Methods and compositions for targeted trans-splicing

Abstract

The present disclosure provides compositions and methods of use for targeting trans-splicing of pre-mRNA in cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 429,031, filed November 30, 2022, the entire contents of which are incorporated herein.

[0002] The present disclosure relates to nucleic acid compositions and related methods for targeting trans-splicing of pre-mRNA in cells.

[0003] Submission of sequence listing via EFS-WEB This application contains a Sequence Listing that has been submitted in XML format via EFS-Web. The contents of the XML copy entitled "AMR-014PC / 134241-5014_Sequence Listing", created on November 30, 2023, is 513,000 bytes in size, and is hereby incorporated by reference in its entirety. [Background technology]

[0004] Gene editing has been widely recognized as a promising approach for treating numerous diseases associated with viral infections, enzyme deficiencies, and inherited myopathies. For example, gene editing using the CRISPR / Cas system can introduce double-strand breaks into a gene of interest, which are repaired by endogenous DNA repair pathways, resulting in gene knockout or mutation repair. Appropriate gene editing can function to eliminate mutations in the encoded protein, reduce the expression of the encoded protein, or alter the function or activity of the encoded protein to provide the desired therapeutic outcome. However, despite significant progress, gene editing approaches remain problematic due to the risk of introducing harmful off-target edits into the genome and packaging constraints for the delivery of system components. An alternative approach for introducing genetic information into cells that avoids the risk of introducing permanent changes to the genome relies on modulating the splicing of endogenous nucleic acids (e.g., RNA transcripts).

[0005] Splicing is a reaction that occurs in the nucleus of eukaryotic cells and is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex. Splicing removes non-coding sequences (introns) from RNA transcripts (pre-mRNA) and joins coding sequences (exons). The spliceosome typically mediates cis-splicing of endogenous RNA transcripts, forming lariat sequences in introns, which are then cleaved to join two exons from the same RNA transcript (see, for example, Matera, et al. (2014) Nat Rev Mol Cell Biol 15(2):108-21; Wilkinson, et al. (2020) Annu Rev Biochem 89:359). The spliceosome can also perform trans-splicing, joining the ends of exons from two different primary RNA transcripts (Lasda, et al. (2011) Wiley Interdiscip Rev RNA 2:417-34). Trans-splicing results in a chimeric molecule that contains one or more exon regions from a first RNA molecule and one or more exon regions from a second RNA molecule.

[0006] Trans-splicing using exogenous nucleic acids encoding desired genetic information is a promising approach for therapeutic nucleic acid editing and other biotechnology applications. For example, it has been demonstrated that artificial RNA introduced into cells can undergo trans-splicing with endogenous pre-mRNA (see, e.g., Puttaraju, et al. (1999) Nat Biotech 17:246). Attempts at such trans-splicing have focused on spliceosome-mediated RNA trans-splicing (SMaRT), in which the activity of pre-mRNA trans-splicing molecules (PTMs) is achieved by RNA-RNA interactions between binding domains hybridized to the target pre-mRNA (Puttaraju, 1999). Some groups have been able to demonstrate in vitro and in vivo activity using SMaRT technology (Mansfield, et al. (2000) Gene therapy 7:1885-1895; Liu, et al. (2002) Nat. Biotechnol. 20:47), but it has not yet been translated into clinical practice due to the relatively inefficient process (Berger, et al. (2016) Wiley Interdisciplinary Reviews: RNA 7:487-98).

[0007] Therefore, new approaches are needed to allow targeted and efficient trans-splicing to introduce desired genetic information into cells. Summary of the Invention

[0008] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising: (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a pre-mRNA target sequence; and (ii) at least one intron sequence comprising a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or contains a sequence motif for directing the one or more binding domains to the pre-mRNA target sequence; (b) a splice acceptor sequence and / or a splice donor sequence; and (c) at least one exon sequence.

[0009] In some embodiments, the one or more binding domain sequences are at least about 5 to about 10, about 5 to about 15, about 5 to about 20, about 10 to about 15, about 10 to about 20, about 15 to about 20, or about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In some embodiments, the one or more binding domain sequences are about 250 to about 300, about 200 to about 300, about 150 to about 300, about 100 to about 300, about 50 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, or less than about 300, 250, 200, 150, 100, or 50 nucleotides in length. In some embodiments, the one or more binding domain sequences are about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

[0010] In some embodiments, the at least one intron sequence comprises one binding domain sequence, in some embodiments, the at least one intron sequence comprises at least two binding domain sequences, in some embodiments, the at least one intron sequence comprises 3, 4, 5, 6, 7, 8, 9, or 10 binding domain sequences.

[0011] In some embodiments, when the nucleic acid is introduced into the cell, the exon of the pre-mRNA is targeted for trans-splicing. In some embodiments, the target sequence is located within the region of the pre-mRNA that contains the exon targeted for trans-splicing. In some embodiments, the target sequence is located near a splice site. In some embodiments, the target sequence is located near a splice donor or splice acceptor.

[0012] In some embodiments, the ncRNA sequence is selected from snRNA, snoRNA, lncRNA, rRNA, ribozyme, sRNA, scaRNA, and vault RNA. In some embodiments, the ncRNA sequence is a snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA. In some embodiments, the ncRNA sequence is a snoRNA. In some embodiments, the snoRNA comprises an H / ACA box or a C / D box.

[0013] In some embodiments, the ncRNA sequence assembles into an RNP. In some embodiments, the ncRNA sequence comprises a sequence motif that assembles into an RNP. In some embodiments, the ncRNA sequence comprises a secondary structure that assembles into an RNP. In some embodiments, the ncRNA sequence comprises a sequence motif and a secondary structure that assembles into an RNP. In some embodiments, the secondary structure comprises one or more stem-loops. In some embodiments, the RNP is selected from a small nuclear RNP (snRNP), a small nucleolar RNP (snoRNP), a Cajal body small RNP (scaRNP), and combinations thereof. In some embodiments, the RNP is a snRNP. In some embodiments, the RNP is selected from U1, U2, U4, U4atac, U5, U6, U6atac, U7, U11, and U12. In some embodiments, the RNP is a snoRNP. In some embodiments, the RNP is selected from a C / D box snoRNP and an H / ACA box snoRNP.

[0014] In some embodiments, the ncRNA comprises an Sm sequence motif. In some embodiments, the Sm sequence motif is assembled into an RNP with an Sm or Lsm protein. In some embodiments, the Sm or Lsm protein is selected from B / B', D3, D2, D1, E, F, G, LSm5, LSm7, LSm4, LSm8, LSm2, LSm3, LSm6, and LSm10 protein.

[0015] In some embodiments, the at least one intron sequence comprises a splice acceptor. In some embodiments, the at least one intron sequence comprises a splice donor. In some embodiments, the at least one intron sequence comprises one or more splicing signals. In some embodiments, the one or more splicing signals are selected from an exonic splicing enhancer (ESE), an intronic splicing enhancer (ISE), an exonic splicing silencer (ESS), an intronic splicing silencer (ISS), a polypyrimidine tract, a branch point, and combinations thereof. In some embodiments, the at least one intron sequence comprises a branch point and a polypyrimidine tract. In some embodiments, the nucleic acid comprising the nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 exons.

[0016] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising, from 5' to 3', (a) (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each complementary to a pre-mRNA target sequence; (ii) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length, which forms a secondary structure and / or contains a sequence motif for directing the one or more binding domains to the pre-mRNA target sequence; and (iii) at least one intron sequence containing one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence. In some embodiments, when the nucleic acid is introduced into the cell, an exon of the pre-mRNA is targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the exon of the pre-mRNA targeted for trans-splicing. In some embodiments, the target sequence is located proximal to a splice site (e.g., a splice acceptor or a splice donor). In some embodiments, the target sequence is located proximal to a splice acceptor. In some embodiments, the target sequence is located proximal to a splice donor. In some embodiments, trans-splicing occurs between a splice donor upstream of an exon of the pre-mRNA and a splice acceptor of the nucleic acid. In some embodiments, trans-splicing results in ligation of the 3' end of an exon upstream of the splice donor of the pre-mRNA to the 5' end of at least one exon sequence of the nucleic acid. In some embodiments, the one or more splicing signals comprise a branch point and a polypyrimidine tract.

[0017] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising, from 5' to 3', a nucleotide sequence including: (a) at least one exon sequence; (b) a splice donor; and (c) (i) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length; and (ii) at least one intron sequence, each of which comprises one or more binding domain sequences of about 4 to about 300 nucleotides, each of which is complementary to a pre-mRNA target sequence, wherein the ncRNA forms a secondary structure and / or comprises a sequence motif for directing the one or more binding domains to the pre-mRNA target sequence. In some embodiments, when the nucleic acid is introduced into the cell, an exon of the pre-mRNA is targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the exon of the pre-mRNA. In some embodiments, the target sequence is located proximal to a splice site (e.g., a splice donor or a splice acceptor). In some embodiments, the target sequence is located proximal to a splice donor. In some embodiments, the target sequence is located proximal to a splice acceptor. In some embodiments, trans-splicing occurs between the splice donor of the nucleic acid and a splice acceptor downstream of an exon of the pre-mRNA. In some embodiments, trans-splicing results in ligation of the 3' end of at least one exon sequence of the nucleic acid to the 5' end of an exon downstream of the splice acceptor of the pre-mRNA.

[0018] In some embodiments, the ncRNA sequence is a snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA. In some embodiments, the snRNA assembles into a snRNP. In some embodiments, the snRNA is a U1 snRNA. In some embodiments, the U1 snRNA assembles into a U1 RNP. In some embodiments, the snRNA is a U11 snRNA. In some embodiments, the U11 snRNA assembles into a U1 RNP. In some embodiments, the snRNA is a U7 snRNA. In some embodiments, the U7 snRNA assembles into a U7 RNP. In some embodiments, the ncRNA sequence comprises an Sm sequence motif. In some embodiments, the ncRNA sequence comprises an Sm sequence motif and a U7 snRNA. In some embodiments, the Sm sequence motif comprises the sequence set forth in SEQ ID NOs: 3 and 4. In some embodiments, the Sm sequence motif assembles into an RNP with an Sm protein. In some embodiments, the Sm protein is selected from the B / B', D3, D2, D1, E, F, and G Sm proteins.

[0019] In some embodiments, the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 9-589, or a portion thereof (e.g., a contiguous portion thereof). In some embodiments, the ncRNA sequence comprises a region about 7 to about 40 nucleotides in length, the region comprising an Sm sequence motif. In some embodiments, the ncRNA sequence comprises a region about 40 to about 300 nucleotides in length, the region comprising a secondary structure and / or an Sm sequence motif. In some embodiments, the Sm sequence motif comprises a sequence selected from SEQ ID NOs: 209-399.

[0020] In some embodiments, the at least one intron sequence comprises one binding domain sequence, which is about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

[0021] In some embodiments, the at least one intron sequence comprises multiple binding domain sequences, each of which is about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

[0022] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence comprising: (i) an ncRNA sequence comprising an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides, each of which is complementary to a pre-mRNA target sequence; (ii) at least one intron sequence comprising one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence. In some embodiments, when the nucleic acid is introduced into the cell, an exon of the pre-mRNA is targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the exon of the pre-mRNA. In some embodiments, the target sequence is located proximal to a splice site. In some embodiments, the target sequence is located proximal to a splice donor or splice acceptor. In some embodiments, trans-splicing occurs between a splice donor upstream of an exon of the pre-mRNA and a splice acceptor of the nucleic acid. In some embodiments, trans-splicing results in ligation of the 3' end of the exon upstream of the splice donor of the pre-mRNA to the 5' end of at least one exon sequence of the nucleic acid. In some embodiments, the one or more splicing signals comprise a branch point and a polypyrimidine tract.

[0023] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising, from 5' to 3', a nucleotide sequence including: (a) at least one exon sequence; (b) a splice donor; and (c) at least one intron sequence, the ncRNA sequence including an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides, each of which is complementary to a pre-mRNA target sequence. In some embodiments, when the nucleic acid is introduced into the cell, the pre-mRNA exon is targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the pre-mRNA exon. In some embodiments, the target sequence is located proximal to a splice site. In some embodiments, the target sequence is located proximal to a splice donor or splice acceptor. In some embodiments, trans-splicing occurs between the splice donor of the nucleic acid and the splice acceptor downstream of the pre-mRNA exon. In some embodiments, trans-splicing results in ligation of the 3' end of at least one exon sequence of the nucleic acid to the 5' end of an exon downstream of the splice acceptor of the pre-mRNA.

[0024] In some embodiments, the ncRNA sequence comprises an H / ACA box comprising, from 5' to 3', an H consensus sequence and an ACA consensus sequence. In some embodiments, the ncRNA sequence comprises at least one binding domain sequence located (i) upstream of the H consensus sequence, (ii) downstream of the ACA consensus sequence, (iii) between the H consensus sequence and the ACA consensus sequence, or (iv) a combination of (i)-(iii).

[0025] In some embodiments, the ncRNA sequence comprises a C / D box comprising, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence. In some embodiments, the ncRNA sequence comprises at least one binding domain located (i) upstream of the C consensus sequence, (ii) between the C consensus sequence and the D' consensus sequence, (iii) between the C' consensus sequence and the D consensus sequence, (iv) downstream of the D consensus sequence, or (iv) a combination of (i)-(iii).

[0026] In some embodiments, the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 590 to 657, or a portion thereof (e.g., a contiguous portion thereof). In some embodiments, the ncRNA sequence comprises a region of about 40 to about 300 nucleotides in length that includes the H consensus sequence and the ACA consensus sequence.

[0027] In some embodiments, the ncRNA sequence comprises one binding domain sequence. In some embodiments, the ncRNA sequence comprises multiple binding domain sequences.

[0028] In some embodiments, the at least one intron sequence comprises at least one binding domain sequence that is fully complementary to the pre-mRNA target sequence. In some embodiments, the at least one intron sequence comprises at least one binding domain sequence that is partially complementary to the pre-mRNA target sequence. In some embodiments, the at least one binding domain sequence comprises one or more mismatches with the pre-mRNA target sequence. In some embodiments, the at least one binding domain sequence is at least 95% complementary to the pre-mRNA target sequence.

[0029] In some embodiments, the nucleic acid comprises a sequence of up to about 20,000 nucleotides in length, or about 50 to about 500, about 50 to about 1,000, about 100 to about 500, about 100 to about 1,000, about 500 to about 1,000, about 500 to about 2,000, about 500 to about 3,000, about 500 to about 4,000, about 500 to about 5,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 5,000 to about 15,000, or about 5,000 to about 20,000 nucleotides in length.

[0030] In some embodiments, the nucleic acid is introduced into the cell as RNA. In some embodiments, the nucleic acid is introduced into the cell as DNA. In some embodiments, the nucleic acid is introduced into the cell by a viral vector. In some embodiments, the viral vector is AAV. In some embodiments, the nucleic acid is introduced into the cell by a non-viral vector.

[0031] In some embodiments, introduction of the nucleic acid into the cell results in trans-splicing efficiency greater than that of a nucleic acid lacking the ncRNA sequence. In some embodiments, the trans-splicing efficiency is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% greater.

[0032] In some embodiments, the nucleic acid is formulated as a lipid nanoparticle.

[0033] In some aspects, the present disclosure provides viral vectors comprising a nucleic acid described herein.

[0034] In some aspects, the present disclosure provides lipid nanoparticles comprising a nucleic acid as described herein.

[0035] In some aspects, the present disclosure provides a cell comprising a nucleic acid described herein, a viral vector described herein, or a lipid nanoparticle described herein.

[0036] In some aspects, the present disclosure provides a pharmaceutical composition comprising a nucleic acid described herein, a viral vector described herein, or a lipid nanoparticle described herein, and a pharmaceutically acceptable carrier.

[0037] In some aspects, the present disclosure provides a pharmaceutical composition comprising a cell described herein and a pharmaceutically acceptable carrier.

[0038] In some aspects, the present disclosure provides methods for targeting trans-splicing of a pre-mRNA in a cell, the method comprising contacting the cell with a nucleic acid described herein, a viral vector described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein, wherein upon contact of the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition with the cell, the one or more binding domain sequences bind to the pre-mRNA, thereby targeting the pre-mRNA for trans-splicing.

[0039] In some aspects, the disclosure provides methods of repairing a mutation in a pre-mRNA in a cell, the method comprising contacting the cell with a nucleic acid described herein, a viral vector described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein, wherein upon contacting the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition with the cell, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation and replace one or more exons of the pre-mRNA containing the mutation by trans-splicing, thereby repairing the mutation.

[0040] In some aspects, the present disclosure provides a method for treating a patient with a disease or disorder associated with a mutation in a pre-mRNA, the method comprising administering to the patient an effective amount of a nucleic acid described herein, a viral vector described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein, wherein upon administration of the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and one or more exons of the pre-mRNA containing the mutation are replaced by trans-splicing, thereby repairing the mutation. In some embodiments, the trans-splicing results in an mRNA that alleviates the disease or that does not cause or contribute to the disease.

[0041] In some aspects, the present disclosure provides a nucleic acid according to any one of the embodiments disclosed herein, a viral vector according to any one of the embodiments disclosed herein, a lipid nanoparticle according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein, for use in treating a patient having a disease or disorder associated with a mutation in a pre-mRNA, the treatment comprising administering the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition to the patient, wherein upon administration of the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation and replace one or more exons of the pre-mRNA containing the mutation by trans-splicing, thereby repairing the mutation.

[0042] In some aspects, the present disclosure provides a nucleic acid according to any one of the embodiments disclosed herein, a viral vector according to any one of the embodiments disclosed herein, a lipid nanoparticle according to any one of the embodiments disclosed herein, or a pharmaceutical composition according to any one of the embodiments disclosed herein for the manufacture of a medicament for use in treating a patient having a disease or disorder associated with a mutation in a pre-mRNA, the treatment comprising administering the medicament to the patient, wherein upon administration of the medicament, one or more binding domain sequences of the nucleic acid bind to the pre-mRNA at a position proximal to the mutation and replace one or more exons of the pre-mRNA containing the mutation by trans-splicing, thereby repairing the mutation.

[0043] In some aspects, the present disclosure provides kits comprising a container containing a nucleic acid described herein, a viral vector described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein, together with instructions for use in repairing a mutation in a pre-mRNA. [Brief explanation of the drawings]

[0044] [Figure 1A] Without wishing to be bound by theory, a schematic diagram showing cis-splicing of pre-mRNA is shown. "SD" refers to the splice donor. "SA" refers to the splice acceptor. [Figure 1B] Without wishing to be bound by theory, a schematic diagram showing trans-splicing of two pre-mRNA molecules is shown. "SD" refers to the splice donor. "SA" refers to the splice acceptor. [Figure 1C] Without wishing to be bound by theory, a schematic diagram showing an exemplary splice editor nucleic acid molecule of the present disclosure for targeted trans-splicing of pre-mRNA is shown. The labels of the splice editor indicate segments corresponding to the RNA-binding domain, non-coding RNA (ncRNA), SA, and exons. The labels of the pre-mRNA indicate segments of the pre-mRNA corresponding to the 5' exon, SD, SA, and 3' exon. [Figure 1D] Without wishing to be bound by theory, a schematic diagram is provided showing the predicted secondary structure of an exemplary splice editor nucleic acid molecule and pre-mRNA of the present disclosure, as well as the interactions between the splice editor and the pre-mRNA that result in a trans-spliced ​​mRNA product. [Figure 1E] Graphs showing the percentage of ncRNAs identified as containing sequence motifs (Sm sequence motif, H / ACA box, and / or C / D box) are shown. [Figure 1F] 1 shows a graph depicting the length in nucleotide numbers for exemplary ncRNAs of the present disclosure. [Figure 2A] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U1 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 2B] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U1 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 2C]Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U1 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 3A] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U11 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 3B] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U11 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 3C] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, U11 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA to initiate a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor (A, B). [Figure 4A]Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, which has, from 5' to 3', an RNA-binding domain, an ncRNA with an Sm sequence motif and U7 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA (A, B, and C), which initiates a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 4B] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, which has, from 5' to 3', an RNA-binding domain, an ncRNA with an Sm sequence motif and U7 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA (A, B, and C), which initiates a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 4C] Without wishing to be bound by theory, a schematic diagram is shown showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, which has, from 5' to 3', an RNA-binding domain, an ncRNA with an Sm sequence motif and U7 snRNA, an intron, an SA, and an exon (A), as well as the interaction of the exemplary splice editor with a target pre-mRNA (A, B, and C), which initiates a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 5A] Without wishing to be bound by theory, a schematic diagram showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, having from 5' to 3' an RNA binding domain, an ncRNA with an Sm sequence motif, an intron, an SA, and an exon is shown. [Figure 5B]Without wishing to be bound by theory, a schematic diagram is shown illustrating the interaction of an exemplary splice editor with a target pre-mRNA, initiating a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 5C] Without wishing to be bound by theory, a schematic diagram is shown illustrating the interaction of an exemplary splice editor with a target pre-mRNA, initiating a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 6A] Without wishing to be bound by theory, a schematic diagram showing the unfolded and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure having, from 5' to 3', a snoRNA with an insertion of two RNA binding domains, an intron, an SA, and an exon is shown. [Figure 6B] Without wishing to be bound by theory, a schematic diagram is shown illustrating the interaction of an exemplary splice editor with a target pre-mRNA, initiating a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 6C] Without wishing to be bound by theory, a schematic diagram is shown illustrating the interaction of an exemplary splice editor with a target pre-mRNA, initiating a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor. [Figure 7A] Without wishing to be bound by theory, an image showing the design of a snoRNA guide construct for exon skipping. [Figure 7B] Graph showing the results of exon skipping of four splice acceptor-targeting snoRNAs (i.e., labeled "snord45a_11_acceptor," "snord45a_7_acceptor," "aca46_acceptor_29," and "aca44_2_acceptor_35," sequences shown in Table 4). [Figure 8]FIG. 11 is a graph showing GFP production as a result of exon skipping from four different splice acceptor-targeting snoRNAs (i.e., labeled "V5_snord45a_11_acceptor", "V4_snord45a_7_acceptor", "V1_aca46_acceptor_29", and "V2_aca44_2_acceptor_35", sequences shown in Table 4). [Figure 9] FIG. 9 is a graph showing the results of exon skipping of four splice acceptor-targeting snoRNAs (i.e., labeled "snord45a_11_acceptor," "snord45a_7_acceptor," "aca46_acceptor_29," and "aca44_2_acceptor_35") compared to the four randomized guides in FIG. [Figure 10] Without wishing to be bound by theory, an image showing the design of a U7 guide construct for trans-splicing. [Figure 11] Graph showing the binding of U7 guide constructs to different target elements and the effect of trans-splicing. [Figure 12] 12 is a graph showing independent validation of the results from Figure 11. The first four bars from the left represent U7 constructs with targeting elements specific for trans-splicing, and the remaining bars on the graph represent U7 constructs with non-targeting elements for trans-splicing. [Figure 13] Graph showing U7-targeted piggybac integrated USH2A (773, hybridizing region and hairpin intact) compared to non-targeted guides (774 and 776), and a mutant U7 hairpin (775, hybridizing region intact, but hairpin region mutated). DETAILED DESCRIPTION OF THE INVENTION

[0045] The present disclosure provides nucleic acid molecules for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell. In some embodiments, the nucleic acid molecules are engineered to contain a nucleotide sequence comprising: (i) at least one non-coding sequence (intron sequence) comprising an RNA-guided domain that binds to one or more target sequences of the target RNA (e.g., the pre-mRNA); (ii) a splice acceptor and / or a splice donor; and (iii) at least one coding sequence (exon sequence). Nucleic acid molecules of the present disclosure are referred to herein as "splice editor nucleic acids" or "splice editor nucleic acid molecules." Without being bound by theory, the binding event brings the splice editor nucleic acid into proximity with a region of the target RNA (e.g., a pre-mRNA) selected for trans-splicing, recruiting a spliceosome to the target RNA (e.g., a pre-mRNA), resulting in efficient trans-splicing. In some embodiments, the target RNA is a pre-mRNA. In some embodiments, the pre-mRNA comprises a nucleotide sequence comprising a disease-causing mutation. In some embodiments, the trans-splicing produces an mRNA that contains a desired change compared to the mRNA produced by cis-splicing of the pre-mRNA, e.g., in some embodiments, the desired change is the repair of a disease-causing mutation in the pre-mRNA.

