RNA-TARGETING SPLICING MODIFIERS FOR TREATMENT OF FOXP3- ASSOCIATED CONDITIONS AND DISEASES

CROSS-REFERENCE

[01] The present application claims priority to and benefit from U.S. Provisional Application No.: 63/348,281, filed on June 2, 2022, the entire contents of which is herein incorporated by reference.

SEQUENCE LISTING

[02] This application incorporates by reference a Sequence Listing XML submitted via the USPTO patent electronic filing system. The Sequence Listing XML, entitled 202883-70560 IPCT.xml, was created on June 1, 2023, and is 51,013,479 bytes in size.

SUMMARY

[03] Disclosed herein is a method of treating a Forkhead Box P3 (FOXP3) associated disease or condition in a human subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a synthetic FOXP3 RNA-targeting splicing modifier (RTSM), thereby treating the disease or condition in the human subject in need thereof; wherein: the synthetic RTSM comprises a binding domain that binds or hybridizes to a target region of a FOXP3 premessenger ribonucleic acid (pre-mRNA); the target region comprises an exon-intron junction comprising a target sequence of Formula (I): DRGUV, wherein D is A, G, or U; wherein R is A or G; wherein V is A, G, or C; and exon skipping is increased as compared to the FOXP3 pre-mRNA spliced in the absence of RTSM as demonstrated by an in vitro assay.

[04] Disclosed herein is a synthetic FOXP3 RTSM that comprises a binding domain that binds or hybridizes to a target region of a FOXP3 pre-mRNA; wherein: the target region comprises an exonintronjunction comprising atarget sequence of Formula (I): DRGUV, wherein D is A, G, or U; wherein R is A or G; wherein V is A, G, or C; and exon skipping is increased as compared to when the FOXP3 pre-mRNA is spliced in the absence of the synthetic RTSM as demonstrated in an in vitro assay.

INCORPORATION BY REFERENCE

[05] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[06] FIG. 1 shows results from RT-PCR analysis of FOXP3 isoforms present in HCC712 cells following treatment with Antisense Morpholino Oligonucleotides (AMOs). Figure discloses SEQ ID NOS: 136-137, respectively, in order of appearance.

[07] FIG. 2 shows results from RT-PCR analysis of FOXP3 isoforms present in MJ (Gi l) cells following treatment with AMOs. Figure discloses SEQ ID NOS: 138-139, respectively, in order of appearance. [08] FIG. 3A and 3B show sequences of FOXP3 isoforms present in HCC712 cells following treatment with AMOs. The sequences were generated from the forward primer (SEQ ID NO: 134).

[09] FIG. 4A and 4B show sequences of FOXP3 isoforms present in MJ (Gi l) cells following treatment with AMOs. The sequences were generated from the reverse primer (SEQ ID NO: 135).

[010] FIG. 5 shows results from Western Blot analysis of FOXP3 protein isoforms present in MJ (G11) cells following treatment with AMOs.

[Oil] FIG. 6 shows quantitation of FOXP3 protein isoforms present in MJ (Gi l) cells following treatment with AMOs.

[012] FIG. 7 shows the activity of FOXP3 protein isoforms present in MJ (Gi l) cells following treatment with AMOs.

[013] FIG. 8 shows results from RT-PCR analysis of FOXP3 isoforms present in MJ (Gi l) cells following treatment with AMOs.

[014] FIG. 9A and FIG. 9B shows sequences of FOXP3 isoforms present in MJ (Gi l) cells following treatment with AMOs. The sequences were generated from the primers set forth in SEQ ID NOS: 140- 141. Figures disclose SEQ ID NOS: 160-161, respectively, in order of appearance.

[015] FIG. 10 shows quantitation of FOXP3 protein isoforms present in MJ (Gi l) cells following treatment with AMOs.

[016] FIG. 11 shows the activity of FOXP3 protein isoforms present in Jurkat cells following treatment with AMOs.

[017] FIG. 12 shows results from RT-PCR analysis of FOXP3 isoforms present in Jurkat cells following treatment with AMOs.

[018] FIG. 13A and FIG. 13B shows sequences of FOXP3 isoforms present in Jurkat cells following treatment with AMOs. The sequences were generated from the primers set forth in SEQ ID NOS: 140- 141. Figures disclose SEQ ID NOS: 162-163, respectively, in order of appearance.

[019] FIG. 14 shows quantitation of FOXP3 protein isoforms present in Jurkat cells following treatment with AMOs.

DETAILED DESCRIPTION

[020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which embodiments herein belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of embodiments herein. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

Definitions

[021] As used herein, the terms "ASO,” “antisense oligonucleotides” and "antisense oligomer" are used interchangeably and can refer to an oligomer, such as a polynucleotide, comprising nucleobases that hybridizes to atarget nucleic acid (e.g., a FOXP3 containing pre-mRNA) sequence by, for example, Watson-Crick base pairing or wobble base pairing (G-U).

[022] The term “binding domain” as used herein can comprise a domain or portion of an RTSM which binds or hybridizes to a region or a portion of a FOXP3 pre-mRNA. The binding/hybridizing can be covalent or non-covalent. Examples of non-covalent binding/hybridizing include binding/hybridizing via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van der Waals interactions.

[023] The terms “complementary” and “complementarity” can refer to polynucleotides (e.g., a sequence of nucleotides) related by base-pairing rules. For example, the sequence “T-G-A (5’-3’),” can be complementary to the sequence “T-C-A (5 ’-3 ’).” Complementarity may be “partial,” in which only some of the nucleic acid’s bases are matched according to base pairing rules. Alternatively, there may be “complete” or “total” complementarity between the nucleic acids. Furthermore, base pairing may be contiguous or non-contiguous. The degree of complementarity between nucleic acid strands can impact efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity can be desired, some embodiments can include one or more 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mismatches with respect to a target RNA. A mismatch can be a mismatch between a base on an RTSM and a base on a target RNA. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer can be within about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the 5’ and/or 3’ terminus. In some embodiments, a base pairing can be a wobble base pairing.

[024] A “CRISPR” (Clustered Regularly Interspersed Short Palindromic Repeat) “CRISPR system,” or “CRISPR nuclease system” and their grammatical equivalents can include a non-coding RNA molecule (e.g., guide RNA) that binds to DNA or RNA and CRISPR-Associated (Cas) proteins (e.g., Cas9) with, at least some or none, nuclease functionality (e.g., two nuclease domains).

[025] The term “exon skipping” can refer to a process by which a portion of an exon, an entire exon, or more than one exon are removed from a pre-processed mRNA so that it or they are not present in a mature RNA, such as an mRNA that is translated into a polypeptide or protein (wherein polypeptide and protein are used interchangeably herein). Accordingly, the portion of the protein that can be otherwise encoded by the skipped exon is not present in the translated form of the protein, and can create a modulated form of the protein. In some embodiments, a modulated protein may be functional, less functional or non-functional. In some embodiments, a modulated protein may be truncated or subjected to nonsense mediated decay. In certain embodiments, a skipped exon can be an aberrant exon from the human FOXP3 gene which may contain a mutation or other alteration in its sequence that otherwise causes mutated forms of the protein. In certain embodiments, an exon being skipped can be a wild-type exon. In certain embodiments, a skipped exon can be a mutated or partially mutated exon. In certain embodiments, a skipped exon can be a non-mutated exon.

[026] A FOXP3 pre-mRNA transcript can comprise 12 transcribed exons which are referred to herein as exon 1 or El, exon 2 or E2, exon 3 or E3, exon 4 or E4, exon 5 or E5, exon 6 or E6, exon 7 or E7, exon 8 or E8, exon 9 or E9, exon 10 or E10, exon 11 or El 1, and exon 12 or E12. In some embodiments, exons 2-11 are coding exons. An exon being skipped can be a target exon. A target exon can be a coding or non-coding exon. An exon being skipped can be a coding or non-coding exon. An exon being skipped can be any one of, or partial fragment thereof, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11 or any combination thereof. As used herein, the symbol “A” followed by an integer indicates the exon being skipped. For example, “FOXP3 A3” indicates that exon 3 is skipped. In another example, “FOXP3A3A8” indicates that exon 3 and exon 8 is skipped. Exon skipping can be seen in either the resulting processed mRNA, translated protein thereof, or any combination thereof. On the other hand, the abbreviation “fl” following FOXP3 indicates that the mRNA or protein is full length, e.g., “FOXP3fl” indicates full length FOXP3.

[027] The terms “identical” and its grammatical equivalents as used herein or “sequence identity” in the context of two nucleobase, nucleic acid sequences or amino acid sequences of polypeptides can refer to the residues in the two sequences which can be the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, can refer to a segment of at least about: 4, 8, 50, 100, 150, to 200 or more contiguous positions can be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Sequence comparison can be conducted by the local homology algorithm or by computerized implementations of these algorithms including, but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignment can be also performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting position in a nucleic acid sequence, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or 100% identical to a reference nucleic acid sequence or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule can be said to have certain percentage of sequence identity with a larger molecule, can mean that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned. The term “substantially identical” and its grammatical equivalents as applied to nucleic acid or amino acid sequences can mean that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98% and at least 99%, compared to a reference sequence using the programs described above, e.g., BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) can use as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. In another example, for amino acid sequences, the BLASTP program can use as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. Percentage of sequence identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (e.g., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. In some embodiments, the substantial identity exists over a region of the sequences that can be at least about 8, at least about 14 residues in length, over a region of at least about 20 residues, and in some embodiments, the sequences can be substantially identical over at least about 25 residues. In some embodiments, the sequences can be substantially identical over the entire length of the coding regions.

[028] The term “nucleobase” can generally refer to nitrogen containing compound that is capable of hydrogen bonding with another nucleobase, for example, as present on a target pre-mRNA. A nucleobase may be any unmodified nucleobase such as adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5, 6- dihydrouracil, 5 -methylcytosine, and 5-hydroxymethoylcytosine.

[029] The term “FOXP3 associated disorder” as used herein refers to a disease or condition that can be associated with an activity, a wild-type activity, a reduced activity, an increased activity, an altered activity, and in some cases, mutant activity, of a FOXP3 protein. [030] As used herein, the term “targeting domain” can comprise a region or portion of an FOXP3 pre-mRNA to which a RTSM can bind or hybridize. The binding/hybridizing can be covalent or non- covalent. Examples of non-covalent binding/hybridizing include binding/hybridizing via hydrogen bonding, Watson-Crick base pairing, wobble-base pairing, and Van der Waals interactions.

[031] As used herein, the terms “Treg” or “regulatory T cell” as used interchangeably herein can refer to specialized subpopulations of T cells that has immunosuppressive activity, for example, Tregs that can maintain resistance to self-antigens by inhibiting the activation of the immune system.

Forkhead Box P3 (FOXP3)

[032] Disclosed herein are RTSMs that can modulate FOXP3 protein production in, for example, a cell, organ or subject, such as a human cell, human organ, or human subject. A FOXP3 protein can be identified by homology search and the sequences of which can be found in genomic and nucleotide databases, such as ENSEMBL, GenBank, or UniProtKB/Swiss-Prot. In some embodiments, a FOXP3 protein can comprise a polypeptide sequence of any of the sequences of SEQ ID NO: 12-16. In some embodiments, a FOXP3 protein not modulated by an RTSM can be a reference protein. In some embodiments, a non-modulated FOXP3 protein can be a full length, an isoform, truncated, functional, wild-type protein or any combination thereof. A full length FOXP3 protein can be referred to herein as FOXP3fl. A truncated FOXP3 protein can be referred to as a FOXP3 A protein.

[033] It was identified that the FOXP3 gene can encode a FOXP3 protein. In one embodiment, a targeted FOXP3 gene comprises the sequence as set forth in RefSeq No. NC_000022.l l (disclosed herein as SEQ ID NO: 1 and incorporated by reference). In some embodiments, a FOXP3 gene comprises one or more gene mutations. In some embodiments, the one or more FOXP3 gene mutations are associated with: gain-of-function of a protein encoded by the gene; reduced or loss-of-function of a protein encoded by the gene; an associated disease or a condition thereof; or more than one of the foregoing.

[034] In some embodiments, the RTSMs disclosed herein can target a FOXP3 pre-mRNA. A FOXP3 pre-mRNA can be the precursor FOXP3 RNA transcribed from the FOXP3 gene, but prior to being spliced into a mature RNA. In some embodiments, a targeted pre-mRNA comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleobases of any one of the sequences as set forth SEQ ID NOS: 2-6 in TABLE 1A. In some embodiments, the pre-mRNA comprises a nucleobase sequence as described herein (e.g., TABLE 1A). Such nucleobase sequences described herein (e.g., TABLE 1A) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a pre-mRNA itself or the sequence that encodes a pre- mRNA, such as a genomic nucleobase strand encoding the pre-mRNA, for example, if a sequence is DNA converting all ‘T’s to ‘U’s, or if a sequence is RNA converting all ‘U’s to ‘T’s. Similarly, disclosure of the nucleobase sequences described herein (e.g., TABLE 1A) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in a pre-mRNA as described herein. In some embodiments, a pre-mRNA nucleobase can be a nucleotide.

[035] In some embodiments, an RTSM disclosed herein can modulate a FOXP3 mRNA transcript produced, for example, a cell, organ or subject. In some embodiments, a FOXP3 mRNA comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleobases of any one of the sequences as set forth in TABLE IB. In some embodiments, the mRNA comprises a nucleobase sequence as described herein (e.g., TABLE IB). Such nucleobase sequences described herein (e.g., TABLE IB) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a mRNA itself, or, the sequence that encodes a mRNA, such as a genomic nucleobase strand encoding the mRNA, for example, if a sequence is DNA converting all ‘T’s to ‘U’s, or if a sequence is RNA converting all ‘U’s to ‘T’s. Similarly, disclosure of the nucleobase sequences described herein (e.g., TABLE IB) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in a mRNA as described herein. In some embodiments, a non-modulated FOXP3 mRNA transcript referred to herein can be a reference mRNA and can comprise a sequence as set forth in any of the sequences in SEQ ID NOS: 7-11 in TABLE IB.

[036] In some embodiments, an RTSM disclosed herein can target a target region of a FOXP3 pre- mRNA. In some embodiments, a target region can comprise part of or all of can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM- RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof.

[037] In some embodiments, upon binding to a target region, RTSMs disclosed herein can increase at least partial exon skipping during splicing of a FOXP3 pre-mRNA. Accordingly, in some embodiments a targeted pre-mRNA comprises one or more exons and one or more introns. A FOXP3 pre-mRNA can comprise 12 exons and 11 introns. Accordingly, in some embodiments a targeted pre- mRNA comprises exons 1-12 and introns 1-11. A FOXP3 pre-mRNA can comprise 10 coding exons, wherein exon 2-11 are coding exons. Accordingly, in some embodiments a targeted pre-mRNA comprises at least exons 2-11 and introns 2-11.

[038] In some embodiments, any one of exons 2-11 can comprise, in part or in full: coding for protein length; coding for protein stability; coding for protein function; binding site coding; coding specific for RTSM-RNA binding; RTSM-RNA duplex formation; coding which limits off-target binding; coding which recruits one or more splicing complex component; or any combination thereof. For example, exon 2 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 3 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 4 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 5 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM- RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 6 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 7 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 8 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM- RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 9 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 10 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM-RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; exon 11 can comprise coding for: protein length, protein stability, protein function, binding site, specific RTSM- RNA binding, RTSM-RNA duplex formation, limiting off-target binding, recruiting one or more splicing complex component, or any combination thereof; or any combination thereof.

