Highly efficient trans-splicing for replacement of target RNA sequences in human cells

Abstract

Disclosed is a composition comprising a trans-splicing nucleic acid comprising: (a) one or more replacement domains encoding therapeutic sequences, operatively linked thereto; (b) one or more intron domains, including a trans-splicing enhancer sequence, that promote RNA splicing of the replacement domains; and (c) one or more antisense domains that promote binding to a target RNA molecule, wherein the RNA trans-splicing reaction promotes highly efficient insertion of the replacement domains into the target RNA. Methods of making and using the compositions of the disclosure are also provided, including, but not limited to, the compositions of the disclosure that can be used to treat a disease or disorder in a patient or subject. Exemplary diseases or disorders of the disclosure include genetic and epigenetic diseases or disorders.

Description

[Technical field]

[0001] This disclosure relates generally to gene therapy, molecular biology, and compositions and methods for altering the sequence composition of RNA molecules. [Background technology]

[0002] There is a long-standing but unmet need in the art to replace disease-causing RNA sequences.The present disclosure provides compositions and methods for highly efficient replacement of specific RNA sequences in RNA molecules in human cells.Specifically, the present disclosure provides compositions and methods for replacing selected RNA sequences in target RNA using RNA trans-splicing molecules that carry sequences that enhance trans-splicing efficiency to treat disease in the context of human gene therapy.

[0003] Effective treatment of human genetic diseases requires efficient replacement of defective gene sequences in human cells. RNA trans-splicing has been proposed as a human gene therapy drug, but has not been successful in clinical trials due to its low efficiency. This disclosure describes an improvement in RNA trans-splicing molecules that can address this long-standing but unmet need. Summary of the Invention

[0004] In some embodiments, the present disclosure provides compositions comprising a trans-splicing RNA molecule comprising (a) at least one domain ("intron domain") that promotes trans-splicing, including one or more trans-splicing enhancer sequences; (b) at least one binding domain ("antisense domain") that includes or consists of a sequence complementary to a pre-mRNA present in a human cell ("target RNA"); and (c) a coding domain ("replacement domain") that is inserted into the target RNA via trans-splicing. The trans-splicing enhancer sequence increases splicing efficiency, such that the trans-splicing RNA molecule can exchange sequences in the target RNA with the replacement domain with high efficiency. In other embodiments, the present disclosure provides compositions comprising a nucleic acid sequence that encodes the trans-splicing RNA molecule.

[0005] In some embodiments, the trans-splicing enhancer sequence comprises 5'-X1X2X3X4X5X6-3', where X1 is uracil (U) or guanine (G), X2 is adenine (A), uracil (U) or guanine (G), X3 is adenine (A), uracil (U) and guanine (G), X4 is adenine (A), uracil (U), cytosine (C) or guanine (G), X5 is adenine (A), cytosine (C), uracil (U) or guanine (G), and X6 is adenine (A), uracil (U) or guanine (G).

[0006] In some embodiments, the trans-splicing enhancer sequence comprises X1X2X3X4X5X6, where X1 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X2 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X3 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X4 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X5 is selected from the group comprising adenine (A), uracil (U) and guanine (G), and X6 is selected from the group comprising uracil (U) and guanine (G).

[0007] In some embodiments, the trans-splicing enhancer sequence comprises X1X2X3X4X5X6, where X1 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X2 is selected from the group comprising uracil (U) and guanine (G), X3 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X4 is selected from the group comprising uracil (U) and guanine (G), X5 is selected from the group comprising uracil (U) and guanine (G), and X6 is selected from the group comprising uracil (U) and guanine (G).

[0008] In some embodiments, the trans-splicing enhancer sequence is immediately adjacent to the replacement domain.

[0009] In some embodiments, the trans-splicing enhancer sequence is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, The substitution domain may be 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between away in the 5' direction from the first nucleotide of the substitution domain.

[0010] In some embodiments, the trans-splicing enhancer sequence is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, The substitution domain may be 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, more than 500 nucleotides, or any number of nucleotides in between in the 3' direction away from the last nucleotide of the substitution domain.

[0011] In some embodiments, the intron domain comprises one trans-splicing enhancer sequence. In some embodiments, the intron domain comprises two or more trans-splicing enhancer sequences. In some embodiments, the intron domain comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 75, 100, 200, 300 or more trans-splicing enhancer sequences.

[0012] In some embodiments, the antisense domain is complementary to a gene encoding a target RNA that occasionally harbors a disease-causing mutation (corresponding accession number in brackets and corresponding disease in parentheses) and is selected from the group consisting of: TNFRSF13B [ENSG00000240505] (common variable immune deficiency); ADA, CECR1 [ENSG00000196839, ENSG00000093072] (adenosine deaminase deficiency); IL2RG [ENSG00000147168] (X-linked severe combined immunodeficiency); HBB [ENSG00000244734] (β-thalassemia); HBA1, HBA2 [ENSG00000206172, ENSG00000188536] (α- thalassemia);U2AF1 [ENSG00000160201] ((myelodysplastic syndromes);SOD1,TARDBP,FUS,MATR3,SOD1,C9ORF72 [ENSG00000142168,ENSG00000120948,ENSG00000089280,ENSG00000015479,ENSG00000142168,ENSG00000147894] (Amyotrophic lateral sclerosis);(amyotrophic lateral sclerosis);MAPT,PGRN[ENSG00000186868,ENSG00000030582](frontotemporal dementia with parkinsonism);CDH23,MYO7A,USH2A[ENSG00000107736,ENSG00000137474,ENSG00000042781](Usher syndrome);GALC[ENSG00000054983]( Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655] (Niemann-Pick disease); PRNP [ENSG00000171867] (prion disease); SCN1A [ENSG00000144285] (Dravet syndrome); PINK1, ATPGAP2 [ENSG00000158828] (early onset Parkinson's disease);ATXN1,ATXN2,ATXN3,PLEKHG4,SPTBN2,CACNA1A,ATXN7,TTBK2,PPP2R2B,KCNC3,PRKCG,ITPR1,TBP,KCND1,FGF14[ENSG00000124788,ENSG00000204 842,ENSG00000066427,ENSG00000196155,ENSG00000173898,ENSG00000141837,ENSG00000163635,ENSG00000128881,ENSG00000156475,ENSG0000 0131398,ENSG00000126583,ENSG00000150995,ENSG00000112592,ENSG00000102057,ENSG00000102466] (Spinocerebellar ataxia);SCN1A,SCN2A,CACNA1A,GRIN2B,GRIN2A,MECP2,FOXG1,SLC6A1,PRRT2,PTEN,KCNQ2,KCNQ3,STARD7,CLRN1 [ENSG00000144285,ENSG00000136531,ENSG00000141837,ENSG000002730 79,ENSG00000183454,ENSG00000169057,ENSG00000176165,ENSG00000157103,ENSG00000167371,ENSG00000171862,ENSG00000075043,ENSG00000184156,ENSG00000084090,ENSG00000163646](inherited epilepsy disorders);ATM[ENSG00000149311](ataxia telangiectasia);GLB1[ENSG00000170266](GM1 gangliosidosis);GBA [ENSG00000177628] (Gaucher disease); GM2A [ENSG00000196743] (GM2 gangliosidosis); UBE3A [ENSG00000114062] (Angelman syndrome); SLC2A1 [ENSG00000117394] (glucose transporter deficiency type 1); LAMP2 [ENSG00000005893] (Danon disease); GLA [ENSG00000102393] (Fabry disease); PKD1, PKD2 [ENSG00000008710, ENSG00000118762] (autosomal dominant polycystic kidney disease);GAA [ENSG00000171298] (Pompe disease); PCSK9, LDLR, APOB, APOE [ENSG00000169174, ENSG00000130164, ENSG00000084674, ENSG00000130203] (familial hypercholesterolemia); MYOC, OPTN, TBK1, WDR36, CYPIB1 [ENSG00000034971, ENSG00000123240, ENSG00000183735, ENSG00000134987, ENSG00000130203] (familial hypercholesterolemia); 000138061] (open-angle glaucoma); IDUA [ENSG00000127415] (Hurler syndrome or mucopolysaccharidosis 1); IDS [ENSG00000010404] (Hunter syndrome or mucopolysaccharidosis 2); CLN3 [ENSG00000188603] (Batten disease); DMD [ENSG00000198947] (Duchenne muscular dystrophy); LMNA [ENSG00000160789] (limb-girdle muscular dystrophy type 1B); DYSF [ENSG00000135636] (limb-girdle muscular dystrophy type 1B); muscular dystrophy type 2B);SGCA[ENSG00000108823](limb-girdle muscular dystrophy type 2D);SGCB[ENSG00000163069](limb-girdle muscular dystrophy type 2E);SGCG[ENSG00000102683](limb-girdle muscular dystrophy type 2C);SGCD[ENSG00000170624](limb-girdle muscular dystrophy type 2F);DUX4[ENSG00000260596](facioscapulohumeral dystrophy);F9[ENSG00000101981](hemophilic muscular dystrophy) disease B); F8 [ENSG00000185010] (hemophilia A); USHA2A, RPGR, RP2, RHO, PRPF31, USH1F, PRPF3, PRPF6 [ENSG00000156313, ENSG00000102218, ENSG00000163914, ENSG00000105618, ENSG00000150275, ENSG00000117360, ENSG00000101161] (retinitis pigmentosa); CFTR [ENSG00000001626] (cystic fibrosis);GJB2, GJB6, STRC, DFNA1, WFS1 [ENSG00000165474, ENSG00000121742, ENSG00000242866, ENSG00000131504, ENSG00000109501] (autosomal dominant hearing loss); POU3F3 [ENSG00000198914] (nonsyndromic hearing loss).

[0013] In some embodiments, the replacement domain is derived from or isolated from the target RNA.

[0014] In some embodiments, the intron domain possesses a binding site that is preferentially targeted by RNA binding proteins that have a disease-causing mutation, and in some embodiments, the dissociation constant of these mutated RNA binding proteins and the intron domain is lower than the dissociation constant of the non-mutated RNA binding protein and the intron domain.

[0015] In some embodiments, the trans-splicing RNA further comprises a 5' untranslated region. In some embodiments, the 5' untranslated region increases the stability of the trans-splicing nucleic acid. In some embodiments, the 5' untranslated region decreases the stability of the trans-splicing nucleic acid. In some embodiments, the 5' untranslated region alters the localization of the trans-splicing nucleic acid. In some embodiments, the 5' untranslated region alters the processing of the trans-splicing nucleic acid.

[0016] In some embodiments, the trans-splicing RNA further comprises a 3' untranslated region. In some embodiments, the 3' untranslated region increases the stability of the trans-splicing nucleic acid. In some embodiments, the 3' untranslated region decreases the stability of the trans-splicing nucleic acid. In some embodiments, the 3' untranslated region alters the localization of the trans-splicing nucleic acid. In some embodiments, the 3' untranslated region alters the processing of the trans-splicing nucleic acid.

[0017] In some embodiments, the replacement domain consists of a sequence derived from or isolated from a human gene. In some embodiments of the compositions of the present disclosure, the sequence comprising the replacement domain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage therebetween identity to the human gene. In some embodiments, the replacement domain has 100% identity to the sequence derived from or isolated from the human gene. In some embodiments, the replacement domain comprises or consists of 2 nucleotides, 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 210 nucleotides, 220 nucleotides, 230 nucleotides, 240 nucleotides, 250 nucleotides, 260 nucleotides, 270 nucleotides, more than 270 nucleotides, or any number of nucleotides in between.

[0018] In some embodiments of the compositions of the present disclosure, the sequence comprising the antisense domain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or any percentage therebetween complementary to the target RNA sequence. In some embodiments, the antisense domain has 100% complementary to the target RNA sequence. In some embodiments, the antisense domain comprises or consists of 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, 160 nucleotides, 170 nucleotides, 180 nucleotides, 190 nucleotides, 200 nucleotides, 210 nucleotides, 220 nucleotides, 230 nucleotides, 240 nucleotides, 250 nucleotides, 260 nucleotides, 270 nucleotides, more than 270 nucleotides, or any number of nucleotides in between, that are complementary to a target RNA sequence.

[0019] In some embodiments of the compositions of the present disclosure, the sequence encoding the trans-spliced ​​RNA further comprises a sequence encoding a promoter capable of expressing the trans-spliced ​​RNA in a eukaryotic cell.

