aspect, disclosed are methods wherein the mutated Py tract is at residue 1 , 2, 3, 4, 5, 6, 7, 8, or 9. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 1 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 2 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 3 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 4 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 5 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 6 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 7 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 8 and the mutated residue is an A, U, C, or G. Also disclosed are methods of engineering a splicing factor variant wherein the Py tract position is position 9 and the mutated residue is an A, U, C, or G.

251. Also disclosed are methods of engineering a splicing factor variant comprising co-cystalizing the Py tract variants with a U2AF⁶⁵ splice factor; comparing the binding affinity of the U2AF⁶⁵ variant to the Py tract variant; identifying contact residues of U2AF⁶⁵ for the particular Py tract variant; selecting amino acid substitutions at contact residues of U2AF⁶⁵ that increase splicing at a target splice site or increases the binding affinity to a mutated residue of the Py tract, and substituting the native amino acid for an identified amino acid.

D. Examples

252. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

RNA Binding Experiments 253. For RNA binding experiments, human U2AF (residues 141-342) was expressed in E. coli as a GST-fusion protein and purified by glutathione affinity, followed by anion-exchange and gel filtration chromatography after protease cleavage of the GST-tag. The U2AF⁶⁵ protein was diluted >10-fold in buffer (100 mM NaCl, 15 mM Hepes, pH 6.8, 0.2 mM TCEP, 0.1 U/μΙ Superase-In (Ambion)) and titrated into 30 nM 5 ^'-fluorescein-labeled RNA in this buffer

(sequences in Table 13). The fluorescence anisotropy changes were fit to obtain the K_D as described in Jenkins et al. "Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF⁶⁵" J. Biol. Chem. 283 : 33641-33649 (2008). The fitted binding curves are shown in Fig. 1.

Table 13

Crystallization and Structure Determination

254. The dU2AF⁶⁵ or dU2AF⁶⁵-D23 IV protein (residues 148-237, 258-336) was expressed, purified, and crystallized as described in Jenkins et al. "U2AF⁶⁵ adapts to diverse pre- mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs," Nucleic Acids Res. 41 : 3859-3873 (2013) and Sickmier et al. "Crystallization and preliminary X-ray analysis of U2AF⁶⁵ variant in complex with a polypyrimidine tract analogue by use of protein engineering. Acta Crystallogr F62:457-459 (2006). The dU2AF⁶⁵ was co- crystallized with 5 ^'-dUdUdUdU(5-Br-dU)dAdU and 5 ^'-dUdUdUdU(5-Br-dU)dGdU. The dU2AF -D231V variant was co-crystallized wtih 5 ^'-dUdUdUdU(5-Br-dU)dUdU. The additives 2% w/v polyethylene glycol 3350 and 10 mM MgCl₂ were included in the dG-containing conditions whereas the dA-containing conditions included 3% v/v ethylene glycol (final concentrations). Crystals were cryo-protected by incremental transfer to 21% v/v glycerol prior to data collection. Structures were determined by difference Fourier using PDB ID 3 VAK as a starting model. Consistent sets of reflections as for 3 VAK were excluded from the refinement for cross-validation. Structures were refined using Phenix, built using COOT, and structure figures were prepared using PYMOL. Crystallographic data and refinement statistics are given in Table 14.

Table 14

Table 14. Cr stallo ra hic Data and Refinement Statistics

5 ^a Values from the highest resolution shell are given in parentheses.

b Rs_ym =∑u_d∑i|Ii ^{~~ <}I^>|/∑hki∑i li where I, is an intensity I for the ith measurement of a reflection with indices hkl and <I> is the weighted mean of all measurements of I.

⁰ Rwork =∑m\\Fobs(hkl)\-\V_C3lc(hkl)\\y∑_hki\F₀bs(hkl)\ for the working set of reflections, Rfr_ee is R_WOrk for randomly-selected 10% of reflections excluded from the refinement. All data from the available resolution ranges were used in the refinement.

