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  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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  • C12N2310/00—Structure or type of the nucleic acid
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
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  • C12N2320/00—Applications; Uses
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  • C12N2320/33—Alteration of splicing
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Definitions

• the present disclosure is directed, generally, to gene therapy, molecular biology, and compositions and methods for altering the sequence composition of RNA molecules.
• compositions and methods for replacement of specific RNA sequences in RNA molecules in human cells with high efficiency are provided.
• present disclosure provides compositions and methods for replacement of chosen RNA sequences within target RNAs using RNA trans-splicing molecules that carry sequences that increase trans-splicing efficiency to treat a disease in the context of a human gene therapy
• RNA trans-splicing has been proposed as a human gene therapeutic but has not experienced success in clinical trials due to low efficiency.
• the present disclosure describes improvements to RNA trans-splicing molecules that could address this long-felt but unmet need.
• the present disclosure provides, in some embodiments, a composition comprising a trans splicing RNA molecule comprising (a) at least one domain that promotes trans-splicing (“Intronic Domain”) that comprises one or more trans-splicing enhancer sequences, (b) at least one binding domain (“Antisense Domain”) that contains or consists of a sequence complementary to a pre- mRNA present in a human cells (“Target RNA”), and (c) a coding domain that is inserted into the Target RNA via trans-splicing (“Replacement Domain”).
• the trans-splicing enhancer sequences increase splicing efficiency so that the trans-splicing RNA molecule can exchange sequences within the Target RNA with the Replacement Domain with high efficiency.
• the present disclosure provides a composition comprising a nucleic acid sequence encoding the trans-splicing RNA molecule.
• the trans-splicing enhancer sequences comprise 5’- X 1 X 2 X 3 X 4 X 5 X 6 -3 wherein Xi is uracil (U) or guanine (G); X 2 is adenine (A), uracil (U) or guanine (G); X 3 is adenine (A), uracil (U) and guanine (G); X 4 is adenine (A), uracil (U), cytosine (C) or guanine (G); X 5 is adenine (A), cytosine (C), uracil (U) or guanine (G); and Xe is adenine (A), uracil (U) or guanine (G).
• the trans-splicing enhancer sequences comprise X 1 X 2 X 3 X 4 X 5 X 6 wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X 2 is selected from the group including adenine (A), uracil (U) and guanine (G); X 3 is selected from the group including adenine (A), uracil (U) and guanine (G); X 4 is selected from the group including adenine (A), uracil (U) and guanine (G); X 5 is selected from the group including adenine (A), uracil (U) and guanine (G); and X ( , is selected from the group including uracil (U) and guanine (G).
• the trans-splicing enhancer sequences comprise X 1 X 2 X 3 X 4 X 5 X 6 wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X 2 is selected from the group including uracil (U) and guanine (G); X 3 is selected from the group including adenine (A), uracil (U) and guanine (G); X4 is selected from the group including uracil (U) and guanine (G); X5 is selected from the group including uracil (U) and guanine (G); and Xe is selected from the group including uracil (U) and guanine (G).
• the trans-splicing enhancer sequences are directly adjacent to the Replacement domain.
• the trans-splicing enhancer sequences are 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,
• the trans-splicing enhancer sequences are 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,
• the Intronic Domain comprises 1 trans-splicing enhancer sequence. In some embodiments, the Intronic Domain comprises 2 or more trans-splicing enhancer sequences. In some embodiments, the Intronic 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.
• the Antisense Domain is complementary to a gene (corresponding accession numbers in brackets, corresponding disease in parentheses) that encodes a Target RNA that sometimes carry disease-causing mutations 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] (Beta-thassalemia); HBA1, HBA2 [ENSG00000206172,
• ENSG00000188536 (alpha-thassalemia); U2AF1 [ENSG00000160201] (myelodysplastic syndrome); SOD1, TARDBP, FUS, MATR3, SOD1, C90RF72 [ENSG00000142168, ENSG00000120948, ENSG00000089280, ENSG00000015479, ENSG00000142168, ENSG00000147894] (Amyotrophic lateral sclerosis); MAPT, PGRN [ENSG00000186868, ENSG00000030582] (Frontotemporal dementia with parkinsonism); CDH23, MY07A, USH2A [ENSG00000107736, ENSG00000137474, ENSG00000042781] (Usher’s syndrome); GALC [ENSG00000054983] (Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655
• the Replacement domain is derived or isolated from the Target RNA.
• the Intronic Domains carry binding sites that are preferentially- targeted by RNA-binding proteins with disease-causing mutations.
• the dissociation constant of these mutated RNA-binding proteins and the Intronic Domain is lower than the dissociation constant of the non-mutated RNA-binding protein and the Intronic Domain.
• the trans-splicing RNA further comprises a 5’ untranslated region.
• the 5’ untranslated region increases the stability of the trans-splicing nucleic acid.
• the 5’ untranslated region reduces the stability of the trans splicing nucleic acid.
• the 5’ untranslated region alters the localization of the trans-splicing nucleic acid.
• the 5’ untranslated region alters the processing of the trans-splicing nucleic acid.
• the trans-splicing RNA further comprises a 3' untranslated region.
• the 3' untranslated region increases the stability of the trans-splicing nucleic acid.
• the 3' untranslated region reduces the stability of the trans splicing nucleic acid.
• the 3' untranslated region alters the localization of the trans-splicing nucleic acid.
• the 3' untranslated region alters the processing of the trans-splicing nucleic acid.
• the Replacement Domain is comprised of sequence derived or isolated from a human gene.
• the sequence comprising the Replacement Domain has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in between of identity with a human gene.
• the Replacement Domain has 100% identity with a sequence derived or isolated from a human gene.
• 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.
• 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 in between of complementarity to the Target RNA sequence.
• the Antisense Domain has 100% complementarity to the Target RNA sequence.
• 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 the complementary to the Target RNA sequence.
• the sequence encoding the trans-splicing RNA further comprises a sequence encoding a promoter capable of expressing the trans-splicing RNA in a eukaryotic cell.
• 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.
• a vector of the disclosure is a viral vector.
• the viral vector comprises a sequence isolated or derived from a retrovirus.
• the viral vector comprises a sequence isolated or derived from a lentivirus.
• the viral vector comprises a sequence isolated or derived from an adenovirus.
• the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
• AAV adeno-associated virus
• the viral vector is replication incompetent.
• the viral vector is isolated or recombinant.