[0020] In some embodiments of the compositions of the present disclosure, the eukaryotic cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell.

[0021] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a viral vector. In some embodiments, the viral vector comprises sequences isolated or derived from a retrovirus. In some embodiments, the viral vector comprises sequences isolated or derived from a lentivirus. In some embodiments, the viral vector comprises sequences isolated or derived from an adenovirus. In some embodiments, the viral vector comprises sequences isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0022] In some embodiments of the compositions and methods of the present disclosure, the viral vector comprises sequences isolated or derived from adeno-associated virus (AAV). In some embodiments, the viral vector comprises inverted terminal repeat or capsid sequences isolated or derived from AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).

[0023] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex, an exosome, or a dendrimer. In some embodiments, the vector is an expression vector or a recombinant expression system. As used herein, the term "recombinant expression system" refers to a genetic construct for the expression of specific genetic material formed by recombination.

[0024] In some embodiments of the compositions and methods of the present disclosure, the expression vectors, viral vectors or non-viral vectors provided herein include, but are not limited to, expression control elements. As used herein, "expression control element" refers to any sequence that regulates the expression of a coding sequence, such as a gene. Exemplary expression control elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, 5' or 3' untranslated regions, and introns.

[0025] Expression control elements can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. A promoter can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments, expression control by a promoter is tissue-specific. Non-limiting exemplary promoters include CMV, CBA, CAG, Cbh, EF-1a, PGK, UBC, GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, nβ2, PPE, ENK, EAAT2, GFAP, MBP, H1, and U6 promoters. In some embodiments, a promoter is a sequence isolated from or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the promoter is isolated or derived from an alanine tRNA promoter, an arginine tRNA promoter, an asparagine tRNA promoter, an aspartic acid tRNA promoter, a cysteine ​​tRNA promoter, a glutamine tRNA promoter, a glutamic acid tRNA promoter, a glycine tRNA promoter, a histidine tRNA promoter, an isoleucine tRNA promoter, a leucine tRNA promoter, a lysine tRNA promoter, a methionine tRNA promoter, a phenylalanine tRNA promoter, a proline tRNA promoter, a serine tRNA promoter, a threonine tRNA promoter, a tryptophan tRNA promoter, a tyrosine tRNA promoter, or a valine tRNA promoter. In some embodiments, the promoter is isolated or derived from a valine tRNA promoter.

[0026] An "enhancer" is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription. Non-limiting exemplary enhancers and post-transcriptional regulatory elements include the CMV enhancer and the WPRE.

[0027] In some embodiments of the compositions and methods of the present disclosure, the expression vectors, viral vectors or non-viral vectors provided herein include, but are not limited to, vector elements such as IRES or 2A peptide sites for the construction of "multicistronic" or "polycistronic" or "bicistronic" or "tricistronic" constructs, i.e., those with two or three or more coding regions or exons, and thus capable of expressing two or more proteins from a single construct from an mRNA. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. The two most widely used strategies for constructing multicistronic configurations are through the use of IRES or 2A self-cleaving sites. "IRES" refers to an internal ribosome entry site or part thereof of viral, prokaryotic or eukaryotic origin used within a polycistronic vector construct. In some embodiments, an IRES is an RNA element that allows translation initiation in a cap-independent manner. The term "self-cleaving peptide" or "sequence encoding a self-cleaving peptide" or "2A self-cleaving site" refers to a linking sequence used within a vector construct to incorporate a site for facilitating ribosomal skipping and thus producing two polypeptides from a single promoter; such self-cleaving peptides include, but are not limited to, sequences encoding T2A and P2A peptides or self-cleaving peptides.

[0028] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenoviral / retroviral chimeric vector, a herpes simplex virus I or II vector, a parvoviral vector, a reticuloendotheliosis virus vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector, or any hybrid or chimeric vector incorporating preferred aspects of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector does not integrate into the host genome, thereby reducing the likelihood of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from 3 kb to 4.75 kb. In some embodiments, exemplary AAV vectors that may be used in any of the compositions, systems, methods and kits described herein may include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32 / 33 vector, a modified AAV.rh32 / 33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh74 vector, a modified AAV.rh74 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector, and any combination or equivalent thereof.In some embodiments, lentiviral vector is an integrase-competent lentiviral vector (ICLV). In some embodiments, lentiviral vector can refer to transgene plasmid vectors, as well as transgene plasmid vectors with associated plasmids (e.g., packaging plasmids, rev expression plasmids, envelope plasmids), and lentivirus-based particles that can introduce exogenous nucleic acid into cells via a viral or viral-like entry mechanism. Lentiviral vectors are well known in the art (see, for example, Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg and Durand et al. (2011) Viruses 3(2):132-159 doi:10.3390 / v3020132). In some embodiments, exemplary lentiviral vectors that may be used in any of the compositions, systems, methods, and kits described herein include human immunodeficiency virus (HIV) 1 vectors, modified human immunodeficiency virus (HIV) 1 vectors, human immunodeficiency virus (HIV) 2 vectors, modified human immunodeficiency virus (HIV) 2 vectors, sooty mangabey simian immunodeficiency virus (SMV) vectors, human immunodeficiency virus (HIV) 3 vectors, human immunodeficiency virus (HIV) 4 vectors, human immunodeficiency virus (HIV) 5 vectors, human immunodeficiency virus (HIV) 6 vectors, human immunodeficiency virus (HIV) 7 vectors, human immunodeficiency virus (HIV) 8 vectors, human immunodeficiency virus (HIV) 9 vectors, human immunodeficiency virus (HIV) 10 vectors, human immunodeficiency virus (HIV) 11 vectors, human immunodeficiency virus (HIV) 12 vectors, human immunodeficiency virus (HIV) 13 vectors, human immunodeficiency virus (HIV) 14 vectors, human immunodeficiency virus (HIV) 15 vectors, human immunodeficiency virus (HIV) 16 vectors, human immunodeficiency virus (HIV) 17 vectors, human immunodeficiency virus (HIV) 18 vectors, human immunodeficiency virus (HIV) 19 vectors, human immunodeficiency virus (HIV) 20 vectors, human immunodeficiency virus (HIV) 21 vectors, human immunodeficiency virus (HIV) 22 vectors, human immunodeficiency virus (HIV) 23 vectors, human immunodeficiency virus (HIV) 24 vectors, human immunodeficiency virus (HIV) 25 vectors, human immunodeficiency virus (HIV) 26 vectors, human immunodeficiency virus (HIV) 27 vectors, human immunodeficiency virus (HIV) 28 vectors, human immunodeficiency virus (HIV) 29 vectors, human immunodef The vector may include a modified Sooty Mangabey Monkey Immunodeficiency Virus (SIVSM) vector, a modified Sooty Mangabey Monkey Immunodeficiency Virus (SIVSM) vector, a modified African Green Monkey Immunodeficiency Virus (SIVAGM) vector, a modified African Green Monkey Immunodeficiency Virus (SIVAGM) vector, an Equine Infectious Anemia Virus (EIAV) vector, a modified Equine Infectious Anemia Virus (EIAV) vector, a feline immunodeficiency virus (FIV) vector, a modified feline immunodeficiency virus (FIV) vector, a visna / maedi virus (VNV / VMV) vector, a modified visna / maedi virus (VNV / VMV) vector, a caprine arthritis-encephalitis virus (CAEV) vector, a modified caprine arthritis-encephalitis virus (CAEV) vector, a bovine immunodeficiency virus (BIV), or a modified bovine immunodeficiency virus (BIV).

[0029] In some embodiments, the trans-splicing nucleic acid is RNA, DNA, a DNA / RNA hybrid, and / or comprises at least one of a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs. As used herein, the term "nucleic acid analog" refers to a compound that has a structural similarity to the standard purine or pyrimidine bases present in DNA or RNA. Nucleic acid analogs may contain modified sugars and / or modified nucleobases compared to the purine or pyrimidine bases naturally occurring in DNA or RNA. In some embodiments, the nucleic acid analog is a 2'-deoxyribonucleoside, 2'-ribonucleoside, 2'-deoxyribonucleotide, or 2'-ribonucleotide, and the nucleic acid base comprises a modified base (e.g., xanthine, uridine, oxanine (oxanosine), 7-methylguanosine, dihydrouridine, 5-methylcytidine, C3 spacer, 5-methyl dC, 5-hydroxybutynl-2'-deoxyuridine, 5-nitroindole, 5-methylisodeoxycytosine, isodeoxyguanosine, deoxyuradine, isodeoxycytidine, other 0-1 purine analogs, N-6-hydroxyaminopurine, nebularine, 7-deazahypoxanthine, other 7-deazapurines, and 2-methylpurines, etc.). In some embodiments, the nucleic acid analog may be selected from the group consisting of inosine, 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In other embodiments, the nucleic acid analog is a nucleic acid mimic, such as, for example, artificial nucleic acids and heterologous nucleic acids (XNAs). [Brief description of the drawings]

[0030] [Figure 1A]Figure 1 illustrates the unmet need addressed by the present invention and provides a schematic diagram of the invention. Figure 1A illustrates the concept of human genetic disease in which mutated ("defective") DNA sequences are either transcribed into RNA that directly contributes to disease ("RNA pathology") or translated into disease-causing proteins ("pathogenic protein translation"). [Figure 1B] Figure 1B shows state-of-the-art RNA trans-splicing technology in which mutation-bearing RNA molecules are targeted by trans-splicing RNA that repairs the mutation but with low efficiency, which is typically insufficient to halt or reverse disease progression. [Figure 1C] 1C illustrates the present invention, in which the trans-splicing molecule carries a trans-splicing enhancer sequence that increases the efficiency of the trans-splicing reaction, helping to halt or reverse disease progression and / or eliminate important disease phenotypes, thereby providing an effective treatment for human genetic diseases.

[0031] [Figure 2A] Figure 2 shows three embodiments of the trans-splicing RNA described in this disclosure. Figure 2A depicts a double trans-splicing molecule that possesses two antisense domains, one replacement domain, two intron domains, and at least two trans-splicing enhancer sequences within the intron domain. This design promotes replacement of an internal sequence within the target RNA while maintaining the adjacent 5' and 3' sequences surrounding the replaced sequence. [Figure 2B] Figures 2B and 2C both describe terminal trans-splicing molecules that contain one antisense domain, one replacement domain, one intron domain, and at least one trans-splicing enhancer sequence within the intron domain. Figure 2B shows the design of a 3' terminal trans-splicing RNA that replaces the 3' end of a target RNA while maintaining the 5' end. [Figure 2C] FIG. 2C shows the design of a 5′-end trans-splicing molecule that replaces the 5′-end of a target RNA while maintaining the 3′-end.

[0032] [Figure 3A] Figure 3 shows an experiment designed to clarify the importance of trans-splicing enhancer sequences in the context of internal trans-splicing mediated production of GFP protein. Figure 3A shows the design of a split GFP reporter that retains the N- and C-terminal portions of GFP ("N-GFP" and "C-GFP") but lacks the internal GFP sequence required for fluorescence. In the reporter, this internal sequence is replaced by a short exon with a stop codon flanked by an intron. The internal sequence ("int-GFP") is a replacement sequence within the RNA trans-splicing molecule that is flanked by two intron sequences and two antisense sequences. [Figure 3B] FIG. 3B shows activity of the reporter alone, where cis-splicing produces a GFP sequence interrupted by a stop codon and therefore produces no GFP signal. [Figure 3C] FIG. 3C shows reporter activity in the presence of trans-splicing molecules that do not contain the trans-splicing enhancer sequence, such that similarly cis-splicing occurs predominantly and no GFP signal is produced. [Figure 3D] FIG. 3D shows the activity of the reporter in the presence of a trans-splicing molecule that contains a trans-splicing enhancer sequence, such that trans-splicing occurs predominantly and a GFP signal is generated.