10 Calculated using the program Molprobity.

Transfection and RT-PCR Analyses

255. The full length human U2AF⁶⁵ cDNA (Origene clone ID BC008740) was mutated to encode the D231V variant. The U2AF⁶⁵ protein was either expressed separately in pCMV6-XL5 (Origene) (Fig. 3B) or subcloned to the bicistronic vector pBI-CMVl (Clontech) for co-expression with the wild-type or mutant RP2 minigenes (Fig. 4A). The RP2 and RP2(U>A) minigenes either were expressed in pEGFP-C2 as described (Fig. 3B) or the RP2-EGFP region was subcloned to pBI-CMVl containing U2AF⁶⁵ (Fig. 4A). HEK293T cells were seeded on 12- well plates (2-4 x 10⁵ cells/well) and grown as monolayers in MEM (Gibco, USA) supplemented with 10%(v/v) of heat- inactivated fetal bovine serum, l%(v/v) L-glutamine, and l%(v/v) penicillin-streptomycin. After 1 day, the cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Grand Island NY) according to the manufacturer's instructions. For transfection of the bicistronic construct shown in Fig. 4, 1 μg was used. Transfection efficiency was monitored by GFP-fluorescence and was comparable for all constructs and replicates. To confirm similar U2AF⁶⁵ and U2AF⁶⁵-D23 IV expression levels, 4xl0⁵ cells were suspended two days post- transfection in 60 μΐ lysis buffer with 20 μΐ SDS-PAGE loading buffer, heated at 90° for two minutes, and the indicated amounts were separated by 12.5% SDS-PAGE and immunoblotted using mouse monoclonal antibodies directed against U2AF⁶⁵ (MC3, Sigma, St. Louis, MO) or as a control for comparison, GAPDH (clone 71.1, Sigma) (Fig. 3 A). Immunoblots were developed using anti-mouse horseradish peroxidase-conjugates (U4758, Sigma) and detected with

SuperSignal WestPico chemi-luminescent substrate (ThermoScientific, Waltham, MA).

256. For real-time reverse transcription PCR (RT-PCR) (Fig. 4), total RNA was isolated 2 days post-transfection using the Cells-to-cDNA II kit (Ambion, Grand Island, NY). RT-PCR was performed for 35 cycles (94 60 s - 60750 s - 72760 s) with forward (5 ^'- CGAGCTGTACAAGTCCGGCC-3 ^') (SEQ ID NO: 50) and reverse (5 ^'-

GGTTCAGGGGGAGGTGTGGG-3 ^') (SEQ ID NO: 51) primers for the RP2 product or forward (5 ^'-CATGTTCGTCATGGGTGTGAACCA-3 ^') (SEQ ID NO: 52) and reverse (5 ^'- ATGGCATGGACTGTGGTCATGAGT-3 ^') (SEQ ID NO: 53) primers for a GAPDH control. Following analyses by 2% agarose gel electrophoresis and ethidium bromide staining, the percentages of exon inclusion were averages of five independent experiments with standard deviation. The quantitative real-time PCR (qRT-PCR) of the RP2 minigenes is detailed in Fig. 3D.

Disease-Causing Mutations in the 5^' Regions of Py tracts Penalize U2AF⁶⁵ Association

257. Although numerous examples of disease-causing mutations in Py tracts have been identified, few studies of the consequences of these mutations for the splice site affinities of U2AF are available. To investigate the involvement of U2AF inhibition, the effects on U2AF⁶⁵ association of mutations in two representative Py tracts: (i) a U— >A

transversion in the Py tract of the neurofibromin 1 (NF1) gene creates a new 3 ' splice site junction (consensus "AG") and leads to familial neurofibromatosis type, and (u) a U— »A transversion in the Py tract of the retinitis pigmentosa-2 (RP2) gene that also introduces an "AG" dinucleotide induces exon skipping and consequently X-linked retinitis pigmentosa, were determined.

Sequences directly preceding the 3 ^' splice site junctions were used, including 13 -nucleotides for NF1, and to rule out the possible influence of flanking sequences, 9-nucleotides for RP2 (Table 13). The Py-tract recognition domain of U2AF⁶⁵ comprising RRM1+RRM2 and bordering residues were titrated into either the wild-type or mutated fluorescein-labeled Py tract RNAs, and the apparent equilibrium dissociation constants (K_D) were fit as described in Jenkins (2008). In both cases, the splice site mutations substantially reduced U2AF⁶⁵ affinity (by 4-5-fold, Table 13). It was concluded that these A-mutations in the 5 ^' regions of Py tracts disrupt splicing not only by introducing an aberrant "AG" consensus that normally dictates the junction of the 3 '-splice site, but also by penalizing U2AF⁶⁵ association.

Purine Substitutions in the 3^' Regions of Py Tracts Have Little Impact on U2AF⁶⁵ Binding