[039] In some embodiments, a targeted exon referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleobases of any one ofthe sequences of SEQ ID NOS: 17-26 as set forth in TABLE

2. In some embodiments, a targeted intron referred to herein comprises a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least about: 4, 5, 6, 7, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300 or more continuous nucleobases of any one ofthe sequences of SEQ ID NOS: 27-36 as set forth in TABLE

3.

[040] In some embodiments, pre-mRNA exon(s), intron(s), or both, may comprise one or more nucleobase alterations at one or more positions in any one of the sequences in TABLE 2 or TABLE 3. Alternative nucleobases can be any one or more of A, C, G or U, a deletion, or an insertion.

[041] Exon and intron nucleobase sequences described herein (e.g., TABLE 2 and TABLE 3) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, as needed, for describing a sequence within an exon or intron itself, or, the sequence that encodes a portion of the exon or intron, such as a genomic nucleobase strand encoding an exon or intron sequence, for example, if a sequence is DNA converting all ‘T’s to ‘U’s, or if a sequence is RNA converting all ‘U’s to ‘T’s. Similarly, disclosure of the exonic and intronic nucleobase sequences described herein (e.g., TABLE 2 and TABLE 3) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in an exon or intron as described herein.

[042] When an intron is contiguous with the 3’ end of an exon, the exon and intron can correspond for splicing purposes and create an exon-intron junction. In some embodiments, a targeted pre-mRNA comprises a target region wherein a target region comprises an exon-intron junction. An exon-intron junction can be at least 2 nucleobases in length, at least 3 nucleobases in length, at least 4 nucleobases in length, at least 5 nucleobases in length, at least 6 nucleobases in length, at least 7 nucleobases in length, at least 8 nucleobases in length, at least 9 nucleobases in length, at least 10 nucleobases in length, at least 11 nucleobases in length, at least 12 nucleobases in length, at least 13 nucleobases in length, at least 14 nucleobases or more in length. For example, exon-intron junctions can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.

[043] In some embodiments, a targeted pre-mRNA comprises a targeted region wherein a targeted region comprises one or more targeted exon-intron junction(s). In some embodiments, a target region comprises a targeted exon-intron junction. In some embodiments, a target region comprises more than one targeted exon-intron junction. In some embodiments, an exon-intron junction can be Exon 2-Intron 2, Exon 3-Intron 3, Exon 4-Intron 4, Exon 5-Intron 5, Exon 6-Intron 6, Exon 7-Intron 7, Exon 8-Intron 8, Exon 9-Intron 9, Exon 10-Intron 10, Exon 11-Intron 11, more than one, or any combination thereof.

TABLE 1A: Exemplary FOXP3 pre-mRNA Transcripts

TABLE IB: Exemplary FOXP3 mRNA Transcripts

TABLE 1C: Exemplary FOXP3 Polypeptides

TABLE 2: Exemplary Exons

TABLE 3: Exemplary Introns

[044] In some embodiments, a targeted region comprises a targeted exon or a portion thereof, a targeted intron or a portion thereof, both, more than one of either, or more than one of both, that is targeted by a binding domain of an RTSM herein. A binding domain of an RTSM in some instances can be from about 2 to about 50 nucleobases in length and can bind or hybridize with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleobases of an intron, any number in between, or any combination thereof. Target region can comprise about 1 to about 25 nucleobases of an exon, 1 to about 25 nucleobases of an intron, or any combination thereof. In some embodiments, a targeted region comprises a sequence that is at least about 2 to about 50 nucleobases long. In some embodiments, a targeted region comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.

[045] In some embodiments, a targeted exon can be any one of, or a portion of any one of Exon 2, Exon

3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, Exon 9, Exon 10, or Exon 11. A targeted exon can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 17-26.

[046] In some embodiments, a targeted intron can be any one of, or a portion of any one of Intron 2, Intron 3, Intron 4, Intron 5, Intron 6, Intron 7, Intron 8, Intron 9, Intron 10, or Intron 11. A targeted intron can comprise a sequence that shares at least about: 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 27-36.

[047] In some embodiments, a targeted exon-intron junction can comprise a sequence that shares at least about: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more sequence identity to: SEQ ID NOS: 17 and 27; SEQ ID NOS: 18 and 28; SEQ ID NOS: 19 and 29; SEQ ID NOS: 20 and 30; SEQ ID NOS: 21 and 31; SEQ ID NOS: 22 and 32; SEQ ID NOS: 23 and 33; SEQ ID NOS: 24 and 34; SEQ ID NOS: 25 and 35; or SEQ ID NOS: 26 and 36.

[048] In some embodiments, a targeted pre-mRNA comprises a targeted sequence. In some embodiments, a target region comprises a targeted sequence. In some embodiments, an exon-intron junction comprises a targeted sequence. In some embodiments, an RTSM targets and binds or hybridizes to a targeted sequence of a FOXP3 pre-mRNA. The term binding can include hybridizing.

[049] In some embodiments, a target sequence comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a target sequence comprises a sequence that can be about: 2,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.

[050] In some embodiments, a targeted sequence comprises a sequence as set forth in Formula (I) below:

Formula (I): Exemplary Targeted Sequence

5’ ... DRGUV ... 3’ wherein D is A, G, or U; wherein R is A or G; wherein V is A, G, or C and wherein 5 ’ . . .3 ’ indicates from the 5 ’ 3 ’ direction.

[051] In some embodiments, a targeted sequence comprises a splice site. In some embodiments, an RTSM specifically binds to a splice site. In some embodiments, an RTSM specifically hybridizes to a splice site. In some embodiments, a splice site comprises a sequence as set forth in Formula (II) below.

Formula (II): Exemplary Splice Site

5’ ... DR|GUV ... 3’ wherein D is A, G, or U; wherein R is A or G; wherein V is A, G, or C; wherein . | ... ” indicates the splice site; and wherein 5 ’ . . .3 ’ indicates from the 5 ’ 3 ’ direction.

[052] In some embodiments, the sequence set forth in Formula (I), Formula (II), or both wherein: D is A, R is G, and V is G; D is A, R is G and V is A; D is A, R is G and V is C; D is A, R is A and V is G; D is U, R is G and V is A; D is U, R is G and V is G; or D is G, R is G and V is A.

[053] In some embodiments, Formula (I), Formula (II), or both, further comprise: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N(s) wherein each N is independently A, G, C, or U. In some embodiments, Formula (I), Formula (II), or both, further comprise: N; NN; NNN; NNNN; NNNNN; NNNNNN; NNNNNNN; NNNNNNNN; NNNNNNNNN; NNNNNNNNNN; NNNNNNNNNNN; NNNNNNNNNNNN; or any combination thereof wherein each N is independently A, G, C, or U.

[054] In some embodiments, an N of a group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous N(s) can be 5’ and adjacent to D or can be 3’ and adjacent to V, wherein each N is independently A, G, C, or U. In some embodiments, Formula (I), Formula (II), or both, further comprise, a N group of: 1, 2, 3, 4, 5, 6, 7 or 8 contiguous N(s) that can be 5’ and adjacent to D; aN group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous N(s), that can be 3’ and adjacent to V; or any combination thereof, wherein each N is independently A, G, C, or U.

[055] In some embodiments, Formula (I), Formula (II), or both, further comprise : N that is 5 ’ and adjacent to D; NN that is 5’ and adjacent to D; NNN that is 5’ and adjacent to D; NNNN that is 5’ and adjacent to D; NNNNN that is 5’ and adjacent to D; NNNNNN that is 5’ and adjacent to D; NNNNNNN that is 5’ and adjacent to D; NNNNNNNN that is 5’ and adjacent to D; N that is 3’ and adjacent to V; NN that is 3’ and adjacent to V ; NNN that is 3 ’ and adjacent to V ; NNNN that is 3 ’ and adjacent to V ; NNNNN that is 3 ’ and adjacent to V; NNNNNN that is 3’ and adjacent to V; NNNNNNN that is 3’ and adjacent to V; NNNNNNNN that is 3’ and adjacent to V; NNNNNNNNN that is 3’ and adjacent to V; NNNNNNNNNN that is 3’ and adjacent to V; NNNNNNNNNNN that is 3’ and adjacent to V; NNNNNNNNNNNN that is 3’ and adjacent to V; more than one of the foregoing; or any combination thereof; or any combination thereof, wherein each N is independently A, G, C or U.

[056] For example, wherein Formula (I), Formula (II), or both, further comprise NN that is 5’ and adjacent to D and NNNNNNN that is 3’ and adjacent to V, the sequence of Formula (I), Formula (II), or both, may comprise NNDRGUVNNNNNNN wherein each N is independently A, G, C or U. For example, wherein Formula (I), Formula (II), or both, further comprise NNNNNNNN that is 5’ and adjacent to R and NNNNNNNNNNNN that is 3’ and adjacent to V, the sequence of Formula (I), Formula (II), or both, may comprise NNNNNNNNDRGUVNNNNNNNNNNNN wherein each N is independently A, G, C or U.

[057] In some embodiments, the sequence set forth in Formula (I), Formula (II), or both, further comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 W(s) wherein each W is independently A or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 B(s) wherein each B is independently G, C, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 H(s) wherein each H is independently A, C, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N(s) wherein each N is independently A, G, C or U; or any combination thereof.

[058] In some embodiments, Formula (I), Formula (II), or both, further comprise: W, wherein W is A or U; V wherein V is A, G, or C; H wherein H is A, C, or U; 1, 2, 3, or 4 B(s) wherein each B is independently G, C, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 N(s) wherein each N is independently A, G, C or U; or any combination thereof. [059] In some embodiments, Formula (I), Formula (II), or both, further comprises 5’ and adjacent to D:

1, 2, 3, 4, 5, or 6 N(s) wherein each N is independently A, G, C or U; B wherein B is G, C or U; H wherein H is A, C, or U; or any combination thereof.

[060] In some embodiments, Formula (I), Formula (II), or both, further comprise 3’ and adjacent to V: W, wherein W is A or U; V wherein V is A, G, or C; 1, 2, or 3 B(s) wherein each B is independently G, C, or U; 1, 2, 3, 4, 5, 6, or 7 N(s) wherein each N is independently A, G, C or U; or any combination thereof.

[061] In some embodiments, Formula (I), Formula (II), or both, further comprise: N that is 5' and adjacent to D; NN that is 5' and adjacent to D; NNN that is 5' and adjacent to D; BNNN that is 5' and adjacent to D; NBNNN that is 5' and adjacent to D; HNBNNN that is 5' and adjacent to D; NHNBNNN that is 5' and adjacent to D; NNHNBNNN that is 5' and adjacent to D; W that is 3' and adjacent to V; WB that is 3' and adjacent to V; WBN that is 3' and adjacent to V; WBNN that is 3' and adjacent to V; WBNNN that is 3' and adjacent to V; WBNNNN that is 3' and adjacent to V; WBNNNNB that is 3' and adjacent to V; WBNNNNBN that is 3' and adjacent to V; WBNNNNBNN that is 3' and adjacent to V; WBNNNNBNNV that is 3' and adjacent to V; WBNNNNBNNVN that is 3' and adjacent to V; WBNNNNBNNVNB that is 3' and adjacent to V; or any combination thereof; wherein each N is independently A, G, C or U, wherein each B is independently G, C or U, wherein each H is independently A, C, or U, wherein each V is A, G, or C, and wherein each W is A or U.

[062] In some embodiments, Formula (I), Formula (II), or both, further comprise: NN that is 5’ and adjacent to D and WBNNNNB that is 3’ and adjacent to V, wherein the sequence of Formula (I), Formula (II), or both, comprise NNDRGUVWBNNNNB wherein each N is independently A, G, C or U, wherein each B is independently G, C or U, and wherein each W is A or U.

[063] In some embodiments, Formula (I), Formula (II), or both, further comprise: NNHNBNNN that is 5 ’ and adj acent to D and WBNNNNBNNVNB that is 3 ’ and adj acent to V, wherein the sequence of Formula (I), Formula (II), or both, comprise NNHNBNNNDRGUVWBNNNNBNNVNB (SEQ ID NO: 158) wherein each N is independently A, G, C or U, wherein each B is independently G, C or U, wherein each H is independently A, C, or U, wherein each V is A, G, or C, and wherein each W is A or U.

[064] Various embodiments of Formula (I) can be seen, for example, in any of the sequences of TABLE 4. Various embodiments of Formula (II) can be seen, for example, in any of the sequences of TABLE 5A.

[065] In some embodiments, a target sequence comprises a sequence that can be at least about 2 to about 50 nucleobases in length. In some embodiments, a target sequence comprises a sequence that can be about:

2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length. In some embodiments, a target sequence can be about 4 nucleobases in length. In some embodiments, a target sequence can be about 14 nucleobases in length. In some embodiments a target sequence can be about 25 nucleobases in length.

[066] In some embodiments, a target sequence comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 37-56. In some embodiments, a target sequence comprises a sequence about 2 to about 50 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 37-56. In some embodiments, a target sequence comprises a sequence about 4 to about 30 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 37-56. In some embodiments, a target sequence comprises a sequence about 14 to about 25 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence of any one of SEQ ID NOS: 37-56.

[067] Target nucleobase sequences described herein (e.g., TABLE 4) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a target region itself, or, the sequence that encodes a portion of the target region, such as a genomic nucleobase strand encoding a target sequence, for example, if a sequence is DNA converting all ‘T’s to ‘U’s, or if a sequence is RNA converting all ‘U’s to ‘T’s. Similarly, disclosure of the target nucleobase sequences described herein (e.g., TABLE 4) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in a target region as described herein.

TABLE 4: Exemplary Targeted Sequences

[068] In some embodiments, a target sequence may comprise one or more nucleobase alterations at one or more positions in any of the sequences in TABLE 4. Alterations can be made singly or in combination with other alterations at other positions. Alternative nucleobases can independently be any one or more of A, C, G or U, a deletion, or an insertion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.

[069] A disclosed RTSM herein can increase exon skipping of a targeted exon during splicing. In some embodiments, an exon can be skipped when an RTSM is bound or hybridized to the target sequence. In some embodiments when an RTSM is bound or hybridized to the target sequence, a portion of, one or more of Exon 2, Exon 3, Exon 4, Exon 5, Exon 6, Exon 7, Exon 8, Exon 9, Exon 10, Exon 11 or any combination thereof, can be skipped during splicing.

[070] In some embodiments, a target region can comprise a splice site. In some embodiments, an exonintron junction can comprise a splice site. In some embodiments, a target sequence can comprise a splice site. In certain embodiments, a splice site can be a 5’ splice site (5’ss). In other embodiments, a splice site can be a 3’ splice site (3’ss). In some embodiments, a 5’ss can be at the 5’ end of an intron, proximal to the 3’ end of an exon, or both, wherein the 5’ end of an intron is contiguous with the 3’ end of the exon. In some embodiments, a 3’ss can be at the 3’ end of an intron, proximal to the 5’ end of an exon, or both, wherein the 3’ end of an intron is contiguous to the 5’ end of the exon. In some embodiments, a 5’ end of an intron is contiguous with a 3’ end of an exon, wherein a splice site is a 5’ss at the 5’ end of the intron.