[0033] [Figure 4A]Figure 4 shows an experiment designed to clarify the importance of trans-splicing enhancer sequences in the context of 5'-end trans-splicing. Figure 4A shows the design of a split GFP reporter that retains the C-terminal portion of GFP ("C-GFP") but lacks the N-terminal GFP sequence required for fluorescence. In the reporter, this N-terminal GFP sequence is replaced by a short exon with a stop codon flanked by an intron. The N-terminal sequence ("N-GFP") is a replacement sequence within the RNA trans-splicing molecule that is flanked by one intron sequence and one antisense sequence. [Figure 4B] FIG. 4B shows activity of the reporter alone, where cis-splicing produces a GFP sequence interrupted by a stop codon and therefore produces no GFP signal. [Figure 4C] FIG. 4C shows reporter activity in the presence of trans-splicing molecules that do not contain the trans-splicing enhancer sequence, such that similarly cis-splicing occurs predominantly and no GFP signal is produced. [Figure 4D] FIG. 4D shows the activity of the reporter in the presence of a trans-splicing molecule that contains a trans-splicing enhancer sequence, such that trans-splicing occurs predominantly and a GFP signal is generated.

[0034] [Figure 5A] Figure 5 shows an experiment designed to clarify the importance of trans-splicing enhancer sequences in the context of 3'-end trans-splicing. Figure 5A shows the design of a split GFP reporter that retains the N-terminal portion of GFP ("N-GFP") but lacks the C-terminal GFP sequence required for fluorescence. In the reporter, this C-terminal GFP sequence is replaced by a short exon with a stop codon flanked by an intron. The C-terminal sequence ("C-GFP") is a replacement sequence within the RNA trans-splicing molecule that is flanked by one intron sequence and one antisense sequence. [Figure 5B]FIG. 5B shows activity of the reporter alone, where cis-splicing produces a GFP sequence interrupted by a stop codon and therefore produces no GFP signal. [Figure 5C] FIG. 5C shows reporter activity in the presence of trans-splicing molecules that do not contain the trans-splicing enhancer sequence, such that similarly cis-splicing occurs predominantly and no GFP signal is produced. [Figure 5D] FIG. 5D shows the activity of the reporter in the presence of a trans-splicing molecule that contains a trans-splicing enhancer sequence, such that trans-splicing occurs predominantly and a GFP signal is generated.

[0035] [Figure 6A] FIG. 6 illustrates the molecular pathology associated with myotonic dystrophy type I and methods of treating this disease including trans-splicing therapeutics. FIG. 6A shows that myotonic dystrophy type I (DM1) is caused by a "CUG" repeat expansion in DNA that is transcribed into RNA composed of "CTG" repeat units. This repeat RNA preferentially binds to a splicing factor protein called MBNL1. The resulting titration of MBNL1 from its typical activity causes widespread dysfunctional RNA splicing in cells. This dysfunctional splicing is responsible for many of the pathologies associated with this disease, including the characteristic myotonia. [Figure 6B] Figure 6B shows the activity of a trans-splicing therapeutic that addresses this molecular pathology via amplification of MBNL1 protein production. Specifically, the trans-splicing RNA binds a gene expression amplification sequence to the 3' end of MBNL1 mRNA, increasing MBNL1 expression. The resulting increase in MBNL1 levels reconstitutes its splicing activity and reverses the disease.

[0036] [Figure 7A]FIG. 7 shows an experiment designed to clarify the importance of trans-splicing enhancer sequences in the context of 3'-end trans-splicing aimed at increasing gene expression via the production of luciferase protein. FIG. 7A illustrates the design of a luciferase reporter carrying Renilla luciferase control ("R. luciferase") and firefly luciferase ("F. luciferase") reporter molecules. The reporter molecule carries exon 9, intron 9, and exon 10 from the human MBNL1 gene. Exon 10 contains regulatory elements that affect MBNL1 gene expression. The trans-splicing RNA targets intron 9 in the 3' trans-splicing process and replaces exon 10 with a translation enhancer. In this way, successful trans-splicing increases the production of firefly luciferase compared to Renilla luciferase. [Figure 7B] FIG. 7B illustrates the activity of the reporter alone where cis-splicing produces a luciferase molecule containing exon 10 of MBNL1. [Figure 7C] FIG. 7C illustrates reporter activity in the presence of trans-splicing molecules without including the trans-splicing enhancer sequence in the trans-splicing molecule, which also results in cis-splicing, resulting in luciferase molecules that predominantly contain exon 10 of MBNL1. [Figure 7D] Figure 7D shows the activity of the reporter in the presence of a trans-splicing molecule containing a trans-splicing enhancer sequence, such that trans-splicing occurs predominantly and exon 10 is replaced by a translational enhancer, thus increasing the firefly luciferase signal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure provides RNA molecules that selectively bind to RNA molecules to efficiently promote trans-splicing reactions. The present disclosure provides vectors, compositions, and cells that contain or encode the trans-splicing RNA molecules. The present disclosure provides methods of using the trans-splicing RNA molecules, vectors, compositions, and cells of the present disclosure to treat diseases or disorders.

[0038] In one aspect, the present invention is a trans-splicing RNA molecule that includes three types of domains (Figure 2). One of the three domain types is a replacement domain that is inserted into a target RNA molecule via a trans-splicing reaction. The second domain type is an antisense domain that is complementary to the target RNA. The third domain type is an intron domain that promotes a trans-splicing reaction between the trans-splicing RNA molecule and the target RNA. The intron domain further includes an intronic trans-splicing enhancing sequence (trans-splicing enhancer sequence) that promotes the trans-splicing reaction. This novel combination of a specific trans-splicing enhancer sequence and an intron domain promotes RNA trans-splicing in a manner that sufficiently replaces disease-causing RNA sequences in human cells to address disease. The present disclosure provides compositions and methods for specifically targeting disease-causing RNA molecules and replacing disease-causing RNA sequences within these RNA molecules with high efficiency. Embodiments of the trans-splicing RNA molecule show utility in a variety of contexts, including replacing disease-causing sequences or inserting engineered sequences into target RNA. The engineered sequence can alter the translation or stability of the target RNA to increase or decrease protein production or target RNA levels. The disclosure provides vectors, compositions and cells that contain or encode the trans-splicing RNA, as well as methods of using the trans-splicing RNA compositions.

[0039] In one aspect, the present invention is an RNA technology that allows the replacement of any sequence within a specific RNA molecule in a living cell. This technology is based on RNA trans-splicing and utilizes the spliceosome, which is naturally present in human cells, to provide the catalytic activity of this trans-splicing process. Typically, RNA splicing occurs within an RNA molecule where exons are joined and introns are removed from an immature messenger RNA molecule (pre-mRNA) to form a mature messenger RNA molecule (mRNA). This process is called cis-splicing. RNA trans-splicing is the process where the spliceosome joins exons from separate RNA molecules. This process occurs rarely in human cells, and state-of-the-art systems that promote RNA trans-splicing are active at low levels. The present invention includes compositions that increase the efficiency of RNA trans-splicing. These improved RNA trans-splicing compositions can be used to replace mutant sequences within target RNA molecules to address human diseases. The replacement of any RNA sequence is a general capability with a myriad of specific applications, some of which are discussed as relevant demonstrations. RNA trans-splicing can insert engineered sequences into a target RNA to confer new activities to the target RNA, such as altered RNA stability or altered RNA translation. This feature can be used to increase protein production by the target RNA. In the broadest sense, this RNA trans-splicing technique can impart any change to both the coding and non-coding regions of the target RNA.

[0040] References describe the activity of splicing-promoting sequences in the context of cis-splicing. However, little is known about 1) the identity of trans-splicing-enhancing sequences, and 2) whether cis-splicing enhancers also enhance trans-splicing. Because the reaction rates of intermolecular (trans-splicing) reactions rather than intramolecular (cis-splicing) reactions are likely to be significantly different, the inventors have conceived a separate group of splicing enhancers that may function in the context of trans-splicing. This has been confirmed by experiments showing that the activity of splicing enhancers in cis-splicing does not necessarily predict activity in trans-splicing. As used herein, these sequences that promote trans-splicing are referred to as "trans-splicing enhancer sequences".

[0041] The compositions comprising the intron trans-splicing enhancing sequences (trans-splicing enhancer sequences) disclosed herein include any sequence that efficiently promotes trans-splicing. Exemplary trans-splicing enhancer sequences include, but are not limited to, TTACGG, TAACGG, GGGTTT, GTTTTG, GGTTTT, GGTTTG, GGTTGG, GTTAGG, TGGTTG, GGGTAG, GGTAGG, GGTAGT, GTAGTT, GTTGGT, GTGGTT, GGTGGT, TGGTGG, TTGGTG, GTAAGG, TAAGGG, TTAGGG, TAGGGG, TTGGGG, GTTGGG, GTAGGG, TATTGG, TGTTGG, TATGGG, TTTGGG, TGTGGG, TTGTGG, GAGTGT, GAGGTA, GGAGGT, TGGGAG, GGG These include GTG, GGGGGA, GGGGGT, GGGGTA, GGGAGG, GGGTGG, GGAGGG, GGTGGG, GAGGGG, GTGGGG, GAGTGG, GTATGG, GGTATT, GTATTT, GTATTG, AGTTTA, AGGTTA, GTAACG, AGGTAA, GGTAAG, TGGGGG, AGGGTT, AGGTTG, AGGTAG, ATTTGG, AGTTGG, TCTGGG, AGAGTG, AGAGGG, AGTGTG, AGAGGT, AGGGAG, AGGGTG, AGGGGG, AGGGGT, AGTGGG, AGTATG, AGGTAT, GTATTC, and GGTAAC.In some embodiments, in the exemplary trans-splicing enhancer sequences above, any of the thymidine bases, a portion of the thymidine bases, or all of the thymidine bases can be substituted with uracil, such that exemplary trans-splicing enhancer sequences include, but are not limited to, UUACGG, UAACGG, GGGUUU, GUUUUG, GGUUUU, GGUUUG, GGUUGG, GUUAGG, UGGUUG, GGGUAG, GGUAGG, GGUAGU, GUAGUU, GUUGGU, GUGGUU, GGUGGU, UGGUGG, UUGGUG, GUAAGG, UAAGGG, UUAGGG, UAGGGG, UUGGGG, GUUGGG, GUAGGG, UAUUGG, UGUUGG, UAUGGG, UUUGG G. Includes UAACG, AGGUAA, GGUAAG, UGGGGG, AGGGUU, AGGUUG, AGGUAG, AUUUGG, AGUUGG, UCUGGG, AGAGUG, AGAGGG, AGUGUG, AGAGGU, AGGGAG, AGGGUG, AGGGGG, AGGGGU, AGUGGG, AGUAUG, AGGUAU, GUAUUC, GGUAAC.

[0042] The RNA trans-splicing technology of the present disclosure, including the inclusion of specific intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences), is the first to demonstrate RNA trans-splicing with high efficiency for multiple RNA targets. Highly efficient RNA trans-splicing has three main advantages over previous RNA trans-splicing systems. First, this improved efficiency can replace defective RNA sequences at a level sufficient to reconstitute the activity of mutant genes to treat recessive genetic disorders. In fact, the treatment of many recessive genetic disorders requires at least 30% efficiency, where 100% is a complete replacement of sequences within the target RNA. Second, this improved efficiency can replace defective RNA sequences at a level sufficient to treat dominant genetic disorders. Since a single mutant allele is sufficient to cause disease, many diseases in this class require highly efficient replacement of mutant sequences, as mutant sequences typically cause toxicity. As a result, even higher efficiencies are required (70%+). Finally, the broad ability of our RNA trans-splicing technology to modify multiple target RNAs demonstrates the first broadly applicable and efficient version of this technology. This is a highly general capability, and the present disclosure provides a demonstration of an RNA trans-splicing system that can efficiently replace sequences with multiple target RNAs.

[0043] The inclusion of an intronic trans-splicing enhancing sequence (trans-splicing enhancer sequence) to form the present RNA trans-splicing technology is a general capability that further allows for the modification of non-coding sequences within the target RNA. By replacing the 5' or 3' untranslated regions of the target RNA with high efficiency, the present invention allows for changes in RNA behavior, such as translation or turnover. The net result of these effects is increased production of protein from the target RNA, or other downstream effects related to changes in RNA levels. Identification of intron trans-splicing enhancer sequences

[0044] RNA sequences that affect RNA cis-splicing are known (Wang, Ma et al. 2012). However, no studies have evaluated the activity of these known splicing enhancers in the context of RNA trans-splicing. We first systematically evaluated whether 50 splicing-enhancing sequences could enhance the efficiency of RNA trans-splicing when placed upstream and downstream of a replacement sequence in a model RNA trans-splicing molecule. These mutant trans-splicing molecules are targeted to a split GFP reporter assay that only fluoresces after successful activity of the RNA trans-splicing molecule (Figures 3-5). Although this assay is qualitative and not fully quantitative, it is useful because it is one that cell biology end users often use when attempting to answer scientific questions regarding the presence, absence, or general magnitude of a transcript. Thus, GFP trans-splicing reporters are widely used in the study of RNA trans-splicing technology. Using a GFP reporter similar to a published system (Koller, Wally et al. 2011), we compared the relative effects of different trans-splicing enhancer sequences on the efficiency of the trans-splicing reaction.