258. Serendipitously, both of the disease-causing U— »A mutations in NF1 and RP2 were located in the 5 ^' regions of the affected Py tracts. Although the region-dependence of disease-causing Py tract mutations has not yet been comprehensively surveyed, it was noted qualitatively that many disease-causing Py tract mutations, such as those in NF1 and RP2 investigated here, are located in the 5 ^' region of this splice site signal. Accordingly, it was previously found that the C-terminal U2AF⁶⁵ RRM2 has a stringent preference for U-nucleotides in the 5 ^' regions of Py tracts, whereas the N-terminal RRM1 is promiscuous for U- or C- nucleotides in the 3 ^' regions (Jenkins 2013). To compare the impact of purine-substitutions in the 3 ^'- with the 5 ^'-regions of the Py tract, artificial purine-substitutions were introduced at the penultimate nucleotide of the NF1 and RP2 Py tracts and U2AF⁶⁵ affinities were determined (Table 13, Fig. 1). The analogous A-substitution in a well-characterized hemoglobin-β splice site (HBB), which is disrupted in cases of β-thalassemia was also compared. In all cases, these purine- mutations in the 3 ^' region of the Py tract had little detectable effect on U2AF⁶⁵ binding. As observed in crystal and NMR structures, it was confirmed that U2AF⁶⁵ directly binds both the 5 ^' and 3 ^' regions of the Py tracts by equivalent UV-crosslinking efficiencies with photo-activated 4- thio-U nucleotides in the 5 ^' versus 3 ^' regions of the RP2 Py tract (Fig. 5). Altogether, these results are in agreement with the respective sequence-stringent and promiscuous U2AF⁶⁵ RRM2 and RRMl bound to the 5 ^'- and 3 ^'-regions of the Py tract. Structures of a "Sweet Spot" on U2AF RRM1 for Binding Purines

259. To address how U2AF⁶⁵ can accommodate purine-substitutions in the 3 ^' regions of degenerate Py tracts, the structures of U2AF⁶⁵ bound to U-tracts containing A- or G- substitutions at the penultimate nucleotide of an otherwise all-U Py tract were determined. A crystallization approach, as described in Jenkins (2008) and Jenkins (2013) was used. The focus of these studies were structure determinations on oligonucleotides containing dA or dG at the penultimate nucleotide and included BrdU as a marker for the sequence registers (Fig. 2A-C, Table 14). The polypeptide and oligonucleotide conformations of the two copies in the asymmetric unit closely match one another and a prior, baseline dU-bound dU2AF⁶⁵ structure (rmsd 0.4-0.6 for matching Ca and CI ^' atoms) (Fig. 2D). With the exception of one alternative dG conformation that engages in crystallographic contacts, the two complexes in each asymmetric unit share similar interactions with the bound purines.

260. The bound adenine or guanine bases stack on the consensus

ribonucleoprotein motif (RNP1) of U2AF⁶⁵ RRM1 and are engaged by hydrogen bonds with the protein backbone as well as the D231, and R150 side chains (Fig. 6B-D). For any type of bound nucleotide base, R150 consistently donates hydrogen bonds to either the pyrimidine-02, dA-Nl, or dG-N7 acceptors. In prior ribose-(r)U or dU-bound structures, the U2AF⁶⁵ H230/D231 backbone amides donate hydrogen bonds to the two lone pairs of the uracil-04 (Fig. 6B). Instead when bound to dA (Fig. 6C), an ordered water molecule mediates these hydrogen bonds with the protein backbone, which is relatively distant from the adenine (6.5 A D231-NH— dA-N7 compared with 3.2 A D231-NH— dU-04 heavy atom distances). However, the carboxylate side chain of U2AF⁶⁵ D231 is newly positioned to accept a direct hydrogen bond from the adenine exocyclic amine. These dA-contacts are reminiscent of the water-mediated and D231 interactions by U2AF⁶⁵ with the exocyclic amine of a bound cytosine. In contrast, the U2AF⁶⁵-bound dG has flipped to the s w-conformer (Fig. 6D), which differs from the awft^'-glycosidic bonds of other types of nucleotides in the U2AF⁶⁵ structures. In this conformation, the guanosine-06 accepts hydrogen bonds from the backbone amides with only slightly less optimal geometry than a uracil-04, whereas in the awft^'-conformer, the exocyclic amine of the guanosine would be expected to sterically interfere with the R150 side chain. Altogether, the structures reveal a "sweet spot" on U2AF⁶⁵ RRMl for binding diverse nucleotides in the 3 ' region of the Py tract.

Design and Structure of a Synthetic U2AF⁶⁵-D231V Variant

261. The relatively weak RNA affinity and promiscuity of U2AF⁶⁵ RRMl raised the hypothesis that these characteristics could be improved in synthetic U2AF⁶⁵ variants. The focus was on optimizing the "sweet spot" on U2AF⁶⁵ RRMl for binding diverse nucleotides at the penultimate position of the Py tract. Based on structure determinations, it was reasoned that replacement of the negatively-charged D231 aspartate with a hydrophobic valine side chain (D23 IV) would specifically increase U2AF⁶⁵ affinity for a U at the corresponding nucleotide position of the Py tract splice site signal. To characterize the modified interactions between the D23 IV mutant and uracil-containing oligonucleotide, we determined the 2.1 A resolution structure of dU2AF⁶⁵-D23 IV bound to a poly-dU oligonucleotide (Table 14). The dU2AF⁶⁵- D23 IV structure demonstrates that the engineered valine packs with the uracil base while maintaining hydrogen bonds with the protein backbone (Fig. 7).