[0046] In some embodiments, the RNA-guided domain comprises (i) one or more binding domains, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), and (ii) a non-coding RNA (ncRNA) sequence. In some embodiments, the ncRNA sequence comprises a secondary structure and / or sequence motif that assembles into a ribonucleoprotein (RNP). Without being bound by theory, assembly of the ncRNA to form an RNP confers one or more desirable properties to the trans-splicing nucleic acid molecule that enable efficient trans-splicing. For example, in some embodiments, the RNP functions (i) to stabilize RNA secondary structures present in the splice editor nucleic acid molecule, the pre-mRNA, or both; (ii) to stabilize RNA-RNA interactions formed between the splice editor nucleic acid molecule and the pre-mRNA; (iii) to protect the splice editor nucleic acid molecule and / or the pre-mRNA from degradation; (iv) to localize the splice editor nucleic acid molecule to the subcellular compartment where the pre-mRNA resides; and (v) a combination of (i)-(iv).

[0047] In some embodiments, the present disclosure provides a method for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the method comprising introducing a splice editor nucleic acid molecule described herein into the cell. In some embodiments, the present disclosure provides a method for repairing a mutation in a target RNA (e.g., a pre-mRNA) in a cell, the method comprising introducing a splice editor nucleic acid molecule described herein into the cell. In some aspects, the introduction occurs in vivo. In some aspects, the introduction occurs ex vivo. In some embodiments, the methods described herein are used to introduce desired edits into a target nucleic acid in a manner that avoids certain drawbacks of gene editing, e.g., gene editing performed using a CRISPR / Cas system. While gene editing is associated with the risk of introducing permanent and disease-causing off-target edits to the genome, the present disclosure provides a method of trans-splicing that avoids genomic DNA modification and allows for transient edits. Thus, without being bound by theory, the methods of the present disclosure can be used to introduce edits into nucleic acids in a cell in a safer manner than gene editing. Additionally, in some embodiments, the methods of the present disclosure are used to inactivate unwanted off-target gene edits introduced into the genome, thereby preventing or ameliorating adverse phenotypes associated with gene editing approaches.

[0048] In some embodiments, the disclosure provides methods of treating a disease or disorder in a subject in need thereof, wherein the disease or disorder is associated with (i) one or more genetic mutations, and / or (ii) an abnormal expression level and / or activity of a gene, or its transcription or translation product, the method comprising administering to the subject one or more splice editor nucleic acid molecules described herein.

[0049] In some embodiments, the disclosure provides methods and compositions for delivery of the splice editor nucleic acid molecule to a cell or a subject. In some embodiments, the splice editor nucleic acid molecule is delivered as DNA. In some embodiments, the splice editor nucleic acid molecule is delivered as RNA. In some aspects, the delivery comprises administering a recombinant expression vector (e.g., a viral vector, e.g., AAV) comprising the splice editor nucleic acid molecule. In some aspects, the delivery comprises administering a non-viral vector (e.g., a lipid particle) comprising the splice editor nucleic acid molecule.

[0050] Splice editor nucleic acid molecules for targeted trans-splicing Accurate pre-mRNA splicing is crucial for proper protein expression. Nuclear pre-mRNA splicing is catalyzed by the spliceosome. Vertebrate gene structures often consist of relatively long introns and short internal exons. Exon-intron boundaries are defined by splice donors (the 5' or 3' splice site of an exon) and splice acceptors (the 3' or 5' splice site of an exon). In addition to recognizing splice sites, the spliceosome relies on various splicing signals, including branchpoint sequences and polypyrimidine tracts, to mediate splicing events. Branchpoint sequences typically contain adenosines located within a consensus sequence and are located approximately 18-40 nucleotides upstream of the 3' splice site. The polypyrimidine tract contains repeated uracil sequences and is proximal to the 3' splice site. Alternative signals can enhance or decrease splicing activity and include exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs). Splicing in cis ("cis splicing") occurs when the 2'OH group of the branched adenosine of the intron attacks the 5' splice site (splice donor) with a nucleophilic attack. This results in cleavage at this site and ligation to the branched adenosine at the 5' end of the intron, forming a lariat structure. The 3' splice site (splice acceptor) is attacked by the 3'OH group of the 5' exon, resulting in ligation of the 5' exon and 3' exon to form mRNA and release the intron lariat (see, for example, Figure 1A).

[0051] In contrast, splicing in trans ("trans-splicing") occurs between two different RNA molecules, in which the 3' splice site (splice acceptor) of a second RNA is attacked by the 3'OH of the 5' exon of a first RNA, resulting in ligation of the 5' exon of the first RNA with the 3' exon of the second RNA, thereby forming a chimeric RNA (see, e.g., Figure 1B).

[0052] The present disclosure provides splice editor nucleic acid molecules for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the splice editor nucleic acid molecule comprising a nucleotide sequence comprising: (i) at least one intron sequence comprising an RNA-guided domain; (ii) one or more splice sites (e.g., splice acceptors and / or splice donors); and (iii) at least one exon sequence. In some embodiments, the RNA-guided domain is designed to bind to a specific region of the target RNA (e.g., a pre-mRNA), thereby enabling splicing between one or more splice sites of the splice editor nucleic acid molecule and one or more splice sites of the target RNA (e.g., a pre-mRNA). In some embodiments, the trans-splicing results in a chimeric mRNA comprising at least one exon sequence of the splice editor nucleic acid and one or more exons of the target RNA (e.g., a pre-mRNA).

[0053] In humans, exon definition is determined by paired splice sites across an exon (e.g., a splice acceptor (3' splice site) at the 5' end of the exon and a splice donor (5' splice site) at the 3' end of the exon). Other splicing signals (e.g., branchpoint sequences, polypyrimidine tracts, intra-exonic (or intronic) splicing enhancers and silencers) contribute to the proper splicing of exons to form mature mRNA. During pre-mRNA splicing, the spliceosome searches for pairs of closely spaced splice sites. Without being bound by theory, the splice editor nucleic acid molecules described herein mediate efficient trans-splicing by bringing a splice site of the target RNA (e.g., a pre-mRNA, e.g., a splice acceptor or splice donor of the target pre-mRNA) into close proximity with a splice site of the splice editor nucleic acid molecule (e.g., a splice acceptor or splice donor of the splice editor nucleic acid molecule), and thus the spliceosome mediates splicing between the splice site of the target pre-mRNA and the splice site of the splice editor nucleic acid molecule.

[0054] In some embodiments, the splice editor nucleic acid molecule comprises a nucleotide sequence comprising, from 5' to 3', (i) at least one intron sequence comprising an RNA-guided domain, (ii) a splice acceptor, and (iii) at least one exon sequence.

[0055] In some embodiments, the splice editor nucleic acid molecule comprises a nucleotide sequence comprising, from 5' to 3', (i) at least one exon sequence, (ii) a splice donor, and (iii) at least one intron sequence comprising an RNA-guided domain.

[0056] In some embodiments, the at least one intron sequence comprises one or more splicing signals (e.g., a branchpoint sequence, a polypyrimidine tract, an ISE, and / or an ISS). In some embodiments, the at least one exon sequence comprises one or more splicing signals (e.g., an ESE and / or an ESS).

[0057] RNA-guided domain In some embodiments, the RNA-guided domain comprises (i) one or more binding domains, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA), and (ii) a nucleotide sequence comprising an ncRNA. In some embodiments, the one or more binding domains mediate binding of the trans-splicing nucleic acid molecule to a target RNA (e.g., pre-mRNA) in a cell. In some embodiments, the ncRNA mediates assembly into an RNP.

[0058] In some embodiments, the RNA-guided domain comprises, from 5' to 3', (i) one or more binding domains, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), and (ii) a nucleotide sequence comprising an ncRNA.

[0059] In some embodiments, the RNA-guided domain comprises, from 5' to 3', a nucleotide sequence having: (i) an ncRNA; and (ii) one or more binding domains, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA).

[0060] In some embodiments, the RNA-guided domain comprises a nucleotide sequence having an ncRNA, wherein one or more binding domains are inserted into or exchanged with consecutive nucleotides of the ncRNA.

[0061] Target sequence In some embodiments, one or more binding domains of the RNA-guiding domain are each complementary to a target sequence of a target RNA (eg, a pre-mRNA) that is targeted for trans-splicing.

[0062] As used herein, the term "target sequence" refers to a sequence of consecutive nucleotides present in a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing. As used herein, the term "consecutive nucleotides" refers to a series of nucleotides that are covalently linked and immediately adjacent to each other. In some embodiments, the target sequence is at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the target sequence is less than about 300, 250, 200, 100, 150, or 50 nucleotides in length. In some embodiments, the target sequence is about 5 to 10, about 5 to 15, about 5 to 20, about 10 to 20, about 10 to 30, about 10 to 40, about 10 to 50, about 10 to 60, about 10 to 70, about 10 to 80, about 10 to 90, about 10 to 100, about 50 to 100, about 50 to 150, about 50 to 200, about 50 to 250, about 50 to 300, about 100 to 200, about 100 to 300, or about 200 to 300 nucleotides in length.

[0063] In some embodiments, the target sequence is in a region containing a splice site in the target RNA (e.g., pre-mRNA) targeted for trans-splicing. As used herein, "splice site of a target RNA (e.g., pre-mRNA) targeted for trans-splicing" refers to a splice site of a target RNA (e.g., pre-mRNA) selected for trans-splicing, where after a splice editor nucleic acid molecule described herein is introduced into a cell containing the target RNA (e.g., pre-mRNA), a trans-splicing event mediates ligation of the splice site of the target RNA (e.g., pre-mRNA) with the splice site of the splice editor nucleic acid molecule. In some embodiments, the target sequence is upstream of the splice site of the target RNA (e.g., pre-mRNA) targeted for trans-splicing. In some embodiments, the target sequence is downstream of the splice site of the target RNA (e.g., pre-mRNA) targeted for trans-splicing. In some embodiments, the target sequence is within a region containing a splice site of a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing, the region spanning at least about 50, about 100, about 150, about 200, about 300, about 400, about 500, about 1,000, about 2,000, about 3,000, about 4,000, or about 5,000 nucleotides.

[0064] In some embodiments, the target sequence is proximal to a splice site of a target RNA (e.g., a pre-mRNA) targeted for trans-splicing. As used herein, the term "proximal to a splice site" refers to a region of less than about 500 nucleotides extending upstream and / or downstream of a splice site of a target RNA (e.g., a pre-mRNA) targeted for trans-splicing.

[0065] In some embodiments, the target sequence is proximal to the splice acceptor targeted for trans-splicing. In some embodiments, the target sequence is upstream of the splice acceptor targeted for trans-splicing. In some embodiments, the target sequence is downstream of the splice acceptor targeted for trans-splicing. In some embodiments, the target sequence overlaps with the splice acceptor targeted for trans-splicing.

[0066] In some embodiments, the target sequence is proximal to the splice donor targeted for trans-splicing. In some embodiments, the target sequence is upstream of the splice donor targeted for trans-splicing. In some embodiments, the target sequence is downstream of the splice donor targeted for trans-splicing. In some embodiments, the target sequence overlaps with the splice donor targeted for trans-splicing.

[0067] In some embodiments, the target sequence is in a region of a target RNA (e.g., a pre-mRNA) that includes the exon targeted for trans-splicing. As used herein, "exon targeted for trans-splicing" refers to an exon of the target RNA that is selected for removal after trans-splicing between a target RNA and a splice editor nucleic acid described herein, where the trans-splicing results in ligation of one or more exons of the target RNA (e.g., a pre-mRNA) with at least one exon sequence of the splice editor nucleic acid to form a chimeric RNA molecule, and the exon targeted for trans-splicing is present in the target RNA but absent in the chimeric RNA molecule formed by the trans-splicing event.

[0068] In some embodiments, the target sequence is upstream of the exon targeted for trans-splicing. In some embodiments, the target sequence is downstream of the exon targeted for trans-splicing. In some embodiments, the target sequence is within the exon targeted for trans-splicing.

[0069] In some embodiments, the target sequence is proximal to the splice acceptor of the exon targeted for trans-splicing. In some embodiments, the target sequence is upstream of the splice acceptor of the exon targeted for trans-splicing. In some embodiments, the target sequence is downstream of the splice acceptor of the exon targeted for trans-splicing. In some embodiments, the target sequence overlaps with the splice acceptor of the exon targeted for trans-splicing.

[0070] In some embodiments, the target sequence is proximal to the splice donor of the exon targeted for trans-splicing. In some embodiments, the target sequence is upstream of the splice donor of the exon targeted for trans-splicing. In some embodiments, the target sequence is downstream of the splice donor of the exon targeted for trans-splicing. In some embodiments, the target sequence overlaps with the splice donor of the exon targeted for trans-splicing.

[0071] RNA-binding domain In some embodiments, the binding domain complementary to the target sequence of the target RNA (e.g., pre-mRNA) is at least 4 nucleotides in length. In some embodiments, the binding domain is less than about 300 nucleotides in length. In some embodiments, the binding domain is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the binding domain is about 250 to about 300, about 200 to about 300, about 150 to about 300, about 100 to about 300, about 50 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, or about 300, 250, 200, 150, 100, or 50 nucleotides in length.

[0072] In some embodiments, the binding domain is about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

[0073] In some embodiments, the binding domain is 10 to 50 nucleotides in length, e.g., 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 20, 11 to 45, 11 to 40, 11 to 35, 11 to 30, 11 to 20, 12 to 45, 12 to 40, 12 to 35, 12 to 30, 12 to 25, 12 to 20, 13 to 45, 13 to 40, 13 to 35, 13 to 30, 13 to 25, 13 to 20, 14 to 45, 14 to 40, 14 to 35, 14 to 30, 14 to 25, 14 to 20, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 16 to 45, 16 to 40, 16 to 35, 16 to 30, 16-25, 16-20, 17-45, 17-40, 17-35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19-30, 19-25, 19-20, e.g., 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0074] In some embodiments, the RNA-guided domain comprises one binding domain. In some embodiments, the RNA-guided domain comprises multiple binding domains. In some embodiments, the RNA-guided domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding domains. In some embodiments, the multiple binding domains are immediately adjacent to each other. In some embodiments, the multiple binding domains are linked by an intervening nucleotide spacer sequence.

[0075] In some embodiments, the one or more binding domains each comprise a sequence sufficiently complementary to the target sequence to enable the splice editor nucleic acid molecule to specifically bind to the target sequence through base pairing. As used herein, the term "base pair" refers to two nucleobases of opposing complementary nucleic acid strands that interact by forming specific hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds). In some embodiments, the base pair is formed by a Watson-Crick base pair. As will be understood by those skilled in the art, a Watson-Crick base pair refers to a set of base-pairing rules in which a purine nucleobase binds to a pyrimidine nucleobase to form a complementary base pair. The nature of the hydrogen bond depends on the particular base pair. For example, a guanosine-cytosine base pair is formed by three hydrogen bonds, while an adenine-thymine or adenine-uracil base pair is formed by two hydrogen bonds. It is understood that analogs or derivatives of canonical nucleobases form base pairing interactions via Watson-Crick base pairing or non-canonical base pairing.

[0076] A binding domain that "specifically binds" to a target sequence of a target RNA (e.g., a pre-mRNA) refers to one that does not significantly bind to a reference sequence, e.g., a nucleic acid lacking the target sequence. For example, a splice editor nucleic acid molecule comprising a binding domain that specifically binds to a target sequence exhibits substantially higher binding affinity to a target RNA (e.g., a pre-mRNA) comprising a nucleotide sequence that includes the target sequence, compared to a target RNA (e.g., a pre-mRNA) that lacks the target sequence. As will be understood by those skilled in the art, the binding affinity between a first nucleic acid strand and a second nucleic acid strand is measured as the melting temperature (Tm), which is the temperature at which half of the first nucleic acid strand is duplexed to the second nucleic acid strand.

[0077] In some embodiments, a binding domain is complementary to a target sequence of a target RNA (e.g., a pre-mRNA) when base-paired to the target sequence under conditions suitable for modulating trans-splicing. Such conditions can be stringent conditions, such as 12-16 hours of incubation of the combination of the target RNA (e.g., a pre-mRNA) and the splice editor nucleic acid molecule in a buffer containing 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA at a temperature of 50°C to 70°C, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions include physiologically relevant conditions that may be encountered in an organism. One of skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0078] non-coding RNA In some embodiments, the RNA-guided domain comprises an ncRNA. As used herein, "ncRNA" refers to an RNA sequence that does not encode a protein and functions in one or more cellular regulatory processes (e.g., RNA splicing, histone modification, translation, RNA pseudouridylation, RNA methylation, RNA cleavage, RNA processing, and RNA modification). For example, certain ncRNAs function in RNA-guided systems that have evolved to (i) stabilize RNA secondary structure and RNA-RNA interactions (Rossi 1996 Journal of Biological Chemistry 271.39(1996):23985-23991; Sabath, et al. (2013) RNA 19:1726-1744; Skrajna, et al. (2017) RNA 23:938-951), (ii) assemble into ribonucleoproteins (RNPs) to encapsulate and protect RNA from degradation (Darzacq 2006), and (iii) localize to relevant subcellular compartments (Roithova, et al. (2018) Nucleic acids research 46:3774-3790). In some embodiments, the ncRNAs are identified according to the methods described herein.

[0079] In some embodiments, the method includes identifying an ncRNA sequence from a database. Databases listing ncRNA sequences are known in the art. For example, in some embodiments, the database is RNAcentral (see, e.g., Nucleic Acids Res 45:D128(2017)). RNAcentral is a searchable database that provides ncRNA sequences with unique identifiers and information about one or more species in which the RNA sequence has been observed.

[0080] In some embodiments, the method includes identifying ncRNAs expressed by a cell or organism. Methods for identifying ncRNAs are known in the art (see, e.g., Huttenhofer, et al. (2006) Nucleic Acids Res 34:635). In some embodiments, intracellular RNA is extracted from a cell or organism, separated by PAGE and elution from the gel, and the ncRNAs are identified by sequence analysis (e.g., via 2D RNA fingerprinting or enzymatic or chemical RNA sequencing). In some embodiments, a cDNA library is generated by reverse transcription of ncRNAs obtained from a cell or organism through a selection process based on size or antibody binding, and then subjected to sequence analysis. In some embodiments, total RNA is harvested from a cell or organism, and ncRNAs are detected using microarray hybridization. In some embodiments, genomic SELEX is used to identify ncRNAs obtained from a cell or organism. In some embodiments, the ncRNA sequences are identified from any known organism. In some embodiments, the organism is a bacterium. In some embodiments, the organism is an archaea. In some embodiments, the organism is a metazoan. In some embodiments, the organism is a vertebrate. In some embodiments, the organism is a mammal, an amphibian, a reptile, a fish, or a bird. In some embodiments, the organism is a human.

[0081] In some embodiments, the ncRNA functions to modify, alter, inhibit, or promote RNP formation and / or canonical processing. In some embodiments, the ncRNA assembles into an RNP. In some embodiments, the RNP functions to stabilize the RNA secondary structure of the splice editor nucleic acid. In some embodiments, the RNP functions to stabilize RNA-RNA interactions within the splice editor nucleic acid and / or with a target RNA (e.g., pre-mRNA). In some embodiments, the RNP functions to protect the splice editor nucleic acid from degradation. In some embodiments, the RNP functions to localize the splice editor nucleic acid to a subcellular compartment containing the target pre-mRNA. Methods for measuring the assembly of one or more nucleic acids (e.g., RNA or DNA) and one or more proteins to form an RNP are known in the art. Such methods include, but are not limited to, electrophoretic mobility shift assays (EMSAs), DNA or RNA pull-down assays, oligonucleotide-targeted RNase H protection assays, fluorescent in situ hybridization colocalization, co-immunoprecipitation assays, and RNA sequencing and cross-linking methods, such as high-throughput sequencing cross-linking immunoprecipitation (HITS-CLIP).

[0082] In some embodiments, the ncRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the entire ncRNA sequence is incorporated into the splice editor nucleic acid. In some embodiments, a portion of the ncRNA sequence is incorporated into the splice editor nucleic acid.

[0083] In some embodiments, the splice editor of the present disclosure comprises an ncRNA sequence or a portion thereof, wherein the ncRNA is selected from an snRNA, a snoRNA, an lncRNA, an rRNA, a ribozyme, a sRNA, a scaRNA, a vault RNA, and combinations thereof.

[0084] In some embodiments, the ncRNA sequence or portion thereof is less than about 500 nucleotides in length. In some embodiments, the ncRNA sequence or portion thereof is less than about 400 nucleotides in length. In some embodiments, the ncRNA sequence or portion thereof is less than about 300 nucleotides in length. In some embodiments, the ncRNA sequence or portion thereof is about 250 to about 300, about 200 to about 300, about 150 to about 300, about 100 to about 300, about 50 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, or about 300, 250, 200, 150, 100, or 50 nucleotides in length.

[0085] In some embodiments, the ncRNA sequence, or portion thereof, is at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length.

[0086] In some embodiments, the ncRNA sequence or portion thereof is about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

[0087] In some embodiments, said ncRNA sequence or its part comprises one or more RNA secondary structures that are assembled into RNP.The method for identifying the secondary structure formed by RNA sequence is known in the art.In some embodiments, said method comprises experimental assay, for example, nuclear magnetic resonance, cryo-electron microscopy or X-ray crystallography. In some embodiments, the method is based on, for example, a thermodynamic model, such as Turner's nearest neighbor model (Schroeder, et al, Methods Enzymol 468, 371-387 (2009); Turner, et al Nucleic Acids Res. 38, D280-2 (2010)) or the Zuker algorithm (Zuker, et al Nucleic Acids Res. 9, 133-148 (1981); Zuker, et al Nucleic Acids Res. 31, 3406-3415 (2003); Markham, et al Methods Mol. Biol. 453, 3-31 (2008); Hofacker, et al Nucleic Acids Res. 31, 3429-3431 (2003); Lorenz, Algorithms Mol. Biol. 6, 26 (2011); Matthews, et al Molecular Modeling of Nucleic Acids. Vol. 682 of ACS Symposium Series. 246-257, Reuther, et al. BMC Bioinform. 11, 129 (2010)), machine learning methods such as CONTRAfold (Do, et al. Bioinformatics 22, e90-8 (2006), Foo, et al. Advances in Neural Information Processing Systems 20, 377-384), ContextFold (Zakov, et al. J. Comput. Biol.18, 1525-1542 (2011)), probabilistic generational models, e.g., stochastic context-free grammars (Rivas, et al. RNA 18, 193-212 (2012)), hybrid models, e.g., SimFold (Andronescu, et al. Bioinformatics 23, i19-28 (2007), Andronescu et al. RNA 16, 2304-2318 (2010)) or MXfold (Akiyama, et al. J. Bioinform. Comput. Biol. 16, 1840025 (2018)), deep learning approaches, e.g., SPOT-RNA (Singh, et al. Nat. Commun. 10, 5407 (2019)) or E2Efold (Chen et al. Proceedings of the 8th International Conference on Learning This includes computational predictions based on the Representations;arXiv:2002.05810(2020)).