[0045] Figures 3-5 include schematic diagrams of the plasmids used in the trans-splicing activity assay. Experiments were performed using either transiently transfected reporter and trans-splicing molecules or lentiviral packaged systems. The inventors observed that several known splicing enhancer sequences (Wang, Ma et al. 2012) that function in the context of cis-splicing do not enhance trans-splicing. The inventors further observed other sequences that may or may not enhance cis-splicing function in the context of trans-splicing. As used herein, these trans-splicing specific enhancers are referred to as "trans-splicing enhancer sequences." Use of intronic trans-splicing enhancer sequences to increase translation of specific target RNAs

[0046] In addition to replacing specific mutated sequences in RNA with non-mutated sequences, another useful manipulation on target mRNA molecules is to increase the protein produced by the mRNA. Many attempts have been made to address this problem of insufficient protein production from specific mRNAs, but each approach has major drawbacks. Indeed, small molecule drugs that increase translation by promoting read-through of stop codons suffer from extensive off-targets due to promotion of read-through on non-target mRNAs (Keeling, Xue et al. 2014). Furthermore, premature stop codons are only one of many causes of insufficient protein levels. tRNAs engineered to block premature stop codons suffer from this same fundamental problem (WO 2018 / 161032). In contrast, RNA trans-splicing systems can replace sequences of any target mRNA with translation-amplifying sequences to increase protein production. The inventors have hypothesized that efficient RNA trans-splicing mediated by intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) could address a long-standing and unmet need for a means to target and promote increased protein production from specific mRNAs.

[0047] Myotonic dystrophy is caused by RNA carrying a repeat "CUG" pathway that binds to the splicing factor MBNL1. Titration of MBNL1 away from its typical targets causes widespread dysfunction of RNA alternative splicing and is responsible for most manifestations of the disease in patients. The inventors reasoned that increasing MBNL1 protein production with an efficient RNA trans-splicing approach could address this disease via production of enough MBNL1 protein to reconstitute its typical activity in alternative splicing regulation (Figure 6).

[0048] To evaluate the ability of an RNA trans-splicing system containing a trans-splicing enhancer sequence to increase protein production from a particular mRNA, we created an RNA trans-splicing system carrying various cis-splicing enhancer sequences and the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) (Figure 7). We also created a reporter containing the firefly luciferase coding sequence and the last two exons and intervening intron of MBNL1 (Figure 7). Although this assay is qualitative and not fully quantitative, it is useful because it is one that cell biology end users often use when trying to answer scientific questions regarding the presence, absence, or general magnitude of a transcript. In fact, this reporter is based on the pMIR-GLO luciferase vector, which is typically used to evaluate stability and protein production from model mRNAs.

[0049] Experiments were performed using either transiently transfected reporter and trans-splicing molecules or lentiviral packaged systems. We observed that some known splicing enhancer sequences (Wang, Ma et al. 2012) that function in the context of cis-splicing do not enhance trans-splicing. We also observed other sequences that may or may not enhance cis-splicing function in the context of trans-splicing. As used herein, these trans-splicing specific enhancers are referred to as "trans-splicing enhancer sequences." These results indicate that the use of trans-splicing enhancer sequences can promote a significant increase in protein production from a specific mRNA. Replacement Domain

[0050] Compositions comprising replacement domains disclosed herein include any strategy in which replacement or insertion of RNA sequences may be an effective therapeutic approach. Exemplary replacement domains include, but are not limited to, sequences derived from or isolated from the following genes, such as (gene accession ID in brackets and associated disease in parentheses): TNFRSF13B [ENSG00000240505] (common variable immune deficiency); ADA, CECR1 [ENSG00000196839, ENSG00000093072] (adenosine deaminase deficiency); IL2RG [ENSG00000147168] (X-linked severe combined immunodeficiency); HBB [ENSG00000244734] (β-thalassemia); HBA1, HBA2 [ENSG00000206172, ENSG00000188536] (α- thalassemia);U2AF1 [ENSG00000160201] ((myelodysplastic syndromes);SOD1,TARDBP,FUS,MATR3,SOD1,C9ORF72 [ENSG00000142168,ENSG00000120948,ENSG00000089280,ENSG00000015479,ENSG00000142168,ENSG00000147894] (Amyotrophic lateral sclerosis);(amyotrophic lateral sclerosis);MAPT,PGRN[ENSG00000186868,ENSG00000030582](frontotemporal dementia with parkinsonism);CDH23,MYO7A,USH2A[ENSG00000107736,ENSG00000137474,ENSG00000042781](Usher syndrome);GALC[ENSG00000054983]( Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655] (Niemann-Pick disease); PRNP [ENSG00000171867] (prion disease); SCN1A [ENSG00000144285] (Dravet syndrome); PINK1, ATPGAP2 [ENSG00000158828] (early onset Parkinson's disease);ATXN1,ATXN2,ATXN3,PLEKHG4,SPTBN2,CACNA1A,ATXN7,TTBK2,PPP2R2B,KCNC3,PRKCG,ITPR1,TBP,KCND1,FGF14[ENSG00000124788,ENSG00000204 842,ENSG00000066427,ENSG00000196155,ENSG00000173898,ENSG00000141837,ENSG00000163635,ENSG00000128881,ENSG00000156475,ENSG0000 0131398,ENSG00000126583,ENSG00000150995,ENSG00000112592,ENSG00000102057,ENSG00000102466] (Spinocerebellar ataxia);SCN1A,SCN2A,CACNA1A,GRIN2B,GRIN2A,MECP2,FOXG1,SLC6A1,PRRT2,PTEN,KCNQ2,KCNQ3,STARD7,CLRN1 [ENSG00000144285,ENSG00000136531,ENSG00000141837,ENSG000002730 79,ENSG00000183454,ENSG00000169057,ENSG00000176165,ENSG00000157103,ENSG00000167371,ENSG00000171862,ENSG00000075043,ENSG00000184156,ENSG00000084090,ENSG00000163646] inherited epilepsy disorders);ATM[ENSG00000149311](ataxia telangiectasia);GLB1[ENSG00000170266](GM1 gangliosidosis);GBA [ENSG00000177628] (Gaucher disease); GM2A [ENSG00000196743] (GM2 gangliosidosis); UBE3A [ENSG00000114062] (Angelman syndrome); SLC2A1 [ENSG00000117394] (glucose transporter deficiency type 1); LAMP2 [ENSG00000005893] (Danon disease); GLA [ENSG00000102393] (Fabry disease); PKD1, PKD2 [ENSG00000008710, ENSG00000118762] (autosomal dominant polycystic kidney disease);GAA [ENSG00000171298] (Pompe disease); PCSK9, LDLR, APOB, APOE [ENSG00000169174, ENSG00000130164, ENSG00000084674, ENSG00000130203] (familial hypercholesterolemia); MYOC, OPTN, TBK1, WDR36, CYPIB1 [ENSG00000034971, ENSG00000123240, ENSG00000183735, ENSG00000134987, ENSG00000130203] (familial hypercholesterolemia); 000138061] (open-angle glaucoma); IDUA [ENSG00000127415] (Hurler syndrome or mucopolysaccharidosis 1); IDS [ENSG00000010404] (Hunter syndrome or mucopolysaccharidosis 2); CLN3 [ENSG00000188603] (Batten disease); DMD [ENSG00000198947] (Duchenne muscular dystrophy); LMNA [ENSG00000160789] (limb-girdle muscular dystrophy type 1B); DYSF [ENSG00000135636] (limb-girdle muscular dystrophy type 1B); muscular dystrophy type 2B);SGCA[ENSG00000108823](limb-girdle muscular dystrophy type 2D);SGCB[ENSG00000163069](limb-girdle muscular dystrophy type 2E);SGCG[ENSG00000102683](limb-girdle muscular dystrophy type 2C);SGCD[ENSG00000170624](limb-girdle muscular dystrophy type 2F);DUX4[ENSG00000260596](facioscapulohumeral dystrophy);F9[ENSG00000101981](hemophilic muscular dystrophy) disease B); F8 [ENSG00000185010] (hemophilia A); USHA2A, RPGR, RP2, RHO, PRPF31, USH1F, PRPF3, PRPF6 [ENSG00000156313, ENSG00000102218, ENSG00000163914, ENSG00000105618, ENSG00000150275, ENSG00000117360, ENSG00000101161] (retinitis pigmentosa); CFTR [ENSG00000001626] (cystic fibrosis);GJB2, GJB6, STRC, DFNA1, WFS1 [ENSG00000165474, ENSG00000121742, ENSG00000242866, ENSG00000131504, ENSG00000109501] (autosomal dominant hearing loss); POU3F3 [ENSG00000198914] (nonsyndromic hearing loss).

[0051] In some embodiments, the replacement domain is codon optimized.

[0052] In addition to sequences derived from human genes, replacement domains can include sequences derived from other organisms to alter the stability, translation, processing, or localization of the target RNA. Exemplary replacement domains derived from non-human sources include, but are not limited to, sequences that increase protein production, such as those derived from or isolated from the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE), triplex from MALAT1, PRE from Hepatitis B virus (HPRE), and iron-responsive elements of the form CAGYCX (Y=U or A; X=U, C, or A). Antisense Domain

[0053] In some embodiments of the compositions of the present disclosure, the pathogenic RNA molecule is a target RNA. In some embodiments, the target RNA comprises a target sequence that is complementary to the antisense domain of a trans-splicing RNA of the present disclosure.

[0054] In some embodiments of the compositions and methods of the present disclosure, the target sequence comprises or consists of 5 to 500 nucleotides. In some embodiments, the target sequence comprises or consists of 50 to 250 nucleotides. In some embodiments, the target sequence comprises or consists of 5 to 50 nucleotides.

[0055] In some embodiments of the disclosed compositions and methods, the target sequence is contained within a single contiguous stretch of the target RNA, in some embodiments, the target sequence may be composed of one or more nucleotides that are not spread among the single contiguous stretch of the target RNA.

[0056] In some embodiments of the present disclosure, the antisense domain of the present disclosure binds to a target sequence.In some embodiments of the present disclosure, the antisense domain of the present disclosure binds to a target RNA.

[0057] In some embodiments of the present disclosure, the antisense domain is selected such that successful trans-splicing causes the removal of the micro open reading frame in the target RNA. In this way, the trans-splicing system removes the micro open reading frame and increases the production of protein from the target RNA. vector

[0058] In some embodiments of the compositions and methods of the present disclosure, the vector comprises or encodes a trans-splicing nucleic acid of the present disclosure. In some embodiments, the vector comprises or encodes at least one trans-splicing nucleic acid of the present disclosure. In some embodiments, the vector comprises or encodes one or more trans-splicing nucleic acids of the present disclosure. In some embodiments, the vector comprises or encodes two or more trans-splicing nucleic acids of the present disclosure.

[0059] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a viral vector. In some embodiments, the viral vector comprises sequences isolated or derived from a retrovirus. In some embodiments, the viral vector comprises sequences isolated or derived from a lentivirus. In some embodiments, the viral vector comprises sequences isolated or derived from an adenovirus. In some embodiments, the viral vector comprises sequences isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0060] In some embodiments of the compositions and methods of the present disclosure, the viral vector comprises sequences isolated or derived from adeno-associated virus (AAV). In some embodiments, the viral vector comprises inverted terminal repeat or capsid sequences isolated or derived from AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. In some embodiments, the viral vector is replication-incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).

[0061] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex, or a dendrimer. In some embodiments, the vector is an expression vector or a recombinant expression system. As used herein, the term "recombinant expression system" refers to a genetic construct for the expression of specific genetic material formed by recombination.

[0062] In some embodiments, the liposome, lipoplex, or nanoparticle can further comprise a non-cationic lipid, a PEG-conjugated lipid, a sterol, or any combination thereof.