• the viral vector is self-complementary.
• the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
• AAV adeno-associated virus
• the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.
• the viral vector is replication incompetent.
• the viral vector is isolated or recombinant (rAAV).
• the viral vector is self-complementary (scAAV).
• a vector of the disclosure is a non-viral vector.
• the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex, an exosome or a dendrimer.
• the vector is an expression vector or recombinant expression system.
• the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.
• an expression vector, viral vector or non-viral vector provided herein includes without limitation, an expression control element.
• An “expression control element” as used herein 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.
• Expression control elements may be constitutive, inducible, repressible, or tissue-specific, for example.
• a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. In some embodiments, expression control by a promoter is tissue-specific.
• Non-limiting exemplary promoters include CMV, CBA, CAG, Cbh, EF-la, PGK, UBC, GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, hb2, PPE, ENK, EAAT2, GFAP, MBP, HI and U6 promoters.
• the promoter is a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA).
• 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
• 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 WPRE.
• an expression vector, viral vector or non-viral vector includes without limitation, vector elements such as an IRES or 2A peptide sites for configuration of “multi cistronic” or “polycistronic” or “bicistronic” or tricistronic” constructs, i.e., having double or triple or multiple coding areas or exons, and as such will have the capability to express from mRNA two or more proteins from a single construct.
• Multi cistronic vectors simultaneously express two or more separate proteins from the same mRNA. The two strategies most widely used for constructing multi cistronic configurations are through the use of an IRES or a 2A self-cleaving site.
• an “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs.
• an IRES is an RNA element that allows for translation initiation in a cap-independent manner.
• self-cleaving peptides or “sequences encoding self-cleaving peptides” or “2A self cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or sequences encoding the self-cleaving peptides.
• the vector is a viral vector.
• the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.
• the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors.
• 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 incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from .3 kb to 4.75 kb.
• exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can 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.rhlO vector, a modified AAV.rhlO 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.rh64Rl vector, and a modified AAV.rh64Rl vector and any
• the lentiviral vector is an integrase-competent lentiviral vector (ICLV).
• the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
• Lentiviral vectors are well-known in the art (see, e.g., Trono D.
• exemplary lentiviral vectors that may be used in any of the herein described compositions, systems, methods, and kits can include a human immunodeficiency virus (HIV) 1 vector, a modified human immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mangabey simian immunodeficiency virus (SIVSM) vector, a modified sooty mangabey simian immunodeficiency virus (SIVSM) vector, a African green monkey simian immunodeficiency virus (SIVAGM) vector, a modified African green monkey simian immunodeficiency virus (SIVAGM) vector, an equine infectious an HIV 1 vector
• a modified human immunodeficiency virus (HIV) 1 vector a human immunodeficiency virus (HIV) 2 vector
• 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.
• nucleic acid analog refers to a compound having structural similarity to a canonical purine or pyrimidine base occurring in DNA or RNA.
• the nucleic acid analog may contain a modified sugar and/or a modified nucleobase, as compared to a purine or pyrimidine base occurring naturally in DNA or RNA.
• the nucleic acid analog is a 2’- deoxyribonucleoside, 2’-ribonucleoside, 2’-deoxyribonucleotide or a T -ribonucleotide
• the nucleobase includes a modified base (such as, for example, xanthine, uridine, oxanine (oxanosine), 7-methlguanosine, dihydrouridine, 5-methylcytidine, C3 spacer, 5-methyl dC, 5- hydroxybutynl-2’-deoxyuridine, 5-nitroindole, 5-methyl iso-deoxy cytosine, iso deoxyguanosine, deoxyuradine, iso deoxycytidine, other 0-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deaza hypoxanthine, other 7-deazapurines, and 2-methyl purines).
• a modified base such as, for example, xanthin
• 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.
• the nucleic acid analog is a nucleic acid mimic (such as, for example, artificial nucleic acids and xeno nucleic acids (XNA).
• FIGURE 1 illustrates the unmet need addressed by the present invention and provides a schematic of the invention.
• FIGURE 1A illustrates the concept of human genetic disease where mutated (“defective”) DNA sequences are transcribed into RNA which directly contribute to disease (“RNA pathogenicity”) or are translated into disease-causing protein (“translation of pathogenic protein”).
• FIGURE IB illustrates the state-of-the-art RNA trans-splicing technology where a mutation-carrying RNA molecule is targeted by a trans-splicing RNA that corrects the mutation but with low efficiency. This low efficiency is typically insufficient to halt or reverse progression of disease.
• FIGURE 1C illustrates the present invention where the trans-splicing molecule carries trans-splicing enhancer sequences that increase the efficiency of the trans splicing reaction. This supports halting or reversal of disease progression and/or elimination of key disease phenotypes thereby providing an effective therapeutic for human genetic disease.
• FIGURE 2 illustrates three embodiments of the trans-splicing RNA described in this disclosure.
• FIGURE 2A describes a double trans-splicing molecule which carries two antisense domains, one replacement domain, two intronic domains, and at least two trans-splicing enhancer sequences within the intronic domains. This design promotes replacement of an internal sequence within the target RNA while maintaining the adjacent 5’ and 3’ sequences around the replaced sequence.
• FIGURES 2B and 2C describe terminal trans-splicing molecules that both contain one antisense domain, one replacement domain, one intronic domain, and at least one trans-splicing enhancer sequence within the intronic domain.
• FIGURE 2B illustrates the design of a 3’ terminal trans-splicing RNA that will replace the 3’ terminal end of a target RNA while maintaining the 5’ end.
• FIGURE 2C illustrates the design of a 5’ terminal trans-splicing molecule that will replace the 5’ terminal end of a target RNA while maintaining the 3’ end.
• FIGURE 3 illustrates an experiment designed to reveal the importance of trans-splicing enhancer sequences in the context of internal trans-splicing via production of GFP protein.
• FIGURE 3 A illustrates the design of a split GFP reporter that carries N- and C-terminal portions of GFP (“N-GFP” and “C-GFP”) but lacks an internal GFP sequence required for fluorescence. In the reporter, this internal sequence is replaced by a short exon with a stop codon that is flanked by introns.
• the internal sequence (“int-GFP”) is the replacement sequence within an RNA trans splicing molecule that is flanked by two intronic sequences and two antisense sequences.
• FIGURE 3B illustrates the activity of the reporter alone so that cis-splicing produces a GFP sequence interrupted by a stop codon therefore producing no GFP signal.