[071] In some embodiments, a targeted exon-intron junction comprises at least a portion of a target exon, at least a portion of a target intron, both, more than one of either, or more than one of both. In some embodiments, a targeted exon-intron junction is located at the 5’ss of a target intron, wherein the targeted exon is downstream of a target intron. In some embodiments, a targeted region can comprise a 5’ss of an intron. In some embodiments, a targeted region can comprise: a 5’ss of intron 2 wherein a targeted exon is exon 2; a 5’ss of intron 3 wherein a targeted exon is exon 3; a5’ss of intron 4 wherein a targeted exon is exon 4; a 5’ss of intron 5 wherein a targeted exon is exon 5; a 5’ss of intron 6 wherein a targeted exon is exon 6; a 5’ss of intron 7 wherein a targeted exon is exon 7; a 5’ss of intron 8 wherein a targeted exon is exon 8; a 5’ss of intron 9 wherein a targeted exon is exon 9; a 5’ss of intron 10 wherein a targeted exon is exon 10; a 5’ss of intron 11 wherein a targeted exon is exon 11; or combinations thereof.

[072] In some embodiments, an RTSM binding domain specifically binds to a splice site. In some embodiments, an RTSM binding domain specifically hybridizes to a splice site. In some embodiments, a splice site comprises a sequence as set forth in Formula (II). Various embodiments of Formula (II) can be seen, for example, in any of the sequences as set forth in TABLE 5A.

[073] In some embodiments, a splice site sequence comprises a sequence that can be at least about 2 to about 50 nucleobases in length. In some embodiments, a splice site sequence comprises a sequence that can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length. In some embodiments, a splice site sequence can be about 4 nucleobases in length. In some embodiments, a splice site sequence can be about 14 nucleobases in length. In some embodiments a splice site sequence can be about 25 nucleobases in length.

[074] In some embodiments, a splice site comprises a sequence that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in SEQ ID NOS: 57-76. In some embodiments, a splice site comprises a sequence about 2 to about 50 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in SEQ ID NOS: 57-76. In some embodiments, a splice site comprises a sequence about 4 to about 30 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in SEQ ID NOS: 57-76. In some embodiments, a splice site comprises a sequence about 14 to about 25 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in SEQ ID NOS: 57-76.

[075] In some embodiments, a splice site sequence can be a 5 ’ splice site sequence. In some embodiments, a 5’ splice site sequence can be the complement of any one of the sequences in in SEQ ID NOS: 57-76. In some embodiments, a 5 ’ splice site sequence can be the inverse complement of any one of the sequences in SEQ ID NOS: 57-76. Splice site nucleobase sequences described herein (e.g., TABLE 5A) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, as needed, for describing a sequence within a splice site itself, or, the sequence that encodes a portion of the splice site, such as a genomic nucleobase strand encoding a splice site sequence, for example, if a sequence is DNA converting all ‘T’s to ‘U’s, or if a sequence is RNA converting all ‘U’sto ‘T’s. Similarly, disclosure of the splice site nucleobase sequences described herein (e.g., TABLE 5A) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in a splice site as described herein.

TABLE 5A: Exemplary Splice Site Sequences

[076] In TABLE 5B, the sequences of SEQ ID NOS: 57-76 are reproduced in the column entitled “Representative Sequence” and in some instances, a splice site can be located where the vertical line is located at each sequence.

TABLE 5B: Exemplary Splice Site Sequences

[077] In some embodiments, a splice site sequence further comprises one or more alterations at one or more positions on either side of a splice site. For example, an alteration can be seen at -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50 or more positions from a splice site in the 3’ to the 5’ direction. In a further example, an alteration can be seen at +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50 or more positions from a splice site in the 5’ to the 3’ direction. In another example, an alteration can be seen at -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, - 7, -6, -5, -4, -3, -2, -1, +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25 positions from a splice site. Alterations can be independently any one of A, C, G or U, a deletion, or an insertion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.

RNA-Targeted Splicing Modifiers (RTSM)

[078] An RTSM disclosed herein can be synthetic RNA targeting splicing modifiers that can increase exon skipping during splicing of FOXP3 pre-mRNA compared to FOXP3 pre-mRNA spliced in the absence of RTSM. An RTSM disclosed herein can increase the level of modulated FOXP3 mRNA transcripts as compared to mRNA transcripts processed in the absence of an RTSM. An RTSM disclosed herein can increase the level of modulated FOXP3 protein production as compared to FOXP3 produced in the absence of an RTSM.

[079] In some cases, a synthetic RTSM can be an engineered RTSM.

[080] As used herein, the term “increase” can mean to induce or to enhance. For example, if no exon skipping occurred in the absence of RTSM, then any incidence of exon skipping or any indication of exon skipping activity resulting from splicing in the presence of RTSM can mean that exon skipping has thereby “increased”. In some instances, an evaluation of an increase in exon skipping can occur in an in vitro assay. In some instances, comparison of the amount of exon skipping in two otherwise substantially identical systems where one system lacks an RTSM and the other system has an RTSM can determine if exon skipping increases in the presence of the RTSM. [081] In some embodiments, an RTSM can be selected from the group consisting of an aptamer, an antisense oligomer (ASO), a CRISPR system, and any combination thereof.

[082] In some embodiments, an RTSM targets and binds or hybridizes to FOXP3 pre-mRNA, wherein binding can be hybridizing. Disclosed herein are RTSMs, wherein RTSM-pre-mRNA binding or hybridizing can prevent recruitment of one or more splicing complex component to the pre-mRNA, decrease the binding affinity of one or more splicing complex component to the pre-mRNA, interfere with splice site signaling, sterically blocks splicing of the pre-MRNA, or any combination thereof. In some embodiments, an RTSM comprises a binding domain that binds or hybridizes to FOXP3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that binds or hybridizes to a target region of a FOXP3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that binds or hybridizes to a target sequence of a FOXP3 pre-mRNA. In some embodiments, an RTSM comprises a binding domain that specifically binds or hybridizes to a splice site sequence of a FOXP3 pre-mRNA.

[083] In some embodiments, a binding domain of an RTSM disclosed herein can be from about 2 to about 50 nucleobases in length and can bind or hybridize with about 1 to about 25 nucleobases of an exon to about 1 to about 25 nucleobases of an intron, any number in between, or any combination thereof. In some embodiments, a binding domain of an RTSM comprises a sequence that can be at least about 2 to about 50 nucleobases long. In some embodiments, a binding domain of an RTSM comprises a sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.

[084] In some embodiments, an RTSM need not to completely bind or hybridize to all nucleobases in a target sequence and the nucleobases to which it does bind or hybridize to may be contiguous or noncontiguous. RTSMs may bind or hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an RTSM binds or hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, a RTSM may bind or hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which an RTSM does not bind. In some embodiments, an RTSM can bind or hybridize to about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 continuous nucleobases of a target pre-mRNA.

[085] In some embodiments, an RTSM binding domain comprises a sequence. In certain embodiments, the RTSM binding domain comprising a binding sequence. In some embodiments, a binding sequence binds or hybridizes to the sequence as set forth in Formula (I). In some embodiments, a binding sequence binds or hybridizes to the sequence as set forth in Formula (II). In some embodiments, a binding sequence comprises a sequence as set forth in Formula (III) below:

Formula (III): Exemplary Binding Sequence

5’ ... BACYH... 3’ wherein B is G, C, T or U; wherein Y is C, T, or U; wherein H is A, C, T, or U and wherein 5 ’ . . .3 ’ indicates from the 5 ’ 3 ’ direction.

[086] In some embodiments, the sequence set forth in Formula (III) wherein: B is C, Y is C and H is T or U; B is C, Y is C, and H is C; B is C, Y is C and H is A; B is T or U, Y is C and H is T or U; B is T or U, Y is C, and H is A; B is T or U, Y is C, and H is C; or B is G, Y is C and H is T or U.

[087] In some embodiments, Formula (III) further comprises: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N(s) wherein each N is independently A, G, C, T or U. In some embodiments, Formula (III) further comprises: N; NN; NNN; NNNN; NNNNN; NNNNNN; NNNNNNN; NNNNNNNN; NNNNNNNNN; NNNNNNNNNN; NNNNNNNNNNN; NNNNNNNNNNNN; or any combination thereof wherein each N is independently A, G, C, T or U. In some embodiments, an N of a group of: 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous N(s) can be 5’ and adjacent to B or can be 3’ and adjacent to H, wherein each N is independently A, G, C, T or U. In some embodiments, Formula (III) further comprises, a N group of: 1, 2, 3, 4, 5, 6, 7 or 8 contiguous N(s) that can be 3’ and adjacent to H; aN group of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 contiguous N(s), that can be 5’ and adjacent to B; or any combination thereof, wherein each N is independently A, G, C, T or U.

[088] In some embodiments, Formula (III) further comprises: N that is 3’ and adjacent to H; NN that is 3’ and adjacent to H; NNN that is 3’ and adjacent to H; NNNN that is 3’ and adjacent to H; NNNNN that is 3’ and adjacent to H; NNNNNN that is 3’ and adjacent to H; NNNNNNN that is 3’ and adjacent to H; NNNNNNNN that is 3’ and adjacent to H; N that is 5’ and adjacent to B; NN that is 5’ and adjacent to B; NNN that is 5 ’ and adjacent to B; NNNN that is 5 ’ and adjacent to B; NNNNN that is 5 ’ and adjacent to B; NNNNNN that is 5’ and adjacent to B; NNNNNNN that is 5’ and adjacent to B; NNNNNNNN that is 5’ and adjacent to B; NNNNNNNNN that is 5 ’ and adjacent to B; NNNNNNNNNN that is 5 ’ and adjacent to B; NNNNNNNNNNN that is 5 ’ and adjacent to B; NNNNNNNNNNNN that is 5 ’ and adjacent to B; more than one of the foregoing; or any combination thereof; or any combination thereof, wherein each N is independently A, G, C, T or U.

[089] For example, wherein Formula (III) further comprises NN that is 3’ and adjacent to H and NNNNNNN that is 5’ and adjacent to B, the sequence of Formula (III) may comprise NNNNNNNBACYH NN wherein each N is independently A, G, C, T or U. For example, wherein Formula (III) further comprises NNNNNNNN that is 3’ and adjacent to H and NNNNNNNNNNNN that is 5’ and adjacent to B, the sequence of Formula (III) may comprise NNNNNNNNNNNNB ACYHNNNNNNNN wherein each N is independently A, G, C, T or U.

[090] In some embodiments, the sequence set forth in Formula (III) further comprising: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 B(s) wherein each B is independently G, C, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N(s) wherein each N is independently A, G, C, T, or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 D(s) wherein each D is independently A, G, T or U; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 W(s) wherein each W is independently A, T, or U; or any combination thereof.

[091] In some embodiments, Formula (III) further comprises: W, wherein W is A, T or U; B wherein B is G, C, T, or U; D wherein D is A, G, T, or U; 1, 2, 3, or 4 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 N(s) wherein each N is independently A, G, C, T or U; or any combination thereof. In some embodiments, Formula (III) further comprises, 3’ and adjacent to H: 1, 2, 3,

4, 5, or 6 N(s) wherein each N is independently A, G, C, T or U; V wherein V is A, G, or C; D wherein D is A, G, T or U; or any combination thereof. In some embodiments, Formula (III) further comprises, 5’ and adjacent to B: W wherein W is A, T or U; B wherein B is G, C, T, or U; 1, 2, or 3 V(s) wherein each V is independently A, G, or C; 1, 2, 3, 4, 5, 6, or 7 N(s) wherein each N is independently A, G, C, T or U; or any combination thereof.

[092] In some embodiments, Formula (III) further comprises: N that is 3' and adjacent to H; NN that is 3' and adjacent to H; NNN that is 3' and adjacent to H; NNNV that is 3' and adjacent to H; NNNVN that is 3' and adjacent to H; NNNVND that is 3' and adjacent to H; NNNVNDN that is 3' and adjacent to H; NNNVNDNN that is 3' and adjacent to H; W that is 5' and adjacent to B; VW that is 5' and adjacent to B; NVW that is 5' and adjacent to B; NNVW that is 5' and adjacent to B; NNNVW that is 5' and adjacent to B; NNNNVW that is 5' and adjacent to B; VNNNNVW that is 5' and adjacent to B; NVNNNNVW that is 5' and adjacent to B; NNVNNNNVW that is 5' and adjacent to B; BNNVNNNNVW that is 5' and adjacent to B; NBNNVNNNNVW that is 5' and adjacent to B; VNBNNVNNNNVW that is 5' and adjacent to B; or any combination thereof; wherein each N is independently A, G, C, T or U, wherein each B is independently G, C, T or U, wherein each D is independently A, G, T or U, wherein each V is independently A, G, or C, and wherein each W is independently A, T or U.

[093] In some embodiments, Formula (III) further comprises NN that is 3’ and adjacent to H and VNNNNVW that is 5’ and adjacent to B, wherein the sequence of Formula (III) comprises VNNNNVWBACYHNN, and wherein each N is independently A, G, C, T or U, wherein each V is independently A, G, or C, and wherein W is A, T or U.

[094] In some embodiments, Formula (III) further comprises NNNVNDNN that is 3' and adjacent to H and VNBNNVNNNNVW that is 5' and adjacent to B, wherein Formula III comprises NN N VN DNN B AC YHVNBNN VNNNNVW (SEQ ID NO: 159), and wherein each N is independently A, G, C, T or U, wherein each B is independently G, C, T or U, wherein each D is independently A, G, T or U, wherein each V is independently A, G, or C, and wherein each W is independently A, T or U.

[095] In some embodiments, a binding sequence comprising a sequence of Formula III can hybridize to a target sequence of a FOXP3 pre-mRNA wherein the target sequence comprises a sequence as set forth in Formula I. In some embodiments, a binding sequence comprising a sequence of Formula III can hybridize to a target sequence of a FOXP3 pre-mRNA wherein the target sequence comprises a sequence as set forth in Formula II. In some embodiments, a binding sequence comprising a sequence of Formula III binds or hybridizes to nucleobases surrounding a splice site in a 3’ to 5’ direction wherein a splice site is indicated by a vertical line in any of the sequences in TABLE 5B.

[096] Various embodiments of Formula (III) can be seen any of the sequences as set forth in TABLE 6 below. In some instances, embodiments of Formula (III) can be modified as described in the methods disclosed herein.

[097] In some embodiments, an RTSM binding domain comprises a sequence that can be at least about 2 to about 50 nucleobases in length. In some embodiments, an RTSM binding sequence comprises a sequence that can be about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length. In some embodiments, an RTSM binding sequence can be about 4 nucleobases in length. In some embodiments, an RTSM binding sequence can be about 14 nucleobases in length. In some embodiments an RTSM binding sequence can be about 25 nucleobases in length.

[098] In some embodiments, an RTSM binding domain comprises a sequence that has at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID

NOS: 37-56. In some embodiments, an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,

55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 37-56. In some embodiments, an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 1%,

5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID

NOS: 37-56. In some embodiments, an RTSM binding domain comprises a sequence about 14 to about 25 nucleobases in length that has at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,

55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 37-56.