[0063] In some embodiments, the liposome, lipoplex, or nanoparticle further comprises a non-cationic lipid, the non-ionic lipid being distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (P ... phosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (DM ... phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidyl diethylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine;Non-cationic lipids are described, for example, in WO 2017 / 099823 or U.S. Patent Application Publication No. 2018 / 0028664.

[0064] In some embodiments, the liposome, lipoplex or nanoparticle further comprises a conjugated lipid, the conjugated lipid being selected from the group consisting of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt.

[0065] In some embodiments, the liposome, lipoplex, or nanoparticle further comprises cholesterol or a cholesterol derivative.

[0066] In some embodiments, the liposome, lipoplex, or nanoparticle further comprises an ionizable lipid, a non-cationic lipid, a complex lipid that inhibits particle aggregation, and a sterol. The amount of the ionizable lipid, the non-cationic lipid, the complex lipid that inhibits particle aggregation, and the sterol can be independently varied. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid present in the particle, a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid present in the particle, a complex lipid that inhibits particle aggregation in an amount of about 0.5 mol% to about 20 mol% of the total lipid present in the particle, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid present in the particle.

[0067] The total lipid to DNA vector ratio can be varied as desired. For example, the total lipid to DNA vector (mass or weight) ratio can be from about 10:1 to about 30:1.

[0068] In some embodiments of the compositions and methods of the present disclosure, the expression vectors, viral vectors or non-viral vectors provided herein include, but are not limited to, expression control elements. As used herein, "expression control elements" refers to any sequence that regulates the expression of a coding sequence, such as a gene. Exemplary expression control elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that is a region of a polynucleotide sequence where the initiation and rate of transcription are controlled. A promoter can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments, expression control by a promoter is tissue-specific. Non-limiting exemplary promoters include CMV, CBA, CAG, Cbh, EF-1a, PGK, UBC, GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, nβ2, PPE, ENK, EAAT2, GFAP, MBP, and U6 promoters. An "enhancer" is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription. Non-limiting exemplary enhancers and post-transcriptional regulatory elements include the CMV enhancer and the WPRE. nucleic acid

[0069] Also provided herein are nucleic acid sequences encoding the trans-splicing nucleic acids disclosed herein for use in the gene transfer and expression techniques described herein. It should be understood, although not always explicitly stated, that the sequences provided herein can be used to provide expression products, as well as substantially identical sequences to produce proteins with the same biological properties. These "biologically equivalent" or "biologically active" or "equivalent" polypeptides are encoded by equivalent polynucleotides as described herein. They may have a nucleic acid sequence that is at least 60%, or alternatively at least 65%, or alternatively at least 70%, or alternatively at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% identical to the reference nucleic acid sequence when compared using sequence identity methods performed under default conditions. Specific sequences are provided as examples of specific embodiments. Furthermore, equivalent polynucleotides are those that hybridize to the reference polynucleotide or its complement under stringent conditions.

[0070] The nucleic acid sequences (e.g., polynucleotide sequences) disclosed herein can be codon-optimized, which is a technique well known in the art. Codon optimization refers to the fact that different cells have different frequencies of usage of certain codons. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a cell type. It is possible to increase expression by changing the codons in the sequence to match the relative abundance of the corresponding tRNA. It is also possible to decrease expression by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. Codon usage tables are known in the art for mammalian cells and various other organisms. Based on the genetic code, nucleic acid sequences encoding various replacement domains can be generated. In some embodiments, such sequences are optimized for expression in a host cell or target cell, such as a host cell used to express a trans-splicing RNA containing a replacement domain in which the disclosed method is performed (e.g., in a mammalian cell, such as a human cell). The codon preference and codon usage table of a particular species can be used to engineer an isolated nucleic acid molecule that encodes a replacement domain that utilizes the codon usage preference of that particular species (e.g., one that encodes a protein that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the corresponding wild-type protein). For example, the replacement domains disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest. In one example, the replacement domain nucleic acid sequence is optimized for expression in human cells, e.g., one that has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or source nucleic acid sequence.In some embodiments, the isolated trans-splicing nucleic acid molecule encoding at least one replacement domain (which may be part of a vector) comprises at least one replacement domain coding sequence that is codon-optimized for expression in eukaryotic cells or at least one replacement domain coding sequence that is codon-optimized for expression in human cells. In one embodiment, such a codon-optimized replacement domain coding sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or source sequence. In another embodiment, the eukaryotic codon-optimized nucleic acid sequence encodes a replacement domain that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with its corresponding wild-type or original protein. In another embodiment, various clones containing functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same replacement domain protein sequence, can be routinely generated. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby two or more codons can encode the same amino acid residue. Thus, for example, leucine may be encoded by CTT, CTC, CTA, CTG, TTA, or TTG, serine may be encoded by TCT, TCC, TCA, TCG, AGT, or AGC, asparagine may be encoded by AAT or AAC, aspartic acid may be encoded by GAT or GAC, cysteine ​​may be encoded by TGT or TGC, alanine may be encoded by GCT, GCC, GCA, or GCG, glutamine may be encoded by CAA or CAG, tyrosine may be encoded by TAT or TAC, and isoleucine may be encoded by ATT, ATC, or ATA.Tables showing the standard genetic code can be found in a variety of sources (see, for example, Stryer, 1988, Biochemistry, 3rd Edition, WH5 Freeman and Co., NY).

[0071] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of the nucleotide residues. The hydrogen bonds may occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex may include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0072] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; and a buffer concentration of about 1xSSC to about 0.1xSSC.

[0073] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the invention. How to use

[0074] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule, comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0075] The present disclosure provides a method for modifying the activity of a protein encoded by an RNA molecule, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0076] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 15% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0077] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 20% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0078] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 30% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0079] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 40% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0080] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 50% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0081] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 60% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0082] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 70% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0083] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 80% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0084] The present disclosure provides a method for modifying the sequence of an RNA molecule or a protein encoded by an RNA molecule with an efficiency of 90% or greater, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0085] The present disclosure provides a method for modifying the sequence of an untranslated region of an RNA molecule, the method comprising contacting the RNA molecule with a composition under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0086] The present disclosure provides a method for increasing expression of an RNA by insertion of a WPRE or a sequence having similar activity, comprising contacting a composition and an RNA molecule under conditions suitable for binding and trans-splicing of one or more trans-splicing RNAs (or portions thereof) to the RNA molecule.

[0087] The present disclosure provides a method for modifying a composition of a protein encoded by a target RNA, the method comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0088] The present disclosure provides a method for modifying a composition of a target RNA with greater than 20% efficiency (100% would constitute a complete replacement of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0089] The present disclosure provides a method for modifying the composition of a protein encoded by a target RNA with an efficiency of 20% or about 20% (100% would constitute a complete substitution of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0090] The present disclosure provides a method for modifying a composition of a target RNA with 60% or about 60% efficiency (100% would constitute a complete replacement of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0091] The present disclosure provides a method for modifying the composition of a protein encoded by a target RNA with an efficiency of 60% or about 60% (100% would constitute a complete substitution of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0092] The present disclosure provides a method for modifying a composition of a target RNA with 70% or about 70% efficiency (100% would constitute a complete replacement of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0093] The present disclosure provides a method for modifying the composition of a protein encoded by a target RNA with 70% or about 70% efficiency (100% would constitute a complete substitution of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0094] The present disclosure provides a method for modifying a composition of a target RNA with 80% or about 80% efficiency (100% would constitute a complete replacement of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0095] The present disclosure provides a method for modifying the composition of a protein encoded by a target RNA with 80% or about 80% efficiency (100% would constitute a complete substitution of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0096] The present disclosure provides a method for modifying a composition of a target RNA with 90% or about 90% efficiency (100% would constitute a complete replacement of a selected sequence within the target RNA), comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0097] The present disclosure provides a method for modifying a composition of a protein encoded by a target RNA with an efficiency of about 90% or greater (100% constituting a perfect substitution of a selected sequence within the target RNA), the method comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA.

[0098] The present disclosure provides a method for modifying a composition of a target RNA with high efficiency, comprising contacting a composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition comprises a vector that includes or encodes a trans-splicing RNA molecule of the present disclosure. In some embodiments, the vector is AAV.

[0099] The present disclosure provides a method for modifying a composition of a protein encoded by a target RNA with high efficiency, comprising contacting the composition with a cell containing the target RNA under conditions suitable for trans-splicing between the composition and the target RNA. In some embodiments, the cell is in vivo, in vitro, ex vivo, or in situ. In some embodiments, the composition comprises a vector that includes or encodes a trans-splicing RNA molecule of the present disclosure. In some embodiments, the vector is AAV.

[0100] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the present disclosure.

[0101] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the present disclosure, wherein the composition comprises a trans-splicing RNA molecule of the present disclosure or a vector encoding the same, and wherein the composition alters the expression level of the RNA molecule or a protein encoded by the RNA molecule of the present disclosure.

[0102] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the present disclosure, wherein the composition comprises a trans-splicing RNA molecule of the present disclosure or a vector encoding the same, and wherein the composition alters the activity of a protein encoded by the RNA molecule.

[0103] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a genetic disease or disorder. In some embodiments, the genetic disease or disorder is a monogenic disease or disorder. In some embodiments, the monogenic disease or disorder is an autosomal dominant disease or disorder, an autosomal recessive disease or disorder, an X-chromosome linked (X-linked) disease or disorder, an X-linked dominant disease or disorder, an X-linked recessive disease or disorder, a Y-linked disease or disorder, or a mitochondrial disease or disorder. In some embodiments, the monogenic disease or disorder is, but is not limited to, common variable immune deficiency. deficiency, adenosine deaminase deficiency, X-linked severe combined immunodeficiency, beta thalassemia, alpha thalassemia, myelodysplastic syndromes, amyotrophic lateral sclerosis, frontotemporal dementia with parkinsonism, Usher syndrome, Krabbe disease, Niemann-Pick disease, prion diseases, Dravet syndrome, early-onset Parkinson's disease, spinocerebellar ataxia, inherited epilepsy disorders, ataxia-telangiectasia, GM1 gangliosidosis, Gaucher disease, GM2 gangliosidosis, Angelman syndrome, glucose transporter deficiency type 1, Danon disease, Fabry disease, chromosomal dominant polycystic kidney disease, Pompe disease, familial hypercholesterolemia, open angle glaucoma, Hurler syndrome or mucopolysaccharidosis 1, Hunter syndrome or mucopolysaccharidosis 2, Batten disease, Duchenne muscular dystrophy, limb-girdle muscular dystrophy type 1B, limb-girdle muscular dystrophy type 2B, limb-girdle muscular dystrophy type 2D, limb-girdle muscular dystrophy type 2E, limb-girdle muscular dystrophy type 2C, limb-girdle muscular dystrophy type 2F, facioscapulohumeral muscular dystrophy, hemophilia B, hemophilia A, retinitis pigmentosa, cystic fibrosis, autosomal dominant hearing impairment, and nonsyndromic hearing loss. In some embodiments, the genetic disease or disorder is a multigenic disease or disorder. In some embodiments, the genetic disease or disorder is a multigenic disease or disorder.In some embodiments, the monogenic disease or disorder is an autosomal dominant disease or disorder, including but not limited to Huntington's disease, neurofibromatosis type 1, neurofibromatosis type 2, Marfan syndrome, hereditary nonpolyposis colorectal cancer, hereditary multiple exotosis, von Willebrand's disease, and acute intermittent porphyria. In some embodiments, the monogenic disease or disorder is an autosomal recessive disease or disorder, including but not limited to albinism, medium-chain acyl-CoA dehydrogenase deficiency, cystic fibrosis, sickle cell disease, Tay-Sachs disease, Niemann-Pick disease, spinal muscular atrophy, and Robert syndrome. In some embodiments, the monogenic disease or disorder is an X-linked disease or disorder, including but not limited to muscular dystrophy, Duchenne muscular dystrophy, hemophilia, adrenoleukodystrophy (ALD), Rett syndrome, and hemophilia A. In some embodiments, the monogenic disease or disorder is a mitochondrial disorder, including but not limited to Leber's hereditary optic neuropathy.