Synthetic U2AF⁶⁵-D231V Prefers Uridine

262. The structural hypothesis that the D23 IV substitution would preferentially increase U2AF⁶⁵ affinity for U over other nucleotides was tested. Unmodified U2AF⁶⁵ or the D23 IV variant binding the NFl, HBB, or RP2 Py tracts were compared. Consistent with a universal U at the nucleotide position that was expected to bind the site bound by D23 IV, the U2AF⁶⁵ affinities for these RNAs increased by more than two-fold in the presence of the D23 IV- substitution. The net free energy gain of approximately -0.5 kcal mol^"1 by the D23 IV mutation agrees with the approximately 100 A² increase in buried hydrophobic surface area, which corresponds to approximately one methyl group (135 A²). Next, the affinities of the unmodified U2AF⁶⁵ or the D23 IV variant for either the A or G-mutations at the penultimate position in the 3 ^' region of the NFl Py tract as well as the corresponding A-mutations of the RP2 and HBB Py tracts were compared. The D23 IV strongly discriminated against the U— >G or U— »A transversions in the NFl Py tract (respectively 4- and 3-fold penalties) or U— »A in the HBB Py tract (2.5-fold penalty), in comparison with little or even slight increases in the affinity of unmodified U2AF⁶⁵ for A-substitutions at this site. The discrimination against the A-mutation in the RP2 Py tract was subtle, possibly due to the shorter length of this oligonucleotide (9mer RP2 compared with 13mer NFl and HBB) and hence potentially flexible location of the now tandem adenosines at the 3 ^' terminus. In conclusion, the D23 IV substitution selectively increases U2AF⁶⁵ affinity for U nucleotides at the penultimate position of Py tracts and discriminates against other nucleotides in the context of longer, pre-mRNA-like sequences.

Synthetic U2AF⁶⁵-D231V Corrects a Representative Splicing Defect in Human Cell Culture

263. The increased U-affinity of the U2AF⁶⁵-D23 IV variant raised the hypothesis that this affinity increase could indirectly overcome the inhibitory effects of other mutations in a Py tract. First, the U2AF⁶⁵-D23 IV affinity for the disease-causing mutant NFl and RP2 Py tracts, which naturally offer U-nucleotides at the D23 IV binding site, was tested. In comparison with unmodified U2AF , the D23 IV substitution improved U2AF -D23 IV affinity for these defective splice sites by approximately 3-fold to near wild-type levels (Table 13). Next, whether the synthetic U2AF⁶⁵-D23 IV variant could correct defective splicing in human cells using a splicing reporter minigene for the retinitis pigmentosa-causing RP2 mutation (RP2(U>A)) (Fig. 4A was tested. Both U2AF⁶⁵-D23 IV and U2AF⁶⁵-D23 IV showed comparable levels of expression in HEK293T cells (Fig. 3A). Co-transfection of the RP2(U>A) minigene with increasing amounts of an U2AF⁶⁵-D231V expression plasmid proportionally improved inclusion of exon following the defective Py tract, whereas co-transfection with unmodified U2AF⁶⁵ had little detectable effect (Fig. 3B and 3C). By including both the splicing minigene and U2AF⁶⁵ on a bicistronic vector that ensures the presence in the same cell, the U2AF⁶⁵-D23 IV variant improved exon inclusion in the mutant RP2(U>A) transcript to levels approaching those of the wild-type transcript (Fig. 4, Fig. 4C). Importantly, this result demonstrates the ability of the synthetic U2AF⁶⁵-D231V variant to compensate for disease-causing Py tract mutations in the presence of a natural U at the penultimate nucleotide of the Py tract.

264. The Py tract splice site signal is recognized by U2AF⁶⁵ (Fig. 6A), which in turn recruits core spliceosome components to a consensus "AG" dinucleotide at a proximal 3 ^' splice site. As shown herein, the penalties of two representative disease-causing Py tract mutations for U2AF⁶⁵ recognition were determined. This can be used to leverage structural information to develop a synthetic U2AF⁶⁵ variant that can relieve the consequences of disease-causing splice site mutations. It was found that neurofibromatosis or retinitis pigmentosa-causing mutations of Py tracts severely reduce U2AF⁶⁵ affinity. A preferred binding site for purine-substitutions in Py tracts in U2AF⁶⁵ RRM1 was determined. Further, high resolution structures of U2AF

recognizing A- or G- at this site of the bound Py tract were determined. Based on structural comparison with prior U- or C-containing structures, a D23 IV mutation was introduced. The crystal structure of dU2AF⁶⁵-D23 IV bound to a U-tract confirms favorable hydrophobic packing between the uracil base and the engineered valine. The D23 IV mutation specifically improves U2AF⁶⁵ affinities for three different U-containing splice sites known to be mutated in human genetic diseases (neurofibromatosis, ^-thalassemia, and retinitis pigmentosa). As shown herein, the U2AF⁶⁵-D23 IV variant improves splicing of a U-containing splice site harboring a retinitis pigmentosa-causing point mutation. This result sets a successful precedent for structure-based tailoring of synthetic U2AF⁶⁵ to match potentially therapeutic splice sites.

265. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made.

Accordingly, other embodiments are within the scope of the following claims.

266. A represenative high-throughput selection strategy (shown in Fig. 8) focuses on derivatives of the fluorescent splicing reporter. In this system, the skipped exon and flanking introns and exons are inserted in-frame between the far-red (fRFP) and yellow fluorescence protein (YFP) coding sequences (Fig. 8A). The central exon length is modified to a non-multiple of three by deletion or insertion of one central nucleotide, such that skipping of the exon leads to a frameshift encoding truncated fRFP without YFP. On the other hand, exon inclusion produces the fRFP-peptide-YFP fusion. Control constructs including only the exon regions (with and without the central target exon), as well as the fRFP and YFP proteins alone, serve as baselines for validation and gating of fluorescence-activated cell sorting results (FACS). To reduce spectral overlap with a blue fluorescent reporter (mBFP) for U2AF65 transfection, we converted the original eGFP coding region of Dr. Lukyanov's vector to YFP. Codons are selected that avoid cryptic splice site signals and confirm that splicing of the original minigene remains unchanged. Minigenes that recapitulate disease-relevant splicing defects have been reported, including SMN2, GAA and DEK. Several additional representative disease-relevant splicing defects that are appropriate for this invention are listed in the tables disclosed herein.

267. The starting library of U2AF⁶⁵ variants is guided by a semi-empirical approach. For each amino acid, the feasibility of improved interactions with the target nucleotide has been checked in silico based on the key interactions of the U2AF⁶⁵/Py tract structures (Fig. 6). The starting library is summarized in Table 1. Each nucleotide binding site of U2AF65 is optimized sequentially, starting with the established D23 IV variant that is expected to target the penultimate U's of the SMN2 and DEK Py tracts. The U2AF⁶⁵ coding region is be carried on the mBFP reporter plasmid (Evogen) to monitor transfection efficiency. Following site-directed mutagenesis, the U2AF⁶⁵ variants is transiently transfected into the stable reporter cell lines (Fig. 8A). Since a practical number of initial variants is tested for each transcript (minimally 25, 22, and 39 variants for SMN2, GAA, and DEK, respectively, see Table 5), each tissue culture well is transfected with a unique U2AF⁶⁵ variant (in replicates). Fluorescence of transfected cells first is screened using in- house inverted microscope with fluorescence attachment (Nikon Diaphot-TMD). Statistics of intensity distributions are obtained using a digital flow cytometer in the URMC Flow Core under direction of Dr. Tim Bushnell. Samples are gated stringently for red and blue intensity cut-offs (respectively for expression of the minigene in the stable cell line and transient co-transfection of the U2AF⁶⁵ variant). The effectiveness of each U2AF⁶⁵ variant in promoting exon inclusion is assessed from 2D plots of the yellow:red vs. blue fluorescence emmission intensities (Fig. 8B). Successful U2AF variants are selected based on increased Y/R ratios at lower B values relative to wild-type U2AF⁶⁵ and empty vector controls, and differences are confirmed by U2AF⁶⁵ immunoblots and RT-PCR of splice variants.

268. Successful RT-PCR reporters for SMN2 and GAA splicing defects have been documented. Similar outcomes are expected for these minigenes in the context of the fluorescent reporter. Several fluorescent splicing reporters have been described in the literature and are available in the unlikely event that an alternative reporter would assist selection. If needed to distinguish the signal of splicing changes due to U2AF⁶⁵-variants relative to the wild-type U2AF⁶⁵-construct, endogenous U2AF⁶⁵ is depleted in the reporter cell line by stable expression of shRNA sequences as described by several groups, and then transiently-transfect U2AF⁶⁵ variants in the context of silent mutations for siRNA-resistance. For higher throughput, a state-of-the-art fluorescence imaging cytometer (CeligoR S, Nexcelom) is available.