[0088] In some embodiments, the one or more RNA secondary structures comprise a single-stranded RNA sequence, a double-stranded RNA sequence, or a combination thereof. In some embodiments, the one or more RNA secondary structures comprise a duplex structure, a stem-loop, a pseudoknot, an internal loop, a multi-branched loop, a bulge loop, an external loop, or a combination thereof. In some embodiments, the ncRNA sequence, or a portion thereof, comprises a sequence motif that assembles into an RNP. In some embodiments, the sequence motif comprises a single-stranded RNA sequence that assembles into an RNP. In some embodiments, the ncRNA sequence, or a portion thereof, comprises a sequence motif and one or more RNA secondary structures that assemble into an RNP. In some embodiments, the secondary structure and / or sequence motif assembles to form an RNP with one or more proteins in a human cell.

[0089] In some embodiments, the ncRNA sequence or portion thereof comprises one or more RNA secondary structures. In some embodiments, the ncRNA sequence or portion thereof comprises one or more sequence motifs. In some embodiments, the ncRNA sequence or portion thereof comprises one or more RNA secondary structures and one or more sequence motifs. In some embodiments, the sequence motif comprises a sequence selected from Table 1. In some embodiments, the sequence motif comprises an H consensus sequence comprising or consisting of a sequence set forth in Table 1. In some embodiments, the H consensus sequence comprises or consists of SEQ ID NO: 1. In some embodiments, the sequence motif comprises an ACA consensus sequence comprising or consisting of a sequence set forth in Table 1. In some embodiments, the ACA consensus sequence comprises or consists of SEQ ID NO: 2. In some embodiments, the sequence motif comprises an H / ACA box, wherein the H / ACA box comprises a sequence comprising an H consensus sequence and an ACA consensus sequence, each comprising a sequence set forth in Table 1. In some embodiments, the H / ACA box comprises a sequence comprising SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the sequence motif comprises a C consensus sequence comprising or consisting of a sequence set forth in Table 1. In some embodiments, the C consensus sequence comprises or consists of SEQ ID NO:5. In some embodiments, the sequence motif comprises a D consensus sequence comprising or consisting of a sequence set forth in Table 1. In some embodiments, the D consensus sequence comprises or consists of SEQ ID NO:6. In some embodiments, the sequence motif comprises a C / D box, the C / D box comprising a C consensus sequence and a D consensus sequence, each comprising a sequence set forth in Table 1. In some embodiments, the C / D box comprises SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, the sequence motif comprises an Sm motif comprising a sequence set forth in Table 1. In some embodiments, the Sm motif comprises SEQ ID NO:3. In some embodiments, the Sm motif comprises SEQ ID NO:4. [Table 1]

[0090] In some embodiments, a splice editor of the disclosure comprises an ncRNA sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, or a portion thereof. In some embodiments, a splice editor of the disclosure comprises an ncRNA sequence selected from SEQ ID NOs: 9-657, or a portion thereof.

[0091] In some embodiments, the ncRNA sequence or portion thereof comprises a contiguous nucleotide sequence at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length, wherein the contiguous nucleotide sequence comprises an Sm sequence motif (e.g., an Sm sequence motif listed in Table 1).

[0092] In some embodiments, the ncRNA sequence or portion thereof comprises a contiguous nucleotide sequence of about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 20 to about 50, about 30 to about 50, or about 40 to about 50 nucleotides in length, wherein the contiguous nucleotide sequence comprises an Sm sequence motif (e.g., an Sm sequence motif listed in Table 1).

[0093] In some embodiments, the ncRNA sequence or portion thereof comprises a contiguous nucleotide sequence of about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, wherein the contiguous nucleotide sequence comprises an Sm sequence motif (e.g., an Sm sequence motif listed in Table 1).

[0094] In some embodiments, the ncRNA sequence or portion thereof comprises a contiguous nucleotide sequence of about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, wherein the contiguous nucleotide sequence comprises (i) an H consensus sequence (e.g., an H consensus sequence listed in Table 1), (ii) an ACA consensus sequence (e.g., an ACA consensus sequence listed in Table 1), or (iii) a combination of (i) and (ii).

[0095] In some embodiments, the ncRNA sequence or a portion thereof comprises a contiguous nucleotide sequence of about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, wherein the contiguous nucleotide sequence comprises (i) a C-box motif described herein (e.g., a C-box motif described herein) (e.g., a C-box motif described herein) (e.g., a C-box motif described herein) (iii) a D-box motif described herein (e.g., a D-box motif described herein) (e.g., a D-box motif described herein) (e.g., a D-box motif described herein) or (v) a combination of (i) to (iv).

[0096] In some embodiments, the ncRNA sequence, or portion thereof, comprises a nucleotide sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, wherein the nucleotide sequence comprises a region of at least about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 nucleotides in length, wherein the region comprises one or more Sm sequence motifs (e.g., one or more Sm sequence motifs listed in Table 1).

[0097] In some embodiments, the ncRNA sequence, or portion thereof, comprises a nucleotide sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, wherein the nucleotide sequence comprises a region of at least about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 20 to about 50, about 30 to about 50, or about 40 to about 50 nucleotides in length, wherein the region comprises one or more Sm sequence motifs (e.g., one or more Sm sequence motifs listed in Table 1).

[0098] In some embodiments, the ncRNA sequence or portion thereof comprises a nucleotide sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, wherein the nucleotide sequence comprises a region of at least about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, wherein the region comprises one or more Sm sequence motifs (e.g., one or more Sm sequence motifs listed in Table 1).

[0099] In some embodiments, the ncRNA sequence or a portion thereof comprises a nucleotide sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, and the nucleotide sequence has at least about 30 to about 100, about 40 to about 100, about 50 to about 60, or about 70 to about 80, or about 90% identity to a sequence selected from SEQ ID NOs: 9-657. The region comprises about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, and the region comprises (i) an H consensus sequence (e.g., an H consensus sequence described in Table 1), (ii) an ACA consensus sequence (e.g., an ACA consensus sequence described in Table 1), or (iii) a combination of (i) and (ii).

[0100] In some embodiments, the ncRNA sequence or a portion thereof comprises a nucleotide sequence having at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 9-657, and the nucleotide sequence is at least about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 10 a region of 0 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length, comprising (i) a C-box motif described herein (e.g., a C-box motif described in Table 1), (ii) a C'-box motif described herein (e.g., a C'-box motif described in Table 1), (iii) a D-box motif described herein (e.g., a D'-box motif described in Table 1), (iv) a D'-box motif described herein (e.g., a D'-box motif described in Table 1), or (v) a combination of (i) to (iv).

[0101] In some embodiments, the ncRNA is an snRNA. In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length snRNA or a portion thereof comprising a nucleotide sequence including one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, an snRNA sequence described herein or an snRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length snRNA sequence described herein or a full-length snRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of an snRNA sequence described herein or an snRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the snRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length snRNA or the portion of the snRNA assembles into a small nuclear RNP (snRNP). In some embodiments, the full-length snRNA or the portion of the snRNA comprises one or more secondary RNA structures that assemble to form a snRNP. In some embodiments, the full-length snRNA or a portion of the snRNA comprises one or more sequence motifs that assemble to form a snRNP. In some embodiments, the full-length snRNA or a portion of the snRNA comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form a snRNP.

[0102] Exemplary metazoan snRNA systems include U1 and U11 snRNAs, which guide spliceosomal RNPs to splice sites (Black, et al. (1985) Cell 42:737-750; Kolossova, et al. (1997) RNA 3:227). Other snRNAs include U2, U4, U4atac, U5, U6, U6atac, and U12, which also form the large and small spliceosomal RNPs (Turunen, et al. (2013) RNA 4:61-76; Nguyen, et al. (2015) Nature 523:47-52; Charenton, et al. (2019) Science 364:362-367). U7 RNA is involved in the cleavage and polyadenylation of histone pre-mRNA (Strub, et al. (1984) EMBO journal 3:2801-2807; Soldati, et al. (1988) Molecular and Cellular Biology 8:1518-1524; Cotton, et al. (1988) The EMBO Journal 7:801-808).