[0104] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, immune diseases or disorders. In some embodiments, the immune disease or disorder is an immune deficiency disease or disorder, including, but not limited to, B cell deficiency, T cell deficiency, neutropenia, asplenia, complement deficiency, acquired immune deficiency syndrome (AIDS), and immune deficiency due to medical intervention (immunosuppression as an intended or adverse effect of medical therapy). In some embodiments, the immune disease or disorder is an autoimmune disease or disorder, including, but not limited to, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune Orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold Agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis cystitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne contralateralis), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, acneic conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucca-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, Ophthalmic cicatricial pemphigoid, Optic neuritis, Relapsing rheumatoid arthritis (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Pars planitis (peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndrome type I, II, III, Polymyalgia rheumatica, Polymyositis, Cardiac Post-myocardial infarction syndrome, Post-pericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt's syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm and testicular autoimmunity, stiff-body syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, or Wegener's granulomatosis.

[0105] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, an inflammatory disease or disorder.

[0106] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a metabolic disease or disorder.

[0107] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a degenerative or progressive disease or disorder. In some embodiments, the degenerative or progressive disease or disorder includes, but is not limited to, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and aging.

[0108] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, an infectious disease or disorder.

[0109] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a childhood or developmental disease or disorder.

[0110] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a cardiovascular disease or disorder.

[0111] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a proliferative disease or disorder. In some embodiments, the proliferative disease or disorder is cancer. In some embodiments, the cancer includes, but is not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, Kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), primary CNS lymphoma (lymphoma), anal cancer, appendix cancer, gastrointestinal carcinoid tumor, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system (brain tumor), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma, brain tumor, breast cancer, Burkitt's sarcoma, osteoporosis ... Lymphoma, Carcinoid Tumor, Carcinoma, Cardiac (Heart) Tumor, Embryonal Tumor, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myeloid Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-cell Lymphoma, Ductal Carcinoma in Situ, Embryonal Tumor, Endometrial Cancer (Uterine Cancer), Ependymoma, Esophageal Cancer, Esthesioneuroblastoma (Head and Neck Cancer), Ewing's Sarcoma (Bone Cancer), Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumors, Eye Cancer, Pediatric Intraocular Melanoma, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Pediatric Gastrointestinal Stromal Tumors, Germ Cell Tumors, Pediatric Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Heart Tumors, Hepatocellular (Liver) Carcinoma, Histiocytosis, Hodgkin's Lymphoma, Hypopharyngeal carcinoma (head and neck cancer), intraocular melanoma, pancreatic islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma (soft tissue sarcoma), kidney (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer (head and neck cancer), leukemia, lip and oral cancer (head and neck cancer), liver cancer, lung cancer (non-small cell and small cell), childhood lung cancer, lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma (skin cancer), mesothelioma, metastatic squamous cell neck cancer (head and neck cancer) of unknown primary origin, midline tract carcinoma with NUT gene alterationChanges), Mouth Cancer (Head and Neck Cancer), Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes, Myelodysplastic / Myeloproliferative Neoplasms, Nasal and Paranasal Sinus Cancer (Head and Neck Cancer), Nasopharyngeal Cancer (Head and Neck Cancer), Neuroblastoma, Non-Hodgkin's Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer Cancer), Lip and oral cavity and oropharyngeal cancer, Osteosarcoma and Malignant fibrous histiocytoma of bone, Ovarian cancer, Pancreatic cancer, Pancreatic neuroendocrine tumors (islet cell tumors), Papillomatosis, Paraganglioma, Parathyroid cancer, Penile cancer, Pharyngeal cancer (Head and neck cancer), Pheochromocytoma, Plasma cell neoplasms / multiple myeloma, Pleuropulmonary blastoma, Pregnancy and breast cancer, Primary central nervous system (CNS) lymphoma, Primary peritoneal cancer, Prostate cancer, Rectal cancer, Recurrent cancer, Renal cell (kidney) cancer, Retinoblastoma, Rhabdomyosarcoma, Children (Soft Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer), Sarcoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Childhood Vascular Tumor (Soft Tissue Sarcoma), Ewing Sarcoma (Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma (Bone Cancer), Uterine Sarcoma, Sezary Syndrome, Lymphoma, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Cutaneous Squamous Cell Carcinoma, Squamous Cell Cervical Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer These include, but are not limited to, nasopharyngeal carcinoma (head and neck cancer), nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, thymoma and thymic carcinoma, thyroid carcinoma, transitional cell carcinoma of the renal pelvis and ureter, renal cell carcinoma, urethral carcinoma, uterine sarcoma, vaginal carcinoma, vascular tumors (soft tissue sarcoma), vulvar carcinoma, Wilms' tumor, and other childhood kidney tumors.

[0112] In some embodiments of the compositions and methods of the present disclosure, the disease or disorder of the present disclosure includes, but is not limited to, a proliferative disease or disorder. In some embodiments, the proliferative disease or disorder is cancer. In some embodiments, the cancer includes the presence of a gene fusion that produces a chimeric RNA with a sequence derived from two genes due to DNA deletion or translocation. Gene fusion pairs include MAN2A1 and FER, DNAJB1 and PRKACA, BCR-ABL1, TMPRSS2 and ERG, EWSR1 and FLI1, PML and RARA, EML4 and ALK, KIAA1549 and BRAF, CCDC6 and RET, SS18 and SSX1, RUNX1 and RUNX1T1, PAX3 and FOXO1, NCOA4 and RET, ETV6 and RUNX1, FUS and DDIT3, SS18 and SSX2, NPM1 and ALK, KMT2A and AFF1, TCF1 and ALK, and ALK. 3 and PBX1, STIL and TAL1, COL1A1 and PDGFB, CRTC1 and MAML2, NAB2 and STAT6, EWSR1 and ATF1, ETV6 and NTRK3, EWSR1 and ERG, EWSR1 and WT1, DNAJB1 and PRKACA, PAX7 and FOXO1, FUS and CREB3L2, CBFA2T3 and GLIS2, PAX8 and PPARG, KMT2A and MLLT1, EWSR1 and NR4A3, KMT2A and MLLT3, ASPSCR1 and TFE3, HM GA2 and LPP, JAZF1 and SUZ12, KIF5B and RET, FUS and ERG, SLC45A3 and ERG, NUP214 and ABL1, SET and NUP214, CD74 and ROS1, ETV6 and ABL1, TPM3 and NTRK1, PRKAR1A and RET, EWSR1 and CREB1, KMT2A and AFDN, EWSR1 and DDIT3, CLTC and ALK, ETV6 and PDGFRB, TPM3 and ALK, KMT2A and MLLT10, TMPRSS2 and ETV1, B RD4 and NUTM1, NUP98 and KDM5A, RANBP2 and ALK, CTNNB1 and PLAG1, KMT2A and ELL, TAF15 and NR4A3, FGFR3 and TACC3, PCM1 and JAK2, YWHAE and NUTM2B, STRN and ALK, CRTC3 and MAML2, CDH11 and USP6, CDKN2D and WDFY2, CIC and DUX4, SLC34A2 and ROS1, ATIC and ALK, CD74 and NRG1, MYB and NFIB, PRCC and TFE3,KIF5B and ALK, TMPRSS2 and ETV4, KMT2A and SEPT9, EWSR1 and POU5F1, FGFR1 and PLAG1, MN1 and ETV6, TBL1XR1 and TP63, KMT2A and EPS15, SLC45A3 and ELK4, DHH and RHEBL1, HEY1 and NCOA2, EZR and ROS1, GOPC and ROS1, HMGA2 and WIF1, KMT2A and CREBBP, SS18 and SSX4B, FAM131B and BRAF, EWSR1 and FEV, EWSR1 and PBX1, TPM4 and ALK, SND1 and BRAF, ACTB and GLI1, KM T2A and KNL1, KMT2A and SEPT6, SDC4 and ROS1, TFG and ALK, HNRNPA2B1 and ETV1, PTPRK and RSPO3, JAZF1 and PHF1, HMGA2 and RAD51B, KMT2A and MLLT11, TPR and NTRK1, AKAP9 and BRAF, FUS and CREB3L1, ETV6 and JAK2, HMGA2 and NFIB, KMT2A and AFF3, CHCHD7 and PLAG1, VTI1A and TCF7L2, LIFR and PLAG1, EWSR1 and ETV1, SRGAP3 and RAF1, KMT2A and AFF4, MEAF6 and PHF 1, PAX3 and NCOA1, HAS2 and PLAG1, EWSR1 and NFATC2, HIP1 and ALK, GOLGA5 and RET, BCR and JAK2, EWSR1 and ETV4, DCTN1 and ALK, MBTD1 and CXorf67, NDRG1 and ERG, CARS and ALK, SFPQ and TFE3, KMT2A and ARHGAP26, KMT2A and EP300, KMT2A and TET1, PAX5 and JAK2, PPFIBP1 and ALK, YWHAE and NUTM2A, LRIG3 and ROS1, TFG and NTRK1, TPM3 and ROS1, SLC45A3 and ETV1, E RC1 and RET, SEC16A and NOTCH1, KTN1 and RET, SEC31A and JAK2, TCEA1 and PLAG1, QKI and NTRK2, RNF130 and BRAF, EIF3E and RSPO2, EWSR1 and ZNF444, LMNA and NTRK1, PPFIBP1 and ROS1, PWWP2A and ROS1, EWSR1 and YY1, FUS and ATF1, PAX3 and NCOA2, ZC3H7B and BCOR, BRD3 and NUTM1, CANT1 and ETV4, CIC and FOXO4, COL1A1 and USP6, EWSR1 and ZNF384, KMT2A and ABI1,KMT2A and ACTN4, KMT2A and CEP170B, KMT2A and FOXO3, KMT2A and GAS7, KMT2A and MLLT6, KMT2A and SEPT2, KMT2A and SEPT5, MSN and ALK, VCL and ALK, EZR and ERBB4, RELCH and RET, SLC3A2 and NRG1, TRIM24 and BRAF, KLC1 and ALK, ARID1A and MAST2, GPBP1L1 and MAST2, NFIX and MAST1, NOTCH1 and GABBR2, TADA2A and MAST1, ZNF700 and MAST1, TRIM24 and RET, TRIM3 3 and RET, SSBP2 and JAK2, KMT2A and EEFSEC, CLCN6 and BRAF, GNAI1 and BRAF, MKRN1 and BRAF, NACC2 and NTRK2, FGFR1 and TACC1, TRIM27 and RET, HMGA2 and FHIT, HOOK3 and RET, PCM1 and RET, CEP89 and BRAF, CLIP1 and ROS1, ERC1 and ROS1, HLA and A and ROS1, LSM14A and BRAF, MYO5A and ROS1, SHTN1 and ROS1, TP53 and NTRK1, TPM3 and ROS1, ZCCHC8 and ROS1, FGFR3 and BAIA P2L1, KLK2 and ETV1, ACSL3 and ETV1, NUP107 and LGR5, HMGA2 and CCNB1IP1, HMGA2 and COX6C, GATM and BRAF, HACL1 and RAF1, HERPUD1 and BRAF, ZSCAN30 and BRAF, SLC45A3 and BRAF, HMGA2 and LHFPL6, COL1A2 and PLAG1, ESRP1 and RAF1, IRF2BP2 and CDX1, TFG and NR4A3, CLTC and TFE3, EWSR1 and MYB, NONO and TFE3, FCHSD1 and BRAF, HMGA2 and EBF1, ACBD6 and RRP1 5, AGPAT5 and MCPH1, AGTRAP and BRAF, ARFIP1 and FHDC1, ATG4C and FBXO38, BBS9 and PKD1L1, CENPK and KMT2A, CNBP and USP6, DDX5 and ETV4, EIF3K and CYP39A1, EPC1 and PHF1, ERO1A and FERMT2, ETV6 and ITPR2, EWSR1 and NFATC1, EWSR1 and PATZ1, EWSR1 and SMARCA5, EWSR1 and SP3, FBXL18 and RNF216, FGFR1 and ZNF703, FN1 and ALK, FUS and FEV, GMDS and PDE8B,HMGA2 and ALDH2, IL6R and ATP8B2, INTS4 and GAB2, JPT1 and USH1G, KLK2 and ETV4, KMT2A and ABI2, KMT2A and ARHGEF12, KMT2A and BTBD18, KMT2A and CASP8AP2, KMT2A and CBL, KMT2A and CIP2A, KMT2A and CT45A2, KMT2A and DAB2IP, KMT2A and FOXO4, KMT2A and FR YL, KMT2A and GMPS, KMT2A and GPHN, KMT2A and LASP1, KMT2A and LPP, KMT2A and MAPRE1, KMT2A and MYO1F, KMT2A and NCKIPSD, KMT2A and NRIP3, KMT2A and PDS5A, KMT2A and PICALM, KMT2A and PRRC1, KMT2A and SARNP, KMT2A and SH3GL1, KMT2A and SORBS2, KMT2A and TOP3A, KMT2A and ZFYVE19, MBOAT2 and PRKCE, MIA2 and GEMIN2, NF1 and ASIC2, NFIA and EHF, NTN1 and ACLY, OMD and USP6, PLA2R1 and RBMS1, PLXND1 and TMCC1, RAF1 and DAZL, RBM14 and PACS1, RGS22 and SYCP1, SEC31A and ALK, SEPT8 and AFF4, SLC22A1 and CUTA, S These include, but are not limited to, LC26A6 and PRKAR2A, SLC45A3 and ETV5, SQSTM1 and ALK, SS18L1 and SSX1, SSH2 and SUZ12, SUSD1 and PTBP3, TCF12 and NR4A3, TECTA and TBCEL, THRAP3 and USP6, TMPRSS2 and ETV5, TPR and ALK, UBE2L3 and KRAS, WDCP and ALK, SS18 and USP6.