[099] In some embodiments, an RTSM binding domain comprises a sequence that shares at least about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 77- 116. In some embodiments, an RTSM binding domain comprises a sequence about 2 to about 50 nucleobases in length that shares at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: SEQ ID NOS: 77-116. In some embodiments, an RTSM binding domain comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: SEQ ID NOS: 77-116. In some embodiments, an RTSM binding domain comprises a sequence about 14 to about 25 nucleobases in length that has at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: SEQ ID NOS: 77-116.

[0100] Binding nucleobase sequences described herein (e.g., TABLE 6) may be described as a nucleobase sequence of either DNA or RNA, however, no matter the form the sequence is described, it is readily understood that such nucleobase sequences can be revised to be RNA or DNA, respectively, by substitution of all ‘T’s with ‘U’s or substitution of all ‘U’s with ‘T’s, as needed, for describing a sequence within binding domain itself, or, the sequence that encodes a portion of a binding domain or a binding sequence, such as a vector encoding a binding sequence or at least a portion of a binding domain. Similarly, disclosure of the binding nucleobase sequences described herein (e.g., TABLE 6) also discloses the complementary nucleobase sequence, the reverse nucleobase sequence, and the reverse complement nucleobase sequence, any one of which can be a nucleobase sequence in a binding domain as described herein.

TABLE 6: Exemplary Binding Sequences

[0101] In some embodiments, a binding sequence may comprise one or more nucleobase alterations at one or more positions in any of the sequences in TABLE 6. Alterations can be made singly or in combination with other alterations at other positions. Alternative nucleobases can be any one or more of A, C, G, T or U, a deletion, or an insertion. In some embodiments, alterations can be obtained through computational predictions and alignment, for example, using SPLIGN, ESEMBL, or Blast.

[0102] In some embodiments, a binding sequence comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one ofthe sequences of SEQ ID NOS: 77-116. In some embodiments, a binding sequence comprises a sequence about 4 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 77-116. In some embodiments, a binding sequence comprises a sequence about 15 to about 25 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 77-116.

[0103] In some embodiments, a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more continuous or non-continuous nucleotides of any one of the sequences of SEQ ID NOS: 37-56. In some embodiments, a binding sequence disclosed herein comprises a sequence that can bind or hybridize to 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more continuous or non-continuous nucleotides of any one of the sequences of SEQ ID NOS: 57-76.

[0104] An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM. In some embodiments, exon skipping modulates a FOXP3 mRNA, a FOXP3 protein, or both. In some embodiments, modulated exon coding can be determined by modulated mRNA transcription, modulated a FOXP3 protein translation, or both. In some embodiments, modulated exon coding comprises: absence of one or more FOXP3 exon(s) in a FOXP3 mRNA, a truncated mRNA, absence of one or more FOXP3 exon(s) in a FOXP3 protein, a non-functional FOXP3 protein, an at least semi-functional FOXP3 protein, a truncated FOXP3 protein, or any combination thereof.

[0105] In some embodiments, an RTSM-modulated FOXP3 mRNA transcript does not comprise a sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to: SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 25; SEQ ID NO: 26; or any combination thereof.

[0106] In some embodiments, wherein RTSM-modulated FOXP3-mRNA transcript can be determined by analysis of a FOXP3 mRNA transcript transcribed in the absence of RTSM, or to a reference FOXP3 mRNA, such as a wild-type FOXP3 mRNA, or both, wherein the reference FOXP3 mRNA can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence as set forth in SEQ ID NOS: 7-11.

[0107] In some embodiments wherein RTSM-modulated FOXP3 protein expression can be determined by analysis of a FOXP3 protein translated in the absence of RTSM, or to a reference FOXP3 protein, such as a wild-type FOXP3 protein, or both, wherein the reference FOXP3 protein can be encoded by a sequence with at least: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences as set forth in SEQ ID NOS: 12-16.

Aptamers

[0108] In some embodiments an RTSM can be an aptamer that binds to a riboswitch on a targeted pre- mRNA. In some embodiments, an RTSM aptamer can be operably linked to a ligand.

[0109] In some embodiments, an RTSM can be operably linked to a ligand. In certain embodiments, a ligand or molecule specific to an aptamer comprises the ability to: bind its ligand-binding aptamer with high affinity; bind independently of other factors; undergo a conformational change or rearrangement upon binding of the ligand; bind under the conditions that support pre-mRNA splicing; avoid off-target splicing interference; or any combinations thereof.

[0110] When an RTSM can be an aptamer-ligand system, the aptamer-ligand comprises a binding domain that targets and binds to a target sequence of a pre-mRNA, wherein the aptamer inserts into the strand and the ligand increases exon skipping. In some embodiments, a ligand can be tobramycin, neomycin, or theophylline.

Antisense Oligonucleotides (ASOs)

[oni] In some embodiments, an RTSM can be an ASO. In some embodiments, an ASO-RTSM disclosed herein comprises a binding domain that targets and binds or hybridizes to a target region of a FOXP3 pre- mRNA In some embodiments, a binding domain of an ASO-RTSM comprises a binding sequence that can be about: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleobases in length.

[0112] In some embodiments, an ASO RTSM comprises a sequence that shares at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 37-56.

[0113] In some embodiments, an ASO-RTSM of the present disclosure comprises a binding sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 43. In some embodiments, an RTSM comprises a binding sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 43. In some embodiments, an RTSM of the present disclosure comprises a binding sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 43.

[0114] In some embodiments, an ASO RTSM of the present disclosure comprises a binding sequence that has at least about 84% sequence complementarity to the sequence of SEQ ID NO: 53. In some embodiments, an ASO RTSM of the present disclosure comprises a binding sequence that has at least about 88% sequence complementarity to the sequence of SEQ ID NO: 53. In some embodiments, an ASO RTSM of the present disclosure comprises a binding sequence that has at least about 92% sequence complementarity to the sequence of SEQ ID NO: 53. In some embodiments, an ASO RTSM of the present disclosure comprises a binding sequence that has at least about 96% sequence complementarity to the sequence of SEQ ID NO: 53. In some embodiments, an ASO RTSM of the present disclosure comprises a binding sequence that has 100% sequence complementarity to the sequence of SEQ ID NO: 53.

[0115] An ASO and a DNA or RNA target binding partner can be complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus in some embodiments, “specifically hybridizable” and “complementary” are terms which can be used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an ASO and a DNA or RNA target. It can be understood that the sequence of an ASO need not be 100% complementary to that of its target sequence to be specifically hybridizable. An ASO can be specifically hybridizable when there are sufficient binding interactions between an ASO and DNA or RNA target such that the ASO, at least temporarily, adheres to the specific region which its targeting. Specific binding can occur under physiological conditions, including but not limited to room temperature, in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. In some embodiments, the above method may be used to select ASOs.

[0116] In some embodiments, an ASO can have exact sequence complementary to a target sequence or near complementarity (e.g., sufficient complementarity to bind or hybridize to a target sequence and modulating splicing at a splice site). Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleobase or nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to a target region within a target nucleobase sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined using BLAST programs (basic local alignment search tools) and PowerBLAST programs.

[0117] ASOs disclosed herein can be designed so that they bind or hybridize to a target nucleobase or nucleic acid (e.g., a targeted region of a pre-mRNA transcript) and remain bound under physiological conditions. In some embodiments, binding as described herein can be hybridizing. In some embodiments, if an ASO binds or hybridizes to a site other than the intended (targeted) nucleic acid sequence, it binds or hybridizes to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of a targeted portion of a pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre-mRNA or transcriptome, such that the likelihood that an ASO will bind or hybridize to other sites and cause "off-target" effects is limited.

[0118] In certain embodiments, ASOs can bind to a target pre-mRNA. In certain embodiments, ASOs can hybridize to a pre-mRNA. In some instances, ASOs can “specifically bind”, "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA. Such hybridization can occur with a T _m (melting temperature) substantially greater than 37°C, at least 50 °C, or between 60 °C to approximately 90 °C. Such hybridization can correspond to stringent hybridization conditions. At a given ionic strength and pH, the T _m is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide. In some embodiments, an ASO can bind to, hybridize to, or specifically hybridize to a splice site sequence in a target pre-mRNA wherein a splice site sequence comprises a sequence that shares at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences of SEQ ID NOS: 57-76.

[0119] In some embodiments, an ASO-RTSM of the present disclosure comprises a binding sequence that has at least about 85% sequence complementary to the sequence of SEQ ID NO: 63. In some embodiments, an RTSM of the present disclosure comprises a binding sequence that has at least about 92% sequence complementary to the sequence of SEQ ID NO: 63. In some embodiments, an RTSM of the present disclosure comprises a binding sequence that has 100% sequence complementary to the sequence of SEQ ID NO: 63.

[0120] In some embodiments, an ASO RTSM comprises a binding sequence that has at least about 84% sequence complementarity to the sequence of SEQ ID NO: 73. In some embodiments, an ASO RTSM comprises a binding sequence that has at least about 88% sequence complementarity to the sequence of SEQ ID NO: 73. In some embodiments, an ASO RTSM comprises a binding sequence that has at least about 92% sequence complementarity to the sequence of SEQ ID NO: 3. In some embodiments, an ASO RTSM comprises a binding sequence that has at least about 96% sequence complementarity to the sequence of SEQ ID NO: 73. In some embodiments, an ASO RTSM comprises a binding sequence that has 100% sequence complementarity to the sequence of SEQ ID NO: 73.

[0121] In some embodiments, an ASO RTSM comprises a sequence that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 77-116.

[0122] In some embodiments, an ASO RTSM comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 83. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 83.

[0123] In some embodiments, an ASO RTSM comprises a sequence that shares at least about 85% sequence identity to the sequence of SEQ ID NO: 93. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 93. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 93.

[0124] In some embodiments, an ASO RTSM comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 103. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 103. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 103. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 103. In some embodiments, an ASO RTSM comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 103.

[0125] In some embodiments, an ASO RTSM comprises a sequence that shares at least about 84% sequence identity to the sequence of SEQ ID NO: 113. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 88% sequence identity to the sequence of SEQ ID NO: 113. In some embodiments, an ASO RTSM of the present disclosure comprises a sequence that shares at least about 92% sequence identity to the sequence of SEQ ID NO: 113. In some embodiments, an ASO RTSM comprises a sequence that shares at least about 96% sequence identity to the sequence of SEQ ID NO: 113. In some embodiments, an ASO RTSM comprises a sequence that has 100% sequence identity to the sequence of SEQ ID NO: 113.

[0126] An ASO disclosed herein can comprise oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of binding to a complementary nucleobase on a target mRNA, but in some embodiments, an ASO does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). An ASO may comprise non-modified nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "modified nucleotides" can include nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of an ASO are modified nucleotides.

[0127] In some embodiments, an ASO described herein further comprise a backbone structure that connects the components of an oligomer. The term "backbone structure" and "oligomer linkages" may be used interchangeably and can refer to the connection between monomers of an ASO. In naturally occurring oligonucleotides, the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of the oligomer. The backbone structure or oligomer linkages of an ASO described herein can include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoramidate, and the like. In some embodiments, the backbone structure of an ASO does not contain phosphorous but rather contains peptide bonds, for example in a peptide nucleic acid (PNA), or linking groups including carbamate, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the backbone modification can be a phosphothioate linkage. In some embodiments, the backbone modification can be a phosphoramidate linkage.

[0128] Any of the ASOs described herein may contain a sugar moiety that comprises ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar moiety or sugar analog, including a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-0-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F; N3'->P5' phosphoramidate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidinidium, 2'-O- guanidinium ethyl, carbamate modified sugars, and bicyclic modified sugars. In some embodiments, the sugar moiety modification can be selected from 2'-0-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification can be an extra bridge bond, such as in a locked nucleic acid (LNA). In some embodiments the sugar analog contains a morpholine ring, such as phosphorodiamidate morpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuransyl or 2'deoxyribofuransyl modification. In some embodiments, the sugar moiety comprises 2'4'-constrained 2'O-methyloxyethyl (cMOE) modifications. In some embodiments, the sugar moiety comprises cEt 2', 4' constrained 2'-0 ethyl BNA modifications. In some embodiments, the sugar moiety comprises tricycloDNA (tcDNA) modifications. In some embodiments, the sugar moiety comprises ethylene nucleic acid (ENA) modifications. In some embodiments, the sugar moiety comprises MCE modifications.

[0129] In some embodiments, one or more monomer, or each monomer of an ASO can be modified in the same way, for example each linkage of the backbone of an ASO comprises a phosphorothioate linkage or each ribose sugar moiety comprises a 2'O-methyl modification. Such modifications that are present on each of the monomer components of an ASO are referred to as "uniform modifications." In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate linkages and sugar moieties comprising morpholine rings (morpholines). Combinations of different modifications to an ASO are referred to as "mixed modifications" or "mixed chemistries."

[0130] In some embodiments, an ASO comprises one or more backbone modifications. In some embodiments, an ASO comprises one or more sugar moiety modification. In some embodiments, an ASO comprises one or more backbone modifications and one or more sugar moiety modifications. In some embodiments, an ASO comprises a 2'MOE modification and a phosphorothioate backbone. In some embodiments, an ASO comprises a phosphorodiamidate morpholino (PMO). In some embodiments, an ASO comprises a peptide nucleic acid (PNA).

[0131] In some embodiments, an ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, an ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O- methoxyethyl moiety. In some embodiments, an ASO comprises at least one modified sugar moiety. In some embodiments, each sugar moiety can be a modified sugar moiety.

[0132] Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be independently modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.

[0133] In some embodiments, an ASO can be comprised of one or more 2'-O-(2 -methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides can be especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein.

[0134] In some embodiments, an ASO comprising one or more modified nucleobases is referred to herein as an “AMO” or an “Antisense Modified Oligonucleotide” or “Antisense Morpholino Oligonucleotide” interchangeably depending on the context. In some embodiments, an Antisense Modified Oligonucleotide can be an Antisense Morpholino Oligonucleotide where one, some, or all sugar molecules are replaced with morpholino group(s).

[0135] ASOs can be synthesized by methods described herein. Alternatively or in addition, ASOs may be obtained from a commercial source. In certain embodiments, an ASO can be prepared by stepwise solidphase synthesis.

[0136] In some cases, it may be desirable to add additional chemical moieties to an ASO, e.g., to enhance pharmacokinetics or to facilitate capture or detection of the compound. Such a moiety may be covalently attached, according to standard synthetic methods. For example, addition of a polyethylene glycol moiety or other hydrophilic polymer, e.g., one having 1-100 monomeric subunits, may be useful in enhancing solubility.

[0137] Further a reporter moiety, such as fluorescein or a radiolabeled group, may be attached for purposes of detection. Alternatively, the reporter label attached to the oligomer may be a ligand, such as an antigen or biotin, capable of binding a labeled antibody or streptavidin. In selecting a moiety for attachment or modification of an antisense compound, can be generally desirable to select chemical compounds of groups that are biocompatible and likely to be tolerated by a subject without undesirable side effects.