• FIGURE 3C illustrates the activity of the reporter in the presence of the trans-splicing molecule without inclusion of trans-splicing enhancer sequences in the trans-splicing molecule so that similarly cis-splicing occurs primarily and GFP signal is not produced.
• FIGURE 3D illustrates the activity of the reporter in the presence of the trans-splicing molecule with inclusion of trans-splicing enhancer sequences so that trans-splicing occurs primarily and GFP signal is produced.
• FIGURE 4 illustrates an experiment designed to reveal the importance of trans-splicing enhancer sequences in the context of 5’ terminal trans-splicing.
• FIGURE 4 A illustrates the design of a split GFP reporter that carries a C-terminal portion of GFP (“C-GFP”) but lacks an 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 that is flanked by introns.
• the N-terminal sequence (“N-GFP”) is the replacement sequence within an RNA trans-splicing molecule that is flanked by one intronic sequence and one antisense sequence.
• FIGURE 4B illustrates the activity of the reporter alone so that cis-splicing produces a GFP sequence interrupted by a stop codon therefore producing no GFP signal.
• FIGURE 4C illustrates the activity of the reporter in the presence of the trans-splicing molecule without inclusion of trans-splicing enhancer sequences in the trans splicing molecule so that similarly cis-splicing occurs primarily and GFP signal is not produced.
• FIGURE 4D illustrates the activity of the reporter in the presence of the trans-splicing molecule with inclusion of trans-splicing enhancer sequences so that trans-splicing occurs primarily and GFP signal is produced.
• FIGURE 5 illustrates an experiment designed to reveal the importance of trans-splicing enhancer sequences in the context of 3’ terminal trans-splicing.
• FIGURE 5 A illustrates the design of a split GFP reporter that carries a N-terminal portion of GFP (“N-GFP”) but lacks an 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 that is flanked by introns.
• the C-terminal sequence (“C-GFP”) is the replacement sequence within an RNA trans-splicing molecule that is flanked by one intronic sequence and one antisense sequence.
• FIGURE 5B illustrates the activity of the reporter alone so that cis-splicing produces a GFP sequence interrupted by a stop codon therefore producing no GFP signal.
• FIGURE 5C illustrates the activity of the reporter in the presence of the trans-splicing molecule without inclusion of trans-splicing enhancer sequences in the trans splicing molecule so that similarly cis-splicing occurs primarily and GFP signal is not produced.
• FIGURE 5D illustrates the activity of the reporter in the presence of the trans-splicing molecule with inclusion of trans-splicing enhancer sequences so that trans-splicing occurs primarily and GFP signal is produced.
• FIGURE 6 illustrates the molecular pathology associated with myotonic dystrophy type I and a treatment method for this disease involving a trans-splicing therapeutic.
• FIGURE 6A illustrates that myotonic dystrophy type I (DM1) is caused by a ‘CTG’ repeat expansion in DNA which is transcribed into an RNA composed of repeating ‘CUG’ units. This repetitive RNA preferentially binds a splicing factor protein called MBNL1. The resulting titration of MBNL1 from its typical activities causes widespread dysfunctional RNA splicing in the cell. This dysfunctional splicing is responsible for many pathologies associated with this disease including the characteristic myotonia.
• FIGURE 6B illustrates the activity of a trans-splicing therapeutic that addresses this molecular pathology via amplification of MBNLl protein production.
• a trans-splicing RNA attaches a gene expression-amplifying sequence to the 3’ terminus of MBNLl mRNA and increases MBNLl expression. The resulting increase in MBNLl levels reconstitutes its splicing activity and reverses disease.
• FIGURE 7 illustrates an experiment designed to reveal the importance of trans-splicing enhancer sequences in the context of 3’ terminal trans-splicing for the purpose of increasing gene expression via production of luciferase protein.
• FIGURE 7A illustrates the design of a luciferase reporter that carries a Renilla luciferase control (“R. luciferase”) and a Firefly luciferase (“F. luciferase”) reporter molecule.
• the reporter molecule carries exon 9, intron 9, and exon 10 from the human MBNL1 gene. Exon 10 contains regulatory elements that influence MBNL1 gene expression.
• trans-splicing RNA targets intron 9 and replaces exon 10 with a translation enhancer in a 3’ trans-splicing process. In this manner, successful trans-splicing increases the production of Firefly luciferase relative to Renilla luciferase.
• FIGURE 7B illustrates the activity of the reporter alone so that cis-splicing yields a luciferase molecule with exon 10 of MBNLl.
• FIGURE 7C illustrates the activity of the reporter in the presence of the trans-splicing molecule without inclusion of trans-splicing enhancer sequences in the trans-splicing molecule so that similarly cis-splicing occurs primarily yielding a luciferase molecule with exon 10 of MBNLl.
• FIGURE 7D illustrates the activity of the reporter in the presence of the trans-splicing molecule with inclusion of trans-splicing enhancer sequences so that trans-splicing occurs primarily and exon 10 is replaced by a translation enhancer, therefore increasing Firefly luciferase signal.
• the disclosure provides an RNA molecule that selectively binds and promotes a trans splicing reaction with an RNA molecule with high efficiency.
• the disclosure provides vectors, compositions and cells comprising or encoding the trans-splicing RNA molecule.
• the disclosure provides methods of using the trans-splicing RNA molecule, vectors, compositions and cells of the disclosure to treat a disease or disorder.
• the invention is a trans-splicing RNA molecule comprising three types of domains (FIGURE 2).
• One of the three domain types is the Replacement Domain which is inserted into a Target RNA molecule via a trans-splicing reaction.
• a second domain type is the Antisense Domain which is complementary to a Target RNA.
• a third domain type is the Intronic Domain which promotes the trans-splicing reaction between the trans-splicing RNA molecule and the Target RNA.
• the Intronic Domain further comprises intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) that promote the trans-splicing reaction.
• This novel combination of specific trans-splicing enhancer sequences and the Intronic Domain promotes RNA trans-splicing in a manner that is sufficiently to replace disease-causing RNA sequences in human cells to address disease.
• the 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.
• the trans-splicing RNA molecule implementations show utility in a variety of contexts including replacement of disease-causing sequences or insertion of engineered sequences into Target RNAs.
• the engineered sequences can alter the translation or stability of Target RNAs to increase or decrease protein production or Target RNA levels.