E. Sequences

SEQ ID NO : 1 : Amino Acid Sequence of U2AF -,65

MSDFDEFERQLNENKQERDKENRHRKRSHSRSRSRDRKRRSRSRDRRNRDQRSASRDRRRRSKP LTRGAKEEHGGLIRSPRHEKKKKVRKYWDVPPPGFEHITPMQYKAMQAAGQIPATALLPTMTPD GLAVTPTPVPWGSQMTRQARRLYVGNIPFGITEEAMMDFFNAQMRLGGLTQAPGNPVLAVQIN QDKNFAFLEFRSVDETTQAMAFDGIIFQGQSLKIRRPHDYQPLPGMSENPSVYVPGVVSTVVPD SAHKLFIGGLPNYLNDDQVKELLTSFGPLKAFNLVKDSATGLSKGYAFCEYVDINVTDQAIAGL NGMQLGDKKLLVQRASVGAKNATLSTINQTPVTLQVPGLMSSQVQMGGHPTEVLCLMNMVLPEE LLDDEEYEEIVEDVRDECSKYGLVKSIEIPRPVDGVEVPGCGKIFVEFTSVFDCQKAMQGLTGR KFANRWVTKYCDPDSYHRRDFW SEQ ID NO: 2: Nucleic Aci [ Sequence of U2AF⁶⁵

ctggagcggg cggcaaggcg aggcgaaagc tgcacagggc cctacgcggc cgcctcagca tgtcggactt cgacgagttc gagcggcagc tcaacgagaa taaacaagag cgggacaagg agaaccggca tcggaagcgc agccacagcc gctctcggag ccgggaccgc aaacgccgga gccggagccg cgaccggcgc aaccgggacc agcggagcgc ctcccgggac aggcgacgac gcagcaaacc tttgaccaga ggcgctaaag aggagcacgg tggactgatt cgttcccccc gccacgagaa gaagaagaag gtccgtaaat actgggacgt gccaccccca ggctttgagc acatcacccc aatgcagtac aaggccatgc aagctgcggg tcagattcca gccactgctc ttctccccac catgacccct gacggtctgg ctgtgacccc aacgccggtg cccgtggtcg ggagccagat gaccagacaa gcccggcgcc tctacgtggg caacatcccc tttggcatca ctgaggaggc catgatggat ttcttcaacg cccagatgcg cctggggggg ctgacccagg cccctggcaa cccagtgttg gctgtgcaga ttaaccagga caagaatttt gcctttttgg agttccgctc agtggacgag actacccagg ctatggcctt tgatggcatc atcttccagg gccagtcact aaagatccgc aggcctcacg actaccagcc gcttcctggc atgtcagaga acccctccgt ctatgtgcct ggggttgtgt ccactgtggt ccccgactct gcccacaagc tgttcatcgg gggcttaccc aactacctga acgatgacca ggtcaaagag ctgctgacat cctttgggcc cctcaaggcc ttcaacctgg tcaaggacag tgccacgggg ctctccaagg gctacgcctt ctgtgagtac gtggacatca acgtcacgga tcaggccatt gcggggctga acggcatgca gctgggggat aagaagctgc tggtccagag ggcgagtgtg ggagccaaga atgccacgct gagcaccatc aatcagacgc ctgtgaccct gcaagtgccg ggcttgatga gctcccaggt gcagatgggc ggccacccga ctgaggtcct gtgcctcatg aacatggtgc tgcctgagga gctgctggac gacgaggagt atgaggagat cgtggaggat gtgcgggacg agtgcagcaa gtacgggctt gtcaagtcca tcgagatccc ccggcctgtg gacggcgtcg aggtgcccgg ctgcggaaag atctttgtgg agttcacctc tgtgtttgac tgccagaaag ccatgcaggg cctgacgggc cgcaagttcg ccaacagagt ggttgtcaca aaatactgtg accccgactc ttatcaccgc cgggacttct ggtagaggcg gctgggggag ggtgggggca gggctggctg ggggcttctc cccactcccg cccccccctt atccccctct gaagacgatg ggcagaggag tgacagccgc agacacacga cagccggcag caactggaat ggcagcaatt aagggtgggg ggcgggggtt ggggggttgg ggggttaggg cagggagggg actggggaag tgcgcacaca gcccacacag acaacacgca cccacacaga cacagaggga aggggttggg atggggacag ggtgcacagc agggcggggt aggaccccag cccctcccaa aacagcctct ccttctccca tagacccctt tcttctcccc ttccccacgg taggaacata gcgtgtttat attttatggc caaactattt tgaattttgt tgtccggccc tcagtgccct gccctctccc ttaccaggac cacagctctg ttccttcggc ctctggtcct ctctggtccc ctcctgggtt tcttacgtag ttgatttttc ctctttagtc tcccccgacc tgcgcccagc cccgtggccc ctgcccctct cctactctct gtggcagttt catatttgct aagacgaatt tgctcattaa acattttgtt gtattttact ttaaaaaaaa aaaaaaaaaa