CRISPR-Cas Systems

[0138] Disclosed herein are RTSMs that can comprise a CRISPR-Cas system (CRISPR system) wherein the system comprises a CRISPR associated protein. The CRISPR system can be designed to target a FOXP3 pre-mRNA, prevent recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, interfere with splice site signaling, sterically block splicing of a pre-mRNA, or any combination thereof. Envisioned herein are CRISPR systems that target RNA. While any suitable CRISPR system may be used for the purposes of the disclosure herein, in some embodiments, systems that target RNA and do not rely on consensus protospacer adjacent motif (PAM) for activity, such as Types III and VI, can be used herein. Accordingly, in some embodiments, the CRISPR systems used herein can rely on RNA protospacer flanking sequences (PFS) or PAM sequences. Hence, in some embodiments, a FOXP3 RNA further comprises a PFS sequence or a PAM sequence.

[0139] Subtypes of suitable CRISPR systems disclosed herein can include, but are not limited Type II class 2, Types III -A, III-B, VI-A, VI -A, VI-C, or VI -D. In other embodiments, Type II RNA-targeting Cas9 systems can also be used as an RTSM disclosed herein.

[0140] In some embodiments, the CRISPR system comprises a guide RNA and a Cas nuclease. In one embodiment, the guide RNA comprises a crispr RNA (crRNA) and a tracr RNA. In another embodiment, the guide RNA comprises a single guide RNA (sgRNA). In some embodiments, the CRISPR system can comprise one or more Cas nuclease. Examples of suitable Cas nucleases include, but are not limited to, Csm3, Cmr4, Csm6, Csxl, Csx27, Csx28, a member of a Cas 7 superfamily, or a Cas9, Casl2, or a Casl3 effector nuclease.

[0141] CRISPR systems that target a FOXP3 pre-mRNA can be computationally identified through determination of a Cas containing signature genes that express RNAse or RNA targeting activity, and transcribed and processed into a CRISPR gRNA.

[0142] In some embodiments, the gRNA of the CRISPR system comprises a binding domain that to a target region of a FOXP3 pre-mRNA. In one embodiment, the gRNA or sgRNA binding domain comprises a sequence about 2 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 37-56. In one embodiment, the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to any one of the sequences of SEQ ID NOS: 37-56.

[0143] In some embodiments, the gRNA or sgRNA binding domain comprises a sequence about 4 to about 50 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 107-116 and 127-136. In some embodiments, the gRNA or sgRNA binding domain comprises a sequence about 14 to about 30 nucleobases in length that has at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the sequences of SEQ ID NOS: 87-96 and 107-116.

[0144] In some embodiments, the gRNA of the CRISPR system targets a FOXP3 pre-mRNA of interest and directs the Cas nuclease to a pre-mRNA. According to some embodiments, the Cas nuclease can be a catalytically dead variant, wherein upon gRNA binding to a target region of a pre-mRNA, the CRISPR system increases exon skipping wherein the system prevents recruitment of a one or more splicing complex component to a pre-mRNA, decrease the binding affinity of one or more splicing complex component to a pre-mRNA, or sterically block mRNA splicing.

[0145] In other embodiments, the Cas nuclease can be selected and or synthesized to interfere with splice site signaling through RNAse activity wherein upon gRNA binding to a target region of a pre-mRNA, the Cas nuclease disrupts splice site signaling sequences of a targeted exon-intron junction thereby inducing exon skipping.

Modifications

[0146] Any of the RTSMs described herein may be modified in order to achieve desired properties or activities of an RTSM or reduce undesired properties or activities of an RTSM. For example, an RTSM or one or more components of any RTSM may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of an RTSM into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the RTSM; and/or modulate the half-life of the RTSM.

[0147] Also included herein are vector delivery systems that are capable of expressing an RTSM sequence herein, such as vectors that express a polynucleotide sequence comprising any one or more of the sequences shown in TABLE 6, as described herein. The term “vector” or “nucleic acid construct” as used herein can mean a polynucleotide molecule, such as a DNA molecule derived from or comprising at least a part of, for example, a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector can contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrated with the genome of the defined host such that the cloned sequence can be reproducible. Accordingly, the vector can be an autonomously replicating vector, e.g., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

[0148] For example, an RTSM of the present disclosure can be conjugated to a cell penetrating peptide. The term “cell penetrating peptide” and “CPP” are used interchangeably and can refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. The peptides, as shown herein, have the capability of inducing cell penetration within 100% of cells of a given cell culture population and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.

[0149] In one embodiment, an RTSM can be an ASO wherein an ASO can comprise an oligonucleotide moiety conjugated to a cell penetrating peptide effective to enhance transport of the compound into cells. In some embodiment, the oligonucleotide moiety can be an arginine-rich peptide transport moiety effective to enhance transport of the compound into cells. The transport moiety can be attached to a terminus of the oligomer. The peptides have the capability of inducing cell penetration within 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of cells of a given cell culture population, including all integers in between, and allow macromolecular translocation within multiple tissues in vivo upon systemic administration. In one embodiment, the cell-penetrating peptide may be an arginine-rich peptide transporter. In another embodiment, the cell-penetrating peptide may be Penetratin or the Tat peptide. In one embodiment, the CPP can be conjugated to an ASO herein and can utilize glycine as the linker between the CPP and the antisense oligonucleotide. For example, a peptide conjugated PMO consists of R5-G-PMO.

[0150] The transport moieties as described above can enhance cell entry of attached oligomers, relative to uptake of the oligomer in the absence of the attached transport moiety. Uptake can be enhanced at least ten fold, and at least twenty fold, relative to the unconjugated compound.

[0151] The use of arginine-rich peptide transporters (e.g., cell-penetrating peptides) can be used herein. For example, when conjugated to an antisense PMO, arginine-rich CPPs can demonstrate an enhanced ability to alter splicing of several gene transcripts. Exemplary peptide transporters, excluding linkers, can be seen in TABLE 7. Linkage portions can be C, G, P, Ahx, B, or AhxB where Ahx and B refer to 6- aminohexanoic acid and beta-alanine, respectively.

TABLE 7: Exemplary Peptide transporters

Assays

[0152] An RTSM disclosed herein can increase exon skipping during pre-mRNA splicing as compared to a pre-mRNA spliced in the absence of an RTSM herein as determined by an in vitro assay. In some embodiments, exon skipping modulates FOXP3 mRNA transcript production. In some embodiments, exon skipping increases production of mRNA transcript excluding coding for one or more exons, a truncated mRNA molecule, or both. In some embodiments, RTSM-modulated mRNA expression can be compared to mRNA processed in the absence of RTSM as determined by an in vitro assay.

[0153] In some embodiments, exon skipping modulates FOXP3 protein production. In certain embodiments, an increase in exon skipping induces an increase in modulated FOXP3 protein expression. In some embodiments, exon skipping increases production of a non-functional FOXP3 protein, a semifunctional FOXP3 protein, a truncated FOXP3 protein, or any combination thereof. In some embodiments, RTSM modulated FOXP3 protein production can be compared to FOXP3 protein production in the absence of RTSM as determined by an in vitro assay.

[0154] In some embodiments, the cell, organ, or subject can be evaluated to determine if appropriate for the methods and compositions described herein. For example, the cell, organ, or subject can be evaluated for the expression of FOXP3, Treg activity or level, or both, immunosuppression, cancer, or any combination thereof. Evaluation methods are described herein, such as in the Examples.

[0155] Methods of determining exon skipping, mRNA modulation, protein expression, or FOXP3 exon expression modulation are described herein, such as in the Examples. Other non-limiting assays to determine gene expression, exon skipping and FOXP3 expression include quantitative PCR (qPCR), including but not limited to PCR, real-time PCR (e.g., Sybr-green), and/or hot PCR. In some cases, expression of one or more genes can be measured by detecting the level of transcripts of the genes. Exon skipping can be measured by detecting the expression of the processed mRNA. Expression of functional FOXP3 protein can be measured by detecting the level or length of the protein, or by an assay that measures its biological activity. For example, expression can be measured by Northern blotting, nuclease protection assays (e.g., RNase protection assays), reverse transcription PCR, quantitative PCR (e.g., real-time PCR such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot-blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarrays, nanostring, and/or sequencing (e.g., next-generation sequencing) Expression can be measured by protein immunostaining, protein immunoprecipitation, electrophoresis (e.g., SDS- PAGE), Western blotting, bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzyme assays (e.g., enzyme-linked immunosorbent assays), immunohistochemistry, flow cytometry, and/or immunocytochemistry. Expression of one or more genes can also be measured by microscopy. The microscopy can be optical, electron, or scanning probe microscopy. Optical microscopy can comprise use of bright field, oblique illumination, cross-polarized light, dispersion staining, dark field, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence (e.g., when particles, e.g., cells, are immunostained), confocal, single plane illumination microscopy, light sheet fluorescence microscopy, deconvolution, or serial time-encoded amplified microscopy.

Methods

[0156] Disclosed herein, among other disclosures, are methods of exon skipping, Treg inhibition, and prophylaxis/treatment of: FOXP3-mediated conditions, immunosuppressive Treg activity, an immunosuppressive microenvironment or matrix, a cancerous disease, or cancer, or any combination thereof.

[0157] In some embodiments, the disclosed compositions and methods result in a truncated FOXP3 protein. In some embodiments, the disclosed compositions and methods result in a decrease in the wildtype FOXP3 protein. In some embodiments, the disclosed compositions and methods result in modulating the splicing of FOXP3 pre-mRNA. In some embodiments, the disclosed compositions and methods result in an FOXP3 mRNA lacking exon 8 or portion thereof. In some embodiments, FOXP3 expression can be modulated compared to a control. A control can be wild-type or non wild-type control. Controls can be positive or negative controls.

Methods of modulating FOXP3 expression

[0158] Disclosed herein are methods to increase exon skipping of an FOXP3 mRNA comprising contacting a FOXP3 pre-mRNA with an RTSM as disclosed herein, and allowing modulated splicing to occur. In some embodiments, increased exon skipping comprises modulated production of FOXP3 mRNA, FOXP3 protein or both.

[0159] In some embodiments, an RTSM herein can increase the level of modulated FOXP3 mRNA transcripts as compared to mRNA transcripts processed in the absence of RTSM. In some embodiments, mRNA transcript modulation can comprise a decrease in full-length FOXP3 mRNA transcript, an increase in truncated FOXP3 mRNA transcript, an increase in an FOXP3 mRNA transcript lacking one or more exons, or any combination thereof. In some embodiments, modulated FOXP3 pre-mRNA can be an increase in FOXP3A8, an increase in FOXP3A3A8, a decrease in FOXP3fl, a decrease in FOXP3A3, or any combination thereof

[0160] In some embodiments, the increase in modulated mRNA transcript production can be about: 0.1%,

0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%,

5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%,

13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60%, 65%, 70%, 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%, or more, or any integer in-between, relative to a non-RTSM contacted control. [0161] In some embodiments, methods further comprise allowing translation of the modulated mRNA to occur. In some embodiments, an RTSM herein can increase the level of modulated FOXP3 protein produced as compared to FOXP3 protein produced in the absence of RTSM. In some embodiments, FOXP3 protein modulation can comprise a decrease in full-length FOXP3 protein, an increase in truncated FOXP3 protein, an increase in an FOXP3 protein lacking one or more exons, inhibition of FOXP3 protein production, or any combination thereof.

[0162] In some embodiments, the increase in modulated FOXP3 protein production can be about: 0.1%,

0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%,

5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%,

13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%,

22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60%, 65%, 70%, 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%, or more, or any integer in-between, relative to a non-RTSM contacted control.

[0163] In some embodiments, increased exon skipping comprises an increase in FOXP3 A8, an increase in FOXP3A3A8, decrease in FOXP3fl, a decrease in FOXP3 A3, or any combination thereof, wherein FOXP3 can be mRNA or protein.

[0164] In some embodiments, methods disclosed herein can increase FOXP3A8, increase FOXP3A3A8, decrease FOXP3H, decrease FOXP3A3, or any combination thereof, by 0.1%, 0.2% 0.3% 0.5%, 0.7%,

0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%,

6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%,

15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,

27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 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%, or more, relative to a non-RTSM contacted control.

Methods of inducing inhibition or formation of Treg cells

[0165] In some embodiments, modulated FOXP3 pre-mRNA transcript processing, modulated FOXP3 protein production or both can at least partially induce inhibition of the number of or activity of Tregs or at least partially prevent or diminish formation of Tregs. In some embodiments, disclosed herein are methods of inducing the inhibition of the number of or activity of Tregs or at least partially preventing or diminishing formation of Tregs comprising contacting a FOXP3 pre-mRNA with an RTSM as disclosed herein, and allowing exon skipping to occur. In some embodiments, an RTSM herein, at least partially as a result of exon skipping, can induce inhibition of the number of or the activity of Tregs or at least partially prevent or diminish formation of Tregs, or both as compared to the number of Tregs or activity or both in the absence of an RTSM. In certain instances, a Treg cell can be, at least initially, a FOXP3 ⁺ cell or a T cell that exhibits immunosuppressive activity or both.

[0166] In some embodiments, Treg activities can be inhibited about: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%,

1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%,

7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%,

15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,

28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60%, 65%, 70%, 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%, or more, or any integer in-between, relative to a non-RTSM contacted control.

[0167] In some embodiments, the number of Treg cells can be reduced about: 0.1%, 0.2% 0.3% 0.5%,

0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%,

14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%,

25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60%, 65%, 70%, 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%, or more, or any integer in-between, relative to a non-RTSM contacted control.

Methods of Treating Diseases and Conditions

[0168] Disclosed herein are methods of prophylaxis/treatment of a FOXP3-associated disease or condition, immunosuppression, or cancer, or any combination thereof. Disclosed herein are methods of treatment comprising administering to a subject in need thereof an effective amount of FOXP3 RTSM, or a pharmaceutical composition comprising the same. A subject in need thereof can be a subject who is suffering from or is at a risk of developing a FOXP3 -associated condition or disease, immunosuppression, or cancer.

[0169] In some embodiments, RTSMs, compositions and methods described herein can reduce or inhibit: the expression of FOXP3 in a T cell or a Treg cell, a tumor microenvironment, tumor stroma, a Treg infdtrated tumor, an immune cell, lymphoid tissue, a lymph node, an intra-tumoral cell, or any combination thereof; an immunosuppression, Treg immunosuppressive activity, amount of Treg cells, or any combination thereof; cancer cell proliferation, tumor growth, metastasis, or any combination thereof; or any combination thereof; and in some embodiments, thereby treating or preventing FOXP3 -associated conditions, immunosuppression, and/or cancer.

[0170] In some embodiments, RTSMs, compositions and methods described herein can induce or activate: anticancer immunity, antitumor immunity, anticancer immune response, antitumor immune response, immune cell activation, immune cell infdtration, inflammatory cell activation, inflammatory cell infdtration, effector immune cell activation, effector immune cell infdtration, T cell activation, T cell infdtration, CD8 ⁺ T cell activation, CD8 ⁺ T cell activation infdtration, NK cell activation, NK cell infdtration, macrophage activation, macrophage infdtration, dendritic cell activation, dendritic cell infdtration, inflammation, inflammatory cytokine expression, inflammatory chemokine expression; or any combination thereof; and in some embodiments, thereby treating or preventing FOXP3 -associated conditions, immunosuppression, and/or cancer.