[0103] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length snRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), and the snRNA can be any of RNU1-1, RNU1-100P, RNU1-101P, RNU1-103P, RNU1-104P, RNU1-105P, RNU1-107P, RNU1-108P, RNU1-109P, RNU1-112P, RNU1-114P, RNU1-115P, RNU1-116P, RNU1-117P, RNU1-118P, RNU1-119P, RNU1-200P, RNU1-201P, RNU1-202P, RNU1-203P, RNU1-204P, RNU1-205P, RNU1-206P, RNU1-207P, RNU1-208P, RNU1-209P, RNU1-210P, RNU1-211P, RNU1-212P, RNU1-214P, RNU1-215P, RNU1-216P, RNU1-217P, RNU1-218P, RNU1-219P, RNU1-220P, RNU1-221P, RNU1-222P, RNU1-223P, RNU1-224P, RNU1-225P, RNU1-226P, RNU1-227P, RNU1-228P, RNU1-229P, RNU1-230P, RNU1-231P, RNU1-232P, RNU1-233P, RNU1-23 7P, RNU1-119P, RNU1-11P, RNU1-123P, RNU1-124P, RNU1-125P, RNU1-128P, RNU1-129P, RNU1-130P, RNU1-131P, RNU1-132P, RNU1-133P, RNU1-134P, RNU 1-136P, RNU1-138P, RNU1-139P, RNU1-140P, RNU1-141P, RNU1-142P, RNU1-143P, RNU1-146P, RNU1-148P, RNU1-149P, RNU1-14P, RNU1-150P, RNU1-151P , RNU1-153P, RNU1-154P, RNU1-155P, RNU1-15P, RNU1-16P, RNU1-17P, RNU1-18P, RNU1-19P, RNU1-2, RNU1-20P, RNU1-21P, RNU1-22P, RNU1-23P, RNU1-2 4P, RNU1-27P, RNU1-28P, RNU1-29P, RNU1-3, RNU1-30P, RNU1-31P, RNU1-32P, RNU1-33P, RNU1-34P, RNU1-35P, RNU1-36P, RNU1-38P, RNU1-39P, RNU1-4, RNU1-40P, RNU1-41P, RNU1-42P, RNU1-43P, RNU1-44P, RNU1-45P, RNU1-46P, RNU1-47P, RNU1-48P, RNU1-49P, RNU1-51P, RNU1-52P, RNU1-54P, RNU1-55P , RNU1-56P, RNU1-57P, RNU1-58P, RNU1-5P, RNU1-61P, RNU1-62P, RNU1-63P, RNU1-64P, RNU1-65P, RNU1-67P, RNU1-68P, RNU1-6P, RNU1-70P, RNU1-72P,RNU1-73P、RNU1-74P、RNU1-75P、RNU1-76P、RNU1-77P、RNU1-78P、RNU1-79P、RNU1-7P、RNU1-80P、RNU1-82P、RNU1-83P、RNU1-84P、RNU1-86P、RNU1-88P、RNU1-89P、RNU1-8P、RNU1-91P、RNU1-93P、RNU1-94P、RNU1-95P、RNU1-96P、RNU1-97P、RNU1-98P、RNU11、RNU11-2P、RNU11-3P、RNU11-4P、RNU11-5P、RNU11-6P、RNU12、RNU12-2P、RNU2-12P、RNU2-13P、RNU2-16P、RNU2-18P、RNU2-19P、RNU2-24P、RNU2-27P、RNU2-30P、RNU2-31P、RNU2-34P、RNU2-35P、RNU2-37P、RNU2-38P、RNU2-41P、RNU2-42P、RNU2-46P、RNU2-50P、RNU2-53P、RNU2-55P、RNU2-60P、RNU2-66P、RNU2-69P、RNU2-70P、RNU2-72P、RNU2-7P、RNU2-9P、RNU4-1、RNU4-10P、RNU4-11P、RNU4-12P、RNU4-13P、RNU4-14P、RNU4-15P、RNU4-16P、RNU4-17P、RNU4-18P、RNU4-2、RNU4-20P、RNU4-21P、RNU4-22P、RNU4-23P、RNU4-24P、RNU4-26P、RNU4-27P、RNU4-28P、RNU4-29P、RNU4-30P、RNU4-31P、RNU4-32P、RNU4-33P、RNU4-34P、RNU4-35P、RNU4-36P、RNU4-37P、RNU4-38P、RNU4-39P、RNU4-40P、RNU4-41P、RNU4-42P、RNU4-43P、RNU4-44P、RNU4-45P、RNU4-46P、RNU4-47P、RNU4-49P、RNU4-4P、RNU4-50P、RNU4-51P、RNU4-52P、RNU4-53P、RNU4-54P、RNU4-55P、RNU4-56P、RNU4-57P、RNU4-58P、RNU4-59P、RNU4-5P、RNU4-60P、RNU4-61P、RNU4-62P、RNU4-63P、RNU4-64P、RNU4-65P、RNU4-66P、RNU4-67P、RNU4-68P、RNU4-69P、RNU4-6P、RNU4-70P、RNU4-71P、RNU4-72P、RNU4-73P、RNU4-74P、RNU4-75P、RNU4-76P、RNU4-77P、RNU4-78P、RNU4-79P、RNU4-7P、RNU4-80P、RNU4-81P、RNU4-82P、RNU4-83P、RNU4-84P、RNU4-85P、RNU4-87P、RNU4-88P、RNU4-89P、RNU4-8P、RNU4-90P、RNU4-91P、RNU4-92P、RNU4-9P、RNU4ATAC、RNU4ATAC10P、RNU4ATAC11P、RNU4ATAC12P、RNU4ATAC13P、RNU4ATAC14P、RNU4ATAC15P、RNU4ATAC16P、RNU4ATAC17P、RNU4ATAC18P、RNU4ATAC2P、RNU4ATAC3P、RNU4ATAC4P、RNU4ATAC5P、RNU4ATAC6P、RNU4ATAC7P、RNU4ATAC8P、RNU4ATAC9P、RNU5A-1、RNU5A-2P、RNU5A-3P、RNU5A-4P、RNU5A-5P、RNU5A-6P、RNU5A-7P、RNU5A-8P、RNU5B-1、RNU5B-2P、RNU5B-3P、RNU5B-4P、RNU5B-6P、RNU5D-1、RNU5D-2P、RNU5E-1、RNU5E-10P、RNU5E-3P、RNU5E-4P、RNU5E-5P、RNU5E-6P、RNU5E-7P、RNU5E-8P、RNU5E-9P、RNU5F-1、RNU5F-2P、RNU5F-3P、RNU5F-4P、RNU5F-6P、RNU5F-7P、RNU5F-8P、RNU6-1、RNU6-1000P、RNU6-1001P、RNU6-1003P、RNU6-1004P、RNU6-1005P、RNU6-1006P、RNU6-1007P、RNU6-1008P、RNU6-1009P、RNU6-100P、RNU6-1010P、RNU6-1011P、RNU6-1012P、RNU6-1013P、RNU6-1014P、RNU6-1015P、RNU6-1016P、RNU6-1017P、RNU6-1018P、RNU6-1019P、RNU6-101P、RNU6-1020P、RNU6-1021P、RNU6-1022P、RNU6-1023P、RNU6-1024P、RNU6-1025P、RNU6-1026P、RNU6-1027P、RNU6-1028P、RNU6-1029P、RNU6-102P、RNU6-1031P、RNU6-1032P、RNU6-1034P、RNU6-1035P、RNU6-1036P、RNU6-1037P、RNU6-1038P、RNU6-1039P、RNU6-103P、RNU6-1040P、RNU6-1041P、RNU6-1042P、RNU6-1043P、RNU6-1044P、RNU6-1045P、RNU6-1046P、RNU6-1047P、RNU6-1048P、RNU6-1049P、RNU6-104P、RNU6-1050P、RNU6-1051P、RNU6-1052P、RNU6-1053P、RNU6-1054P、RNU6-1055P、RNU6-1056P、RNU6-1057P、RNU6-1059P、RNU6-105P、RNU6-1060P、RNU6-1061P、RNU6-1062P、RNU6-1064P、RNU6-1065P、RNU6-1066P、RNU6-1067P、RNU6-1068P、RNU6-1069P、RNU6-106P、RNU6-1071P、RNU6-1072P、RNU6-1073P、RNU6-1074P、RNU6-1075P、RNU6-1076P、RNU6-1077P、RNU6-1078P、RNU6-1079P、RNU6-107P、RNU6-1080P、RNU6-1081P、RNU6-1082P、RNU6-1083P、RNU6-1084P、RNU6-1085P、RNU6-1086P、RNU6-1087P、RNU6-1088P、RNU6-1089P、RNU6-108P、RNU6-1090P、RNU6-1091P、RNU6-1092P、RNU6-1093P、RNU6-1094P、RNU6-1095P、RNU6-1096P、RNU6-1097P、RNU6-1098P、RNU6-1099P、RNU6-109P、RNU6-10P、RNU6-1100P、RNU6-1101P、RNU6-1102P、RNU6-1103P、RNU6-1104P、RNU6-1105P、RNU6-1106P、RNU6-1107P、RNU6-1108P、RNU6-1109P、RNU6-110P、RNU6-1110P、RNU6-1111P、RNU6-1112P、RNU6-1113P、RNU6-1114P、RNU6-1115P、RNU6-1116P、RNU6-1117P、RNU6-1118P、RNU6-1119P、RNU6-111P、RNU6-1120P、RNU6-1121P、RNU6-1122P、RNU6-1123P、RNU6-1124P、RNU6-1125P、RNU6-1126P、RNU6-1127P、RNU6-1128P、RNU6-1129P、RNU6-112P、RNU6-1130P、RNU6-1131P、RNU6-1132P、RNU6-1133P、RNU6-1134P、RNU6-1135P、RNU6-1136P、RNU6-1137P、RNU6-1138P、RNU6-113P、RNU6-1140P、RNU6-1141P、RNU6-1143P、RNU6-1144P、RNU6-1145P、RNU6-1146P、RNU6-1147P、RNU6-1148P、RNU6-1149P、RNU6-114P、RNU6-1150P、RNU6-1151P、RNU6-1152P、RNU6-1153P、RNU6-1154P、RNU6-1155P、RNU6-1156P、RNU6-1157P、RNU6-1158P、RNU6-1159P、RNU6-115P、RNU6-1160P、RNU6-1161P、RNU6-1162P、RNU6-1163P、RNU6-1164P、RNU6-1165P、RNU6-1167P、RNU6-1168P、RNU6-1169P、RNU6-116P、RNU6-1170P、RNU6-1171P、RNU6-1172P、RNU6-1174P、RNU6-1175P、RNU6-1176P、RNU6-1177P、RNU6-1178P、RNU6-1179P、RNU6-117P、RNU6-1180P、RNU6-1181P、RNU6-1183P、RNU6-1184P、RNU6-1186P、RNU6-1187P、RNU6-1188P、RNU6-1189P、RNU6-118P、RNU6-1190P、RNU6-1191P、RNU6-1192P、RNU6-1193P、RNU6-1194P、RNU6-1195P、RNU6-1196P、RNU6-1197P、RNU6-1198P、RNU6-1199P、RNU6-119P、RNU6-11P、RNU6-1200P、RNU6-1201P、RNU6-1203P、RNU6-1204P、RNU6-1205P、RNU6-1206P、RNU6-1、 207P、RNU6-1208P、RNU6-1209P、RNU6-120P、RNU6-1210P、RNU6-1211P、RNU6-1212P、RNU6-1213P、RNU6-1214P、RNU6-1215P、RNU6-1216P、RNU6-1217P、RNU6-1218P、RNU6-1219P、RNU6-121P、RNU6-1220P、RNU6-1222P、RNU6-1223P、RNU6-1224P、RNU6-1225P、RNU6-1226P、RNU6-1227P、RNU6-1228P、RNU6-1229P、RNU6-122P、RNU6-1230P、RNU6-1231P、RNU6-1232P、RNU6-1233P、RNU6-1234P、RNU6-1235P、RNU6-1236P、RNU6-1237P、RNU6-1238P、RNU6-1239P、RNU6-123P、RNU6-1240P、RNU6-1241P、RNU6-1242P、RNU6-1243P、RNU6-1244P、RNU6-1245P、RNU6-1246P、RNU6-1247P、RNU6-1248P、RNU6-1249P、RNU6-1250P、RNU6-1251P、RNU6-1252P、RNU6-1254P、RNU6-1255P、RNU6-1256P、RNU6-1257P、RNU6-1258P、RNU6-125P、RNU6-1260P、RNU6-1261P、RNU6-1262P、RNU6-1263P、RNU6-1264P、RNU6-1265P、RNU6-1266P、RNU6-1267P、RNU6-1268P、RNU6-1269P、RNU6-126P、RNU6-1270P、RNU6-1271P、RNU6-1272P、RNU6-1273P、RNU6-1274P、RNU6-1275P、RNU6-1276P、RNU6-1277P、RNU6-1278P、RNU6-1279P、RNU6-127P、RNU6-1280P、RNU6-1281P、RNU6-1282P、RNU6-1283P、RNU6-1284P、RNU6-1285P、RNU6-1286P、RNU6-1287P、RNU6-1288P、RNU6-1289P、RNU6-128P、RNU6-1290P、RNU6-1291P、RNU6-1292P、RNU6-1293P、RNU6-1294P、RNU6-1296P、RNU6-1297P、RNU6-1298P、RNU6-1299P、RNU6-129P、RNU6-12P、RNU6-1300P、RNU6-1301P、RNU6-1303P、RNU6-1304P、RNU6-1305P、RNU6-1306P、RNU6-1307P、RNU6-1308P、RNU6-1309P、RNU6-130P、RNU6-1310P、RNU6-1311P、RNU6-1312P、RNU6-1313P、RNU6-1314P、RNU6-1315P、RNU6-1316P、RNU6-1317P、RNU6-1318P、RNU6-1319P、RNU6-131P、RNU6-1320P、RNU6-1321P、RNU6-1322P、RNU6-1323P、RNU6-1324P、RNU6-1325P、RNU6-1326P、RNU6-1327P、RNU6-1328P、RNU6-1329P、RNU6-132P、RNU6-1330P、RNU6-1331P、RNU6-1332P、RNU6-1333P、RNU6-1334P、RNU6-1335P、RNU6-1336P、RNU6-1337P、RNU6-1338P、RNU6-1339P、RNU6-133P、RNU6-1340P、RNU6-135P、RNU6-136P、RNU6-137P、RNU6-138P、RNU6-139P、RNU6-13P、RNU6-140P、RNU6-141P、RNU6-142P、RNU6-143P、RNU6-144P、RNU6-145P、RNU6-146P、RNU6-147P、RNU6-148P、RNU6-14P、RNU6-150P、RNU6-151P、RNU6-152P、RNU6-153P、RNU6-154P、RNU6-155P、RNU6-156P、RNU6-157P、RNU6-158P、RNU6-159P、RNU6-15P、RNU6-160P、RNU6-161P、RNU6-162P、RNU6-163P、RNU6-164P、RNU6-165P、RNU6-166P、RNU6-167P、RNU6-168P、RNU6-169P、RNU6-16P、RNU6-170P、RNU6-171P、RNU6-172P、RNU6-173P、RNU6-174P、RNU6-175P、RNU6-176P、RNU6-177P、RNU6-178P、RNU6-179P、RNU6-17P、RNU6-180P、RNU6-181P、RNU6-182P、RNU6-183P、RNU6-184P、RNU6-185P、RNU6-187P、RNU6-188P、RNU6-189P、RNU6-18P、RNU6-190P、RNU6-191P、RNU6-192P、RNU6-193P、RNU6-194P、RNU6-195P、RNU6-196P、RNU6-197P、RNU6-198P、RNU6-199P、RNU6-19P、RNU6-2、RNU6-200P、RNU6-201P、RNU6-202P、RNU6-203P、RNU6-204P、RNU6-205P、RNU6-206P、RNU6-207P、RNU6-208P、RNU6-209P、RNU6-20P、RNU6-210P、RNU6-211P、RNU6-212P、RNU6-213P、RNU6-214P、RNU6-215P、RNU6-216P、RNU6-217P、RNU6-218P、RNU6-219P、RNU6-21P、RNU6-220P、RNU6-221P、RNU6-222P、RNU6-223P、RNU6-224P、RNU6-225P、RNU6-226P、RNU6-227P、RNU6-228P、RNU6-229P、RNU6-22P、RNU6-230P、RNU6-231P、RNU6-232P、RNU6-233P、RNU6-234P、RNU6-235P、RNU6-236P、RNU6-237P、RNU6-238P、RNU6-239P、RNU6-23P、RNU6-240P、RNU6-241P、RNU6-242P、RNU6-243P、RNU6-244P、RNU6-245P、RNU6-246P、RNU6-247P、RNU6-248P、RNU6-249P、RNU6-24P、RNU6-250P、RNU6-251P、RNU6-252P、RNU6-253P、RNU6-254P、RNU6-255P、RNU6-256P、RNU6-257P、RNU6-258P、RNU6-259P、RNU6-25P、RNU6-260P、RNU6-261P、RNU6-262P、RNU6-263P、RNU6-264P、RNU6-266P、RNU6-267P、RNU6-268P、RNU6-269P、RNU6-26P、RNU6-270P、RNU6-271P、RNU6-272P、RNU6-273P、RNU6-274P、RNU6-275P、RNU6-276P、RNU6-277P、RNU6-278P、RNU6-279P、RNU6-27P、RNU6-280P、RNU6-281P、RNU6-282P、RNU6-283P、RNU6-284P、RNU6-285P、RNU6-286P、RNU6-287P、RNU6-288P、RNU6-289P、RNU6-28P、RNU6-290P、RNU6-291P、RNU6-293P、RNU6-294P、RNU6-295P、RNU6-296P、RNU6-297P、RNU6-298P、RNU6-299P、RNU6-29P、RNU6-300P、RNU6-301P、RNU6-302P、RNU6-303P、RNU6-304P、RNU6-306P、RNU6-307P、RNU6-308P、RNU6-309P、RNU6-30P、RNU6-310P、RNU6-311P、RNU6-312P、RNU6-313P、RNU6-314P、RNU6-315P、RNU6-316P、RNU6-317P、RNU6-318P、RNU6-319P、RNU6-31P、RNU6-320P、RNU6-321P、RNU6-322P、RNU6-323P、RNU6-324P、RNU6-325P、RNU6-326P、RNU6-327P、RNU6-328P、RNU6-329P、RNU6-32P、RNU6-330P、RNU6-331P、RNU6-332P、RNU6-333P、RNU6-334P、RNU6-335P、RNU6-336P、RNU6-337P、RNU6-338P、RNU6-339P、RNU6-33P、RNU6-340P、RNU6-341P、RNU6-342P、RNU6-343P、RNU6-344P、RNU6-345P、RNU6-346P、RNU6-347P、RNU6-348P、RNU6-349P、RNU6-34P、RNU6-351P、RNU6-352P、RNU6-353P、RNU6-354P、RNU6-355P、RNU6-356P、RNU6-358P、RNU6-359P、RNU6-35P、RNU6-360P、RNU6-361P、RNU6-362P、RNU6-363P、RNU6-364P、RNU6-365P、RNU6-366P、RNU6-367P、RNU6-368P、RNU6-369P、RNU6-36P、RNU6-370P、RNU6-371P、RNU6-373P、RNU6-374P、RNU6-375P、RNU6-376P、RNU6-377P、RNU6-378P、RNU6-379P、RNU6-37P、RNU6-380P、RNU6-381P、RNU6-382P、RNU6-383P、RNU6-384P、RNU6-386P、RNU6-387P、RNU6-388P、RNU6-389P、RNU6-38P、RNU6-390P、RNU6-391P、RNU6-392P、RNU6-393P、RNU6-394P、RNU6-395P、RNU6-396P、RNU6-397P、RNU6-398P、RNU6-399P、RNU6-39P、RNU6-3P、RNU6-400P、RNU6-401P、RNU6-402P、RNU6-403P、RNU6-405P、RNU6-406P、RNU6-407P、RNU6-408P、RNU6-409P、RNU6-40P、RNU6-410P、RNU6-411P、RNU6-412P、RNU6-413P、RNU6-414P、RNU6-415P、RNU6-416P、RNU6-417P、RNU6-418P、RNU6-419P、RNU6-41P、RNU6-420P、RNU6-421P、RNU6-422P、RNU6-424P、RNU6-425P、RNU6-426P、RNU6-428P、RNU6-429P、RNU6-42P、RNU6-430P、RNU6-431P、RNU6-432P、RNU6-433P、RNU6-434P、RNU6-435P、RNU6-436P、RNU6-437P、RNU6-438P、RNU6-439P、RNU6-43P、RNU6-440P、RNU6-441P、RNU6-442P、RNU6-444P、RNU6-445P、RNU6-446P、RNU6-447P、RNU6-448P、RNU6-449P、RNU6-44P、RNU6-450P、RNU6-451P、RNU6-452P、RNU6-453P、RNU6-454P、RNU6-455P、RNU6-456P、RNU6-457P、RNU6-458P、RNU6-45P、RNU6-460P、RNU6-461P、RNU6-462P、RNU6-463P、RNU6-464P、RNU6-465P、RNU6-466P、RNU6-467P、RNU6-468P、RNU6-469P、RNU6-46P、RNU6-470P、RNU6-471P、RNU6-472P、RNU6-473、 P、RNU6-474P、RNU6-475P、RNU6-476P、RNU6-477P、RNU6-478P、RNU6-479P、RNU6-47P、RNU6-480P、RNU6-481P、RNU6-482P、RNU6-483P、RNU6-484P、RNU6-485P、RNU6-486P、RNU6-487P、RNU6-488P、RNU6-489P、RNU6-48P、RNU6-490P、RNU6-491P、RNU6-492P、RNU6-493P、RNU6-494P、RNU6-495P、RNU6-496P、RNU6-497P、RNU6-498P、RNU6-499P、RNU6-49P、RNU6-4P、RNU6-500P、RNU6-501P、RNU6-502P、RNU6-503P、RNU6-504P、RNU6-505P、RNU6-506P、RNU6-507P、RNU6-508P、RNU6-509P、RNU6-50P、RNU6-510P、RNU6-511P、RNU6-512P、RNU6-513P、RNU6-514P、RNU6-516P、RNU6-517P、RNU6-518P、RNU6-519P、RNU6-520P、RNU6-521P、RNU6-522P、RNU6-523P、RNU6-524P、RNU6-525P、RNU6-526P、RNU6-527P、RNU6-528P、RNU6-529P、RNU6-530P、RNU6-531P、RNU6-532P、RNU6-533P、RNU6-534P、RNU6-535P、RNU6-536P、RNU6-537P、RNU6-538P、RNU6-539P、RNU6-53P、RNU6-540P、RNU6-541P、RNU6-542P、RNU6-543P、RNU6-544P、RNU6-545P、RNU6-546P、RNU6-547P、RNU6-548P、RNU6-549P、RNU6-54P、RNU6-550P、RNU6-551P、RNU6-552P、RNU6-553P、RNU6-554P、RNU6-555P、RNU6-556P、RNU6-557P、RNU6-558P、RNU6-559P、RNU6-55P、RNU6-560P、RNU6-561P、RNU6-562P、RNU6-563P、RNU6-564P、RNU6-565P、RNU6-566P、RNU6-567P、RNU6-56P、RNU6-570P、RNU6-571P、RNU6-572P、RNU6-573P、RNU6-574P、RNU6-575P、RNU6-576P、RNU6-577P、RNU6-578P、RNU6-579P、RNU6-57P、RNU6-580P、RNU6-581P、RNU6-582P、RNU6-583P、RNU6-584P、RNU6-586P、RNU6-587P、RNU6-588P、RNU6-589P、RNU6-58P、RNU6-590P、RNU6-591P、RNU6-592P、RNU6-593P、RNU6-595P、RNU6-596P、RNU6-597P、RNU6-598P、RNU6-599P、RNU6-59P、RNU6-5P、RNU6-600P、RNU6-601P、RNU6-602P、RNU6-603P、RNU6-604P、RNU6-605P、RNU6-606P、RNU6-607P、RNU6-608P、RNU6-609P、RNU6-60P、RNU6-610P、RNU6-611P、RNU6-612P、RNU6-613P、RNU6-614P、RNU6-615P、RNU6-616P、RNU6-617P、RNU6-618P、RNU6-619P、RNU6-61P、RNU6-620P、RNU6-621P、RNU6-622P、RNU6-623P、RNU6-624P、RNU6-625P、RNU6-626P、RNU6-627P、RNU6-628P、RNU6-629P、RNU6-62P、RNU6-630P、RNU6-631P、RNU6-632P、RNU6-633P、RNU6-634P、RNU6-635P、RNU6-636P、RNU6-637P、RNU6-638P、RNU6-639P、RNU6-63P、RNU6-640P、RNU6-641P、RNU6-642P、RNU6-643P、RNU6-644P、RNU6-645P、RNU6-646P、RNU6-647P、RNU6-648P、RNU6-649P、RNU6-64P、RNU6-650P、RNU6-651P、RNU6-652P、RNU6-653P、RNU6-654P、RNU6-655P、RNU6-656P、RNU6-657P、RNU6-658P、RNU6-659P、RNU6-65P、RNU6-660P、RNU6-661P、RNU6-662P、RNU6-663P、RNU6-664P、RNU6-665P、RNU6-666P、RNU6-667P、RNU6-668P、RNU6-669P、RNU6-66P、RNU6-670P、RNU6-672P、RNU6-673P、RNU6-674P、RNU6-675P、RNU6-677P、RNU6-678P、RNU6-679P、RNU6-67P、RNU6-680P、RNU6-681P、RNU6-682P、RNU6-684P、RNU6-685P、RNU6-686P、RNU6-687P、RNU6-689P、RNU6-68P、RNU6-690P、RNU6-692P、RNU6-693P、RNU6-694P、RNU6-695P、RNU6-696P、RNU6-697P、RNU6-698P、RNU6-699P、RNU6-6P、RNU6-7、RNU6-700P、RNU6-701P、RNU6-702P、RNU6-703P、RNU6-704P、RNU6-705P、RNU6-706P、RNU6-707P、RNU6-708P、RNU6-709P、RNU6-70P、RNU6-710P、RNU6-711P、RNU6-712P、RNU6-713P、RNU6-714P、RNU6-715P、RNU6-716P、RNU6-717P、RNU6-718P、RNU6-719P、RNU6-71P、RNU6-720P、RNU6-721P、RNU6-722P、RNU6-723P、RNU6-724P、RNU6-725P、RNU6-726P、RNU6-727P、RNU6-728P、RNU6-729P、RNU6-72P、RNU6-730P、RNU6-731P、RNU6-732P、RNU6-733P、RNU6-735P、RNU6-737P、RNU6-738P、RNU6-739P、RNU6-73P、RNU6-740P、RNU6-741P、RNU6-742P、RNU6-743P、RNU6-744P、RNU6-745P、RNU6-746P、RNU6-747P、RNU6-748P、RNU6-749P、RNU6-74P、RNU6-750P、RNU6-751P、RNU6-752P、RNU6-753P、RNU6-754P、RNU6-755P、RNU6-756P、RNU6-757P、RNU6-758P、RNU6-759P、RNU6-75P、RNU6-760P、RNU6-761P、RNU6-762P、RNU6-763P、RNU6-764P、RNU6-765P、RNU6-766P、RNU6-767P、RNU6-768P、RNU6-769P、RNU6-76P、RNU6-770P、RNU6-771P、RNU6-772P、RNU6-774P、RNU6-775P、RNU6-776P、RNU6-777P、RNU6-778P、RNU6-77P、RNU6-780P、RNU6-781P、RNU6-782P、RNU6-783P、RNU6-784P、RNU6-785P、RNU6-786P、RNU6-787P、RNU6-788P、RNU6-789P、RNU6-78P、RNU6-790P、RNU6-791P、RNU6-792P、RNU6-793P、RNU6-794P、RNU6-795P、RNU6-796P、RNU6-797P、RNU6-798P、RNU6-799P、RNU6-79P、RNU6-8、RNU6-800P、RNU6-801P、RNU6-803P、RNU6-804P、RNU6-805P、RNU6-806P、RNU6-807P、RNU6-808P、RNU6-809P、RNU6-80P、RNU6-810P、RNU6-811P、RNU6-812P、RNU6-813P、RNU6-815P、RNU6-816P、RNU6-817P、RNU6-818P、RNU6-819P、RNU6-81P、RNU6-820P、RNU6-821P、RNU6-822P、RNU6-823P、RNU6-824P、RNU6-826P、RNU6-827P、RNU6-828P、RNU6-829P、RNU6-82P、RNU6-830P、RNU6-831P、RNU6-832P、RNU6-833P、RNU6-834P、RNU6-835P、RNU6-836P、RNU6-837P、RNU6-838P、RNU6-839P、RNU6-83P、RNU6-840P、RNU6-841P、RNU6-842P、RNU6-843P、RNU6-844P、RNU6-845P、RNU6-847P、RNU6-848P、RNU6-849P、RNU6-84P、RNU6-850P、RNU6-851P、RNU6-853P、RNU6-854P、RNU6-855P、RNU6-856P、RNU6-857P、RNU6-858P、RNU6-859P、RNU6-85P、RNU6-860P、RNU6-861P、RNU6-862P、RNU6-863P、RNU6-864P、RNU6-865P、RNU6-866P、RNU6-867P、RNU6-869P、RNU6-86P、RNU6-871P、RNU6-873P、RNU6-874P、RNU6-875P、RNU6-876P、RNU6-877P、RNU6-878P、RNU6-879P、RNU6-87P、RNU6-880P、RNU6-881P、RNU6-882P、RNU6-883P、RNU6-884P、RNU6-885P、RNU6-886P、RNU6-887P、RNU6-888P、RNU6-889P、RNU6-88P、RNU6-890P、RNU6-891P、RNU6-892P、RNU6-893P、RNU6-894P、RNU6-895P、RNU6-896P、RNU6-897P、RNU6-898P、RNU6-899P、RNU6-89P、RNU6-9、RNU6-900P、RNU6-901P、RNU6-902P、RNU6-903P、RNU6-904P、RNU6-905P、RNU6-906P、RNU6-907P、RNU6-908P、RNU6-909P、RNU6-90P、RNU6-910P、RNU6-911P、RNU6-912P、RNU6-913P、RNU6-914P、RNU6-915P、RNU6-916P、RNU6-917P、RNU6-918P、RNU6-919P、RNU6-91P、RNU6-920P、RNU6-921P、RNU6-922P、RNU6-923P、RNU6-924P、RNU6-925P、RNU6-926P、RNU6-927P、RNU6-928P、RNU6-929P、RNU6-92P、RNU6-930P、RNU6-931P、RNU6-932P、RNU6-933P、RNU6-934P、RNU6-935P、RNU6-936P、RNU6-937P、RNU6-938P、RNU6-939P、RNU6-940P、RNU6-941P、RNU6-942P、RNU6-943P、RNU6-944P、RNU6-945P、RNU6-946P、RNU6-947P、RNU6-948P、RNU6-949P、RNU6-94P、RNU6-950P, RNU6-951P, RNU6-952P, RNU6-953P, RNU6-954P, RNU6-955P, RNU6-956P, RNU6-957P, RNU6-958P, RNU6-959P, RNU6-95P, RNU6-960P, RNU6-961P, RNU6-964P 、RNU6-965P、RNU6-966P、RNU6-967P、RNU6-968P、RNU6-969P、RNU6-970P、RNU6-971P、RNU6-972P、RNU6-973P、RNU6-974P、RNU6-975P、RNU6-976P、RNU6-977P、RNU6-978P、RNU6-979P、RNU6-97P、RNU6-980P、RNU6-982P、RNU6-983P、RNU6-984P、RNU6-985P、RNU6-986P、RNU6-987P、RNU6-988P、RNU6-989P、RNU6-98P、RNU6-990P、RNU6-991P、RNU6-992P、RNU6-993P、RNU6-994P、RNU6-995P、RNU6-996P、RNU6-997P、RNU6-998P、RNU6-999P、RNU6-99P、RNU6ATAC、RNU6ATAC10P、RNU6ATAC11P、RNU6ATAC12P、RNU6ATAC13P、RNU6ATAC14P、RNU6ATAC15P、RNU6ATAC16P、RNU6ATAC17P、RNU6ATAC18P、RNU6ATAC19P、RNU6ATAC20P、RNU6ATAC21P、RNU6ATAC22P、RNU6ATAC23P、RNU6ATAC24P、RNU6ATAC25P、RNU6ATAC26P、RNU6ATAC27P、RNU6ATAC28P、RNU6ATAC29P、RNU6ATAC2P、RNU6ATAC30P、RNU6ATAC31P、RNU6ATAC32P、RNU6ATAC33P、RNU6ATAC34P、RNU6ATAC36P、RNU6ATAC37P、RNU6ATAC38P、RNU6ATAC39P、RNU6ATAC3P、RNU6ATAC40P、RNU6ATAC41P、RNU6ATAC42P、RNU6ATAC4P、RNU6ATAC5P、RNU6ATAC6P、RNU6ATAC7P、RNU6ATAC8P、RNU6ATAC9P、RNU6V、RNU7-1、RNU7-102P、RNU7-103P、RNU7-104P、RNU7-105P、RNU7-106P、RNU7-107P、RNU7-10P、RNU7-110P、RNU7-111P、RNU7-113P、RNU7-115P、RNU7-116P、RNU7-119P、RNU7-11P、RNU7-120P、RNU7-121P、RNU7-123P、RNU7-124P、RNU7-125P、RNU7-126P、RNU7-127P、RNU7-128P、RNU7-129P、RNU7-12P、RNU7-130P、RNU7-133P、RNU7-134P、RNU7-136P、RNU7-137P、RNU7-138P、RNU7-13P、RNU7-140P、RNU7-141P、RNU7-143P、RNU7-144P、RNU7-147P、RNU7-148P、RNU7-149P、RNU7-14P、RNU7-151P、RNU7-152P、RNU7-153P、RNU7-154P、RNU7-155P、RNU7-156P、RNU7-157P、RNU7-159P、RNU7-160P、RNU7-161P、RNU7-164P、RNU7-165P、RNU7-167P、RNU7-169P、RNU7-170P、RNU7-171P、RNU7-172P、RNU7-173P、RNU7-174P、RNU7-175P、RNU7-176P、RNU7-179P、RNU7-180P、RNU7-181P、RNU7-182P、RNU7-183P、RNU7-185P、RNU7-186P、RNU7-187P、RNU7-188P、RNU7-18P、RNU7-190P、RNU7-192P、RNU7-193P、RNU7-194P、RNU7-195P、RNU7-196P、RNU7-197P、RNU7-19P、RNU7-200P、RNU7-20P、RNU7-21P、RNU7-22P、RNU7-23P、RNU7-24P、RNU7-25P、RNU7-26P、RNU7-27P、RNU7-28P、RNU7-29P、RNU7-2P、RNU7-30P、RNU7-34P、RNU7-35P、RNU7-37P、RNU7-38P、RNU7-3P、RNU7-40P、RNU7-41P、RNU7-43P、RNU7-45P、RNU7-46P、RNU7-47P、RNU7-48P、RNU7-49P、RNU7-4P、RNU7-50P、RNU7-51P、RNU7-52P、RNU7-53P、RNU7-54P、RNU7-55P、RNU7-56P、RNU7-57P、RNU7-59P、RNU7-60P、RNU7-61P、RNU7-62P、RNU7-63P、RNU7-65P, RNU7-66P, RNU7-67P, RNU7-69P, RNU7-6P, RNU7-70P, RNU7-71P, RNU7-73P, RNU7-74P, RNU7-75P, RNU7-77P, RNU7-79P, RNU7-7P, RNU7-80P, RNU7-81 P, RNU7-82P, RNU7-84P, RNU7-85P, RNU7-87P, RNU7-88P, RNU7-8P, RNU7-90P, RNU7-92P, RNU7-93P, RNU7-94P, RNU7-95P, RNU7-96P, RNU7-97P, RNU7-99P, RNU7 -9P, RNVU1-1, RNVU1-14, RNVU1-15, RNVU1-17, RNVU1-18, RNVU1-19, RNVU1-2, RNVU1-21, RNVU1-22, RNVU1-23, RNVU1-24, RNVU1-25, RNVU1-26, RNVU1-27, RNV Selected from U1-28, RNVU1-29, RNVU1-2A, RNVU1-3, RNVU1-30, RNVU1-31, RNVU1-32, RNVU1-33, RNVU1-34, RNVU1-4, RNVU1-6, RNVU1-7, RNVU1-8, U1, U2, U4, U6, U7. ,

[0104] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length snRNA or a portion thereof comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), wherein the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0105] In some embodiments, the snRNA is U1 snRNA. In some embodiments, the U1 snRNA assembles into U1 RNP. In some embodiments, the snRNA is U2 snRNA. In some embodiments, the U2 snRNA assembles into U2 RNP. In some embodiments, the snRNA is U4 snRNA. In some embodiments, the U1 snRNA assembles into U4 RNP. In some embodiments, the snRNA is U4atac snRNA. In some embodiments, the U1 snRNA assembles into U4atac RNP. In some embodiments, the snRNA is U5 snRNA. In some embodiments, the U1 snRNA assembles into U5 RNP. In some embodiments, the snRNA is U6 snRNA. In some embodiments, the U1 snRNA assembles into U6 RNP. In some embodiments, the snRNA is U6atac snRNA. In some embodiments, the U1 snRNA assembles into U6atac RNP. In some embodiments, the snRNA is U7 snRNA. In some embodiments, the U1 snRNA assembles into U7 RNP. In some embodiments, the snRNA is U11 snRNA. In some embodiments, the U1 snRNA assembles into U11 RNP. In some embodiments, the snRNA is U12 snRNA. In some embodiments, the U1 snRNA assembles into U12 RNP.

[0106] In some embodiments, the ncRNA comprises an Sm sequence motif. In some embodiments, the Sm sequence motif assembles with an Sm protein to form an RNP. In some embodiments, the Sm protein is a B / B', D3, D2, D1, E, F, or G Sm protein.