[0113] In some embodiments of the methods of the present disclosure, the subject of the present disclosure has been diagnosed with a disease or disorder. In some embodiments, the subject of the present disclosure exhibits at least one sign or symptom of the disease or disorder. In some embodiments, the subject has a biomarker that predicts the risk of developing the disease or disorder. In some embodiments, the biomarker is a genetic mutation.

[0114] In some embodiments of the method of the present disclosure, the subject of the present disclosure is a female.In some embodiments of the method of the present disclosure, the subject of the present disclosure is a male.In some embodiments, the subject of the present disclosure has two XX or XY chromosomes.In some embodiments, the subject of the present disclosure has two XX or XY chromosomes and a third chromosome that is either X or Y.

[0115] In some embodiments of the methods of the present disclosure, the subject of the present disclosure is a newborn, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the present disclosure, the subject of the present disclosure is at least 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, or 31 days old. In some embodiments of the methods of the present disclosure, the subject of the present disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old. In some embodiments of the methods of the present disclosure, the subject of the present disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 years old, or any age or subage in between.

[0116] In some embodiments of the methods of the present disclosure, the subject of the present disclosure is a mammal. In some embodiments, the subject of the present disclosure is a non-human mammal.

[0117] In some embodiments of the methods of the present disclosure, the subject of the present disclosure is a human.

[0118] In some embodiments of the method of the present disclosure, the therapeutically effective amount comprises a single dose of the composition of the present disclosure. In some embodiments, the therapeutically effective amount comprises at least one dose of the composition of the present disclosure. In some embodiments, the therapeutically effective amount comprises one or more doses of the composition of the present disclosure.

[0119] In some embodiments of the disclosed methods, the therapeutically effective amount eliminates a sign or symptom of the disease or disorder, hi some embodiments, the therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder.

[0120] In some embodiments of the methods of the present disclosure, the therapeutically effective amount eliminates the disease or disorder.

[0121] In some embodiments of the disclosed methods, the therapeutically effective amount prevents the onset of a disease or disorder. In some embodiments, the therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, the therapeutically effective amount reduces the severity of a sign or symptom of a disease or disorder. In some embodiments, the therapeutically effective amount improves the prognosis of a subject.

[0122] In some embodiments of the methods of the present disclosure, the compositions of the present disclosure are administered to the subject systemically. In some embodiments, the compositions of the present disclosure are administered to the subject via an intravenous route. In some embodiments, the compositions of the present disclosure are administered to the subject by injection or infusion.

[0123] In some embodiments of the method of the present disclosure, the composition of the present disclosure is administered to the subject locally. In some embodiments, the composition of the present disclosure is administered to the subject by intraosseous, intraocular, intracerebrospinal or intraspinal route. In some embodiments, the composition of the present disclosure is administered directly to the cerebrospinal fluid of the central nervous system. In some embodiments, the composition of the present disclosure is administered directly to the tissue or fluid of the eye and does not have bioavailability outside the eye structure. In some embodiments, the composition of the present disclosure is administered to the subject by injection or infusion.

[0124] In some embodiments, the compositions comprising the trans-splicing RNA disclosed herein are formulated as pharmaceutical compositions. Briefly, the pharmaceutical compositions for use disclosed herein may comprise fusion protein(s) or polynucleotides encoding fusion protein(s) that may be included in AAV, which may be immuno-orthogonal, in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise a buffer, such as neutral buffered saline, phosphate buffered saline; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); preservatives. The compositions of the present disclosure may be formulated for oral, intravenous, topical, enteral, intraocular, and / or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intravenous administration. Exemplary embodiments

[0125] Embodiment 1: A composition comprising a trans-splicing nucleic acid comprising: (a) one or more replacement domains encoding a therapeutic sequence, operably linked to (b) one or more intron domains that facilitate RNA splicing of the replacement domain, the replacement domain comprising(s) an intron trans-splicing enhancing sequence(s), and (c) one or more antisense domains that facilitate binding to a target RNA molecule.

[0126] Embodiment 2: The intron trans-splicing enhancing sequence (trans-splicing enhancer sequence) is selected from the group consisting of TTACGG, TAACGG, GGGTTT, GTTTTG, GGTTTT, GGTTTG, GGTTGG, GTTAGG, TGGTTG, GGGTAG, GGTAGG, GGTAGT, GTAGTT, GTTGGT, GTGGTT, GGTGGT, TGGTGG, TTGGTG, GTAAGG, TAAGGG, TTAGGG, TAGGGG, TTGGGG, GTTGGG, GTAGGG, TATTGG, TGTTGG, TATGGG, TTTGGG, TGTGGG, TTGTGG, GAGTGT, GAGGTA, GGAGGT, TGGGAG, GGGGTG, GGGG 2. The composition of embodiment 1, wherein the composition is derived from or isolated from the group of sequences consisting of: GA, GGGGGT, GGGGTA, GGGAGG, GGGTGG, GGAGGG, GGTGGG, GAGGGG, GTGGGG, GAGTGG, GTATGG, GGTATT, GTATTT, GTATTG, AGTTTA, AGGTTA, GTAACG, AGGTAA, GGTAAG, TGGGGG, AGGGTT, AGGTTG, AGGTAG, ATTTGG, AGTTGG, TCTGGG, AGAGTG, AGAGGG, AGTGTG, AGAGGT, AGGGAG, AGGGTG, AGGGGG, AGGGGT, AGTGGG, AGTATG, AGGTAT, GTATTC, GGTAAC.In some embodiments, in the exemplary trans-splicing enhancer sequences above, any of the thymidine bases, a portion of the thymidine bases, or all of the thymidine bases can be substituted with uracil, such that exemplary trans-splicing enhancer sequences include, but are not limited to, UUACGG, UAACGG, GGGUUU, GUUUUG, GGUUUU, GGUUUG, GGUUGG, GUUAGG, UGGUUG, GGGUAG, GGUAGG, GGUAGU, GUAGUU, GUUGGU, GUGGUU, GGUGGU, UGGUGG, UUGGUG, GUAAGG, UAAGGG, UUAGGG, UAGGGG, UUGGGG, GUUGGG, GUAGGG, UAUUGG, UGUUGG, UAUGGG, UUUGG G. Includes UAACG, AGGUAA, GGUAAG, UGGGGG, AGGGUU, AGGUUG, AGGUAG, AUUUGG, AGUUGG, UCUGGG, AGAGUG, AGAGGG, AGUGUG, AGAGGU, AGGGAG, AGGGUG, AGGGGG, AGGGGU, AGUGGG, AGUAUG, AGGUAU, GUAUUC, GGUAAC.

[0127] Embodiment 3: The intron trans-splicing enhancing sequence (trans-splicing enhancer sequence) has the formula X1X2X3X4X5X6, where X1 is selected from the group comprising adenine (A), uracil (U) and guanine (G), X2 is selected from the group comprising adenine (A), uracil (U) and guanine (G), and X3 is selected from the group comprising adenine (A), uracil (U) and guanine (G). , X4 is selected from the group comprising adenine (A), uracil (U), cytosine (C) and guanine (G), X5 is selected from the group comprising adenine (A), cytosine (C), uracil (U) and guanine (G), and X6 is selected from the group comprising adenine (A), uracil (U) and guanine (G).

[0128] Embodiment 4: The composition of embodiment 1, wherein the trans-splicing enhancer sequence consists of a strand of RNA nucleobases comprising at least one RNA motif having the formula X1X2X3X4X5X6, where X1 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X2 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X3 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X4 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X5 is selected from the group comprising adenine (A), uracil (U) and guanine (G); and X6 is selected from the group comprising uracil (U) and guanine (G).

[0129] Embodiment 5: The composition of embodiment 1, wherein the trans-splicing enhancer sequence consists of a strand of RNA nucleobases comprising at least one RNA motif having the formula X1X2X3X4X5X6, where X1 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X2 is selected from the group comprising uracil (U) and guanine (G); X3 is selected from the group comprising adenine (A), uracil (U) and guanine (G); X4 is selected from the group comprising uracil (U) and guanine (G); X5 is selected from the group comprising uracil (U) and guanine (G); and X6 is selected from the group comprising uracil (U) and guanine (G).

[0130] Embodiment 6: The replacement domain is selected from the group consisting of GLB1 (GM1 gliosis); GBA (Gaucher disease); GM2A (GM2 gangliosidosis); PCSK9, LDLR, APOB, APOE (familial hypercholesterolemia); GAA (Pompe disease); MYOC, OPTN, TBK1, WDR36, CYPIB1 (open angle glaucoma); IDS (Hunter syndrome or mucopolysaccharidosis 2); IDUA (Hurler syndrome or mucopolysaccharidosis 1); CLN3 (Batten disease); F9 (hemophilia B); F8 (hemophilia A), LAMP2 (Danon disease); GLA (Fabry disease); SLC2A1 (glucose transporter deficiency type 1); UBE3A (Angelman syndrome); MYOC, OPTN, TBK1, WDR36, CYPIB1 (open angle glaucoma); IDUA (Hurler syndrome or mucopolysaccharidosis 1); The composition of embodiments 1 to 5, wherein the gene is derived from or isolated from a human gene selected from the group consisting of: IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-49, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48 ...

[0131] Embodiment 7: The composition of embodiments 1-5, wherein the replacement domain is derived from or isolated from an expression-enhancing sequence selected from the group consisting of Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE), triplex from MALAT1, PRE of Hepatitis B Virus (HPRE), and iron response element.

[0132] Embodiment 8: The antisense domain is selected from the group consisting of TNFRSF13B (common variable immune deficiency), ADA, CECR1 (adenosine deaminase deficiency), IL2RG (X-linked severe combined immunodeficiency), HBB (beta thalassemia), HBA1, HBA2 (alpha thalassemia), U2AF1 (myelodysplastic syndrome), SOD1, TARDBP, FUS, MATR3, SOD1, C9ORF72 (amyotrophic lateral sclerosis), MAPT, PGRN (frontotemporal dementia with parkinsonism), CDH23, MYO7A, USH2A (Usher syndrome), GALC (Krabbe disease), SMPD1, NPC1, NPC2 (Niemann-Pick disease), PRNP (prion disease), SCN1A (Dravet syndrome), PINK1, ATPGAP2 (early onset Parkinson's disease), ATXN1, ATXN2, ATXN3, PLEKHG4, SPTBN2, CACNA1A, ATXN7, TTBK2, PPP2R2B, KCNC3, PRKCG, ITRP1, TBP, KCND1, FGF14 (spinocerebellar ataxia), SCN1A, SCN2A, CACNA1A, GRIN2B, GRIN2A, MECP2, FOXG1, SLC6A1, PR RT2, PTEN, KCNQ2, KCNQ3, STARD7, CLRN1 (inherited epilepsy disorder), ATM (ataxia telangiectasia), GLB1 (GM1 gangliosidosis), GBA (Gaucher disease), GM2A (GM2 gangliosidosis), UBE3A (Angelman syndrome), SLC2A1 (glucose transporter deficiency type 1), LAMP2 (Danon disease), GLA (Fabry disease), PKD1, PKD2 (autosomal dominant polycystic kidney disease), GAA (Pompe disease), PCSK9, LDLR, APOB, APOE ( familial hypercholesterolemia), MYOC, OPTN, TBK1, WDR36, CYPIB1 (open angle glaucoma), IDUA (Hurler syndrome or mucopolysaccharidosis 1), IDS (Hunter syndrome or mucopolysaccharidosis 2), CLN3 (Batten disease), DMD (Duchenne muscular dystrophy), LMNA (limb-girdle muscular dystrophy type 1B), DYSF (limb-girdle muscular dystrophy type 2B), SGCA (limb-girdle muscular dystrophy type 2D), SGCB (limb-girdle muscular dystrophy type 2E), SGCG (limb-girdle muscular dystrophy type 2C),The composition according to any one of the preceding embodiments, which is derived from or isolated from a human gene selected from the group consisting of SGCD (limb-girdle muscular dystrophy type 2F), DUX4, D4Z4 (facioscapulohumeral muscular dystrophy), F9 (hemophilia B), F8 (hemophilia A), USHA2A, RPGR, RP2, RHO, PRPF31, USH1F, PRPF3, PRPF6 (retinitis pigmentosa), CFTR (cystic fibrosis), GJB2, GJB6, STRC, DFNA1, DFNA14 (autosomal dominant hearing impairment), POU3F3 (nonsyndromic hearing loss).