[0171] Examples of FOXP3-associated conditions or diseases can include conditions or disorders comprising a: FOXP3 ⁺ Treg, FOXP3 ⁺ and CD25 ⁺ Treg, or both. In some embodiments, a FOXP3 associated condition or disease can comprise FOXP3 ⁺ Treg, FOXP3 ⁺ and CD25 ⁺ Treg, or both in a microenvironment, a matrix, stroma, a lymph node or combinations thereof. A microenvironment as referred to herein can be a cancer or tumor microenvironment. A matrix as referred to herein can be an extracellular matrix.

[0172] In some embodiments, a FOXP3-associated condition or disease comprises an immunosuppression. Examples of an immunosuppression can include a Treg exhibiting immunosuppressive activity, presence of an immunosuppressive microenvironment, presence of an immunosuppressive matrix, or any combination thereof.

[0173] In some embodiments, a FOXP3 -associated condition or disease comprises a cancer. In some embodiments, cancer can include conditions that generate tumors as well as conditions that do not produce tumors such as hematological malignancies. The tumors may be present in a variety of locations in a subject including breast, lungs, liver, stomach, skin, pancreas and the like. Examples of cancer include a: lung cancer, non-small cell lung carcinoma (NSCLC), small-cell lung carcinoma (SCLC), squamous cell carcinoma (SCC), head and neck cancer, head and neck squamous cell carcinoma (HNSCC), gastrointestinal cancer, large intestinal cancer, small intestinal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, liver cancer, hepatocellular carcinoma (HCC), esophageal cancer, pancreatic cancer, pancreatic adenocarcinoma, biliary tract cancer, gastric cancer, urothelial cancer, breast cancer, triple -negative breast cancer (TNBC), ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, mesothelioma, sarcomas (e.g. epitheloid, rhabdoid and synovial), chordoma, renal cancer, renal cell carcinoma (RCC), brain cancer, neuroblastoma, glioblastoma, skin cancer, melanoma, basal cell carcinoma, merkel cell carcinoma, blood cancer, hematopoetic cancer, myeloma, multiple myeloma (MM), B cell malignancies, lymphoma, B cell lymphoma, Hodgkin lymphoma, T cell lymphoma, leukemia, or acute lymphocytic leukemia (ALL), or any combination thereof.

[0174] In certain embodiments, the B-cell lymphoma is a non-Hodgkin's B-cell lymphoma. Examples of non-Hodgkin's B-cell lymphoma of certain embodiments that can be treated with compositions herein include, but are not limited to, diffuse large B cell lymphoma (DLBCL), activated B-cell lymphoma (ABC- DLBCL), germinal center B-cell lymphoma (GCB DLBCL), follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis.

[0175] In certain embodiments, the T-cell lymphoma that can be treated with compositions herein include, but are not limited to, peripheral T-cell lymphoma, and anaplastic large cell lymphoma (ALCL).

[0176] In certain embodiments, the leukemia that can be treated with compositions herein includes, but is not limited to, acute lymphocytic leukemia (ALL).

[0177] In certain embodiments, breast cancer can have more of the following characteristics: Androgen Receptor positive, dependent on androgen for growth; Estrogen Receptor (ER) negative, independent of estrogen for growth; Progesterone Receptor (PR) negative, independent of progesterone for growth; or Her2/neu negative. In certain embodiments, the breast cancer is ER, PR, and HER2 triple negative (ER“, PR”, HER2“). In certain embodiments, the breast cancer can be triple negative and AR positive (ER“, PR“, HER2“, AR ⁺ ). In certain embodiments, the breast cancer can be ER negative and AR positive (ER“, AR ⁺ ). In certain embodiments, the breast cancer can be ER positive and AR positive (ER ⁺ , AR ⁺ ). In certain embodiments, the breast cancer can be apocrine. Apocrine breast cancers can be “triple negative”, meaning that the cells do not express ER, PR, or HER2 receptors, and occasionally, AR positive. In certain embodiments, an apocrine breast cancer can be ER, PR, and HER2 triple negative and AR positive (ER“, PR”, HER2“, AR ⁺ ). In certain embodiments, an apocrine breast cancer can be ER negative and AR positive (ER“, AR ⁺ ). In certain embodiments, an apocrine breast cancer can originate from the sweat gland of the breast. In certain embodiments, an apocrine breast cancer can be a ductal cancer or cancer cell of the breast. In certain embodiments, an apocrine breast cancer can have any one or more of the following features: a large amount of eosinophilic granular cytoplasm, well-defined margins, large vesicular nuclei, a nuclear to cytoplasmic ratio of about 1:2, and/or accumulations of secreted granules in the apical cytoplasm known as apical snouts. In certain embodiments, the breast cancer can be an ER negative and AR positive (ER“, AR ⁺ ) molecular apocrine breast cancer. In certain aspects, an ER negative and AR positive (ER“, AR ⁺ ) molecular apocrine breast cancer can further be PR positive, PR negative, HER2 negative, or HER2 positive. In certain embodiments, the breast cancer can be HER2 positive. In certain embodiments, the breast cancer can be PR positive. In certain embodiments, the breast cancer can be ER positive. Breast cancer can be identified as positive or negative with respect to hormone receptors, such as ER, PR, or HER2 by standard histological techniques. For example, in some embodiments histological breast cancer samples can be classified as “triple negative” (ER“, PR“, HER2“) when less than 1% of cells demonstrate nuclear staining for estrogen and progesterone receptors, and immunohistochemical staining for HER2 shows a 0, 1-fold, or a 2-fold positive score and a FISH ratio (HER2 gene signals to chromosome 17 signals) of less than 1.8 according to the relevant American Society of Clinical Oncology (ASCO) and/or College of American Pathologists (CAP) guidelines.

[0178] In some embodiments, cancers treated by compositions and methods disclosed herein can include breast cancer, cervical cancer, esophageal cancer, hepatocellular carcinoma, lung cancer, melanoma, multiple myeloma, pancreatic adenocarcinoma, renal cell carcinoma, a kidney cancer, or any combination thereof. In some embodiments, cancers treated by compositions and methods disclosed herein can include a breast cancer, a cervical cancer, a melanoma, a kidney cancer, or any combination thereof.

[0179] In some embodiments, methods disclosed herein can inhibit the expression of FOXP3 in a: Treg cell, microenvironment, stroma, Treg infdtrated tumor, immune cell, lymphoid tissue, lymph node, intra- tumoral cell, or any combination thereof by: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%,

2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,

9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%,

17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,

32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 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%, or more, relative to a non-RTSM treated control.

[0180] In some embodiments, methods disclosed herein can reduce or inhibit an: immunosuppression,

Treg immunosuppressive activity, immunosuppressive microenvironment, immunosuppressive matrix, or any combination thereof by: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%,

11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%,

19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,

40%, 45%, 50%, 55%, 60%, 65%, 70%, 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%, or more, relative to anon-

RTSM treated control.

10181] In some embodiments, methods disclosed herein can reduce or inhibit the number of Treg cells by:

0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%,

4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%,

12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%,

21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%,

55%, 60%, 65%, 70%, 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%, or more, relative to a non-RTSM treated control. In certain instances, a Treg cell can be, at least initially, a FOXP3 ⁺ cell or a T cell that exhibits immunosuppressive activity or both.

[0182] In some embodiments, methods disclosed herein can reduce or inhibit a cancer, cancer cell proliferation, tumor growth, metastasis, or any combination thereof, by: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3%, 3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%,

6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%,

15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,

27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 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%, or more, relative to a non-RTSM treated control.

[0183] In some embodiments, methods disclosed herein can increase or activate can induce or activate: anticancer immunity, antitumor immunity, anticancer immune response, antitumor immune response, immune cell activation, immune cell infdtration, inflammatory cell activation, inflammatory cell infdtration, effector immune cell activation, effector immune cell infdtration, T cell activation, T cell infiltration, CD8 ⁺ T cell activation, CD8 ⁺ T cell activation infiltration, NK cell activation, NK cell infiltration, macrophage activation, macrophage infiltration, dendritic cell activation, dendritic cell infiltration, inflammation, inflammatory cytokine expression, inflammatory chemokine expression, or any combination thereof by: 0.1%, 0.2% 0.3% 0.5%, 0.7%, 0.9%, 1%, 1.01%, 1.5%, 2%, 2.01%, 2.5%, 3% _;

3.01%, 3.5%, 4%, 4.01%, 4.5%, 5%, 5.01%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%,

11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,

40%, 45%, 50%, 55%, 60%, 65%, 70%, 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%, or more, relative to anon- RTSM treated control.

[0184] In some embodiments, methods disclosed herein further comprises evaluating the subject prior to RTSM administration to determine whether the subject is suitable for the treatment. In some embodiments, a subject suitable for treatment can be a subject comprising a FOXP3 ⁺ Treg, an immunosuppression, or a cancer, or any combination thereof.

Methods of Administration

[0185] As disclosed in further detail below, the formulations or preparations herein may be given orally, parenterally, systemically, topically, rectally or intramuscular administration. They can be given in a form suitable for each administration route. For example, they can be administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.

[0186] Regardless of the route of administration selected, formulations herein may conveniently be presented in unit dosage form. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0. 1 percent to about ninety-nine percent of active ingredient, from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.

[0187] The selected dosage level will depend upon a variety of factors including the activity of the particular RTSM herein, the route of administration, the time of administration, the rate of excretion or metabolism of the particular RTSM being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular oligomer employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0188] In some embodiments, a threshold dose can be a dose at which dose of an RTSM at which exon skipping, for example in FOXP3, for example skipping exon 8, can be observed. In some of these instances, increasing the dosage of the RTSM can result in the production of mRNA which can lack the skipped exon, for example exon 8 in a FOXP3 mRNA. In some instances, a dose of RTSM will be reached beyond which no additional production of exon skipped mRNA, for example exon 8 skipped FOXP3 mRNAs are produced. In some embodiments, administration of an RTSM can be in a dose dependent manner.

[0189] In general, a suitable daily dose of a compound herein will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular, intramuscular and subcutaneous doses of the compounds herein for a subject, when used for the indicated effects, will range from about 0.0001 to about 100 mg per kilogram of body weight of the subject per day. [0190] In some embodiments wherein an RTSM is an ASO, doses of an ASO herein can be generally administered from about 0.001 mg/kg to about 1000 mg/kg, wherein mg is mg of RTSM and kg is the body weight of the subject. For example, 0.001 mg/kg to about 1 mg/kg, 1-20 mg/kg, 20-40 mg/kg, 40-60mg.kg, 60-80 mg/kg, or 80-100 mg/kg. For i.v. administration, doses can be from about 0.5 mg to 100 mg/kg. In some embodiments, an ASO can be administered at doses of about: 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 21 mg/kg, 22mg/kg, 23 mg/kg, 24mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg 50 mg/kg, 51 mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57 mg/kg, 58 mg/kg, 59 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, including all integers in between.

[0191] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain situations, dosing is one administration per day. In certain embodiments, dosing is one or more administration per every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, as needed, to maintain the desired expression of a FOXP3 pre-mRNA and/or a FOXP3 protein. An RTSM may be administered in continuously or in cycles.

[0192] In some embodiments, an RTSM of the present can be administered, generally at regular intervals (e.g., daily, weekly, biweekly, monthly, bimonthly). An RTSM may be administered at regular intervals, e.g., daily; once every two days; once every three days; once every 3 to 7 days; once every 3 to 10 days; once every 7 to 10 days; once every week; once every two weeks; once monthly. For example, an RTSM may be administered once weekly by intravenous infusion. An RTSM may be administered intermittently over a longer period of time, e.g., for several weeks, months or years. For example, an RTSM may be administered once every one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months. In addition, an RTSM may be administered once every one, two, three, four or five years. Administration may be followed by, or concurrent with, co-administration with a second agent, for example with an antibiotic, steroid or other therapeutic agent. The treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.

[0193] When co-administered with one or more other therapies, an RTSM of the disclosure may be administered either simultaneously with the other treatment(s), or sequentially in any order and can be temporally spaced up to several days apart.

Pharmaceutical Compositions and Dosage Forms

[0194] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising an RTSM described herein and a carrier thereof for administration in a subject.

[0195] In certain embodiments, the pharmaceutically acceptable compositions comprise a therapeutically- effective amount of one or more of an RTSM, formulated together with one or more pharmaceutically acceptable: carriers (additives) and/or diluents. In some embodiments, when an RTSM herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99%, or 10 to 30% of an RTSM in combination with a pharmaceutically acceptable carrier.

[0196] A pharmaceutical composition of the present disclosure can be delivered, e.g., subcutaneously or intravenously with a standard needle and syringe or a pen delivery device. The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. The injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying an RTSM herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be fdled in an appropriate ampoule.

[0197] Compositions of the present disclosure can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The amount of an RTSM disclosed herein contained can be about 5 to about 500 mg per dosage form in a unit dose. In one embodiment, an RTSM can be contained in about in about 5 to about 100 mg, for example for a parental dosage form. In other embodiments, an RTSM can be contained in about 10 to about 250 mg for the other dosage forms.

[0198] For example, oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets can be used as solid dosage forms. These can be prepared, for example, by mixing the RTSM, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0199] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. In some embodiments, pharmaceutical formulations and medicaments may be prepared as liquid suspensions or aqueous solutions, for example, using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, or any combination of these. In some embodiments, pharmaceutical compositions can be prepared in a lyophilized form. The lyophilized preparations can comprise a cryoprotectant known in the art. The term “cryoprotectants” as used herein generally includes agents, which provide stability to the protein from freezing-induced stresses. Examples of cryoprotectants include polyols such as, for example, mannitol, and include saccharides such as, for example, sucrose, as well as including surfactants such as, for example, polysorbate, poloxamer or polyethylene glycol, and the like. Cryoprotectants also contribute to the tonicity of the formulations. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or par- enteral administration.

[0200] As noted above, suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, com oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, iso- propyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly (ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.

[0201] For nasal administration, the pharmaceutical formulations and medicaments may be a spray or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bio- availability modifiers or any combination of these. A propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

[0202] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which can be prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. Alternatively, sterile oils may be employed as solvents or suspending agents. In some embodiments, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri -glycerides.

[0203] For injection, the pharmaceutical formulation and/ or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers or any combination of these.

[0204] For rectal administration, the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum. Rectal suppositories are prepared by mixing one or more compounds herein with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories. Water, saline, aqueous dextrose and related sugar solutions, and glycerols may be employed in the preparation of suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.

[0205] The concentration of an RTSM in these compositions can vary widely, e.g., from less than about 10%, least about 25% to as much as 75% or 90% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

[0206] In some embodiments, pharmaceutical compositions comprising an RTSM described herein can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0207] Pharmaceutical compositions are optionally manufactured such as, by way of example only, by means of mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0208] In certain embodiments, compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. In other embodiments, compositions may also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0209] In some embodiments, sustained-release preparations can be used. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing an RTSM of the present disclosure, in which the matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thiodisulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

[0210] In some embodiments, an RTSM can be administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier. In some embodiments, an RTSM can be linked with a viral vector, e.g., to render an RTSM more effective or increase transport across the blood-brain barrier. For example, delivery of agents can be by administration of an adenovirus vector to motor neurons in muscle tissue. Delivery of vectors directly to the brain, include but are not limited to the striatum, the thalamus, the hippocampus, or the substantia nigra.