SEQ ID NO: 3

MSDFDEFERQLNENKQERDKENRHRKRSHSRSRSRDRKRRSRSRDRRNRDQRSASRDRRRRSKP LTRGAKEEHGGLIRSPRHEKKKKVRKYWDVPPPGFEHI TPMQYKAMQAAGQI PATALLPTMTPD GLAVTPTPVPWGSQMTRQARRLYVGNI PFGITEEAMMDFFNAQMRLGGLTQAPGNPVLAVQIN QDKNFAFLEFRSVDETTQAMAFDGI I FQGQSLKIRRPHDYQPLPGMSENPSVYVPGVVSTVVPD SAHKLFIGGLPNYLNDDQVKELLTSFGPLKAFNLVKDSATGLSKGYAFCEYVDINVTDQAIAGL NGMQLGDKKLLVQRASVGAKNATLVSPPSTINQTPVTLQVPGLMSSQVQMGGHPTEVLCLMNMV LPEELLDDEEYEEIVEDVRDECSKYGLVKS IEI PRPVDGVEVPGCGKI FVEFTSVFDCQKAMQG LTGRKFANRVWTKYCDPDSYHRRDFW

SEQ ID NO: 4

ATGTCGGACTTCGACGAGTTCGAGCGGCAGCTCAACGAGAATAAACAAGAGCGGGACAAGGAGA ACCGGCATCGGAAGCGCAGCCACAGCCGCTCTCGGAGCCGGGACCGCAAACGCCGGAGCCGGAG CCGCGACCGGCGCAACCGGGACCAGCGGAGCGCCTCCCGGGACAGGCGACGACGCAGCAAACCT TTGACCAGAGGCGCTAAAGAGGAGCACGGTGGACTGATTCGTTCCCCCCGCCACGAGAAGAAGA AGAAGGTCCGTAAATACTGGGACGTGCCACCCCCAGGCTTTGAGCACATCACCCCAATGCAGTA CAAGGCCATGCAAGCTGCGGGTCAGATTCCAGCCACTGCTCTTCTCCCCACCATGACCCCTGAC GGTCTGGCTGTGACCCCAACGCCGGTGCCCGTGGTCGGGAGCCAGATGACCAGACAAGCCCGGC GCCTCTACGTGGGCAACATCCCCTTTGGCATCACTGAGGAGGCCATGATGGATTTCTTCAACGC CCAGATGCGCCTGGGGGGGCTGACCCAGGCCCCTGGCAACCCAGTGTTGGCTGTGCAGATTAAC CAGGACAAGAATTTTGCCTTTTTGGAGTTCCGCTCAGTGGACGAGACTACCCAGGCTATGGCCT TTGATGGCATCATCTTCCAGGGCCAGTCACTAAAGATCCGCAGGCCTCACGACTACCAGCCGCT TCCTGGCATGTCAGAGAACCCCTCCGTCTATGTGCCTGGGGTTGTGTCCACTGTGGTCCCCGAC TCTGCCCACAAGCTGTTCATCGGGGGCTTACCCAACTACCTGAACGATGACCAGGTCAAAGAGC TGCTGACATCCTTTGGGCCCCTCAAGGCCTTCAACCTGGTCAAGGACAGTGCCACGGGGCTCTC CAAGGGCTACGCCTTCTGTGAGTACGTGGACATCAACGTCACGGATCAGGCCATTGCGGGGCTG AACGGCATGCAGCTGGGGGATAAGAAGCTGCTGGTCCAGAGGGCGAGTGTGGGAGCCAAGAATG CCACGCTGGTGAGCCCCCCGAGCACCATCAATCAGACGCCTGTGACCCTGCAAGTGCCGGGCTT GATGAGCTCCCAGGTGCAGATGGGCGGCCACCCGACTGAGGTCCTGTGCCTCATGAACATGGTG CTGCCTGAGGAGCTGCTGGACGACGAGGAGTATGAGGAGATCGTGGAGGATGTGCGGGACGAGT GCAGCAAGTACGGGCTTGTCAAGTCCATCGAGATCCCCCGGCCTGTGGACGGCGTCGAGGTGCC CGGCTGCGGAAAGATCTTTGTGGAGTTCACCTCTGTGTTTGACTGCCAGAAAGCCATGCAGGGC CTGACGGGCCGCAAGTTCGCCAACAGAGTGGTTGTCACAAAATACTGTGACCCCGACTCTTATC ACCGCCGGGACTTCTGGTAG

Claims

V. CLAIMS What is claimed is:

1. A U2AF splice factor variant comprising one or more amino acid substitution at a contact residue of SEQ ID NO: 1 or a corresponding residue of U2AF⁶⁵ for a pre-mRNA Py tract splice site, wherein the variant increases splicing for a target Py tract splice site.