[0133] Embodiment 9: The composition of any one of embodiments 1 to 5, wherein the trans-splicing RNA comprises an untranslated region that alters the localization, processing or transport of the trans-splicing nucleic acid.

[0134] Embodiment 10: A composition described in any one of embodiments 1 to 13, wherein the sequence comprising the trans-splicing nucleic acid comprises a sequence that is bound by an RNA-binding protein that increases the trans-splicing efficiency.

[0135] Embodiment 11: The composition of any one of embodiments 1 to 13, wherein the trans-splicing nucleic acid is RNA, DNA, a DNA / RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs.

[0136] Embodiment 12: The composition of any one of embodiments 1 to 1, wherein the trans-splicing nucleic acid molecule further comprises a heterologous promoter.

[0137] Embodiment 13: The composition of any one of embodiments 1 to 13, wherein the promoter is isolated from or derived from a promoter capable of driving expression of a transfer RNA (tRNA). Cells and tissues

[0138] In some embodiments of the compositions and methods of the present disclosure, the cell of the present disclosure is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell. In some embodiments, the cell is a non-human mammalian cell, such as a non-human primate cell. In some embodiments, the cell of the present disclosure is a somatic cell. In some embodiments, the cell of the present disclosure is a germline cell. In some embodiments, the germline cell of the present disclosure is not a human cell.

[0139] In some embodiments of the compositions and methods of the present disclosure, the cell of the present disclosure is a stem cell. In some embodiments, the cell of the present disclosure is an embryonic stem cell. In some embodiments, the embryonic stem cell of the present disclosure is not a human cell. In some embodiments, the cell of the present disclosure is a pluripotent stem cell or a multipotent stem cell. In some embodiments, the cell of the present disclosure is an adult stem cell. In some embodiments, the cell of the present disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, the cell of the present disclosure is a hematopoietic stem cell (HSC).

[0140] In some embodiments of the compositions and methods of the present disclosure, the immune cells of the present disclosure are lymphocytes. In some embodiments, the immune cells of the present disclosure are T lymphocytes (also referred to herein as T cells). Exemplary T cells of the present disclosure include, but are not limited to, naive T cells, effector T cells, helper T cells, memory T cells, regulatory T cells (Tregs), and gamma delta T cells. In some embodiments, the immune cells of the present disclosure are B lymphocytes. In some embodiments, the immune cells of the present disclosure are natural killer cells. In some embodiments, the immune cells of the present disclosure are antigen presenting cells.

[0141] In some embodiments of the compositions and methods of the present disclosure, the muscle cells of the present disclosure are myoblasts or muscle cells. In some embodiments, the muscle cells of the present disclosure are cardiomyocytes, skeletal muscle cells, or smooth muscle cells. In some embodiments, the muscle cells of the present disclosure are striated cells.

[0142] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are epithelial cells. In some embodiments, the epithelial cells of the present disclosure form squamous epithelium, cuboidal cell epithelium, columnar cell epithelium, stratified cell epithelium, pseudostratified columnar cell epithelium, or transitional cell epithelium. In some embodiments, the epithelial cells of the present disclosure form glands, including but not limited to the pineal gland, thymus, pituitary gland, thyroid gland, adrenal gland, apocrine gland, holocrine gland, merocrine gland, serous gland, mucous gland, and sebaceous gland. In some embodiments, the epithelial cells of the present disclosure contact the exterior surface of an organ, including but not limited to the lung, spleen, stomach, pancreas, bladder, intestine, kidney, gallbladder, liver, larynx, or pharynx. In some embodiments, the epithelial cells of the present disclosure contact the exterior surface of a blood vessel or vein.

[0143] In some embodiments of the compositions and methods of the present disclosure, the brain cells of the present disclosure are neuronal cells. In some embodiments, the neuronal cells of the present disclosure are neurons of the central nervous system. In some embodiments, the neuronal cells of the present disclosure are neurons of the brain or spinal cord. In some embodiments, the neuronal cells of the present disclosure are neurons of the cranial nerves or optic nerves. In some embodiments, the neuronal cells of the present disclosure are neurons of the peripheral nervous system. In some embodiments, the neuronal cells of the present disclosure are glial cells or glial cells. In some embodiments, the glia of the present disclosure are glial cells of the central nervous system, including but not limited to oligodendrocytes, astrocytes, ependymal cells, and microglia. In some embodiments, the glia of the present disclosure are glial cells of the peripheral nervous system, including but not limited to Schwann cells and satellite cells.

[0144] In some embodiments of the compositions and methods of the present disclosure, the liver cells of the present disclosure are hepatocytes. In some embodiments, the liver cells of the present disclosure are hepatic stellate cells. In some embodiments, the liver cells of the present disclosure are Kupffer cells. In some embodiments, the liver cells of the present disclosure are sinusoidal endothelial cells.

[0145] In some embodiments of the compositions and methods of the present disclosure, the retinal cells of the present disclosure are photoreceptors. In some embodiments, the photoreceptor cells of the present disclosure are rods. In some embodiments, the retinal cells of the present disclosure are cones. In some embodiments, the retinal cells of the present disclosure are bipolar cells. In some embodiments, the retinal cells of the present disclosure are ganglion cells. In some embodiments, the retinal cells of the present disclosure are horizontal cells. In some embodiments, the retinal cells of the present disclosure are amacrine cells.

[0146] In some embodiments of the compositions and methods of the present disclosure, the cardiac cells of the present disclosure are cardiomyocytes, hi some embodiments, the cardiac cells of the present disclosure are cardiac pacemaker cells.

[0147] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are primary cells.

[0148] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are cultured cells.

[0149] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are in vivo, in vitro, ex vivo, or in situ.

[0150] In some embodiments of the compositions and methods of the present disclosure, the somatic cells of the present disclosure are autologous or allogeneic. Incorporation by Reference

[0151] All documents cited herein, including cross-references or related patents or applications, are incorporated herein by reference in their entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or embodied herein, or that it alone, or in combination with any other reference or references, teaches, suggests, or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control. Other embodiments

[0152] While particular embodiments of the present disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the present disclosure, and the appended claims include all such changes and modifications that are within the scope of the present disclosure. References Keeling, KM, X. Xue, G. Gunn and DMBedwell (2014). “Therapeutics based on stop codon readthrough.” Annu Rev Genomics Hum Genet 15:371-394. Koller, U., V. Wally, LGMitchell, A. Klausegger, EMMurauer, E. Mayr, C. Gruber, S. Hainzl, H. Hintner and JWBauer (2011). “A novel screening system improves genetic correction by internal exon replacement.” Nucleic Acids Res 39(16):e108. Wang,Y.,M.Ma,X.Xiao and Z.Wang(2012).“Intronic splicing enhancers,cognate splicing factors and context-dependent regulation rules.”Nat Struct Mol Biol 19(10):1044-1052.

Claims

1. (a) one or more replacement domains encoding a therapeutic sequence, operably linked to: (b) one or more intron domains that facilitate RNA splicing of the one or more replacement domains, each of the one or more intron domains comprising a trans-splicing enhancer sequence; (c) one or more antisense domains that facilitate binding to a target RNA molecule; A composition comprising a trans-splicing nucleic acid comprising:

2. The trans-splicing enhancer sequence is represented by the formula X １ X ２ X ３ X ４ X ５ X ６ (In the formula, X １ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ２ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ３ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ４ is selected from the group consisting of adenine (A), uracil (U), cytosine (C) and guanine (G); ５ is selected from the group consisting of adenine (A), cytosine (C), uracil (U) and guanine (G); ６ The composition of claim 1, wherein the nucleic acid sequence is selected from the group consisting of adenine (A), uracil (U) and guanine (G).

3. The trans-splicing enhancer sequence is represented by the formula X １ X ２ X ３ X ４ X ５ X ６ (In the formula, X １ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ２ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ３ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ４ is selected from the group consisting of adenine (A), uracil (U), and guanine (G); ５ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ６ The composition of claim 1, wherein the nucleic acid sequence is selected from the group consisting of uracil (U) and guanine (G).

4. The trans-splicing enhancer sequence is represented by the formula X １ X ２ X ３ X ４ X ５ X ６ (In the formula, X １ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ２ is selected from the group consisting of uracil (U) and guanine (G); ３ is selected from the group consisting of adenine (A), uracil (U) and guanine (G); ４ is selected from the group consisting of uracil (U) and guanine (G); ５ is selected from the group consisting of uracil (U) and guanine (G); ６ The composition of claim 1, wherein the nucleic acid sequence is selected from the group consisting of uracil (U) and guanine (G).

5. The composition of any one of claims 1 to 4, wherein the trans-splicing enhancer sequence is flanked by RNA motifs that further increase trans-splicing efficiency.

6. The composition of any one of claims 1 to 5, wherein the trans-splicing enhancer sequence is less than 300 bases from the 3' splice site of the trans-splicing nucleic acid.

7. The composition of any one of claims 1 to 5, wherein the trans-splicing enhancer sequence is less than 300 bases from the 5' splice site of the trans-splicing nucleic acid.

8. The composition of any one of claims 1 to 7, wherein each of the one or more intron domains comprises two or more trans-splicing enhancer sequences.

9. The composition described in any one of claims 1 to 8, wherein the trans-splicing nucleic acid includes a 3' untranslated region that increases trans-splicing efficiency.

10. The composition of any one of claims 1 to 9, wherein the trans-splicing nucleic acid comprises a 5' untranslated region that increases trans-splicing efficiency.

11. The composition of any one of claims 1 to 10, wherein each of the one or more substituted domains comprises a gene expression enhancing element.

12. 12. The composition of claim 11, wherein the gene expression enhancing element comprises a sequence derived from or isolated from the group consisting of Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE), MALAT1-derived triplex, Hepatitis B virus PRE (HPRE), and iron response element.

13. The composition of any one of claims 1 to 12, further comprising an RNA binding protein that strengthens the interaction between the trans-splicing nucleic acid and the target RNA molecule, increasing trans-splicing efficiency.

14. The composition of any one of claims 1 to 13, wherein the trans-splicing nucleic acid is RNA, DNA, a DNA / RNA hybrid, a nucleic acid analog, a chemically modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs.

15. The composition of any one of claims 1 to 14, wherein the trans-splicing nucleic acid further comprises a heterologous promoter.

16. A vector comprising or encoding the composition of any one of claims 1 to 15.

17. 17. The vector of claim 16, wherein the vector is selected from the group consisting of an adeno-associated viral vector, a retroviral vector, a lentiviral vector, an adenoviral vector, a nanoparticle, a micelle, a liposome, a lipoplex, a polymersome, a polyplex, and a dendrimer.

18. A cell comprising a composition described in any one of claims 1 to 15, or a vector described in claim 16 or claim 17.

19. Use of a composition according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17, in the manufacture of a medicament for treating a disease in a subject in need of such treatment.

20. Use of a composition according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17, in the manufacture of a medicament for repairing a genetic defect in a subject in need of repair of the genetic defect.

21. A composition comprising a composition according to any one of claims 1 to 15, or a vector according to claim 16 or claim 17, for treating a disease in a subject in need of such treatment.

22. A composition comprising a composition described in any one of claims 1 to 15, or a vector described in claim 16 or claim 17, for repairing a genetic defect in a subject in need of repair of the genetic defect.

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