[0211] In embodiments, an RTSM can be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In some embodiments, an RTSM can be coupled to a substance that promotes penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In some embodiments, osmotic blood brain barrier disruption can be assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myoinositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. In some embodiments, the composition can be encapsulated in glucose -coated polymeric nanocarriers.

Second Agent

[0212] The compositions herein may be administered alone or in combination with another therapeutic. The additional therapeutic may be administered prior, concurrently, consecutively, or subsequently to the administration of the composition.

[0213] The compositions disclosed herein, comprising an RTSM, described herein, can also contain more than one active agent as necessary for the particular indication being treated, such as those with complementary activities that do not adversely affect each other. For example, the composition can further comprise an anti-inflammatory, a therapeutic protein, a steroid, an analgesic, a non-steroidal antiinflammatory, a corticosteroid, an immune system modulator, an additional RTSM, more than one of the foregoing, or any combination thereof. [0214] In certain embodiments, a second agent can be an additional RTSM. In some embodiments, a second RTSM may target the same target sequence, or a portion of the same target sequence, as a first RTSM. In some embodiments, a second RTSM may target a different target sequence, or at least a portion of a different target sequence, as a first RTSM. In some embodiments a second RTSM can target pre-mRNA or mRNA.

[0215] In certain embodiments, a second agent can be an immune system modulator including but not limited to TLR agonists (e.g. MEDI9197) and STING agonists (e.g. MK-1454); inhibitors of immunoinhibitory mediators including but not limited to CD39 and CD73 inhibitors (e.g. oleclumab), IDO1 inhibitors (e.g. epacadostat), and arginase inhibitors (e.g. INCB001158); activators of T cell costimulatory receptors including but not limited to CD 137 agonists (e.g. urelumab, utomilumab), CD27 agonists (e.g. varlimumab), and CD40 agonists (e.g. MEDI5083); inhibitors of T cell inhibitory receptors including but not limited to LAG3 inhibitors (e.g. relatlimab), TIM3 inhibitors (e.g. LY3321367), and TIGIT inhibitors (e.g. tiragolumab); activators of Treg inhibitory receptors including but not limited to GITR agonists (e.g. MEDI1873); NK cell activation strategies including but not limited to NKG2a (e.g. monalizumab); cancer vaccines (e.g. Sipuleucel-T); immune checkpoint inhibitors; and immunogenic killing of the tumor including but not limited to oncolytic viruses, CAR T-cells, radiation, photodynamic therapy, and chemotherapy (e.g. anthracyclines, oxaliplatin etc).

[0216] In certain embodiments, the RTSM described herein can be used to treat a second agent in addition to or instead of being used to treat a subject, wherein the RTSM treated second agent can therefore be used to treat the subject. For example, an RTSM described herein can be used to treat CAR T-cells, thereby inducing one or more CAR T-cells with downregulated FOXP3 to be used in treating a subject in need thereof. In certain embodiments, the RTSM and second agent are synergistic.

[0217] In certain embodiments, an immune system modulator can also be immune checkpoint inhibitors that directly or indirectly inhibits, partially or completely, an immune checkpoint pathway. Immune checkpoint pathways include PD-1/PD-L1, CTLA4/B7-1, TIM-3, LAG3, By-He, H4, HAVCR2, ID01, and CD276 and VTCN1. Non-limiting examples of immune checkpoint inhibitors include fully human monoclonal antibodies, such as BMS-936558/MDX-1106, BMS-936559/MDX-1105, ipilimumab/Yervoy, and tremelimumab; humanized antibodies, such as CT-011 and MK-3475; and fusion proteins, such as AMP -224. Examples can also include pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq).

[0218] In some embodiments, the second agent may be formulated with the compositions described herein or separately co-administered.

[0219] Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients of the compositions thereof described herein can be trapped in drug delivery systems which maximize delivery into a cell, or into a cellular component, such as nuclear delivery. The active ingredients of the compositions thereof described herein can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microparticle, microemulsions, nano-particles and nanocapsules) or in macroemulsions. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome. Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vehicles to target the embodiments to a particular tissue as well as to increase the half life of the composition. Liposomes containing the RTSM, the second active compound or both can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through fdters of defined pore size to yield liposomes with the desired diameter. RTSMs herein can be conjugated to liposomes via a disulfide interchange reaction. In another example, the drug delivery system can be a synthetic nanoparticles such as, lipid-based nanoparticles (e.g, comprising a cationic or ionizable lipid, cholesterol, a helper lipid, and a polyethylene glycol (PEG)-lipid and include, but are not limited to cKK- E12, Cl 2-200, ALC-0315, SM-102, FTT5, DLin-MC3-DMA, Intellia LP01, DLin-KC2DMA)) or polymer- or dendrimer- based nanoparticles (e.g., poly(ethylenimine) (PEI), poly(l-lysine) (PLL), poly(beta-amino-ester) (PBAE), poly(lactic-co-glycolic acid) (PLGA), or PAMAM, poly (amidoamine). The second agent can also be optionally contained within the drug delivery system.

Further Embodiments

[0220] In some embodiments, described herein, is a method of decreasing expression of full length FOXP3 protein comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent.

[0221] In some embodiments, described herein, is a method of treating a cancer comprising administering a therapeutic agent that binds or hybridizes to a portion of a FOXP3 pre-mRNA to a subject, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent.

[0222] In some embodiments, described herein, is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds or hybridizes to a portion of a FOXP3 pre-mRNA.

[0223] In some embodiments, described herein, is a method of producing a nonfunctional form of FOXP3 protein comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent.

[0224] In some embodiments, described herein, is a method of inhibiting regulatory T cells (Tregs) comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre- mRNA that is spliced in the absence of the therapeutic agent, wherein the Tregs are inhibited.

[0225] As used herein, the terms "ASO" and "antisense oligomer" are used interchangeably and refer to an oligomer such as a polynucleotide, comprising nucleobases that hybridizes to a target nucleic acid (e.g., a FOXP3 pre-mRNA) sequence by Watson-Crick base pairing or wobble base pairing (G-U). The ASO may have exact sequence complementary to the target sequence or near complementarity (e.g., sufficient complementarity to bind or hybridize to the target sequence and modulating splicing at a splice site). ASOs are designed so that they bind (or hybridize) to a target nucleic acid (e.g., a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. In some cases, if they hybridize to a site other than the intended (targeted) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (to a few sites other than a target nucleic acid). Design of an ASO can take into consideration the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence in other locations in the genome or cellular pre- mRNA or transcriptome, such that the likelihood the ASO will bind or hybridize other sites and cause "off- target" effects is limited.

[0226] In some embodiments, ASOs "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA. In some embodiments, ASO specifically hybridize at 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 37 °C or more. In some cases, such hybridization occurs with a Tm substantially greater than 37 °C, at least 50 °C, or between 60 °C to approximately 90 °C. Such hybridization can correspond to stringent hybridization conditions. As used herein "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non- specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide. For example, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ⁺ ion, such as, about 0.01 to 1.0 M Na ⁺ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. lx SSC at 60°C.

[0227] Oligomers, such as oligonucleotides, are "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double -stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree to which one polynucleotide is complementary with another) is quantifiable in terms of the proportion (e.g., the percentage) of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs can comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an ASO in which 18 of 20 nucleobases of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered together or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs.

[0228] An ASO need not hybridize to all nucleobases in a target sequence and the nucleobases to which it does hybridize may be contiguous or noncontiguous. ASOs may hybridize over one or more segments of a pre-mRNA transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure may be formed). In certain embodiments, an ASO hybridizes to noncontiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobase(s) to which the ASO does not hybridize.

[0229] The ASOs described herein comprise nucleobases that are complementary to nucleobases present in a targeted portion of a pre-mRNA. The term ASO embodies oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridizing to a complementary nucleobase on a target mRNA but does not comprise a sugar moiety, such as a peptide nucleic acid (PNA). The ASOs may comprise naturally-occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the preceding. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and/or having a modified backbone. In some embodiments, all of the nucleotides of the ASO are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions are, for example, as described herein.

[0230] One or more nucleobases of an ASO may be any naturally occurring, unmodified nucleobase, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase such that it is capable of hydrogen bonding with a nucleobase present on a target pre-mRNA.

[0231] Any of the ASOs or any component of an ASO (e.g., a nucleobase, sugar moiety, backbone) described herein may be modified in order to achieve desired properties or activities of the ASO or reduce undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity to a target sequence on a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (e.g., RNase H); improve uptake of the ASO into a cell and/or into the nucleus of a cell; alter the pharmacokinetics or pharmacodynamics of the ASO; and/or modulate the half-life of the ASO.

[0232] In some embodiments, the ASOs are comprised of 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOs comprised of such nucleotides are especially well-suited to the methods disclosed herein; oligomers having such modifications have been shown to have significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable, for example, for oral delivery in some embodiments described herein.

[0233] Methods of synthesizing ASOs will be known to one of skill in the art. Alternatively or in addition, ASOs may be obtained from a commercial source.

[0234] Described, herein are compositions and methods useful for treating cancer. In some embodiments, these compositions and methods result in a truncated FOXP3 protein. In some embodiments, these compositions and methods result in a decrease in the wild-type FOXP3 protein. In some embodiments, the compositions and methods result in modulating the splicing of FOXP3 RNA. In some embodiments, the compositions and methods result in a FOXP3 RNA lacking exon 8.

[0235] In some embodiments, disclosed herein is a method of decreasing expression of full length FOXP3 protein comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent. In some embodiments, the therapeutic agent causes skipping of exon 8 in the pre-mFOXP3 RNA. In some embodiments, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some embodiments, the 5’ splice site sequence is in intron 8 of the FOXP3 pre-mRNA. In some embodiments, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some embodiments, the therapeutic agent is an antisense oligonucleotide (ASO). The ASO comprises a sequence that is at least about 80% identity to SEQ ID NO: 123. In some embodiments, the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 123. In some embodiments, the ASO comprises a sequence that is at least about 90% identity to SEQ ID NO: 123. In some embodiments, the 5’ splice site is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to SEQ ID NO: 93. In some embodiments, the 5’ splice site or the ASO comprises a sequence with at least 95% sequence identity to the reverse complement of SEQ ID NO: 93.

[0236] In some embodiments, disclosed herein is a method of treating a cancer comprising administering a therapeutic agent that binds or hybridizes to a portion of a FOXP3 pre-mRNA to a subject, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent. In some embodiments, the cancer is selected from the group consisting of breast cancer, cervical cancer, esophageal cancer, hepatocellular carcinoma, lung cancer, melanoma, multiple myeloma, pancreatic adenocarcinoma, and renal cell carcinoma.

[0237] In some embodiments, disclosed herein is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds or hybridizes to a portion of a FOXP3 pre-mRNA. In some embodiments, the therapeutic agent is an antisense oligomer (ASO) and wherein the ASO comprises a sequence that is at least about 80%, 85%, 90%, 95%, 97%, or 100% identity to SEQ ID NO: 123.

[0238] In some embodiments, the ASO comprises a backbone modification comprising a phosphorothioate linkage or a phosphorodiamidate linkage. In some embodiments, the ASO comprises a phosphorodiamidate morpholino, a locked nucleic acid, a peptide nucleic acid, a 2'-O-methyl, a 2'-Fluoro, or a 2'-O- methoxyethyl moiety. In some embodiments, the ASO comprises at least one modified sugar moiety. In some embodiments, each sugar moiety is a modified sugar moiety. In some embodiments, the ASO consists of from 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases, or 12 to 15 nucleobases. In some embodiments, the ASO is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, complementary to the targeted portion of the FOXP3 pre-mRNA or mRNA encoding the FOXP3 protein. In some embodiments, the method further comprises assessing FOXP3 pre-mRNA or mRNA or FOXP3 protein expression. In some embodiments, the cells are ex vivo.

[0239] In some embodiments, the therapeutic agent is administered to the subject by intravitreal injection, intrathecal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, subretinal injection, intracerebroventricular injection, intramuscular injection, topical application, or implantation.

[0240] In some embodiments, the therapeutic agent is administered with one or more agents capable of promoting penetration of the subject antisense oligonucleotide across the blood-brain barrier. In some embodiments, the therapeutic agent is linked with a viral vector, e.g., to render the therapeutic agent more effective or increase transport across the blood-brain barrier. For example, delivery of agents can be by administration of an adenovirus vector to motor neurons in muscle tissue. Delivery of vectors directly to the brain, e.g., the striatum, the thalamus, the hippocampus, or the substantia nigra, can also be by administration of an adenovirus vector into cells of the central nervous system, including the brain.

[0241] In embodiments, the therapeutic agent is linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In some embodiments, the therapeutic agent is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, e.g., an antibody to the transferrin receptor. In some embodiments, osmotic blood brain barrier disruption is assisted by infusion of sugars, e.g., meso erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, dulcitol, myo-inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) ribose, adonitol, D(+) arabitol, L(-) arabitol, D(+) fucose, L(-) fucose, D(-) lyxose, L(+) lyxose, and L(-) lyxose, or amino acids, e.g., glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood brain barrier penetration may differ depending on the administration route, the neuron type, and the material being delivered. [0242] In some embodiments, the therapeutic agent is encapsulated in glucose-coated polymeric nanocarriers.

[0243] In some embodiments, disclosed herein is a method of decreasing expression of full length FOXP3 protein comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent. In some methods disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre-mRNA. In some methods disclosed herein, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some methods disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre- mRNA. In some methods disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some methods disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some methods disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some methods disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123.

[0244] In some methods disclosed herein is a method of treating a cancer in a subject comprising administering a therapeutic agent in a therapeutic amount to the subject that binds or hybridizes to a portion of a FOXP3 pre-mRNA in a subject, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent, which can, for example, be shown in an in vitro assay. In some methods disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre-mRNA. In some methods disclosed herein, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some methods disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre-mRNA. In some methods disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some methods disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some methods disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some methods disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123. In some methods disclosed herein, the cancer is selected from the group consisting of a: breast cancer, cervical cancer, esophageal cancer, hepatocellular carcinoma, lung cancer, melanoma, multiple myeloma, pancreatic adenocarcinoma, renal cell carcinoma, or any combination thereof. In some methods disclosed herein, the cancer comprises a breast cancer.

[0245] In some embodiments, disclosed herein is a pharmaceutical composition comprising a therapeutic agent and a pharmaceutically acceptable excipient, wherein the therapeutic agent binds or hybridizes to a portion of a FOXP3 pre-mRNA. In some compositions disclosed herein, the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent. In some compositions disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre- mRNA. In some compositions disclosed herein, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some compositions disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre-mRNA. In some compositions disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some compositions disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some compositions disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some compositions disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123.

[0246] In some embodiments, disclosed herein is a method of producing a nonfunctional form of FOXP3 protein comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent. In some methods disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre-mRNA. In some methods disclosed herein, the therapeutic agent binds or hybrizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some methods disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre- mRNA. In some methods disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some methods disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some methods disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some methods disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123.