[0107] In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length snoRNA or a portion thereof comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, a snoRNA sequence described herein or identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length snoRNA sequence described herein or identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of a snoRNA sequence described herein or identified according to the methods described herein (e.g., a region of consecutive nucleotides of the snRNA) is incorporated into a splice editor nucleic acid of the present disclosure.

[0108] In some embodiments, the full-length snoRNA or a portion thereof is assembled into a small nucleolar RNP (snoRNP). snoRNAs are involved in RNA methylation and RNA pseudouridylation (Bachellerie 2002, Kiss 2004). There are two classes of snoRNAs: (i) H / ACA box snoRNAs, which are involved in pseudouridylation, and (ii) C / D box snoRNAs, which are involved in 2'-O-ribose methylation (Jorjani 2016, Kufel 2019). snoRNAs can also form RNPs called snoRNPs (Khanna 2006) and hybridize to their RNA targets via Watson-Crick base pairing (Jin 2007).

[0109] In some embodiments, the full-length snoRNA or a portion thereof comprises an H / ACA box. In some embodiments, the H / ACA box comprises a nucleotide sequence comprising, from 5' to 3', an H consensus sequence (e.g., an H consensus sequence comprising the sequence set forth in SEQ ID NO: 1) and an ACA consensus sequence (e.g., an ACA consensus sequence comprising the sequence set forth in SEQ ID NO: 2). In some embodiments, the H / ACA box snoRNA assembles to form an H / ACA snoRNP. In some embodiments, the full-length snoRNA or a portion thereof comprises a C / D box. In some embodiments, the C / D box comprises, from 5' to 3', a nucleotide sequence comprising a C consensus sequence (e.g., a C consensus sequence comprising the sequence set forth in SEQ ID NO: 5), a D' consensus sequence (e.g., a D' consensus sequence comprising the sequence set forth in SEQ ID NO: 8), a C' consensus sequence (e.g., a C' consensus sequence comprising the sequence set forth in SEQ ID NO: 7), and a D consensus sequence (e.g., a D consensus sequence comprising the sequence set forth in SEQ ID NO: 6). In some embodiments, the C / D box snoRNAs assemble to form a C / D snoRNP.

[0110] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length snoRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), such as SCARNA18, SCARNA18B, SNORA1, SNORA10, SNORA108, SNORA10B, SNORA11, SNORA11B, SNORA11C, SNORA11D, SNORA11E, SNORA11F, SNORA11G, SNORA12, SNORA13, SNORA14, SNORA15, SNORA16, SNORA17, SNORA18A, SNORA18B, SNORA1, SNORA10B, SNORA11C, SNORA17D, SNORA17E, SNORA17F, SNORA17G, SNORA18, SNORA18H, SNORA18H, SNORA18H, SNORA18H, SNORA18H, SNORA18H, SNORA18H, SNORA18H, SNORA10 ... NORA14A, SNORA14B, SNORA15, SNORA15B-1, SNORA15B-2, SNORA16A, SNORA16B, SNORA17A, SNORA17B, SNORA18, SNORA19, SNORA1B, SNORA20, SNORA20B, SNORA21, SNORA21B, SNORA22, SNORA22B, SNORA22C, SNORA24, SNORA24B, SNORA25, SNORA25B, SNORA26, SNORA27, SNORA28, SNORA29, SNORA2A, SNORA2B, SNORA2C, SNORA30, SNORA30B, SNORA31, SNORA31B, SNORA32, SNORA33, SNORA35, SNORA35B, SNORA36A, SNORA36B, SNORA36C, SNORA37, SNORA38, SNORA3 8B, SNORA3A, SNORA3B, SNORA3C, SNORA4, SNORA40, SNORA40B, SNORA40C, SNORA41, SNORA41B, SNORA44, SNORA46, SNORA47, SNORA48, SNORA48B, SNORA49 , SNORA50A, SNORA50B, SNORA50C, SNORA50D, SNORA51, SNORA52, SNORA54, SNORA55, SNORA56, SNORA57, SNORA58, SNORA58B, SNORA59A, SNORA5A, SNORA5 B, SNORA5C, SNORA6, SNORA60, SNORA61, SNORA62, SNORA63, SNORA63B, SNORA63C, SNORA63D, SNORA63E, SNORA64, SNORA65, SNORA66, SNORA67, SNORA68,SNORA68B、SNORA69、SNORA70、SNORA70B、SNORA70C、SNORA70D、SNORA70E、SNORA70F、SNORA70G、SNORA70H、SNORA70I、SNORA70J、SNORA71、SNORA71A、SNORA71C、SNORA71D、SNORA71E、SNORA72、SNORA73、SNORA74、SNORA74D、SNORA75、SNORA75B、SNORA77、SNORA77B、SNORA78、SNORA79、SNORA79B、SNORA7A、SNORA7B、SNORA8、SNORA80A、SNORA80B、SNORA80C、SNORA80D、SNORA80E、SNORA81、SNORA84、SNORA9、SNORA9B、SNORD10、SNORD100、SNORD101、SNORD102、SNORD104、SNORD105、SNORD105B、SNORD107、SNORD108、SNORD109A、SNORD109B、SNORD11、SNORD110、SNORD111、SNORD111B、SNORD112、SNORD113-1、SNORD113-2、SNORD113-3、SNORD113-4、SNORD113-5、SNORD113-6、SNORD113-7、SNORD113-8、SNORD113-9、SNORD114-1、SNORD114-10、SNORD114-11、SNORD114-12、SNORD114-13、SNORD114-14、SNORD114-15、SNORD114-16、SNORD114-17、SNORD114-18、SNORD114-19、SNORD114-2、SNORD114-20、SNORD114-21、SNORD114-22、SNORD114-23、SNORD114-24、SNORD114-25、SNORD114-26、SNORD114-27、SNORD114-28、SNORD114-29、SNORD114-3、SNORD114-30、SNORD114-31、SNORD114-4、SNORD114-5、SNORD114-6、SNORD114-7、SNORD114-9、SNORD115、SNORD115-1、SNORD115-10、SNORD115-11、SNORD115-12、SNORD115-13、SNORD115-14、SNORD115-15、SNORD115-16、SNORD115-17、SNORD115-18、SNORD115-19、SNORD115-2、SNORD115-20、SNORD115-21、SNORD115-22、SNORD115-23、SNORD115-24、SNORD115-25、SNORD115-26、SNORD115-27、SNORD115-28、SNORD115-29、SNORD115-3、SNORD115-30、SNORD115-31、SNORD115-32、SNORD115-33、SNORD115-34、SNORD115-35、SNORD115-36、SNORD115-37、SNORD115-38、SNORD115-39、SNORD115-4、SNORD115-40、SNORD115-41、SNORD115-42、SNORD115-43、SNORD115-44、SNORD115-45、SNORD115-46、SNORD115-47、SNORD115-48、SNORD115-5、SNORD115-6、SNORD115-7、SNORD115-8、SNORD115-9、SNORD116、SNORD116-1、SNORD116-10、SNORD116-11、SNORD116-12、SNORD116-13、SNORD116-14、SNORD116-15、SNORD116-16、SNORD116-17、SNORD116-18、SNORD116-19、SNORD116-2、SNORD116-20、SNORD116-21、SNORD116-22、SNORD116-23、SNORD116-24、SNORD116-25、SNORD116-26、SNORD116-27、SNORD116-28、SNORD116-29、SNORD116-3、SNORD116-30、SNORD116-4、SNORD116-5、SNORD116-6、SNORD116-7、SNORD116-8、SNORD116-9、SNORD117、SNORD118、SNORD11B、SNORD12、SNORD121A、SNORD121B、SNORD123、SNORD124、SNORD125、SNORD126、SNORD127、SNORD12B、SNORD12C、SNORD13、SNORD13D、SNORD13E、SNORD13P1、SNORD13P3、SNORD14、SNORD14A、SNORD14B、SNORD14C、SNORD14D、SNORD14E、SNORD15A、SNORD15B、SNORD16、SNORD18、SNORD18A、SNORD18B、SNORD18C、SNORD19、SNORD19B、SNORD19C、SNORD1A、SNORD1B、SNORD1C、SNORD2、SNORD20、SNORD21、SNORD22、SNORD23、SNORD24、SNORD25、SNORD26、SNORD27、SNORD28、SNORD28B、SNORD29、SNORD30、SNORD31B、SNORD32A、SNORD32B、SNORD33、SNORD34、SNORD35A、SNORD35B、SNORD36、SNORD36A、SNORD36B、SNORD36C、SNORD37、SNORD38A、SNORD38B、SNORD38C、SNORD38D、SNORD39、SNORD41、SNORD42、SNORD42A、SNORD42B、SNORD43、SNORD45A、SNORD45B、SNORD45C、SNORD46、SNORD48、SNORD49A、SNORD49B、SNORD4A、SNORD4B、SNORD5、SNORD50B、SNORD51、SNORD52、SNORD53、SNORD53B、SNORD54、SNORD55、SNORD56、SNORD56B、SNORD57、SNORD58、SNORD58A、SNORD58B、SNORD58C、SNORD59A、SNORD6、SNORD60、SNORD61、SNORD62、SNORD62A、SNORD62B、SNORD63、SNORD63B、SNORD64、SNORD65、SNORD65B、SNORD65C、SNORD66、SNORD67、SNORD68、SNORD69、SNORD7、SNORD70、SNORD70B、SNORD71、SNORD72、SNORD73A、SNORD73B、SNORD74B、SNORD77B、SNORD79、SNORD8、SNORD81、SNORD82、SNORD83、SNORD83A、SNORD83B、SNORD84、SNORD86、Selected from SNORD87, SNORD88A, SNORD88B, SNORD88C, SNORD89, SNORD9, SNORD90, SNORD92, SNORD93, SNORD94, SNORD95, SNORD96A, SNORD96B, SNORD97, SNORD98, SNORD99, U8, snoZ196.

[0111] In some embodiments, the ncRNA is a scaRNA. In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length scaRNA or a portion thereof comprising a nucleotide sequence including one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, a scaRNA sequence described herein or a scaRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length scaRNA sequence described herein or a full-length scaRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of a scaRNA sequence described herein or a portion of a scaRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the scaRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length scaRNA or a portion thereof assembles into a Cajal body small RNP (scaRNP). In some embodiments, the full-length scaRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form a scaRNP. In some embodiments, the full-length scaRNA or portion thereof comprises one or more sequence motifs that assemble to form a scaRNP. In some embodiments, the full-length scaRNA or portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, where (i), (ii), or both, assemble to form a scaRNP. In some embodiments, the full-length scaRNA or portion thereof comprises an H / ACA box, which comprises, from 5' to 3', a nucleotide sequence that includes an H consensus sequence (e.g., an H consensus sequence comprising the sequence set forth in SEQ ID NO: 1) and an ACA consensus sequence (e.g., an ACA consensus sequence comprising the sequence set forth in SEQ ID NO: 2).

[0112] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length scaRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), wherein the scaRNA is selected from SCARNA1, SCARNA11, SCARNA14, SCARNA15, SCARNA17, SCARNA20, SCARNA21, SCARNA21B, SCARNA22, SCARNA23, SCARNA3, SCARNA4, SCARNA8.

[0113] In some embodiments, the ncRNA is a lncRNA. In some embodiments, the splice editor nucleic acid molecule of the present disclosure comprises a full-length lncRNA or a portion thereof comprising a nucleotide sequence including one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, a lncRNA sequence described herein or a lncRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length lncRNA sequence described herein or a full-length lncRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of a lncRNA sequence described herein or a portion of a lncRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the lncRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length lncRNA or a portion thereof assembles into an RNP. In some embodiments, the full-length lncRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form an RNP. In some embodiments, the full-length lncRNA or a portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length lncRNA or portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form an RNP.

[0114] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length lncRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), wherein the lncRNA is selected from the group consisting of AADACL2-AS1, ARHGEF26-AS1, ARMC2-AS1, BCAR3-AS1, C4B, CABIN1, CAPN15, CARS1-AS1, CASC19, CELF2-AS2, CPB2-AS1, EPHA5-AS1, ETV7-AS1, F11-AS1, FLG-AS1, GATA6-AS1, GLYCTK-AS1, HCG17, HCG27, HCG9, HHATL-AS1, HOTAIRM1, KIFC1, LIN C00511, LINC00824, LINC01060, LINC01358, LINC01378, LINC01409, LINC01606, LINC01676, LINC01943, LINC02276, LINC02301, LINC02690, LINC02695, LINC02790, LINC02805, LRIG3-DT, LY6E-DT, MALAT1, MAP3K14, MAPK4, MEIOB, OR12D3, PCDH9-AS2, PHF1, PSMB1, SLC8A1-AS1, SNHG25, SPRY4-AS1, TEX41, TTTY17A, TTTY17B, UST-AS2, ZEB2-AS1, hsa-mir-1253, hsa-mir-423.

[0115] In some embodiments, the ncRNA is a miscRNA. In some embodiments, the splice editor nucleic acid molecule of the present disclosure comprises a full-length miscRNA or a portion thereof comprising a nucleotide sequence including one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, a miscRNA sequence described herein or a miscRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length miscRNA sequence described herein or a full-length miscRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of a miscRNA sequence described herein or a portion of a miscRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the miscRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length miscRNA or a portion thereof assembles into an RNP. In some embodiments, the full-length miscRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form an RNP. In some embodiments, the full-length miscRNA or a portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length miscRNA or a portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form an RNP.

[0116] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length miscRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), and the miscRNA is selected from the group consisting of RN7SKP12, RN7SKP223, RN7SKP233, RN7SKP260, RN7SKP295, RN7SKP298, RN7SKP35, RN7SKP83, RN7SKP98, RNY1, RNY1P1, RNY1P10, RNY1P11, RNY1P12, RNY1P13, RNY1P14, RNY1P15, RNY1P16, RNY1P2, RNY1P3, RNY1P4, RNY1P5, RNY1P6, RNY1P7, RNY1P8, RNY1P9, RNY3, RNY3P1, RNY3 P10, RNY3P11, RNY3P12, RNY3P13, RNY3P14, RNY3P15, RNY3P16, RNY3P2, RNY3P3, RNY3P4, RNY3P5, RNY3P7 , RNY3P8, RNY3P9, RNY4, RNY4P10, RNY4P13, RNY4P14, RNY4P16, RNY4P17, RNY4P18, RNY4P19, RNY4P20, RNY 4P23, RNY4P24, RNY4P25, RNY4P27, RNY4P28, RNY4P29, RNY4P3, RNY4P30, RNY4P34, RNY4P36, RNY4P37, RNY4P6, RNY4P7, RNY4P9, VTRNA1-1, VTRNA1-2, VTRNA1-3, VTRNA2-2P, VTRNA3-1P, and vault, Y_RNA.

[0117] In some embodiments, the ncRNA is an Mt tRNA. In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length Mt tRNA or a portion thereof comprising a nucleotide sequence including one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, an Mt tRNA sequence described herein or an Mt tRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length Mt tRNA sequence described herein or a full-length Mt tRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of an Mt tRNA sequence described herein or an Mt tRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the Mt tRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length Mt tRNA or a portion thereof assembles into an RNP. In some embodiments, the full-length Mt tRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form an RNP. In some embodiments, the full-length Mt tRNA or portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length Mt tRNA or portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form an RNP.

[0118] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length Mt RNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), wherein the Mt tRNA is selected from MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, and MT-TY.

[0119] In some embodiments, the ncRNA is an rRNA. In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length rRNA or a portion thereof comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, an rRNA sequence described herein or an rRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length rRNA sequence described herein or a full-length rRNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of an rRNA sequence described herein or a portion of an rRNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the rRNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length rRNA or a portion thereof assembles into an RNP. In some embodiments, the full-length rRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form an RNP. In some embodiments, the full-length rRNA or a portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length rRNA or portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form an RNP.

[0120] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a full-length rRNA or a portion thereof comprising a nucleotide sequence that includes one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1), wherein the rRNA is selected from the group consisting of RNA5S1, RNA5S2, RNA5S3, RNA5S4, RNA5S5, RNA5S6, RNA5S7, RNA5S8, RNA5S9, RNA5S10, RNA5S11, RNA5S12, RNA5S13, RNA5S14, RNA5S15, RNA5S 16, RNA5S17, RNR1, RNR2, RNR3, RNR4, RNR5, RNA18SN1, RNA18SN2, RNA18SN3, RNA18SN4, RNA18SN5, RNA28SN1, RNA28SN2, RNA28SN3, RNA28SN4, RNA28SN5, RNA45SN1, RNA45SN2, RNA45SN3, RNA45SN4, RNA45SN5, RNA5-8SN1, RNA5-8SN2, RNA5-8SN3, RNA5-8SN4, and RNA5-8SN5.

[0121] In some embodiments, the ncRNA is a vault RNA. In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length vault RNA, or a portion thereof, comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs described in Table 1). In some embodiments, a vault RNA sequence described herein or a vault RNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a full-length vault RNA sequence described herein or a full-length vault RNA sequence identified according to the methods described herein is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, a portion of a vault RNA sequence described herein or a portion of a vault RNA sequence identified according to the methods described herein (e.g., a region of consecutive nucleotides of the vault RNA) is incorporated into a splice editor nucleic acid of the present disclosure. In some embodiments, the full-length vault RNA, or a portion thereof, assembles into an RNP. In some embodiments, the full-length vault RNA, or a portion thereof, comprises one or more secondary RNA structures that assemble to form an RNP. In some embodiments, the full-length vault RNA or portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length vault RNA or portion thereof comprises (i) one or more secondary RNA structures and (ii) one or more sequence motifs, and (i), (ii), or both, assemble to form an RNP.

[0122] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a vault RNA or a portion thereof, wherein the vault RNA is VTRNA2-1.

[0123] Methods for engineering splice editor nucleic acids of the present disclosure The present disclosure provides methods for engineering a splice editor nucleic acid described herein. In some embodiments, the method comprises: (A) identifying one or more candidate ncRNAs; (B) obtaining an ncRNA sequence from the one or more candidate ncRNAs; and (C) producing a splice editor nucleic acid comprising: (i) (a) the ncRNA sequence; and (b) a nucleotide sequence comprising an intron sequence comprising one or more binding domains described herein, (ii) a splice acceptor and / or a splice donor, and (iii) one or more exon sequences, thereby providing a splice editor nucleic acid for targeting trans-splicing of the target RNA (e.g., a target pre-mRNA). In some embodiments, the method comprises introducing the splice editor nucleic acid into a cell or population of cells and determining the efficiency of trans-splicing of the target RNA (e.g., a target pre-mRNA) according to the methods described herein. In some embodiments, the trans-splicing efficiency of the splice editor nucleic acid is compared to that of a control nucleic acid. In some embodiments, the control nucleic acid comprises (i), (ii), and (iii) and lacks an ncRNA sequence.

[0124] Methods for identifying candidate ncRNA sequences In some embodiments, identifying one or more candidate ncRNA sequences involves (i) obtaining one or more ncRNA sequences from a database and / or by experimental analysis of RNA expressed by a cell or organism described herein; (ii) predicting the secondary structure formed by the one or more ncRNA sequences according to the methods described herein; (iii) comparing the secondary structure predicted in (ii) with a reference secondary structure (e.g., a secondary structure present in an ncRNA known in the art); and (iv) selecting one or more candidate ncRNA sequences having a predicted secondary structure that has substantial similarity to the reference secondary structure. Computational methods for predicting secondary structures formed by ncRNA sequences are known in the art (see, e.g., Lorenz, et al. Vienna RNA Package 2.0 Algorithms for Molecular Biology, 6:1 26, 2011). Methods for performing RNA sequence analysis by comparing predicted secondary structures with reference secondary structures are also known in the art (see, e.g., Eddy, et al. (1994) Nucleic Acids Res 22:2079).

[0125] In some embodiments, identifying one or more candidate ncRNA sequences includes (i) obtaining one or more ncRNA sequences from a database and / or by experimental analysis of RNA expressed by a cell or organism described herein; (ii) predicting a secondary structure formed by the one or more ncRNA sequences according to the methods described herein; (iii) comparing the secondary structure predicted in (ii) with a reference secondary structure (e.g., a secondary structure present in an ncRNA known in the art); and (iv) selecting one or more candidate ncRNA sequences having a predicted secondary structure that has substantial similarity to the reference secondary structure and includes a sequence motif described herein (e.g., the sequence motif includes one or more sets of sequences listed in Table 1).

[0126] In some embodiments, the one or more candidate ncRNA sequences are selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657.

[0127] How to obtain ncRNA sequences In some embodiments, obtaining ncRNA sequences for inclusion in a splice editor nucleic acid of the disclosure comprises (i) identifying one or more candidate ncRNA sequences described herein, and (ii) selecting an ncRNA sequence from the one or more candidate ncRNA sequences.

[0128] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 7 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 8 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 9 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 10 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 11 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1). In some embodiments, the ncRNA sequence is about 12 nucleotides in length and comprises an Sm motif described herein (e.g., an Sm motif described in Table 1).

[0129] In some embodiments, obtaining the ncRNA sequence includes selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and includes an Sm motif described herein (e.g., an Sm motif described in Table 1).

[0130] In some embodiments, obtaining the ncRNA sequences includes selecting ncRNA sequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of one or more candidate ncRNA sequences or portions thereof (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequences are at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contain an Sm motif described herein (e.g., an Sm motif described in Table 1).

[0131] In some embodiments, obtaining the ncRNA sequence includes selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and includes an H-box motif described herein (e.g., an H-box motif described in Table 1).

[0132] In some embodiments, obtaining the ncRNA sequences includes selecting ncRNA sequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of one or more candidate ncRNA sequences or portions thereof (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657 or portions thereof), wherein the ncRNA sequences are at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contain an H-box motif described herein (e.g., an H-box motif described in Table 1).

[0133] In some embodiments, obtaining the ncRNA sequence includes selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contains an ACA box motif described herein (e.g., an ACA box motif described in Table 1).

[0134] In some embodiments, obtaining the ncRNA sequences includes selecting ncRNA sequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of one or more candidate ncRNA sequences or portions thereof (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657 or portions thereof), wherein the ncRNA sequences are at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contain an ACA box motif described herein (e.g., an ACA box motif described in Table 1).

[0135] In some embodiments, obtaining the ncRNA sequence includes selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contains an H box and an ACA box motif described herein (e.g., an H box and an ACA box motif described in Table 1).

[0136] In some embodiments, obtaining the ncRNA sequences includes selecting ncRNA sequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of one or more candidate ncRNA sequences or portions thereof (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657 or portions thereof), wherein the ncRNA sequences are at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length and contain the H box and ACA box motifs described herein (e.g., the H box and ACA box motifs described in Table 1).

[0137] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence from the one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one of the sequences set forth in SEQ ID NOs:9-657, or any combination thereof), wherein the ncRNA sequence is at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length and comprises (i) a C-box motif described herein (e.g., a C-box motif described herein (e.g., a C-box motif described herein (e.g., a C-box motif described herein (e.g., a C-box motif described herein (e.g., a C-box motif described herein (e.g., a D ... combination of (i)-(iv))).

[0138] In some embodiments, obtaining the ncRNA sequences includes selecting ncRNA sequences that have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of one or more candidate ncRNA sequences or portions thereof (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences set forth in SEQ ID NOs: 9-657 or portions thereof), and the sequences have at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the sequence. The amino acid sequence of the present invention is about 0, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides in length, and comprises (i) a C-box motif described herein (e.g., a C-box motif described in Table 1), (ii) a C'-box motif described herein (e.g., a C'-box motif described in Table 1), (iii) a D-box motif described herein (e.g., a D'-box motif described in Table 1), (iv) a D'-box motif described herein (e.g., a D'-box motif described in Table 1), or (v) a combination of (i)-(iv).