• This disclosure provides vectors, compositions and cells comprising or encoding the trans-splicing RNA and methods of using the trans-splicing RNA compositions.
• the invention is an RNA technology that enables replacement of arbitrary sequences within specific RNA molecules in living cells.
• the technology based on RNA trans splicing, utilizes the naturally-existing spliceosome in human cells to provide the catalytic activity for this trans-splicing process
• RNA splicing occurs within RNA molecules where exons are concatenated and introns removed from immature messenger RNA molecules (pre- mRNAs) to form mature messenger RNA molecules (mRNAs). This process is referred to as cis- splicing.
• RNA trans-splicing is a process by which the spliceosome concatenates exons derived from distinct and separate RNA molecules.
• RNA trans-splicing can insert engineered sequences into a target RNA to impart new activities to the target RNA such as altered RNA stability or altered RNA translation. This feature can be used to increase production of protein by a target RNA. In the broadest sense, this RNA trans-splicing technology can impart arbitrary changes to both coding and non-coding regions of target RNAs.
• trans-splicing enhancer sequences are termed “trans-splicing enhancer sequences”.
• compositions comprising intronic trans-splicing enhancing sequences include any sequences that promote trans-splicing in an efficient manner.
• exemplary trans-splicing enhancer sequences include without limitation: TTACGG, TAACGG, GGGTTT, GTTTTG, GGTTTT, GGTTTG, GGTTGG, GTTAGG, TGGTTG, GGGTAG, GGTAGG, GGTAGT, GTAGTT, GTTGGT, GTGGTT, GGTGGT, TGGTGG, TTGGTG, GTAAGG, TAAGGG, TTAGGG, TAGGGG, TTGGGG, GTTGGG,
• 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.
• the exemplary trans-splicing enhancer sequences include without limitation: 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, UUUGGG, UGUGGG, UUGUGG, GAGUGU, GAGGUA, GGAGGU, UGGGAG, GGGGUG, GGGGGA, GGGGGU, GGGGGGGG
• RNA trans-splicing technology which involves the inclusion of specific intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences), is the first to show RNA-trans-splicing with high efficiency against multiple RNA targets.
• Highly efficient RNA trans-splicing has three primary advantages over previous RNA trans-splicing systems. First, this improved efficiency can replace defective RNA sequences at levels sufficient to reconstitute the activity of mutated genes to treat recessive genetic disorders. Indeed, treatment of many recessive gene disorders require at least 30% efficiency where 100% is complete replacement of a sequence within a Target RNA. Second, this improved efficiency can replace defective RNA sequences at levels sufficient to treat dominant genetic disorders.
• RNA trans-splicing technology As a single mutated allele is sufficient to cause disease, many diseases in this class require highly-efficient replacement of mutated sequences as the mutated sequences typically cause toxicity. As a result, even higher efficiency is required (70%+).
• 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 very general capability, with this disclosure providing demonstrations of RNA trans-splicing system that can efficiently replace sequences with multiple target RNAs.
• RNA trans-splicing enhancer sequences intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) to form the present RNA trans-splicing technology is a general capability that further allows the alteration of non-coding sequences within target RNAs.
• this invention allows the alteration of RNA behaviors such as translation or turnover. The net result of these effects is increased production of protein from Target RNAs or other downstream effects associated with altered RNA levels.
• RNA sequences that influence RNA cis-splicing have been known (Wang, Ma et al.
• FIGURES 3-5 contain a schematic of the plasmids used in the trans-splicing activity assays. Experiments were conducted with either a transiently-transfected reporter and trans splicing molecule or systems packaged in lentivirus. The present inventor observed that some known splicing enhancing sequences that function in the context of cis-splicing (Wang, Ma et al.
• trans-splicing enhancer sequences are termed “trans-splicing enhancer sequences”.
• trans-splicing Enhancing Sequences to increase the translation of specific target RNAs
• RNA trans-splicing system in contrast, could replace sequences in any target mRNA with translation-amplifying sequences to increase protein production.
• the present inventor conceived of efficient RNA trans-splicing mediated by intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) could address this long-felt but unmet need of a means to promote targeted amplification of protein production from specific mRNAs.
• Myotonic dystrophy is caused by RNAs that carry repetitive ‘CUG’ tracts that bind 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 disease in patients.
• the present inventor conceived of increasing MBNL1 protein production with an efficient RNA trans-splicing approach could address this disease via production of sufficient MBNLl protein to reconstitute its typical activities in alternative splicing regulation (FIGURE 6).
• RNA trans-splicing system carrying various cis-splicing enhancer sequences and a Woodchuck Hepatitis Virus (WHV) post-transcriptional Regulatory Element (WPRE) (FIGURE 7).
• WPRE Woodchuck Hepatitis Virus
• the present inventor also created a reporter that contains a firefly luciferase coding sequence and the last 2 exons and intervening intron of MBNLl (FIGURE 7).
• This assay is qualitative, not fully quantitative, but is useful because it is what end-users in cell biology often use when attempting to answer scientific questions about the presence, absence, or general magnitude of a transcript. Indeed, this reporter is based on the pMIR-GLO luciferase vector that is typically used to assess the stability and protein production from a model mRNA.
• trans-splicing enhancer sequences are termed “trans-splicing enhancer sequences”.
• compositions comprising replacement domains disclosed herein include any strategies where replacement or insertion of RNA sequences could be an effective therapy.
• exemplary replacement domains include without limitation sequences derived or isolated from the following genes (with gene accession IDs in brackets and associated diseases in parentheses) such as TNFRSF13B [ENSG00000240505] (common variable immune deficiency); ADA, CECR1 [ENSG00000196839, ENSG00000093072] (Adenosine deaminase deficiency); IL2RG [ENSG00000147168] (X-linked severe combined immunodeficiency); HBB [ENSG00000244734] (Beta-thassalemia); HBA1, HBA2 [ENSG00000206172,
• ENSG00000188536 (alpha-thassalemia); U2AF1 [ENSG00000160201] (myelodysplastic syndrome); SOD1, TARDBP, FUS, MATR3, SOD1, C90RF72 [ENSG00000142168, ENSG00000120948, ENSG00000089280, ENSG00000015479, ENSG00000142168, ENSG00000147894] (Amyotrophic lateral sclerosis); MAPT, PGRN [ENSG00000186868, ENSG00000030582] (Frontotemporal dementia with parkinsonism); CDH23, MY07A, USH2A [ENSG00000107736, ENSG00000137474, ENSG00000042781] (Usher’s syndrome); GALC [ENSG00000054983] (Krabbe disease); SMPD1, NPC1, NPC2 [ENSG00000166311, ENSG00000141458, ENSG00000119655
• the replacement domain is codon optimized.