[0247] In some embodiments, disclosed herein is a method of inhibiting regulatory T cells (Tregs) activity comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre- mRNA that is spliced in the absence of the therapeutic agent, wherein the Treg activity is inhibited relative to Treg activity in the absence of the therapeutic agent. In some methods disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre-mRNA. In some methods disclosed herein, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some methods disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre- mRNA. In some methods disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some methods disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some methods disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some methods disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123.

[0248] In some embodiments, disclosed herein is a method of reducing a number of regulatory T cells (Tregs) comprising contacting a FOXP3 pre-mRNA with a therapeutic agent that binds or hybridizes to a portion of the FOXP3 pre-mRNA, whereby the therapeutic agent causes skipping of an exon in the FOXP3 pre-mRNA that is spliced in the absence of the therapeutic agent, wherein the number of Tregs is reduced relative to the number of Tregs in the absence of the therapeutic agent. In some methods disclosed herein, the therapeutic agent causes skipping of exon 8 in the FOXP3 pre-mRNA. In some methods disclosed herein, the therapeutic agent binds or hybridizes to a 5’ splice site sequence in the FOXP3 pre-mRNA. In some methods disclosed herein, at least a portion of the 5’ splice site sequence is in intron 8 of the FOXP3 pre-mRNA. In some methods disclosed herein, the 5’ splice site comprises a sequence with at least 95% sequence identity to SEQ ID NO: 93. In some methods disclosed herein, the therapeutic agent is an antisense oligonucleotide (ASO). In some methods disclosed herein, the ASO comprises a sequence that is at least about 80% identical to SEQ ID NO: 123. In some methods disclosed herein, the ASO comprises a sequence that is at least about 90% identical to SEQ ID NO: 123.

[0249] In some embodiments, nucleobases corresponding to the abbreviations in various nucleobase sequences disclosed herein can be found in TABLE 8A below.

TABLE 8A: Nucleobase Abbreviations

[0250] In some embodiments, amino acids corresponding to the abbreviations in various polypeptide sequences disclosed herein can be found, for example, in TABLE 8B below.

TABLE 8B: Amino Acid Abbreviations

EXAMPLES

[0251] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. ASOs described herein can be synthesized using synthetic techniques, using methods described herein or combinations of both. Alternatively, ASOs are available commercially from various sources, including Integrated DNA Technologies (IDT) and GeneTools, LLC.

Example 1: FOXP3 splicing modulation in breast cancer cells and cutaneous T cell lymphoma

[0252] HCC712 cells were plated at a concentration of 800,000 cells/well in a 6-well plate and cultured according to ATCC recommendations for 24 hours.

[0253] ASOs with morpholino-modified backbones (i.e., AMOs) were transfected individually into each well using EndoPorter at 6pM. Each AMO was added to the wells to achieve final concentrations of IpM, 5pM, and 20pM of the FOXP3 E8 5’ss AMO and IpM, 5pM, and 20pM of the CONT AMO. FOXP3 E8 5’ss is an AMO that targets the 5’ splice site of intron 8 of the FOXP3 pre-mRNA. CONT is an AMO that targets a human beta-globin intron mutation that causes the disease beta-thalassemia. The sequences of the AMOs used are set forth below in TABLE 9, noting that each sugar in the sequence in TABLE 9 is replaced with a morpholino group.

TABLE 9: Example 1 AMO sequences

[0254] MJ (Gi l) cells were electroporated at 0.75xl0 ⁶ cells per electroporation reaction with increasing concentrations 1 pM, 5 pM, and 20 pM final concentration) of the FOXP3 E8 5’ss and CONT AMOs using Lonza 4D-Nucleofector™ X Unit and Lonza Cell Line Optimization 4D-Nucleofector X Kit The cells from each electroporation reaction were then plated in 1 well/reaction of a 24-well tissue culture plate.

[0255] Samples from both the HCC712 cells and the MJ (Gi l) cells were harvested after 48hrs. RNA was prepared from each sample, using RNeasy plus mini kits. RNA was quantitated and cDNA was made with Ipg total RNA using SuperScriptTM III First-Strand Synthesis SuperMix.

[0256] RT-PCR reactions were performed using Invitrogen Platinum SuperFI DNA Polymerase, with the primers set forth in TABLE 10.

TABLE 10: Example 1 RT-PCR Primers

[0257] After the initial denaturation step (98°C, 30s), 30 cycles were performed (98°C 10s, 59.5°C 10s, 72°C 15s), followed by a final extension step (72°C 5min). The PCR products from both the HCC712 and the MJ (G11) samples were run on an agarose gel (2%) shown in FIG. 1 and FIG. 2 respectively.

[0258] The band corresponding to the expected FOXP3 E7E8E9 isoform was cut from lanes 4 and 5 (upper band) and the band corresponding to the expected FOXP3 E7E9 isoform was cut from lane 7 (lower band) from AMO-treated HCC712 and MJ (Gi l) cells (FIG. 1 and FIG. 2 respectively). Gel-extracted purified DNA samples were sent for sequencing with the forward and reverse primers identified above (SEQ ID NOs: 134 and 135). Sequencing shown in FIG. 3A and 3B was performed in HCC712 cells as seen in FIG. 1 and sequencing from FIG. 4A and 4B was performed on MJ (G11) cells as seen in FIG. 2. Isoform FOXP3 E7E8E9 corresponds to the expected product that contains exons 7, 8 and 9. The RT sequences of isoform FOXP3 E7E8E9 can be seen in SEQ ID NO: 136 (Forward) and SEQ ID NO: 138 (Reverse). Isoform FOXP3 E7E9 corresponds to the expected product that contains exons 7 and 9, exon 8 having been skipped. The RT sequences of isoform FOXP3 E7E9 can be seen in SEQ ID NO: 137 (Forward) and SEQ ID NO: 139 (Reverse). Visual representation of the sequences set forth in TABLE 11 are shown in FIG. 3A, 3B, 4A and FIG. 4B. The sequence corresponding to exon 8 is in bolded underline in FIG. 3A and FIG. 4A. Absence of exon 8 can be seen in FIG. 3B and FIG. 4B.

TABLE 11: Example 1 FOXP3 Isoforms

Example 2: FOXP3 protein modulation in cutaneous T cell lymphoma

[0259] MJ (G11) cells were electroporated at 0.75xl0 ⁶ cells per electroporation reaction with increasing concentrations (1 pM, 5 pM, and 20 pM final concentration) of the FOXP3 E8 5’ss (SEQ ID NO: 123) and CONT ASOs (SEQ ID NO: 133) using Lonza 4D-Nucleofector™ X Unit and Lonza Cell Line Optimization 4D-Nucleofector X Kit. The cells from each electroporation reaction were then plated in 1 well/reaction of a 24-well tissue culture plate.

[0260] Samples were harvested in RIPA buffer after 48hrs. Protein was quantitated by BCA assays and 30pg of each sample was loaded on an SDS-PAGE gel. The following antibodies were used to probe the SDS-PAGE gel: Mouse anti-human FOXP3 (clone 236A/E8) antibody was used to detect all isoforms of human FOXP3 (FOXP3fl, FOXP3A3, and FOXP3A3A8) and Rabbit anti-human Vinculin (clone E2E10V) antibody was used as a loading control. The results from the BCA Assay are shown in FIG. 5. Quantitation of the FOXP3 isoforms are shown in FIG. 6.

Example 3: AMO FOXP3 protein modulation in cutaneous T cell lymphoma cells

[0261] MJ (Gi l) cells were transfected at 0.75x10 ⁶ cells per well in a 24-well tissue culture plate with 1 pg of plasmid DNA of a gain-of-signal FOXP3-Nanoluc minigene-Firefly luciferase dual reporter (pGL4.50[luc2/P2A/Hygro/FOXP3 E7E8fsE9 WT 5’ss-Nanoluc] minigene) or control construct (pGL4.50[luc2/P2A/Hygro/FOXP3 E7E8fsE9 Null 5’ss-Nanoluc] minigene) for 24 hours using X- tremeGENE™ HP DNA Transfection Reagent. The cells transfected with the gain-of-signal FOXP3- Nanoluc minigene reporter were electroporated at 0.75xl0 ⁶ cells per electroporation reaction with increasing concentrations (1 pM, 5 pM, and 20 pM final concentration) of the FOXP3 E8 5’ss AMO (SEQ ID NO: 113) and CONT (Standard Control) (SEQ ID NO: 133) AMOs using Lonza 4D-NucleofectorTM X Unit and Lonza Cell Line Optimization 4D-Nucleofector X Kit. The cells from each electroporation reaction were then plated in 1 well/reaction of a 24-well tissue culture plate. The cells were harvested 48 hours after electroporation and plated at O. lxlO ⁶ cells per well in an opaque white 96-well assay plate. Nanoluc and Firefly luciferase signal was read using the Nano-Gio® Dual-Luciferase® Reporter Assay System. Nanoluc signal values were normalized to Firefly luciferase values. FOXP3-Nanoluc gain of signal in AMO transfected can be seen in FIG. 7.

[0262] Some cells from FIG. 7 were also harvested for RNA isolation using RNeasy plus mini. RNA was quantitated and cDNA was made with Ipg total RNA using SuperScriptTM III First-Strand Synthesis SuperMix. RT-PCR reactions were performed using Invitrogen Platinum SuperFI DNA Polymerase, with the primers set forth in TABLE 12.

TABLE 12: Example 3 RT-PCR Primers

[0263] After the initial denaturation step (98°C, 30s), 30 cycles were performed (98°C 10s, 59.5°C 10s, 72°C 15s), followed by a final extension step (72°C 5min). The PCR products were run on an agarose gel (2%) as depicted in FIG. 8. The band corresponding to the expected minigene-derived canonical FOXP3 isoform (FOXP3 E7E8fsE9-Nanoluc) was cut from lane 3 (upper band) and the band corresponding to the expected minigene-derived skipped FOXP3 isoform (FOXP3 E7E9-Nanoluc) was cut from lane 9 (lower band) from ASO-treated transfected MJ (Gi l) cells. Gel-extracted purified DNA samples were sent for sequencing with the forward and reverse primers identified in Table 12 (SEQ ID NOS: 140 and 141) above.

[0264] Isoform FOXP3 E7E8fsE9-Nanoluc corresponds to the expected product that contains minigene- derived exons 7, 8, and 9. The RT sequence of isoform FOXP3 E7E8fsE9-Nanoluc can be seen in SEQ ID NO: 142. FOXP3 E7E9-Nanoluc corresponds to the expected product that contains minigene-derived exons 7 and 9, minigene-derived exon 8 having been skipped. The RT sequence of isoform FOXP3 E7E9-Nanoluc can be seen in SEQ ID NO: 143. Visual representation of the sequences as set forth in TABLE 13 are shown in FIG. 9A and FIG. 9B. The sequence corresponding to exon 8 is in bolded underline in FIG. 9A. Absence of exon 8 can be seen in FIG. 9B. TABLE 13: Example 3 FOXP3 Isoforms

[0265] Next, cDNA obtained from untransfected and empty vector samples, and samples transfected with no AMO, CONT AMO, FOXP3 E8 5’ss AMO, Null 5’ss WT 5’ss minigene from the indicated bands in FIG. 8, was used to perform qPCR reactions using TaqMan™ Fast Advanced Master Mix utilizing the qPCR primers and probes in TABLE 14 below. After the initial denaturation (95°C, 2 minutes), 40 cycles were performed (95 °C Is, 60°C 20s). Relative concentration of cDNA encoding FOXP3 exon 8 can be seen in FIG. 10.

TABLE 14: Example 3 qPCR Primers/Probes Example 4: AMO FOXP3 protein modulation in Jurkat Cells

[0266] Jurkat cells were transfected at 0.75x10 ⁶ cells per well in a 24-well tissue culture plate with 1 pg of plasmid DNA of a loss-of-signal FOXP3-Nanoluc minigene reporter (WT 5’ss minigene), Null 5’ss minigene, or control construct (Expression construct) for 24 hours using X-tremeGENE™ HP DNA Transfection Reagent. The cells transfected with the loss-of-signal FOXP3-Nanoluc minigene reporter were electroporated at 0.75xl0 ⁶ cells per electroporation reaction with increasing concentrations (1 pM, 5 pM, and 20 pM final concentration) of the FOXP3 E8 5’ss AMO (SEQ ID NO: 113) and CONT (Standard Control) (SEQ ID NO: 133) AMOs using Lonza 4D-Nucleofector™ X Unit and Lonza Cell Line Optimization 4D-Nucleofector X Kit. The cells from each electroporation reaction were then plated in 1 well/reaction of a 24-well tissue culture plate. Cells were harvested 48 hours after electroporation and plated at O.lxlO ⁶ cells per well in an opaque white 96-well assay plate. Nanoluc signal was read using the Nano- Glo® Luciferase® Reporter Assay. Nanoluc signal values were normalized to the Nanoluc values of the corresponding Standard Control ASO conditions. FOXP3-Nanoluc loss of signal can be seen in FIG. 11.

[0267] Some cells from test groups as indicated in FIG. 11 were also harvested for RNA isolation. Using RNeasy plus mini kits. RNA was quantitated and cDNA was made with Ipg total RNA using SuperScriptTM III First-Strand Synthesis SuperMix. RT-PCR reactions were performed using Invitrogen Platinum SuperFI DNA Polymerase, with the primer set forth in TABLE 15.

TABLE 15: Example 4 RT-PCR Primers

[0268] After the initial denaturation step (98°C, 30s), 30 cycles were performed (98°C 10s, 59.5°C 10s, 72°C 15s), followed by a final extension step (72°C 5min). As seen in FIG. 12, the PCR products were run on an agarose gel (2%). The band corresponding to the expected minigene -derived canonical FOXP3 isoform (FOXP3 E7E8insTE9delC-Nanoluc) was cut from lane 3 (upper band) and the band corresponding to the expected minigene -derived skipped FOXP3 isoform (FOXP3 E7E9delC-Nanoluc) was cut from lane 9 (lower band) from ASO-treated transfected Jurkat cells. Gel-extracted purified DNA samples were sent for sequencing with the forward and reverse primers identified in TABLE 15 above.

[0269] Isoform FOXP3 E7E8insTE9delC-Nanoluc corresponds to the expected product that contains minigene-derived exons 7, 8 and 9. The RT sequence of isoform FOXP3 E7E8insTE9delC-Nanoluc can be seen in SEQ ID NO 153. Isoform FOXP3 E7E9delC-Nanoluc corresponds to the expected product that contains minigene-derived exons 7 and 9, minigene-derived exon 8 having been skipped. The RT sequence of isoform FOXP3 E7E9delC-Nanoluc can be seen in SEQ ID NO: 154. Visual representation of the sequences as set forth in TABLE 16 are shown in FIG. 13A and FIG. 13B. The sequence corresponding to exon 8 is in bolded underline in FIG. 13A. Absence of exon 8 can be seen in FIG. 13B. TABLE 16: Example 4 FOXP3 Isoforms

[0270] cDNA obtained from lanes in FIG. 12, was used to perform qPCR reactions using TaqMan™ Fast Advanced Master Mix using the qPCR primers and probes as indicated in TABLE 17. After the initial denaturation (95°C, 2 minutes), 40 cycles were performed (95°C Is, 60°C 20s). Relative concentration of cDNA encoding FOXP3 exon 8 can be seen in FIG. 14.

TABLE 17: Example 4 qPCR Primers/Probes