[0139] Methods for Producing Splice Editor Nucleic Acids The splice editor nucleic acids provided by the present disclosure are produced by any suitable nucleic acid synthesis method or means known in the art. In some embodiments, the splice editor nucleic acid is produced as RNA. In some embodiments, the splice editor nucleic acid is produced as DNA. The present disclosure further provides delivery systems comprising the splice editor nucleic acid, such as vectors comprising the splice editor nucleic acid and lipid particles comprising the splice editor nucleic acid.

[0140] Methods for producing the splice editor nucleic acid include, but are not limited to, in vitro transcription (IVT), synthetic and / or chemical synthesis methods, or combinations thereof. In some embodiments, enzymatic methods (e.g., IVT), solid-phase methods, liquid-phase methods, combinatorial synthesis methods, small-region synthesis methods, and ligation methods are used.

[0141] In some embodiments, the present disclosure provides splice editor nucleic acids produced using IVT enzymatic synthesis. Methods for producing polynucleotides by IVT are known in the art and are described in International Application No. PCT / US2013 / 30062.

[0142] In some embodiments, the present disclosure provides splice editor nucleic acids that are chemically synthesized by any means described in the art. In some embodiments, the splice editor nucleic acid is produced by oligonucleotide synthesis. Oligonucleotide synthesis is the chemical synthesis of relatively short fragments or chains of single-stranded nucleic acids with a defined chemical structure (sequence). Methods of oligonucleotide synthesis are known in the art (see, e.g., Reese (2005) Organic & Biomolecular Chemistry 3(2l):385l). While chemical synthesis methods are constantly expanding, purification of such nucleic acids by procedures such as high-performance liquid chromatography (HPLC, avoiding the use of gels such as PAGE) tends to become more difficult as the length of polynucleotides increases significantly beyond about 100 nucleotides. One approach used to generate longer nucleic acids is to produce two or more molecules and combine them.

[0143] Methods for determining the trans-splicing efficiency of splice editor nucleic acids In some embodiments, the present disclosure provides methods for determining the efficiency of trans-splicing of a target RNA (e.g., a pre-mRNA) using the splice editor nucleic acid molecules described herein.

[0144] In some embodiments, the method comprises the use of a fluorescence-based splicing reporter assay. In some embodiments, the assay comprises contacting a reporter cell or population of cells with a splice editor nucleic acid molecule according to a method described herein (e.g., via transfection with a viral vector encoding the splice editor nucleic acid molecule), wherein the splice editor nucleic acid molecule comprises at least one exon encoding a reporter molecule, wherein a trans-splicing event is indicated by the presence of a fluorescent signal from the reporter molecule, detected using methods known in the art. For example, in some embodiments, the reporter molecule is a fluorescent protein detected using fluorescence-activated cell sorting (FACS). For example, in some embodiments, the splice editor nucleic acid molecule comprises a nucleotide sequence encoding a first portion of a fluorescent protein, the target RNA comprises a nucleotide sequence encoding a second portion of a fluorescent protein, the trans-splicing produces an RNA comprising a nucleotide sequence encoding a full-length fluorescent protein, and the trans-splicing event is detected using a fluorometric method (e.g., FACS).

[0145] In some embodiments, the splice editor nucleic acid is introduced into cells for a period of time (e.g., via a viral or non-viral vector) using methods described herein, after which RNA is extracted from the cells and trans-splicing products are detected. For example, mRNA spliced ​​from a target RNA (e.g., a target pre-mRNA) is analyzed by a suitable method known in the art (e.g., end-point or quantitative RT-PCR or RNA sequencing). In some embodiments, a cell or population of cells is contacted with the splice editor nucleic acid molecule, and next-generation sequencing (NGS) technology is used to determine the degree of trans-splicing. For example, in some embodiments, mRNA extracted from cells treated or contacted with a splice editor nucleic acid provided by the present disclosure is enzymatically converted into cDNA, which is further analyzed by NGS analysis to determine the degree of mRNA molecules containing exon sequences incorporated from the splice editor nucleic acid molecule.

[0146] In some embodiments, trans-splicing is determined by protein sequence analysis of a polypeptide translated from an mRNA spliced ​​from the pre-mRNA. In some embodiments, the RNA-guided molecule repairs the mutation by incorporation of a repaired exon, where translation of the mRNA resulting from trans-splicing of the pre-mRNA and the splice editor nucleic acid produces a polypeptide comprising the amino acid sequence encoded by the repaired exon. The protein sequence analysis is performed using techniques including, but not limited to, Sanger sequencing, mass spectrometry, a functional assay measuring the enzymatic activity of the polypeptide, or immunoblotting using an antibody reactive to the repaired amino acid sequence.

[0147] In some embodiments, trans-splicing is determined by measuring the activity of a protein translated from an mRNA spliced ​​from the pre-mRNA. For example, in some embodiments, the protein is an enzyme, and a method for measuring trans-splicing comprises measuring enzyme activity using a functional ELISA.

[0148] In some embodiments, methods for measuring the efficiency of trans-splicing using the splice editor nucleic acids of the present disclosure are described in U.S. Application No. 16 / 994,230, which is incorporated herein by reference.

[0149] In some embodiments, methods for measuring the efficiency of trans-splicing using splice editor nucleic acids of the disclosure are any of those described in Chen, et al. (2009) Gene Ther 16:211; Rindt, et al. (2012) Cell Mol Life Sci 69:4191; Monjaret, et al. (2014) Mol Ther 22:1176; Berger, et al. (2015) Mol Ther 23:918.

[0150] In some embodiments, the methods described herein are used to measure the efficiency of trans-splicing of a pre-mRNA using a splice editor nucleic acid molecule described herein.

[0151] In some embodiments, a splice editor nucleic acid molecule described herein comprising a nucleotide sequence comprising (i) at least one intron sequence comprising one or more binding domains and an ncRNA, each having complementarity to a target sequence of the pre-mRNA, (ii) one or more splice sites (e.g., a splice acceptor and / or a splice donor), and (iii) at least one exon sequence, results in a higher trans-splicing efficiency than a splice editor nucleic acid molecule that does not comprise the ncRNA, as measured using methods described herein. In some embodiments, the splice editor nucleic acid molecule described herein results in a trans-splicing efficiency that is at least about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 20-fold higher than the trans-splicing efficiency of a splice editor nucleic acid molecule that does not comprise the ncRNA.

[0152] Exemplary Splice Editor Nucleic Acids In some embodiments, the disclosure provides Subgroup I nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', a nucleotide sequence comprising: (a) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure and / or comprises a sequence motif for directing the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0153] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA), (ii) an ncRNA sequence that forms a secondary structure and / or contains a sequence motif for directing the one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and (iii) at least one intron sequence comprising one or more splicing signals, wherein the ncRNA is a snRNA, (b) a splice acceptor, and (c) at least one exon sequence. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0154] In some embodiments, the subgroup I nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence comprising a sequence motif selected from an Sm sequence motif and an Lsm sequence motif for directing the one or more binding domain sequences to the target RNA (e.g., pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0155] In some embodiments, the subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure to direct the one or more binding domain sequences to the target RNA (e.g., pre-mRNA) and comprises a sequence motif selected from an Sm sequence motif and an Lsm sequence motif; and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0156] In some embodiments, the subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP that directs the binding domains to the target RNA (e.g., pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0157] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA), (ii) an ncRNA sequence that is an snRNA, forming a secondary structure and / or comprising a sequence motif that assembles to form an RNP that directs the one or more binding domains to the target RNA (e.g., pre-mRNA), and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0158] In some embodiments, the subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence comprising sequence motifs selected from Sm sequence motifs and Lsm sequence motifs, which assemble to form an RNP that directs the binding domains to the target RNA (e.g., pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0159] In some embodiments, the subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure that assembles to form an RNP that directs the binding domains to the target RNA (e.g., pre-mRNA), and that comprises a sequence motif selected from an Sm sequence motif and an Lsm sequence motif; and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0160] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or contains a sequence motif for directing the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) at least one intron sequence that includes one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0161] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each complementary to a target sequence of the target RNA (e.g., pre-mRNA), (ii) an ncRNA sequence that is an snRNA of about 7 to about 300 nucleotides in length, that forms a secondary structure and / or contains a sequence motif for directing the one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0162] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, comprising a sequence motif selected from an Sm sequence motif and an Lsm sequence motif, for directing the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0163] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, which forms a secondary structure to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA) and which comprises a sequence motif selected from an Sm sequence motif and an Lsm sequence motif; and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0164] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or comprises sequence motifs that assemble to form an RNP that directs the binding domains to the target RNA (e.g., a pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0165] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each complementary to a target sequence of the target RNA (e.g., pre-mRNA); (ii) an ncRNA sequence that is an snRNA of about 7 to about 300 nucleotides in length, that forms a secondary structure and / or contains sequence motifs that assemble to form an RNP that directs the binding domain to the target RNA (e.g., pre-mRNA); and (iii) at least one intron sequence that includes one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0166] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides comprising a sequence motif selected from an Sm sequence motif and an Lsm sequence motif, which assemble to form an RNP that directs the binding domains to the target RNA (e.g., a pre-mRNA); and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0167] In some embodiments, the Subgroup I nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, which forms a secondary structure that assembles to form an RNP that directs the binding domains to the target RNA (e.g., a pre-mRNA), and which comprises a sequence motif selected from an Sm sequence motif and an Lsm sequence motif; and (iii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0168] In some embodiments, the Subgroup I nucleic acids comprise one binding domain. In some embodiments, the Subgroup I nucleic acids comprise two binding domains. In some embodiments, the Subgroup I nucleic acids comprise three binding domains. In some embodiments, the Subgroup I nucleic acids comprise four binding domains. In some embodiments, the Subgroup I nucleic acids comprise five binding domains.

[0169] In some embodiments, the target RNA is a pre-mRNA. In some embodiments, the pre-mRNA comprises, from 5' to 3', a 5' exon, a splice donor, an intron, a splice acceptor, and a 3' exon, wherein the 3' exon comprises a mutation. In some embodiments, each of the one or more binding domains of the subgroup I nucleic acid is complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the target sequence is located in the 5' exon of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice donor of the pre-mRNA. In some embodiments, the target sequence is within an intron of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice acceptor of the pre-mRNA. In some embodiments, the target sequence is located in the 3' exon of the pre-mRNA. In some embodiments, trans-splicing occurs between the splice donor of the pre-mRNA and the splice acceptor of the subgroup I nucleic acid. In some embodiments, the trans-splicing results in ligation of the 3' end of the 5' exon of the pre-mRNA to the 5' end of at least one exon sequence of the subgroup I nucleic acid.

[0170] In some embodiments, one or more splicing signals of the Subgroup I nucleic acids comprise a branchpoint. In some embodiments, one or more splicing signals of the Subgroup I nucleic acids comprise a polypyrimidine tract. In some embodiments, one or more splicing signals of the Subgroup I nucleic acids comprise a branchpoint and a polypyrimidine tract. In some embodiments, the one or more splicing signals further comprise an ISE. In some embodiments, the one or more splicing signals further comprise an ISS.

[0171] In some embodiments, at least one exon sequence of the subgroup I nucleic acids comprises an ESE. In some embodiments, at least one exon sequence of the subgroup I nucleic acids comprises an ESS.

[0172] In some embodiments, the disclosure provides a nucleic acid for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence; and (ii) at least one intron sequence, each comprising one or more binding domain sequences complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure for directing the one or more binding domains to the target RNA and / or comprising a sequence motif.

[0173] In some embodiments, the disclosure provides subgroup II nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence, and (ii) a nucleotide sequence comprising at least one intron sequence, each comprising one or more binding domain sequences complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure for directing the one or more binding domains to the target RNA and / or comprises a sequence motif, and the ncRNA is an snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0174] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence; and (ii) at least one intron sequence, each comprising one or more binding domain sequences complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA comprises a sequence motif for directing the one or more binding domains to the target RNA, the sequence motifs being selected from an Sm sequence motif and an Lsm sequence motif.

[0175] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence; and (ii) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure to guide the one or more binding domains to the target RNA, and the sequence motif comprises at least one intron sequence, the sequence motif being selected from an Sm sequence motif and an Lsm sequence motif.

[0176] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence, and (ii) at least one intron sequence comprising a sequence motif and / or forming a secondary structure that assembles to form an RNP, each comprising one or more binding domain sequences that are complementary to a target sequence of the target RNA (e.g., a pre-mRNA), and that directs the one or more binding domains to the target RNA.

[0177] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure that assembles to form an RNP that guides the one or more binding domains to the target RNA, and / or comprises a sequence motif, and the ncRNA is an snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0178] In some embodiments, the subgroup II nucleic acids comprise a nucleotide sequence comprising, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence, and (ii) at least one intron sequence, each of which comprises one or more binding domain sequences complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequences comprise sequence motifs that assemble to form an RNP that guides the one or more binding domains to the target RNA, and the sequence motifs are selected from an Sm sequence motif and an Lsm sequence motif.

[0179] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence; and (ii) one or more binding domain sequences, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure that assembles to form an RNP that directs the one or more binding domains to the target RNA; and at least one intron sequence comprising a sequence motif, wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0180] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length; and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure for directing the one or more binding domains to the target RNA and / or at least one intron sequence comprising a sequence motif.

[0181] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or contains a sequence motif for directing the one or more binding domains to the target RNA, and wherein the ncRNA is an snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0182] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising at least one intron sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length; and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif for directing the one or more binding domains to the target RNA, the sequence motif being selected from an Sm sequence motif and an Lsm sequence motif.

[0183] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length; and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure to guide the one or more binding domains to the target RNA, and the sequence motif comprises at least one intron sequence selected from an Sm sequence motif and an Lsm sequence motif.

[0184] In some embodiments, the subgroup II nucleic acids comprise, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure that assembles to form an RNP that guides the one or more binding domains to the target RNA, and / or a nucleotide sequence comprising at least one intron sequence comprising a sequence motif.

[0185] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure that assembles to form an RNP that guides the one or more binding domains to the target RNA, and / or a nucleotide sequence containing at least one intron sequence, wherein the ncRNA is an snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0186] In some embodiments, the Subgroup II nucleic acids comprise a nucleotide sequence comprising, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif that assembles to form an RNP that guides the one or more binding domains to the target RNA, and the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif, and the ncRNA sequence comprises at least one intron sequence.

[0187] In some embodiments, the Subgroup II nucleic acids comprise, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, (c) (i) an ncRNA sequence from about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences from about 4 to about 300 nucleotides, each having complementarity to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure that assembles to form an RNP that guides the one or more binding domains to the target RNA, and the sequence motif comprises a nucleotide sequence comprising at least one intron sequence, the sequence motif being selected from an Sm sequence motif and an Lsm sequence motif.

[0188] In some embodiments, the subgroup II nucleic acids comprise one binding domain. In some embodiments, the subgroup II nucleic acids comprise two binding domains. In some embodiments, the subgroup II nucleic acids comprise three binding domains. In some embodiments, the subgroup II nucleic acids comprise four binding domains. In some embodiments, the subgroup II nucleic acids comprise five binding domains.

[0189] In some embodiments, the target RNA is a pre-mRNA. In some embodiments, the pre-mRNA comprises, from 5' to 3', a 5' exon, a splice donor, an intron, a splice acceptor, and a 3' exon, wherein the 5' exon comprises a mutation. In some embodiments, each of the one or more binding domains of the subgroup II nucleic acid is complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the target sequence is located in the 5' exon of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice donor of the pre-mRNA. In some embodiments, the target sequence is within an intron of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice acceptor of the pre-mRNA. In some embodiments, the target sequence is located in the 3' exon of the pre-mRNA. In some embodiments, trans-splicing occurs between the splice donor of the subgroup II nucleic acid and the splice acceptor of the pre-mRNA. In some embodiments, the trans-splicing results in ligation of the 3' end of the 5' exon of at least one exon sequence of the subgroup II nucleic acid to the 5' end of the 3' exon of the pre-mRNA.

[0190] In some embodiments, at least one exon sequence of the subgroup II nucleic acids comprises an ESE. In some embodiments, at least one exon sequence of the subgroup II nucleic acids comprises an ESS.

[0191] In some embodiments, the disclosure provides subgroup III nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence comprising: (i) an snoRNA sequence comprising an H / ACA box or a C / D box and one or more binding domain sequences, each having complementarity to a pre-mRNA target sequence; and (ii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0192] In some embodiments, the disclosure provides subgroup III nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', (a) a nucleotide sequence comprising: (i) an snoRNA sequence comprising an H / ACA box or a C / D box that directs the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA) and one or more binding domain sequences, each having complementarity to a pre-mRNA target sequence; and (ii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0193] In some embodiments, the subgroup III nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) an snoRNA sequence comprising one or more binding domain sequences, each having complementarity to a pre-mRNA target sequence, and an H / ACA box or a C / D box that assemble to form an RNP that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and (ii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0194] In some embodiments, the Subgroup III nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) an snoRNA sequence comprising an H / ACA box or a C / D box that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA) and one or more binding domain sequences about 4 to about 30 nucleotides in length, each having complementarity to a pre-mRNA target sequence; and (ii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0195] In some embodiments, the Subgroup III nucleic acids comprise, from 5' to 3', (a) a nucleotide sequence comprising: (i) an snoRNA sequence comprising an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides in length, each having complementarity to a pre-mRNA target sequence, that assemble to form an RNP that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and (ii) at least one intron sequence comprising one or more splicing signals, (b) a splice acceptor, and (c) at least one exon sequence.

[0196] In some embodiments, the target RNA is a pre-mRNA. In some embodiments, the pre-mRNA comprises, from 5' to 3', a 5' exon, a splice donor, an intron, a splice acceptor, and a 3' exon, wherein the 3' exon comprises a mutation. In some embodiments, each of the one or more binding domains of the subgroup III nucleic acid is complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the target sequence is located in the 5' exon of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice donor of the pre-mRNA. In some embodiments, the target sequence is within an intron of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice acceptor of the pre-mRNA. In some embodiments, the target sequence is located in the 3' exon of the pre-mRNA. In some embodiments, trans-splicing occurs between the splice donor of the pre-mRNA and the splice acceptor of the subgroup III nucleic acid. In some embodiments, the trans-splicing results in ligation of the 3' end of the 5' exon of the pre-mRNA to the 5' end of at least one exon sequence of the subgroup III nucleic acid.

[0197] In some embodiments, one or more splicing signals of the subgroup III nucleic acids comprise a branch point. In some embodiments, one or more splicing signals of the subgroup III nucleic acids comprise a polypyrimidine tract. In some embodiments, one or more splicing signals of the subgroup III nucleic acids comprise a branch point and a polypyrimidine tract. In some embodiments, the one or more splicing signals further comprise an ISE. In some embodiments, the one or more splicing signals further comprise an ISS.

[0198] In some embodiments, at least one exon sequence of the subgroup III nucleic acids comprises an ESE. In some embodiments, at least one exon sequence of the subgroup III nucleic acids comprises an ESS.

[0199] In some embodiments, the present disclosure provides subgroup IV nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, and (c) A nucleotide sequence containing at least one intron sequence, including an snoRNA sequence containing an H / ACA box or a C / D box and one or more binding domain sequences, each of which has complementarity to a pre-mRNA target sequence.

[0200] In some embodiments, the subgroup IV nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; and (c) at least one intron sequence comprising an H / ACA box or a C / D box that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA) and an snoRNA sequence that each comprises one or more binding domain sequences complementary to a pre-mRNA target sequence.

[0201] In some embodiments, the subgroup IV nucleic acids comprise a nucleotide sequence comprising, from 5' to 3', (a) at least one exon sequence, (b) a splice donor, and (c) at least one intron sequence comprising an H / ACA box or a C / D box and an snoRNA sequence each comprising one or more binding domain sequences complementary to a pre-mRNA target sequence that assemble to form an RNP that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA).

[0202] In some embodiments, the Subgroup IV nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; and (c) at least one intron sequence, including an H / ACA box or a C / D box that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA) and an snoRNA sequence that includes one or more binding domain sequences about 4 to about 30 nucleotides in length, each complementary to a pre-mRNA target sequence.

[0203] In some embodiments, the Subgroup IV nucleic acids comprise, from 5' to 3', a nucleotide sequence comprising: (a) at least one exon sequence; (b) a splice donor; and (c) at least one intron sequence, including an H / ACA box or a C / D box that assemble to form an RNP that directs the one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and an snoRNA sequence that includes one or more binding domain sequences about 4 to about 30 nucleotides in length, each complementary to a pre-mRNA target sequence.

[0204] In some embodiments, the target RNA is a pre-mRNA. In some embodiments, the pre-mRNA comprises, from 5' to 3', a 5' exon, a splice donor, an intron, a splice acceptor, and a 3' exon, wherein the 5' exon comprises a mutation. In some embodiments, each of the one or more binding domains of the subgroup IV nucleic acid is complementary to a target sequence of the target RNA (e.g., a pre-mRNA), wherein the target sequence is located in the 5' exon of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice donor of the pre-mRNA. In some embodiments, the target sequence is within an intron of the pre-mRNA. In some embodiments, the target sequence is proximal to the splice acceptor of the pre-mRNA. In some embodiments, the target sequence is located in the 3' exon of the pre-mRNA. In some embodiments, trans-splicing occurs between the splice donor of the subgroup IV nucleic acid and the splice acceptor of the pre-mRNA. In some embodiments, the trans-splicing results in ligation of the 3' end of the 5' exon of at least one exon sequence of the subgroup IV nucleic acid to the 5' end of the 3' exon of the pre-mRNA.

[0205] In some embodiments, at least one exon sequence of the subgroup IV nucleic acids comprises an ESE. In some embodiments, at least one exon sequence of the subgroup IV nucleic acids comprises an ESS.

[0206] In some embodiments, the subgroup III or subgroup IV nucleic acids comprise an H / ACA box, which comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located upstream of the H consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located downstream of the ACA consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located between the H consensus sequence and the ACA consensus sequence.

[0207] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises, from 5' to 3', an H / ACA box comprising a nucleotide sequence having an H consensus sequence and an ACA consensus sequence, and one binding domain. In some embodiments, the one binding domain sequence is located upstream of the H consensus sequence. In some embodiments, the one binding domain sequence is located downstream of the ACA consensus sequence. In some embodiments, the one binding domain sequence is located between the H consensus sequence and the ACA consensus sequence.

[0208] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises, from 5' to 3', an H / ACA box comprising a nucleotide sequence having an H consensus sequence and an ACA consensus sequence, a first binding domain, and a second binding domain. In some embodiments, the first binding domain sequence and the second binding domain sequence are each located upstream of the H consensus sequence. In some embodiments, the first binding domain sequence and the second binding domain sequence are each located downstream of the ACA consensus sequence. In some embodiments, the first binding domain sequence and the second binding domain sequence are each located between the H consensus sequence and the ACA consensus sequence. In some embodiments, the first binding domain sequence is located upstream of the H consensus sequence and the second binding domain sequence is located between the H consensus sequence and the ACA consensus sequence or downstream of the ACA consensus sequence. In some embodiments, the first binding domain sequence is located upstream of the H consensus sequence or between the H consensus sequence and the ACA consensus sequence, and the second binding domain sequence is located between the H consensus sequence and the ACA consensus sequence or downstream of the ACA consensus sequence.