• Replacement Domains can comprise sequences derived from other organisms in order to alter the stability, translation, processing, or localization of a target RNA.
• a pathogenic RNA molecule is a target RNA.
• the target RNA comprises a target sequence that is complementary to an antisense domain of the trans-splicing RNA of the disclosure.
• the target sequence comprises or consists of between 5 and 500 nucleotides. In some embodiments, the target sequence comprises or consists of between 50 and 250 nucleotides. In some embodiments, the target sequence comprises or consists of between 5 and 50 nucleotides.
• a target sequence is contained within a single contiguous stretch of the target RNA.
• the target sequence may consist of comprise of one or more nucleotides that are not spread among a single contiguous stretch of the target RNA.
• an Antisense Domain of the disclosure binds to a target sequence. In some embodiments of the disclosure, an antisense domain of the disclosure binds to a target RNA. [57] In some embodiments of the disclosure, the Antisense Domain is chosen so that successful trans-splicing causes removal of micro open reading frames in the Target RNA. In this manner, the trans-splicing system removes micro open reading frames and increases the production of protein from the target RNA.
• a vector comprises or encodes a trans-splicing nucleic acid of the disclosure. In some embodiments, the vector comprises or encodes at least one trans-splicing nucleic acid of the disclosure. In some embodiments, the vector comprises or encodes one or more trans-splicing nucleic acid(s) of the disclosure. In some embodiments, the vector comprises or encodes two or more trans-splicing nucleic acids of the disclosure.
• a vector of the disclosure is a viral vector.
• the viral vector comprises a sequence isolated or derived from a retrovirus.
• the viral vector comprises a sequence isolated or derived from a lentivirus.
• the viral vector comprises a sequence isolated or derived from an adenovirus.
• the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
• AAV adeno-associated virus
• the viral vector is replication incompetent.
• the viral vector is isolated or recombinant.
• the viral vector is self-complementary.
• the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
• AAV adeno-associated virus
• the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.
• the viral vector is replication incompetent.
• the viral vector is isolated or recombinant (rAAV).
• the viral vector is self-complementary (scAAV).
• a vector of the disclosure is a non-viral vector.
• the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
• the vector is an expression vector or recombinant expression system.
• the term“recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.
• the liposome, lipoplex, or nanoparticle can further comprise a non- cationic lipid, a PEG conjugated lipid, a sterol, or any combination thereof.
• the liposome, lipoplex, or nanoparticle further comprises a non- cationic lipid, wherein the non-ionic lipid is selected from the group consisting of distearoyl-sn- glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane- 1 -
• the liposome, lipoplex, or nanoparticle further comprises a conjugated lipid, wherein the conjugated lipid, wherein the conjugated-lipid is selected from the group consisting of PEG-diacyl glycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)- 2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcar
• DAG PEG-
• the liposome, lipoplex, or nanoparticle further comprises cholesterol or a cholesterol derivative.
• the liposome, lipoplex, or nanoparticle further comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol.
• the amount of the ionizable lipid, the non-cationic lipid, the conjugated lipid that inhibits aggregation of particles, and the sterol can be varied independently.
• the lipid nanoparticle comprises an ionizable lipid in an amount from about 20 mol % to about 90 mol % of the total lipid present in the particle, a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipid present in the particle, a conjugated lipid that inhibits aggregation of particles in an amount from about 0.5 mol % to about 20 mol % of the total lipid present in the particle, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipid present in the particle.
• the ratio of total lipid to DNA vector can be varied as desired.
• the total lipid to DNA vector (mass or weight) ratio can be from about 10: 1 to about 30: 1.
• an expression vector, viral vector or non-viral vector provided herein includes without limitation, an expression control element.
• An“ expression control element” as used herein 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 may be constitutive, inducible, repressible, or tissue-specific, for example.
• A“promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled.
• Non-limiting exemplary promoters include CMV, CBA, CAG, Cbh, EF-la, PGK, UBC, GUSB, UCOE, hAAT, TBG, Desmin, MCK, C5-12, NSE, Synapsin, PDGF, MecP2, CaMKII, mGluR2, NFL, NFH, hb2, 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 posttranscriptional regulatory elements include the CMV enhancer and WPRE.
• nucleic acid sequences encoding the trans-splicing nucleic acids disclosed herein for use in 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 the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or“ equivalent” polypeptides are encoded by equivalent polynucleotides as described herein.
• nucleic acid sequences may be compared using sequence identity methods run under default conditions. Specific sequences are provided as examples of particular embodiments. Additionally, an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement.
• the nucleic acid sequences may be codon-optimized which is a technique well known in the art. Codon optimization refers to the fact that different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in a particular cell type. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.
• nucleic acid sequences coding for various replacement domains can be generated.
• such a sequence is optimized for expression in a host or target cell, such as a host cell used to express the trans-splicing RNA containing a replacement domain in which the disclosed methods are practiced (such as in a mammalian cell, e.g., a human cell).
• Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a replacement domain (such as one encoding a protein having 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 protein) that takes advantage of the codon usage preferences of that particular species.
• a replacement domain such as one encoding a protein having 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 protein
• the replacement domains disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest.
• a replacement domain nucleic acid sequence is optimized for expression in human cells, such as one having 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 originating nucleic acid sequence.
• an isolated trans-splicing nucleic acid molecule encoding at least one replacement domain (which can be part of a vector) includes at least one replacement domain coding sequence that is codon optimized for expression in a eukaryotic cell, or at least one replacement domain coding sequence codon optimized for expression in a human cell.
• 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 originating sequence.
• a eukaryotic cell codon optimized nucleic acid sequence encodes a replacement domain having 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 originating protein.
• a variety of clones containing functionally equivalent nucleic acids may be routinely generated, such as nucleic acids which differ in sequence but which encode the same replacement domain protein sequence.
• Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue.
• leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG
• serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC
• asparagine can be encoded by AAT or AAC
• aspartic acid can be encoded by GAT or GAC
• cysteine can be encoded by TGT or TGC
• alanine can be encoded by GCT, GCC, GCA, or GCG
• glutamine can be encoded by CAA or CAG
• tyrosine can be encoded by TAT or TAC
• isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3.sup
• Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
• the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
• the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded 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.
• Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
• Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
• Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.lx SSC;
• Homology or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A 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 alternatively less than 25% identity, with one of the sequences of the present invention.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 15% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 20% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 30% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 40% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 50% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 60% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 70% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 80% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an RNA molecule or a protein encoded by the RNA molecule with 90% or more efficiency comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the sequence of an untranslated region of an RNA molecule comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans-splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of increasing the expression of an RNA by insertion of WPRE or sequences with similar activity comprising contacting the composition and the RNA molecule under conditions suitable for binding and trans-splicing of one or more of the trans splicing RNAs (or a portion thereof) to the RNA molecule.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with efficiency exceeding 20% where 100% constitutes complete replacement of a chosen sequence within the target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with efficiency at or about 20% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with efficiency at or about 60% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with efficiency at or about 60% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with efficiency at or about 70% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with efficiency at or about 70% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with efficiency at or about 80% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with efficiency at or about 80% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with efficiency at or about 90% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with efficiency at or about 90% where 100% constitutes complete replacement of a chosen sequence within the Target RNA comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the disclosure provides a method of modifying the composition of a target RNA with high efficiency comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the cell is in vivo, in vitro, ex vivo or in situ.
• the composition comprises a vector comprising or encoding a trans-splicing RNA molecule of the disclosure.
• the vector is an AAV.
• the disclosure provides a method of modifying the composition of a protein encoded by a target RNA with high efficiency comprising contacting the composition and a cell comprising the target RNA under conditions suitable for trans-splicing among the composition and the target RNA.
• the cell is in vivo, in vitro, ex vivo or in situ.
• the composition comprises a vector comprising or encoding a trans-splicing RNA molecule of the disclosure.
• the vector is an AAV.
• the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the disclosure.
• the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the disclosure, wherein the composition comprises a vector comprising or encoding a trans-splicing RNA molecule of the disclosure, and wherein the composition modifies a level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule.
• the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the disclosure, wherein the composition comprises a vector comprising or encoding a trans-splicing RNA molecule of the disclosure and wherein the composition modifies an activity of a protein encoded by an RNA molecule.
• a disease or disorder of the disclosure includes, but is not limited to, a genetic disease or disorder.
• the genetic disease or disorder is a single-gene disease or disorder.
• the single-gene 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.
• the singe-gene disease or disorder is, but not limited to, common variable immune deficiency, Adenosine deaminase deficiency, X-linked severe combined immunodeficiency, Beta- thassalemia, alpha-thassalemia, myelodysplastic syndrome, Amyotrophic lateral sclerosis, Frontotemporal dementia with parkinsonism, Usher’s syndrome, Krabbe disease, Niemann Pick disease, prion disease, Dravet syndrome, early-onset Parkinson’s disease, spinocerebellar ataxias, genetic epilepsy disorders, Ataxia-telangiectasia, GM1 gangliosidosis, Gaucher disease, GM2 gangliosidosis, Angelman syndrome, glucose transporter deficiency type 1, Danon disease, Fabry disease, Autosomal dominant polycystic kidney disease, Pompe disease, Familial hypercholesterolemia, Open Angle Glaucoma, Hurler syndrome or Mucopo
• the genetic disease or disorder is a multiple-gene disease or disorder. In some embodiments, the genetic disease or disorder is a multiple-gene disease or disorder. In some embodiments, the single-gene 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 exostoses, Von Willebrand disease, and acute intermittent porphyria.
• the single-gene 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 Roberts syndrome.
• the single-gene disease or disorder is X-linked disease or disorder including, but not limited to, muscular dystrophy, Duchenne muscular dystrophy, Hemophilia, Adrenoleukodystrophy (ALD), Rett syndrome, and Hemophilia A.
• the single-gene disease or disorder is a mitochondrial disorder including, but not limited to, Leber's hereditary optic neuropathy.
• a disease or disorder of the disclosure includes, but is not limited to, an immune disease or disorder.
• the immune disease or disorder is an immunodeficiency disease or disorder including, but not limited to, B-cell deficiency, T-cell deficiency, neutropenia, asplenia, complement deficiency, acquired immunodeficiency syndrome (AIDS) and immunodeficiency due to medical intervention (immunosuppression as an intended or adverse effect of a medical therapy).
• 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 dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Cha
• a disease or disorder of the disclosure includes, but is not limited to, an inflammatory disease or disorder.
• a disease or disorder of the disclosure includes, but is not limited to, a metabolic disease or disorder.
• a disease or disorder of the disclosure includes, but is not limited to, a degenerative or a progressive disease or disorder.
• the degenerative or a progressive disease or disorder includes, but is not limited to, amyotrophic lateral sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, and aging.
• a disease or disorder of the disclosure includes, but is not limited to, an infectious disease or disorder.
• a disease or disorder of the disclosure includes, but is not limited to, a pediatric or a developmental disease or disorder.
• a disease or disorder of the disclosure includes, but is not limited to, a cardiovascular disease or disorder.
• a disease or disorder of the disclosure includes, but is not limited to, a proliferative disease or disorder.
• the proliferative disease or disorder is a cancer.
• the cancer includes, but is not limited to, Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Appendix Cancer, Gastrointestinal Carcinoid Tumors, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Central Nervous System (Brain Cancer), Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma, Osteosarcoma, Malignant Fi
• Lip and Oral Cavity 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 Neck Cancer with Occult Primary (Head and Neck Cancer), Midline Tract Carcinoma With NUT Gene Changes, Mouth Cancer (Head and Neck Cancer), Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes,
• Neoplasms Nasal Cavity and Paranasal Sinus Cancer (Head and Neck Cancer), Nasopharyngeal Cancer (Head and Neck Cancer), Neuroblastoma, Non- Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Lip and Oral Cavity Cancer 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 Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate
• Soft Tissue Sarcoma Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer (Head and Neck Cancer), Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic Carcinoma , Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Renal Cell Cancer, Urethral Cancer, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma), Vulvar Cancer, Wilms Tumor and Other Childhood Kidney Tumors.
• a disease or disorder of the disclosure includes, but is not limited to, a proliferative disease or disorder.
• the proliferative disease or disorder is a cancer.
• the cancer involves the present of a gene fusion that produces a chimeric RNA with sequences derived from two genes due to a deletion or translocation of DNA.