2. The U2AF⁶⁵ variant of claim 1, wherein the substituted amino acid of U2AF⁶⁵ is at

residue 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, or 339 of SEQ ID NO: 1 or at a residue corresponding to residue 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, or 339 of SEQ ID NO: 1.

3. The U2AF⁶⁵ variant of claim 2, wherein the variant comprises two or more substitutions.

4. The U2AF⁶⁵ variant of claim 3, wherein the variant comprises substitutions at residues 289 and 254, 256 and 260, 215 and 252, 253 and 287, or 272 and 292 of SEQ ID NO: 1 or at a residue corresponding to residue 289 and 254, 256 and 260, 215 and 252, 253 and 287, or 272 and 292 of SEQ ID NO: 1.

5. A composition comprising any one or more of the U2AF⁶⁵ variants of any of claims 1-4.

6. A polypeptide comprising the U2AF⁶⁵ variant of any of claims 1 -4.

7. A nucleic acid encoding the polypeptide of claim 6.

8. A method of decreasing defective splicing of an mRNA comprising contacting the

mRNA with the U2AF⁶⁵ variant of any of claims 1-6.

9. A method of treating a disorder associated with an aberrantly spliced mRNA in a subject, comprising administering to the subject the U2AF⁶⁵ variant of any of claims 1-6.

10. The method of claim 9, wherein the U2AF⁶⁵ variant is administered as a polynucleotide sequence encoding the U2AF⁶⁵ variant.

1 1. The method of claim 9, wherein the disorder is retinitis pigmentosa, β-thalassemia, or neurofribormatosis .

12. The method of claim 9, wherein the U2AF⁶⁵ variant comprises a substitution of at

residue 231 of SEQ ID NO: 1 or at corresponding residue of U2AF⁶⁵.

13. A method of treating a disorder associated with an aberrantly spliced mRNA in a subject, comprising administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF⁶⁵ variant comprises a substitution of at a contact residue for a pre-mRNA Py tract splice site.

14. The method of claim 13, wherein the aberrant splicing of mRNA is due to a substitution in the pre-mRNA Py tract causing defective binding of U2AF⁶⁵.

15. The method of claim 14, wherein the disorder is retinitis pigmentosa, β-thalassemia, or neurofribormatosis .

16. The method of claim 14, wherein the substitution in the Py tract is at position 1, 2, 3, 4,

5, 6, 7, 8, or 9 of the Py tract.

17. The method of claim 13, wherein the U2AF⁶⁵ splicing factor variant comprises a

substitution at residue wherein the substituted amino acid of U2AF⁶⁵ is at residue 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, or 339 of SEQ

ID NO: 1 or at a residue corresponding to residue 145, 147, 150, 155, 196, 197, 199, 215, 227. 229, 230, 231, 252, 253, 254, 256, 260, 262, 268, 272, 287, 289, 292, 293, 296, 298, 300, 328, 329, 330, 331, 333, 335, 336, or 339 of SEQ ID NO: 1.

18. A method of decreasing defective splicing of an mRNA in a subject, comprising

administering to the subject a therapeutically effective amount of a polynucleotide encoding a U2AF⁶⁵ splicing factor variant; wherein the U2AF⁶⁵ variant comprises a substitution of at a contact residue for a pre-mRNA Py tract splice site.

19. A method of engineering a splicing factor variant comprising mutating one of the nine nucleotide residues of a pre-mRNA splice site polypyrimidine (Py) tract of a gene to generate dU, dC, dG, and dA Py tract mutants; co-cystalizing the mutated Py tract mutants comprising a deoxy-ribose oligonucleotide backbone with a U2AF⁶⁵ splice factor variant wherein the U2AF⁶⁵ variant is a deletion mutant of the amino acids corresponding to residues 238-257 of SEQ ID NO: 1 ; comparing the binding affinity of the U2AF⁶⁵ variant to the Py tract mutants; identifying contact residues of U2AF⁶⁵ for a Py tract mutant; selecting amino acid substitutions at contact residues of U2AF⁶⁵ that increase the binding affinity to a mutated residue of the Py tract, and substituting the native amino acid for an identified amino acid.

20. A method of engineering a splicing factor variant comprising co-cystalizing Py tract variants with a U2AF⁶⁵ splice factor variant of SEQ ID NO: 1 ; comparing the binding affinity of the U2AF⁶⁵ variant to the Py tract variants; identifying contact residues of

U2AF⁶⁵ for a Py tract variant; selecting amino acid substitutions at contact residues of U2AF⁶⁵ that increase splicing at the target splice site, and substituting the native amino acid for an identified amino acid