[0209] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises an H / ACA box and one binding domain, wherein the H / ACA box comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence, and the one binding domain is located upstream of the H consensus sequence, downstream of the ACA consensus sequence, or between the H consensus sequence and the ACA consensus sequence.

[0210] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises an H / ACA box and two binding domains, wherein the H / ACA box comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence, and the two binding domains are each independently located upstream of the H consensus sequence, downstream of the ACA consensus sequence, and / or between the H consensus sequence and the ACA consensus sequence.

[0211] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises an H / ACA box and three binding domains, wherein the H / ACA box comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence, and the three binding domains are each independently located upstream of the H consensus sequence, downstream of the ACA consensus sequence, and / or between the H consensus sequence and the ACA consensus sequence.

[0212] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises an H / ACA box and four binding domains, wherein the H / ACA box comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence, and the four binding domains are each independently located upstream of the H consensus sequence, downstream of the ACA consensus sequence, and / or between the H consensus sequence and the ACA consensus sequence.

[0213] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises an H / ACA box and five binding domains, wherein the H / ACA box comprises a nucleotide sequence having, from 5' to 3', an H consensus sequence and an ACA consensus sequence, and the five binding domains are each independently located upstream of the H consensus sequence, downstream of the ACA consensus sequence, and / or between the H consensus sequence and the ACA consensus sequence.

[0214] In some embodiments, the subgroup III or subgroup IV nucleic acids comprise a C / D box, the C / D box comprising a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located upstream of the C consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located between the C consensus sequence and the D' consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located between the C' consensus sequence and the D consensus sequence. In some embodiments, one or more binding domain sequences of the subgroup III or subgroup IV nucleic acids are located downstream of the D consensus sequence.

[0215] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises a C / D box and a binding domain, wherein the C / D box comprises a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence, and the binding domain is located upstream of the C consensus sequence, between the C consensus sequence and the D' consensus sequence, between the C' consensus sequence and the D consensus sequence, or downstream of the D consensus sequence.

[0216] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises a C / D box and two binding domains, wherein the C / D box comprises a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence, and the two binding domains are each independently located upstream of the C consensus sequence, between the C and D' consensus sequences, between the C' and D consensus sequences, and / or downstream of the D consensus sequence.

[0217] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises a C / D box and three binding domains, wherein the C / D box comprises a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence, and the three binding domains are each independently located upstream of the C consensus sequence, between the C and D' consensus sequences, between the C' and D consensus sequences, and / or downstream of the D consensus sequence.

[0218] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises a C / D box and four binding domains, wherein the C / D box comprises a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence, and the four binding domains are each independently located upstream of the C consensus sequence, between the C and D' consensus sequences, between the C' and D consensus sequences, and / or downstream of the D consensus sequence.

[0219] In some embodiments, the subgroup III or subgroup IV nucleic acid comprises a C / D box and five binding domains, wherein the C / D box comprises a nucleotide sequence having, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence, and the five binding domains are each independently located upstream of the C consensus sequence, between the C and D' consensus sequences, between the C' and D consensus sequences, and / or downstream of the D consensus sequence.

[0220] In some embodiments, a nucleic acid of the disclosure (e.g., a nucleic acid of any one of subgroups I-IV) comprises at least one binding domain sequence that is fully complementary to the target sequence. In some embodiments, the nucleic acid comprises at least one binding domain sequence that is partially complementary to the target sequence (e.g., comprising at least 95% complementarity to the target sequence). In some embodiments, the nucleic acid comprises at least one binding domain sequence that is fully complementary to the target sequence and at least one binding domain sequence that is partially complementary to the target sequence (e.g., comprising at least 95% complementarity to the target sequence).

[0221] In some embodiments, a nucleic acid of the disclosure (e.g., a nucleic acid of any one of subgroups I-IV) has a sequence up to about 20,000 nucleotides in length. In some embodiments, the sequence is up to about 10,000 nucleotides in length. In some embodiments, the sequence is up to about 9,000 nucleotides in length. In some embodiments, the sequence is up to about 8,000 nucleotides in length. In some embodiments, the sequence is up to about 7,000 nucleotides in length. In some embodiments, the sequence is up to about 6,000 nucleotides in length. In some embodiments, the sequence is up to about 5,000 nucleotides in length. In some embodiments, the sequence is about 50 to about 500 nucleotides in length. In some embodiments, the sequence is about 50 to about 1,000 nucleotides in length. In some embodiments, the sequence is about 100 to about 500 nucleotides in length. In some embodiments, the sequence is about 100 to about 1,000 nucleotides in length. In some embodiments, the sequence is about 500 to about 1,000 nucleotides in length. In some embodiments, the sequence is about 500 to about 2,000 nucleotides in length. In some embodiments, the sequence is about 500 to about 3,000 nucleotides in length. In some embodiments, the sequence is about 500 to about 4,000 nucleotides in length. In some embodiments, the sequence is about 500 to about 5,000 nucleotides in length. In some embodiments, the sequence is about 1,000 to about 5,000 nucleotides in length. In some embodiments, the sequence is about 1,000 to about 10,000 nucleotides in length. In some embodiments, the sequence is about 5,000 to about 15,000 nucleotides in length. In some embodiments, the sequence is about 5,000 to about 20,000 nucleotides in length.

[0222] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a sequence selected from Table 3, or a portion thereof. Table 3 shows exemplary nucleotide sequences of splice editor nucleic acids of the disclosure. As shown in the table, regions of the sequence are separated by a hyphen (-), and the region identities are designated, from 5' to 3', as Region 1, Region 2, Region 3, and optionally Region 4. In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises a sequence having the formula 5'-[A]-[B]-3', where [A] is a nucleotide sequence selected from Table 3, and [B] is a sequence comprising, from 5' to 3', a splice acceptor and one or more exon sequences. In some embodiments, [A] comprises a nucleotide sequence selected from Table 3, and the RNA binding domain is replaced with an RNA binding domain described herein.

[0223] In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises, from 5' to 3', one or more RNA binding domains described herein, an ncRNA sequence, an intron sequence, a splice acceptor, and one or more exon sequences, wherein the ncRNA sequence has about 90%, 95%, 98%, 99%, or 100% of the ncRNA sequence identified in Table 3. In some embodiments, a splice editor nucleic acid molecule of the disclosure comprises, from 5' to 3', one or more exon sequences, a splice donor, an intron sequence, an ncRNA sequence, and one or more RNA binding domains described herein, wherein the ncRNA sequence has about 90%, 95%, 98%, 99%, or 100% of the ncRNA sequence identified in Table 3. In some embodiments, the intron sequence has about 90%, 95%, 98%, 99%, or 100% of the intron sequence identified in Table 3.

[0224] vector In some embodiments, the present disclosure provides vectors comprising one or more splice editor nucleic acids described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is circular. In some embodiments, the vector is linear. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[0225] In some embodiments, the vector is an expression vector, which is capable of directing the expression of a nucleic acid to which it is operably linked. As used herein, "expression vector" or "recombinant expression vector" refers to a replicon, e.g., a plasmid, phage, virus, or cosmid, to which another DNA segment, or "insert," is attached so that replication of the attached segment occurs within a cell.

[0226] In some embodiments, the vector or expression vector is a plasmid. As used herein, "plasmid" refers to a circular double-stranded DNA loop into which additional nucleic acid segments are ligated.

[0227] In some embodiments, the vector or expression vector is a viral vector, in which an additional nucleic acid segment is ligated into the viral genome. Non-limiting exemplary viral vectors include viral vectors based on vaccinia virus, poliovirus, adenovirus and adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, and picornavirus. Non-limiting exemplary viral vectors also include viral vectors based on retroviruses, such as murine leukemia virus and spleen necrosis virus, as well as vectors derived from retroviruses, such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. In some embodiments, the vector is used in eukaryotic target cells, and includes, but is not limited to, pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).

[0228] In some embodiments, the vector comprises one or more transcriptional and / or translational control elements. In some embodiments, the one or more transcriptional and / or translational control elements used depend on the target cell population and the vector system. In some embodiments, a variety of suitable transcriptional and translational control elements are used in the expression vector, such as those further described below, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc.

[0229] In some embodiments, a vector comprising a splice editor nucleic acid of the disclosure is operably linked to a control element, e.g., a transcription control element such as a promoter. In some embodiments, the transcription control element functions in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the splice editor nucleic acid sequence is operably linked to one or more control elements that allow expression in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.

[0230] In some embodiments, the expression vector comprises a promoter that is an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). Examples of inducible promoters include, but are not limited to, a T7 RNA polymerase promoter, a T3 RNA polymerase promoter, an isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose-inducible promoter, a heat shock promoter, a tetracycline-regulated promoter (e.g., Tet-ON, Tet-OFF, etc.), a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc. In some embodiments, the inducible promoter is regulated by a molecule, including, but not limited to, doxycycline, an RNA polymerase, e.g., T7 RNA polymerase, an estrogen receptor, an estrogen receptor fusion, etc.

[0231] In some embodiments, the promoter is a constitutive promoter (eg, a CMV promoter, a UBC promoter).

[0232] In some embodiments, the promoter is a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). A spatially restricted promoter may also be referred to as an enhancer, a transcriptional control element, a regulatory sequence, etc. Any convenient spatially restricted promoter is suitable for use in the present disclosure, and the selection of an appropriate promoter (e.g., a liver-specific promoter, a brain-specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in the germline, a promoter that drives expression in the lung, a promoter that drives expression in muscle, a promoter that drives expression in pancreatic islet cells, etc.) depends on the organism. For example, a variety of spatially restricted promoters are known for plants, flies, worms, mammals, mice, etc. Thus, spatially restricted promoters can be used to regulate expression of a splice editor nucleic acid in a wide variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also temporally restricted, such that the promoter is in an "on" or "off" state at specific stages of embryonic development or at specific stages of a biological process. By way of illustration, examples of spatially restricted promoters include, but are not limited to, liver-specific promoters, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, and the like.

[0233] Suitable promoters for use in the present disclosure include those derived from viruses, herein referred to as viral promoters, or those derived from organisms, including prokaryotes or eukaryotes. In some embodiments, suitable promoters for use in the present disclosure include any promoter that drives expression by an RNA polymerase (e.g., pol I, pol II, pol III).

[0234] Exemplary promoters include, but are not limited to, the SV40 early promoter, the mouse mammary tumor virus long terminal repeat (LTR) promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the cytomegalovirus (CMV) promoter, e.g., the CMV immediate early promoter region (CMVIE), the Rous sarcoma virus (RSV) promoter, the human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), the enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1; 31 (17)), the human H1 promoter (H1), and the like.

[0235] Exemplary eukaryotic promoters (i.e., promoters that function in eukaryotic cells) include, but are not limited to, cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, human elongation factor 1 promoter (EF1), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

[0236] In some embodiments, the present disclosure provides vectors comprising a splice editor nucleic acid described herein and an RNA polymerase III promoter (e.g., U6 and H1). Descriptions and parameters for improving the use of such promoters are known in the art, and further information and approaches have been described elsewhere. See, e.g., Ma, H. et al., Molecular Therapy-Nucleic Acids 3, e161 (2014).

[0237] In some embodiments, the expression vector comprises a ribosome binding site for translation initiation and a transcription terminator. In some embodiments, the expression vector comprises sequences appropriate for amplifying expression. In some embodiments, the expression vector comprises, for example, a nucleotide sequence encoding a non-native tag (e.g., a histidine tag, a hemagglutinin tag, green fluorescent protein, etc.) operably linked to the splice editor nucleic acid.

[0238] Methods for introducing nucleic acids into host cells or populations of host cells are known in the art, and any known method can be used to introduce a nucleic acid (e.g., an expression construct) into cells. In some embodiments, a splice editor nucleic acid molecule or a vector comprising the splice editor nucleic acid molecule is delivered to a population of cells using well-developed transfection techniques, see, e.g., Angel and Yanik (2010) PLoS ONE 5(7):e 11756, and commercially available TransMessenger® reagent from Qiagen, Stemfect™ RNA transfection kit from Stemgent, and TranslT®-mRNA transfection kit from Mims Bio LLC (see also Beumer et al. (2008). PNAS 105(50):19821-19826).

[0239] In some embodiments, the splice editor nucleic acid molecule is introduced into the cell or population of cells as RNA. In some embodiments, the RNA has chemical properties suitable for intracellular delivery, tolerability, and stability, e.g., after in vivo or in vitro administration. In some embodiments, the RNA is modified, e.g., comprises modified sugar moieties, modified internucleoside linkages, modified nucleosides, modified nucleotides, and / or combinations thereof. In some embodiments, the modified RNA exhibits one or more of the following properties: is not immunostimulatory, is nuclease resistant, has improved cellular uptake, has extended half-life, has increased translation efficiency, and / or is not toxic to a cell or mammal, e.g., after contact with cells in vivo, ex vivo, or in vitro.

[0240] Delivery Agent In some embodiments, delivery of the splice editor nucleic acid described herein is carried out by one or more of the methods described herein. In some embodiments, the splice editor nucleic acid is delivered by a viral vector, a lipid nanoparticle (LNP), a synthetic polymer, or a combination thereof. In some embodiments, the delivery methods described herein are suitable for administering a splice editor nucleic acid of the present disclosure to a target cell population or target tissue to target pre-mRNA for trans-splicing in the target cell or tissue intracellularly, ex vivo, or in vivo.

[0241] In some embodiments, the delivery comprises administering the splice editor nucleic acid as RNA or DNA. In some embodiments, the delivery comprises administering the splice editor nucleic acid as DNA formulated as LNPs or polymeric nanoparticles. In some embodiments, the delivery comprises administering the splice editor nucleic acid as RNA formulated as LNPs or polymeric nanoparticles.

[0242] In some aspects, the delivery comprises administering a recombinant expression vector (e.g., a plasmid, a viral vector) comprising the splice editor nucleic acid. In some embodiments, the recombinant expression vector is a non-viral vector (e.g., a plasmid). In some embodiments, the recombinant expression vector is a viral vector (e.g., AAV). In some embodiments, the delivery comprises formulation of one or more recombinant expression vectors using LNPs or polymeric nanoparticles. In some embodiments, a combination of a viral vector and a non-viral delivery vehicle is used.

[0243] In some embodiments, the splice editor nucleic acid molecule is delivered by a non-viral delivery vehicle, including, but not limited to, a nanoparticle, a liposome, a ribonucleoprotein, a positively charged peptide, a small molecule-RNA conjugate, an aptamer-RNA chimera, and an RNA-fusion protein complex. Non-limiting exemplary non-viral delivery vehicles include those described in Peer and Lieberman, Gene Therapy, 18:1127-1133 (2011), which focuses on non-viral delivery vehicles for siRNA that are also useful for delivering other polynucleotides.

[0244] Viral delivery In some embodiments, the splice editor nucleic acid molecule is delivered by a viral delivery vehicle, e.g., AAV. In some embodiments, the viral vector (e.g., AAV vector) comprises one or more splice editor nucleic acids described herein. In some embodiments, the cloning capacity of the viral vector is sufficient to deliver the splice editor nucleic acid.

[0245] In some embodiments, recombinant adeno-associated virus (rAAV) vectors are used for delivery. The technology for producing rAAV particles, in which a cell is supplied with a polynucleotide to be delivered (e.g., a nucleic acid encoding one or more gRNAs and / or a site-specific endonuclease), the rep and cap genes, and a packaged AAV genome containing helper virus functions, is standard in the art. rAAV production typically requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from the rAAV genome (i.e., not within the rAAV), and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which recombinant virus can be obtained and can be derived from an AAV serotype different from the ITRs of the rAAV genome, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, and tropism-altered AAV vectors. The production of pseudotyped rAAV is disclosed, for example, in U.S. Patent No. 7,056,602.

[0246] In some embodiments, methods for producing packaging cells involve creating a cell line that stably expresses all of the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, an AAV rep and cap gene distinct from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line can then be infected with a helper virus, such as adenovirus. The advantages of this method are that the cells are selectable and that it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0247] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Ratschin et al., Mol. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828), U.S. Pat. No. 5,173,414, WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776, WO 95 / 13392, WO 96 / 17947, PCT / US98 / 18600, WO 97 / 09441(PCT / US96 / 14423), WO 97 / 08298(PCT / US96 / 13872), WO 97 / 21825(PCT / US96 / 20777), WO 97 / 06243(PCT / FR96 / 01064), WO 99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595.

[0248] In addition to adeno-associated virus vectors, other viral vectors can be used, including, but not limited to, adenovirus, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein-Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.

[0249] Nanoparticle Composition In some embodiments, a splice editor nucleic acid of the disclosure, or a recombinant expression vector comprising the splice editor nucleic acid, is delivered to a host cell (e.g., ex vivo) or a subject via a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the nucleic acid or expression vector is formulated in a nanoparticle or other delivery vehicle (e.g., a polymeric nanoparticle) to facilitate cellular uptake and / or protect it from degradation upon delivery to a subject.

[0250] In some embodiments, the nanoparticle composition comprises a lipid. Lipid nanoparticles include, but are not limited to, liposomes and micelles. A variety of lipids may be present, including cationic and / or ionic lipids, anionic lipids, neutral lipids, amphipathic lipids, complex lipids (e.g., PEGylated lipids), and / or structured lipids. Such lipids may be used alone or in combination.

[0251] Nanoparticles are typically ultrafine particles ranging in size from 1 to 100-500 nanometers (nm), including the surrounding interfacial layer, and often exhibit size-related or size-specific properties. Nanoparticle compositions vary widely and include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, nanoparticle compositions can be liposomes with lipid bilayers of 500 nm or less in diameter. In some embodiments, nanoparticle compositions are vesicles containing one or more lipid bilayers. In certain embodiments, nanoparticle compositions contain two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can contain one or more ligands, proteins, or channels.

[0252] In some embodiments, the nanoparticle composition comprises a splice editor nucleic acid and / or a recombinant expression vector comprising the splice editor nucleic acid.

[0253] In some embodiments, the disclosure provides LNP compositions comprising (a) a splice editor nucleic acid molecule described herein or an expression vector comprising the splice editor nucleic acid molecule, and (b) one or more lipid moieties selected from the group consisting of an amino lipid, a helper lipid, a structural lipid, a phospholipid, an ionic lipid, a PEG-lipid, a lipid, and cholesterol or a cholesterol derivative. In some embodiments, the disclosure provides LNP compositions comprising (a) a splice editor nucleic acid molecule described herein or an expression vector comprising the splice editor nucleic acid molecule, and (b) one or more lipid moieties selected from the group consisting of an ionic lipid, an amino lipid, an anionic lipid, a neutral lipid, an amphipathic lipid, a helper lipid, a structural lipid, a PEG-lipid, and a lipid, and, optionally, (c) a targeting moiety.

[0254] In some embodiments, the LNP composition comprises one or more lipid moieties that promote or enhance cellular uptake via the apolipoprotein E (apoE)-low density lipoprotein receptor (LDLR) pathway. For example, certain ionic lipids are known in the art to increase cellular uptake of LNPs via the apoE-LDLR pathway (see, e.g., Semple, et al. (2010) NAT BIOTECH 28:172). In some embodiments, the LNP composition comprises one or more lipid moieties that promote or enhance cellular uptake via an apoE-LDLR-independent pathway.

[0255] In some embodiments, the LNPs of the present disclosure are formed by any method known in the art, including, but not limited to, continuous mixing, direct dilution, and in-line dilution processes. Additional techniques and methods suitable for preparing the LNPs described herein include coacervation, microemulsion, supercritical fluid techniques, and phase inversion temperature (PIT) techniques.

[0256] Pharmaceutical Composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising a splice editor nucleic acid, recombinant expression vector, or delivery system described herein in combination with a suitable pharmaceutically acceptable carrier or diluent.

[0257] In some embodiments, the pharmaceutical composition comprises (1) one or more splice editor nucleic acids described herein, and (2) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises (1) an expression vector comprising a splice editor nucleic acid described herein, and (2) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises one or more splice editor nucleic acids, or a recombinant expression vector (e.g., AAV) comprising one or more splice editor nucleic acids formulated as a lipid composition (e.g., LNP), and (2) a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the one or more splice editor nucleic acids or recombinant expression vectors.

[0258] Exemplary pharmaceutically acceptable excipients, such as carriers, solvents, stabilizers, adjuvants, diluents, etc., may be used depending on the particular method of administration and dosage form. Contemplated pharmaceutical compositions generally may be formulated to achieve a physiologically compatible pH depending on the formulation and route of administration. In some embodiments, the composition comprises a therapeutically effective amount of the one or more splice editor nucleic acids or recombinant expression vectors together with one or more pharmaceutically acceptable excipients.

[0259] Suitable excipients can include, for example, carrier molecules, including large, slowly metabolized macromolecules. Other exemplary excipients can include antioxidants, chelating agents, carbohydrates, stearic acid, liquids such as oils, water, saline, glycerol and ethanol, wetting or emulsifying agents, pH buffering substances, etc.

[0260] Pharmaceutical compositions may be formulated into solutions, suppositories, or injectable preparations. In some embodiments, the pharmaceutical compositions are formulated to provide systemic administration of the one or more splice editor nucleic acids or recombinant expression vectors, for example, after enteral or parenteral administration. In some embodiments, the pharmaceutical compositions are formulated to provide localized administration of the one or more splice editor nucleic acids or recombinant expression vectors, for example, after topical administration or implantation. In some embodiments, the pharmaceutical compositions are formulated for immediate activity or sustained release of the one or more splice editor nucleic acids or recombinant expression vectors.

[0261] Typically, an effective amount of a splice editor nucleic acid, recombinant expression vector, or delivery system described herein can be provided for use in, for example, a method of treating a subject having a disease or disorder. Methods for calculating the effective amount or dose are within the skill of one of ordinary skill in the art. The final dose will depend on the route of administration and the nature of the disorder being treated. A competent clinician will be able to determine the effective amount of a splice editor nucleic acid, recombinant expression vector, or delivery system described herein to administer to a patient to halt or reverse the progression of the disorder.

[0262] In some embodiments, based on animal data and other information available about the trans-splicing system, a clinician can determine the maximum safe dose for an individual depending on the route of administration. For example, an intravenous dose may be higher than an intrathecal dose, given the larger volume of fluid into which the therapeutic composition is administered. Similarly, compositions that are rapidly cleared from the body may be administered at higher or repeated doses to maintain therapeutic concentrations. Using routine techniques, a competent clinician will be able to optimize the dosage of a particular therapeutic agent during the course of routine clinical trials.

[0263] For inclusion in pharmaceutical preparations, the splice editor nucleic acids, recombinant expression vectors, or delivery systems described herein can be obtained from a suitable commercial source. In some embodiments, therapies based on the splice editor nucleic acids, recombinant expression vectors, or delivery systems described herein used for therapeutic administration must be sterile. Therapeutic compositions will generally be placed in a container having a sterile access port, for example, an infusion bag or a vial having a stopper pierceable by a hypodermic injection needle. In some embodiments, the therapeutic components are stored in unit-dose or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.

[0264] How to use In some embodiments, the disclosure provides intracellular, ex vivo, and in vivo methods comprising the use of a splice editor nucleic acid, recombinant expression vector, or delivery system described herein to target trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell. In some embodiments, the methods comprise the use of a splice editor nucleic acid, recombinant expression vector, or delivery system described herein to repair a mutation in a target RNA (e.g., a pre-mRNA). In some embodiments, the disclosure provides methods of treating a patient having a disease or disorder, the methods comprising administering a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein to target trans-splicing of a target RNA (e.g., a pre-mRNA) in a target cell population and / or target tissue, thereby treating the disease or disorder.