• Gene fusions pairs include but are not limited to: MAN2A1 and FER, DNAJB1 and PRKACA, BCR-ABL1, TMPRSS2 and ERG , EWSR1 and FLU, PML and RARA, EML4 and ALK, KIAA1549 and BRAF, CCDC6 and RET, SS18 and SSXl, RUNX1 and RUNX1T1, PAX3 and FOXOl, NCOA4 and RET, ETV6 and RUNX1, FUS and DDIT3, SS18 and SSX2, NPMl and ALK, KMT2A and AFFl, TCF3 and PBXl, STIL and TALI, COL1A1 and PDGFB, CRTC1 and MAML2, NAB2 and STAT6, EWSR1 and ATFl, ETV6 and NTRK3, EWSR1 and ERG, EWSR1 and WTl, DNAJB1 and PRKACA, PAX7 and FOXOl, FUS and
• a subject of the disclosure has been diagnosed with the disease or disorder.
• the subject of the disclosure presents at least one sign or symptom of the disease or disorder.
• the subject has a biomarker predictive of a risk of developing the disease or disorder.
• the biomarker is a genetic mutation.
• a subject of the disclosure is female. In some embodiments of the methods of the disclosure, a subject of the disclosure is male. In some embodiments, a subject of the disclosure has two XX or XY chromosomes. In some embodiments, a subject of the disclosure has two XX or XY chromosomes and a third chromosome, either an X or a Y.
• a subject of the disclosure is a neonate, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the disclosure, a subject of the 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 disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months old.
• a subject of the 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 or any number of years or partial years in between of age.
• a subject of the disclosure is a mammal. In some embodiments, a subject of the disclosure is a non-human mammal.
• a subject of the disclosure is a human.
• a therapeutically effective amount comprises a single dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises at least one dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises one or more dose(s) of a composition of the disclosure.
• a therapeutically effective amount eliminates a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount reduces a severity of a sign or symptom of the disease or disorder.
• a therapeutically effective amount eliminates the disease or disorder.
• a therapeutically effective amount prevents an onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount improves a prognosis for the subject.
• a composition of the disclosure is administered to the subject systemically. In some embodiments, the composition of the disclosure is administered to the subject by an intravenous route. In some embodiments, the composition of the disclosure is administered to the subject by an injection or an infusion.
• a composition of the disclosure is administered to the subject locally.
• the composition of the disclosure is administered to the subject by an intraosseous, intraocular, intracerebrospinal or intraspinal route.
• the composition of the disclosure is administered directly to the cerebral spinal fluid of the central nervous system.
• the composition of the disclosure is administered directly to a tissue or fluid of the eye and does not have bioavailability outside of ocular structures.
• the composition of the disclosure is administered to the subject by an injection or an infusion.
• compositions comprising the trans-splicing RNAs disclosed herein are formulated as pharmaceutical compositions.
• pharmaceutical compositions for use as disclosed herein may comprise a fusion protein(s) or a polynucleotide encoding the fusion protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
• compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
• buffers such as neutral buffered saline, phosphate buffered saline and the like
• carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
• proteins polypeptides or amino acids
• antioxidants e.g., antioxidants
• chelating agents such as EDTA or glutathione
• adjuvants e.g., aluminum hydroxide
• preservatives e.g., aluminum hydroxide
• Embodiment 1 A composition comprising a trans-splicing nucleic acid, comprising: (a) one or more replacement domains that encode a therapeutic sequence operably linked to; (b) one or more intronic domains that promote RNA splicing of the replacement domain comprising intronic trans-splicing enhancing sequence(s); and (c) one or more antisense domains that promote binding to a target RNA molecule.
• Embodiment 2 The composition of embodiment 1, wherein the intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) are derived or isolated from the group of sequences 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, GGTG, GGGGGA, GGGGGT, GGGGTA, GGGAGG, GGGTGG, GGAGGG, GG, GGAG
• the exemplary trans-splicing enhancer sequences include without limitation: 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, UUUGGG, UGUGGG, UUGUGG, GAGUGU, GAGGUA, GGAGGU, UGGGAG, GGGGUG, GGGGGA, GGGGGU, GGGGGGGG
• Embodiment 3 The composition of embodiment 1, wherein the intronic trans-splicing enhancing sequences (trans-splicing enhancer sequences) consist of a chain of RNA nucleobases comprising at least one RNA motif having the formula X 1 X 2 X 3 X 4 X 5 X 6 wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X 2 is selected from the group including adenine (A), uracil (U) and guanine (G); X 3 is selected from the group including adenine (A), uracil (U) and guanine (G); X 4 is selected from the group including adenine (A), uracil (U), cytosine (C) and guanine (G); X 5 is selected from the group including adenine (A), cytosine (C), uracil (U) and guanine (G); and
• Embodiment 4 The composition of embodiment 1, wherein the trans-splicing enhancer sequence consists of a chain of RNA nucleobases comprising at least one RNA motif having the formula X 1 X 2 X 3 X 4 X 5 X 6 wherein; Xi is selected from the group including adenine (A), uracil (El) and guanine (G); X2 is selected from the group including adenine (A), uracil (U) and guanine (G); X3 is selected from the group including adenine (A), uracil (U) and guanine (G); X4 is selected from the group including adenine (A), uracil (U) and guanine (G); X5 is selected from the group including adenine (A), uracil (U) and guanine (G); and Xe is selected from the group including uracil (U) and guanine (G).
• Xi is selected
• Embodiment 5 The composition of embodiment 1, wherein the trans-splicing enhancer sequence consists of a chain of RNA nucleobases comprising at least one RNA motif having the formula X1X2X3X4X5X6 wherein; Xi is selected from the group including adenine (A), uracil (U) and guanine (G); X2 is selected from the group including uracil (U) and guanine (G); X3 is selected from the group including adenine (A), uracil (U) and guanine (G); X4 is selected from the group including uracil (U) and guanine (G); X5 is selected from the group including uracil (U) and guanine (G); and X ( , is selected from the group including uracil (U) and guanine (G).
• Xi is selected from the group including adenine (A), uracil (U) and guanine (
• Embodiment 6 The composition of embodiments 1-5, wherein the replacement domain is derived or isolated from a human gene selected from the group consisting of: GLB1 (GM1 gangliosidosis); GBA (Gaucher disease); GM2A (GM2 gangliosidosis); PCSK9, LDLR, APOB, APOE (Familial hypercholesterolemia); GAA (Pompe disease); MYOC, OPTN, TBK1, WDR36, CYPIBl (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); MY
• Embodiment 7 The composition of embodiments 1-5, wherein the replacement domain is derived or isolated from an expression-enhancing sequence selected from the group consisting of: Woodchuck Hepatitis Virus (WHV) Post-transcriptional Regulatory Element (WPRE), triplex from MALATl, the PRE of Hepatitis B virus (HPRE), and an iron response element.
• WV Woodchuck Hepatitis Virus
• WPRE Post-transcriptional Regulatory Element
• HPRE Hepatitis B virus
• iron response element an iron response element
• Embodiment 8 The composition of any one of embodiments 1-5, wherein the antisense domain is complementary to sequences derived or isolated from a human gene selected from the group consisting of: TNFRSF13B (common variable immune deficiency), ADA, CECR1 (Adenosine deaminase deficiency), IL2RG (X-linked severe combined immunodeficiency), HBB (Beta-thassalemia), HBA1, HBA2 (alpha-thassalemia), U2AF1 (myelodysplastic syndrome), SOD1, TARDBP, FUS, MATR3, SOD1, C90RF72 (Amyotrophic lateral sclerosis), MAPT, PGRN (Frontotemporal dementia with parkinsonism), CDH23, MY07A, USH2A (Usher’s syndrome), GALC (Krabbe disease), SMPD1, NPC1, NPC2 (Niemann Pick disease), PRNP (prion disease), SCN
• Embodiment 9 The composition of any one of embodiments 1-5, wherein the trans splicing RNA comprises an untranslated region that alters the localization, processing, or transport of the trans-splicing nucleic acid.
• Embodiment 10 the composition of any one of embodiments 1-13, 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.
• Embodiment 11 the composition of any of one embodiments 1-13, wherein the trans splicing nucleic acid is RNA, DNA, a DNA/RNA hybrid, nucleic acid analog, a chemically- modified nucleic acid, or a chimera composed of two or more nucleic acids or nucleic acid analogs.
• Embodiment 12 the composition of any of one embodiments 1-1, wherein the wherein the trans-splicing nucleic acid molecule further comprises a heterologous promoter.
• Embodiment 13 the composition of any of one embodiments 1-13, wherein the promoter is isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA).
• tRNA transfer RNA
• a cell of the disclosure is a eukaryotic cell.
• the cell is a mammalian cell.
• the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell.
• the cell is a non-human mammalian cell such as a non-human primate cell.
• a cell of the disclosure is a somatic cell.
• a cell of the disclosure is a germline cell. In some embodiments, a germline cell of the disclosure is not a human cell.
• a cell of the disclosure is a stem cell.
• a cell of the disclosure is an embryonic stem cell.
• an embryonic stem cell of the disclosure is not a human cell.
• a cell of the disclosure is a multipotent stem cell or a pluripotent stem cell.
• a cell of the disclosure is an adult stem cell.
• a cell of the disclosure is an induced pluripotent stem cell (iPSC).
• a cell of the disclosure is a hematopoietic stem cell (HSC).
• an immune cell of the disclosure is a lymphocyte.
• an immune cell of the disclosure is a T lymphocyte (also referred to herein as a T-cell).
• T-cells of the 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.
• an immune cell of the disclosure is a B lymphocyte.
• an immune cell of the disclosure is a natural killer cell.
• an immune cell of the disclosure is an antigen-presenting cell.
• a muscle cell of the disclosure is a myoblast or a myocyte.
• a muscle cell of the disclosure is a cardiac muscle cell, skeletal muscle cell or smooth muscle cell.
• a muscle cell of the disclosure is a striated cell.
• a somatic cell of the disclosure is an epithelial cell.
• an epithelial cell of the disclosure forms a squamous cell epithelium, a cuboidal cell epithelium, a columnar cell epithelium, a stratified cell epithelium, a pseudostratified columnar cell epithelium or a transitional cell epithelium.
• an epithelial cell of the disclosure forms a gland including, but not limited to, a pineal gland, a thymus gland, a pituitary gland, a thyroid gland, an adrenal gland, an apocrine gland, a holocrine gland, a merocrine gland, a serous gland, a mucous gland and a sebaceous gland.
• an epithelial cell of the disclosure contacts an outer surface of an organ including, but not limited to, a lung, a spleen, a stomach, a pancreas, a bladder, an intestine, a kidney, a gallbladder, a liver, a larynx or a pharynx.
• an epithelial cell of the disclosure contacts an outer surface of a blood vessel or a vein.
• a brain cell of the disclosure is a neuronal cell.
• a neuron cell of the disclosure is a neuron of the central nervous system.
• a neuron cell of the disclosure is a neuron of the brain or the spinal cord.
• a neuron cell of the disclosure is a neuron of a cranial nerve or an optic nerve.
• a neuron cell of the disclosure is a neuron of the peripheral nervous system.
• a neuron cell of the disclosure is a neuroglial or a glial cell.
• a glial of the disclosure is a glial cell of the central nervous system including, but not limited to, oligodendrocytes, astrocytes, ependymal cells, and microglia.
• a glial of the disclosure is a glial cell of the peripheral nervous system including, but not limited to, Schwann cells and satellite cells.
• a liver cell of the disclosure is a hepatocytes. In some embodiments, a liver cell of the disclosure is a hepatic stellate cell. In some embodiments, a liver cell of the disclosure is Kupffer cell. In some embodiments, a liver cell of the disclosure is a sinusoidal endothelial cells.
• a retinal cell of the disclosure is a photoreceptor.
• a photoreceptor cell of the disclosure is a rod.
• a retinal cell of the disclosure is cone.
• a retinal cell of the disclosure is a bipolar cell.
• a retinal cell of the disclosure is a ganglion cell.
• a retinal cell of the disclosure is a horizontal cell.
• a retinal cell of the disclosure is an amacrine cell.
• a heart cell of the disclosure is a cardiomyocyte. In some embodiments, a heart cell of the disclosure is a cardiac pacemaker cell.
• a somatic cell of the disclosure is a primary cell.
• a somatic cell of the disclosure is a cultured cell.
• a somatic cell of the disclosure is in vivo, in vitro, ex vivo or in situ.
• a somatic cell of the disclosure is autologous or allogeneic.

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