[0265] Intracellular RNA editing In some embodiments, the method comprises introducing a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein into a cell or population of cells. In some embodiments, the method comprises contacting the cell with a splice editor nucleic acid, expression vector, delivery system, or pharmaceutical composition described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the eukaryotic cell is a rodent cell. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the cell is a cell derived from a patient.

[0266] The splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein can be introduced into the cell via any method known in the art, such as, for example, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, shear-driven cell permeation, fusion to a cell-penetrating peptide followed by cell contact, microinjection, and nanoparticle-mediated delivery. In some embodiments, the vector system can be introduced into the cell via viral infection.

[0267] In some embodiments, the disclosure provides methods for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the method comprising contacting the cell with a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein upon contacting the cell with the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition, one or more binding domains of the splice editor nucleic acid bind to the target RNA (e.g., a pre-mRNA) and trans-splicing results in ligation of one or more exons of the target RNA (e.g., a pre-mRNA) to one or more exons of the splice editor nucleic acid.

[0268] In some embodiments, the disclosure provides methods for targeting trans-splicing of a pre-mRNA in a cell or population of cells that contains a disease-causing mutation, the method comprising contacting the cell or population of cells with a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein upon contacting the cell with the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition, one or more binding domains of the splice editor nucleic acid bind to the pre-mRNA and trans-splicing results in ligation of one or more exons of the pre-mRNA to one or more exons of the splice editor nucleic acid, thereby resulting in an mRNA that lacks the disease-causing mutation.

[0269] In some embodiments, the disclosure provides methods for targeting trans-splicing of a pre-mRNA in a cell or population of cells derived from a patient having a disease or disorder, the method comprising contacting the cell or population of cells with a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein upon contacting the cell or population of cells with the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition, one or more binding domains of the splice editor nucleic acid bind to the target RNA (e.g., pre-mRNA) and trans-splicing results in ligation of one or more exons of the target RNA (e.g., pre-mRNA) to one or more exons of the splice editor nucleic acid, and reintroducing the cell or population of cells into the patient, thereby treating or ameliorating the disease or disorder.

[0270] In vivo RNA editing The present disclosure provides methods for treating a patient having a disease or disorder using a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein. In some embodiments, the disease or disorder is associated with one or more mutations in a target RNA, and the method targets trans-splicing of the target RNA to remove the one or more mutations.

[0271] In some embodiments, the disclosure provides methods of treating a patient having a disease or disorder, the method comprising administering to the patient a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein.

[0272] In some embodiments, the disclosure provides methods of treating a patient having a disease or disorder by targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a target tissue or cell population, the method comprising administering to the patient a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein upon administration of the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition, the splice editor nucleic acid binds to the target RNA (e.g., a pre-mRNA) and trans-splicing results in ligation of one or more exons of the target RNA (e.g., the pre-mRNA) to one or more exons of the splice editor nucleic acid, thereby treating or ameliorating the disease or disorder.

[0273] In some embodiments, the disclosure provides methods of treating a patient having a disease or disorder associated with one or more mutations in a pre-mRNA in a target tissue or cell population, the method comprising administering to the patient a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein upon administration of the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition, the splice editor nucleic acid binds to a pre-mRNA and results in ligation, by trans-splicing, of one or more exons of the pre-mRNA with one or more exons of the splice editor nucleic acid, resulting in an mRNA that lacks the disease-causing mutation, thereby treating or ameliorating the disease or disorder.

[0274] In some embodiments, the route of administration is any route identified by one of skill in the art as sufficient to deliver a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein to the target tissue or cell population.

[0275] In some embodiments, administration of a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein repairs the pre-mRNA mutation in the target tissue or cell population of the patient.

[0276] The term "treatment" refers to the application of one or more of the methods described herein for the amelioration of disease. In some embodiments, the specific procedure is the administration of a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein. "Treatment" of an individual (e.g., a mammal such as a human) or cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, and can be performed either prophylactically or after the initiation of a pathological event or contact with a pathogenic agent. Treatment includes any desired effect on the symptoms or pathology of a disease or condition, and may include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition, e.g., a minimal change or improvement in one or more measurable markers of the disease or condition being treated.

[0277] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal suitable for the methods described herein. In some embodiments, the patient, subject, or individual is a human.

[0278] kit The present disclosure provides kits for performing the methods described herein, in some embodiments, the kits comprise a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein.

[0279] In some embodiments, the kit comprises a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, and reagents for reconstituting and / or diluting the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition.

[0280] In some embodiments, the kit comprises one or more additional reagents selected from buffers, buffers for introducing the splice editor nucleic acid, recombinant expression vector, delivery system into cells, wash buffers, control reagents, control vectors, control polynucleotides, reagents for in vitro production of the recombinant expression vector or delivery system, adaptors for sequencing, etc. The buffers can be stabilization buffers, reconstitution buffers, dilution buffers, etc. The kit can also comprise one or more components that can be used to promote or enhance on-target binding or trans-splicing of the splice editor nucleic acid.

[0281] In addition to the above-mentioned components, the kit may further include instructions for using the components of the kit to practice the method. The instructions for practicing the method may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be included in the kit as a package insert, on the label of the container of the kit or its components (i.e., associated with the package or subpackage), or the like. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not included in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this case is a kit that includes a web address where the instructions can be viewed and / or downloaded. Like the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0282] In some embodiments, the kit comprises a container containing a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, and instructions for use to target trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell or population of cells.

[0283] In some embodiments, the kit comprises a container containing a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, and instructions for administering the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition to a patient in need thereof to target trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell or population of cells of the patient.

[0284] definition As used herein, the term "pre-mRNA" refers to precursor mRNA, which is RNA that contains both exons and intron(s). Pre-mRNA is a type of primary transcript that is processed into messenger RNA. It is synthesized by transcription from a DNA template in the cell nucleus. In some embodiments, the RNA is derived from a mammalian cell. In other embodiments, the RNA is derived from the mitochondria of a mammalian cell.

[0285] As used herein, the term "RNA binding" is used to describe a molecule, protein, nucleic acid, or complex that specifically binds to RNA.

[0286] As used herein, a "disease" is a state of health of an animal in which the animal is unable to maintain homeostasis and, if the disease is not improved, the animal's health continues to deteriorate. In contrast, an animal's "disorder" is a state of health in which the animal is able to maintain homeostasis, but the animal's health is not better than it would be if the disorder did not exist. A disorder, if left untreated, does not necessarily lead to further deterioration of the animal's health. [Example]

[0287] Example 1: ncRNA selection This example describes the method used to identify ncRNAs for inclusion in a splice editor capable of targeting pre-mRNAs and producing trans-splicing events. As shown in Figure 1E, nearly all ncRNAs retrieved from public databases were confirmed to contain the sequence motifs identified in Table 1. Using RNAlib-2.5.1 software, the secondary structures of ncRNA sequences identified in the public domain, including metazoan U1 snRNA, U11 snRNA, U7 snRNA, Sm, and H / ACA snRNA sequences, were predicted. The predicted secondary structures were compared with known secondary structures using an RNA covariance model (see, e.g., Eddy, et al. (1994) Nucleic Acids Research 22:2079-2088). ncRNA sequences with predicted secondary structures similar to known secondary structures were selected for further evaluation. This approach provided over 120,000 candidate ncRNA sequences. As shown in Figure 1F, these candidate ncRNA sequences ranged in length from approximately 7 nucleotides to over 300 nucleotides. Exemplary candidate ncRNA sequences identified by this computational analysis are set forth in SEQ ID NOs: 9-657.

[0288] Example 2: Design and testing of a splice editor for trans-splicing Splice editor nucleic acid molecules were designed for targeted trans-splicing. The nucleic acid molecules comprise (a) a nucleotide sequence comprising (i) at least one binding domain sequence complementary to a target sequence of a pre-mRNA, and (ii) an intron sequence comprising an ncRNA sequence, (b) a splice site, and (c) at least one exon sequence. These ncRNA sequences were selected from candidate ncRNAs identified as described in Example 1. The splice editor nucleic acid molecules were engineered to incorporate all or part of the candidate ncRNA sequence, including secondary structure, and / or sequence motifs identified in Table 1.

[0289] A first set of nucleic acid molecules was designed to contain snRNA-derived ncRNA sequences and undergo trans-splicing with the splice donor of a pre-mRNA (to repair mutations at the 5' end of an exon). These nucleic acid molecules contained, from 5' to 3', nucleotide sequences including: (i) a binding domain sequence, (ii) snRNA sequence (U1 snRNA, U11 snRNA, Sm sequence motif, and U7 snRNA, or Sm sequence motif), (iii) a branch point, and (iv) an intron with a polypyrimidine tract, (b) a splice acceptor, and (c) an exon. Schematics of exemplary U1-based splice editor nucleic acid molecules are shown in Figures 2A, 2B, and 2C, and their sequences are listed in Table 3 (in the column of Table 3, the column titled "Region 2" is labeled "U1_X_Y," where X and Y are integers). Schematics of exemplary U11-based splice editor nucleic acid molecules are shown in Figures 3A, 3B, and 3C, and their sequences are shown in Table 3 (in Table 3, the column entitled "Region 2" is assigned a label of "U11_X_Y," where X and Y are integers). Schematics of exemplary U7-based splice editor nucleic acid molecules are shown in Figures 4A, 4B, and 4C, and their sequences are shown in Table 3 (in Table 3, the column entitled "Region 2" is assigned a label beginning with "Sm_Z," where Z is an integer, and the column entitled "Region 3" is assigned a label beginning with "U7_X," where X is an integer). Schematics of exemplary Sm-based splice editor nucleic acid molecules are shown in Figures 5A, 5B, and 5C, and the sequences are shown in Table 3. (In Table 3, the column titled "Region 2" is assigned a label beginning with "Sm_Z," where Z is an integer, and the column titled "Region 3" is assigned a label beginning with "adeno_intron.")

[0290] A second set of nucleic acid molecules was designed to contain ncRNA sequences derived from H / ACA snoRNAs and undergo trans-splicing at the splice donor of pre-mRNAs (to repair mutations at the 5' end of the exon). These nucleic acid molecules contained, from 5' to 3', the following nucleotide sequences: (i) the first and second binding domains inserted into the snoRNA sequence of the H / ACA box; (ii) a branch point; (iii) an intron with a polypyrimidine tract; (b) a splice acceptor; and (c) an exon. Schematics of exemplary snoRNA-based splice editor nucleic acid molecules are shown in Figures 6A, 6B, and 6C, and their sequences are listed in Table 3 (in the column of Table 3, the column titled "Region 1" is assigned a label beginning with "sno," "SNO," or "SCARNA").

[0291] These nucleic acid molecules are evaluated for trans-splicing using reporter cells, where correct RNA editing generates mRNA that produces a fluorescent protein. The splice editor is introduced into the reporter cells via viral or non-viral methods. Viral methods include, but are not limited to, lentivirus, AAV, and adenovirus. Non-viral methods include, but are not limited to, transfection or electroporation. Cells are first transfected with a splice donor reporter construct encoding a pre-mRNA under the control of a CMV promoter. This pre-mRNA contains blue fluorescent protein (BFP), a self-cleaving p2A linker, a cleavable GFP (5'GFP), and a splice donor. BFP is used to confirm stable expression of the reporter construct. This reporter construct contains intron 1 and exon 2 of matrix metallopeptidase 9 (MMP9) downstream of the splice donor to ensure splicing events occur, followed by a bovine growth hormone polyadenylation signal (bGHpA), which allows stable expression of the construct. These splice editor nucleic acids contain an exon that is the second half of a truncated GFP (3'GFP), and trans-splicing results in expression of the full-length GFP. Reporter cells containing the correct edits generate a signal via the fluorescent reporter and are sorted via FACS. Sorted cells are sequenced to identify active splice editors.

[0292] Example 3: Design and testing of snoRNAs for exon skipping In this experiment, we developed snoRNA guide constructs with modified hybridizing regions that allow the snoRNA to function as a guide for the RNP complex, and tested the snoRNA guide constructs for exon skipping.

[0293] In these experiments, snoRNA guide constructs were designed in a plasmid with sequences complementary to the target sequence to produce snoRNPs that close the splice site, thereby enabling exon skipping. To test for exon skipping, splice acceptor-targeting snoRNA guide molecules were designed in a plasmid (i.e., pA0077, Figure 7A, Table 2). This included intron and exon sites of the MMP9 gene as well as two GFP sites (Figure 7A). The elements were then introduced into another target plasmid (i.e., pA0016), which included a U6 promoter and a polyT terminator (Figure 7A). The design of these constructs and plasmids allowed the snoRNA to bind to the splice site of the MMP9 gene, thereby closing the splice site and resulting in the production of GFP, as shown in the volcano plot in Figure 7B and the graph in Figure 8.

[0294] Four splice acceptor-targeting snoRNA candidates (i.e., "snord45a_11_acceptor," "snord45a_7_acceptor," "aca46_acceptor_29," and "aca44_2_acceptor_35" in Figure 7B, which were obtained from a high-throughput exon skipping screen and whose sequences are shown in Table 4) were synthesized as IDT eBlocks and cloned into the pA0016 vector. This vector was transfected into a HEK293FT cell line stably expressing a PiggyBAC-integrated MMP9 exon skipping reporter (pA0077). This cell line was established by FACS sorting of high-expressing single-cell clones expressing the reporter BFP. 100 ng of the element vector was transfected into HEK293FT cells seeded in 96-well plates using Lipofectamine 2000 so that cells were 90% confluent at the time of transfection. 48 hours after transfection, cells were analyzed by flow cytometry for % GFP compared to non-targeting control (ntc) or pUC19-treated cells. Figure 8 shows GFP production as a result of exon skipping from four different splice acceptor-targeting snoRNAs (i.e., snord is a C / D snoRNA and snora or aca is an H / ACA, labeled "V5_snord45a_11_acceptor," "V4_snord45a_7_acceptor," "V1_aca46_acceptor_29," and "V2_aca44_2_acceptor_35"; sequences are shown in Table 4) compared to four randomized guides (i.e., "V10ntc_random_9," "V11ntc_aca42_1_acceptor_106," "V12ntc_snord51_123_acceptor," and pUC19 (plasmid alone)). The results in Figures 7A, 7B, 8 and 9 demonstrate that the snoRNAs disclosed herein induce exon skipping. [Table 2-1] [Table 2-2]

[0295] Example 4: Design and testing of U7 snRNA for trans-splicing In this experiment, a U7 snRNA guide construct was developed and tested for trans-splicing. The U7 snRNA guide construct was designed in a plasmid with a sequence complementary to the target sequence to produce a U7 snRNA construct that closes the splice site, thereby enabling trans-splicing. To test for trans-splicing, a U7 snRNA guide was designed in a plasmid (i.e., pA0120, Figure 10, Table 2), which also included intron and exon regions of the USH2A gene and two GFP sites (Figure 10).

[0296] The elements were then introduced into another plasmid target (i.e., pA0177), which included a pCMV promoter and a bGHpA promoter (Figure 10), and contained an additional GFP site. In these experiments, trans-splicing is monitored by the expression of GFP. This is because GFP

[0297] The elements were then introduced into another plasmid target (i.e., pA0177), which was included after the pCMV promoter and before bGHpA (Figure 10), and contained an additional GFP site. In these experiments, trans-splicing was monitored by GFP expression. This is because the GFP site in the plasmid target (i.e., pA0177) replaced and restored the GFP site in the pA0120 plasmid, thereby demonstrating trans-splicing by GFP expression. Figure 11 is a graph showing the binding of U7 snRNA guide constructs to different targeting elements and the effect of trans-splicing. Figure 12 is a graph showing independent validation of the results from Figure 11. The first four bars from the left represent U7 snRNA constructs with targeting elements specific for trans-splicing, while the remaining bars in the graph represent U7 snRNA constructs with non-targeting elements for trans-splicing. These experiments demonstrate, inter alia, that trans-splicing is enhanced by targeting the vicinity of the splice donor. Figure 13 is a graph showing U7 snRNA-targeted piggybac-integrated USH2A (773, hybridizing region and hairpin intact) compared to non-targeted guides (774 and 776) and a mutant U7 snRNA hairpin (775, hybridizing region intact, but hairpin region mutated). These experiments further demonstrate, inter alia, that targeting a complementary region with a U7 snRNA construct having both a hairpin and a hybridizing region leads to significantly higher trans-splicing compared to the non-targeted control. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3] Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 Table 4-25 Table 4-26 Table 4-27 Table 4-28 Table 4-29 Table 4-30 Table 4-31 Table 4-32 Table 4-33 Table 4-34 Table 4-35 Table 4-36 Table 4-37 Table 4-38 Table 4-39 Table 4-40 Table 4-41 Table 4-42

Claims

1. 1. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, comprising: (a) at least one intron sequence comprising: (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a pre-mRNA target sequence; and (ii) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or contains a sequence motif to direct the one or more binding domains to the pre-mRNA target sequence; (b) a splice acceptor sequence and / or a splice donor sequence, and (c) at least one exon sequence The nucleic acid comprising a nucleotide sequence comprising:

2. 2. The nucleic acid of claim 1, wherein the one or more binding domain sequences are at least about 5 to about 10, about 5 to about 15, about 5 to about 20, about 10 to about 15, about 10 to about 20, about 15 to about 20, or about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length.

3. 3. The nucleic acid of claim 1, wherein the one or more binding domain sequences are about 250 to about 300, about 200 to about 300, about 150 to about 300, about 100 to about 300, about 50 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 50 to about 250, about 50 to about 200, about 50 to about 150, about 50 to about 100, or less than about 300, 250, 200, 150, 100, or 50 nucleotides in length.

4. 4. The nucleic acid of any one of claims 1 to 3, wherein the one or more binding domain sequences are about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

5. The nucleic acid according to any one of claims 1 to 4, comprising one binding domain sequence.

6. The nucleic acid according to any one of claims 1 to 4, comprising at least two binding domain sequences.

7. The nucleic acid of claim 6, comprising 3, 4, 5, 6, 7, 8, 9, or 10 binding domain sequences.

8. The nucleic acid of any one of claims 1 to 7, wherein an exon of the pre-mRNA is targeted for trans-splicing when the nucleic acid is introduced into the cell.

9. 9. The nucleic acid of claim 8, wherein the target sequence is located within a region of the pre-mRNA that contains the exon targeted for trans-splicing.

10. The nucleic acid of claim 9 , wherein the target sequence is located proximal to a splice site.

11. 11. The nucleic acid of claim 9 or 10, wherein the target sequence is located proximal to a splice donor or splice acceptor.

12. 12. The nucleic acid of any one of claims 1 to 11, wherein the ncRNA sequence is selected from snRNA, snoRNA, lncRNA, rRNA, ribozyme, sRNA, scaRNA, and vault RNA.

13. The nucleic acid of claim 12 , wherein the ncRNA sequence is snRNA.

14. 14. The nucleic acid of claim 13, wherein the snRNA is selected from U7 snRNA, U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, and U12 snRNA.

15. The nucleic acid of claim 12, wherein the ncRNA sequence is a snoRNA.

16. The nucleic acid of claim 15, wherein the snoRNA comprises an H / ACA box or a C / D box.

17. The nucleic acid of any one of claims 1 to 16, wherein the ncRNA sequence is assembled into an RNP.

18. The nucleic acid of any one of claims 1 to 17, wherein the ncRNA sequence comprises a sequence motif that assembles into an RNP.

19. The nucleic acid of any one of claims 1 to 17, wherein the ncRNA sequence comprises a secondary structure that assembles into an RNP.

20. The nucleic acid of any one of claims 1 to 17, wherein the ncRNA sequence comprises sequence motifs and secondary structures that assemble into RNPs.

21. 21. The nucleic acid of claim 19 or 20, wherein the secondary structure comprises one or more stem loops.

22. 22. The nucleic acid of any one of claims 17 to 21, wherein the RNP is selected from small nuclear RNPs (snRNPs), small nucleolar RNPs (snoRNPs), small Cajal body RNPs (scaRNPs), and combinations thereof.

23. 23. The nucleic acid of claim 22, wherein the RNP is selected from U1, U2, U4, U4atac, U5, U6, U6atac, U7, U11, and U12.

24. 23. The nucleic acid of claim 22, wherein the RNP is selected from a C / D box snoRNP and an H / ACA box snoRNP.

25. The nucleic acid of any one of claims 1 to 24, wherein the ncRNA comprises an Sm sequence motif.

26. 26. The nucleic acid of claim 25, wherein the Sm sequence motif is assembled into an RNP with an Sm or Lsm protein.

27. 26. The nucleic acid of claim 25, wherein the Sm or Lsm protein is selected from B / B', D3, D2, D1, E, F, G, LSm5, LSm7, LSm4, LSm8, LSm2, LSm3, LSm6, and LSm10 proteins.

28. The nucleic acid according to any one of claims 1 to 27, comprising a splice acceptor.

29. The nucleic acid according to any one of claims 1 to 27, comprising a splice donor.

30. 30. The nucleic acid of any one of claims 1 to 29, wherein the at least one intron sequence comprises one or more splicing signals.

31. 31. The nucleic acid of claim 30, wherein the one or more splicing signals are selected from an exonic splicing enhancer (ESE), an intronic splicing enhancer (ISE), an exonic splicing silencer (ESS), an intronic splicing silencer (ISS), a polypyrimidine tract, a branch point, and combinations thereof.

32. 32. The nucleic acid of any one of claims 1 to 31, wherein the at least one intron sequence comprises a branch point and a polypyrimidine tract.

33. 33. The nucleic acid of any one of claims 1 to 32, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 exons.

34. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, comprising, 5' to 3': (a) (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a pre-mRNA target sequence; (ii) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length, which forms a secondary structure and / or contains a sequence motif for directing the one or more binding domains to the pre-mRNA target sequence; and (iii) at least one intron sequence containing one or more splicing signals; (b) a splice acceptor, and (c) at least one exon sequence The nucleic acid comprising a nucleotide sequence comprising:

35. 35. The nucleic acid of claim 34, wherein an exon of the pre-mRNA is targeted for trans-splicing when the nucleic acid is introduced into the cell.

36. 36. The nucleic acid of claim 34 or 35, wherein the target sequence is located upstream of the exon of the pre-mRNA targeted for trans-splicing.

37. 37. The nucleic acid of claim 36, wherein the target sequence is located proximal to a splice site.

38. 38. The nucleic acid of claim 36 or 37, wherein the target sequence is located proximal to a splice acceptor or splice donor.

39. 39. The nucleic acid of any one of claims 35 to 38, wherein trans-splicing occurs between a splice donor upstream of the exon of the pre-mRNA and the splice acceptor of the nucleic acid.

40. 40. The nucleic acid of any one of claims 35 to 39, wherein trans-splicing results in ligation of the 3' end of the exon upstream of the splice donor of the pre-mRNA to the 5' end of the at least one exon sequence of the nucleic acid.

41. 41. The nucleic acid of any one of claims 34 to 40, wherein the one or more splicing signals comprise a branch point and a polypyrimidine tract.

42. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, comprising, 5' to 3': (a) at least one exon sequence; (b) a splice donor; (c) (i) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length, and (ii) at least one intron sequence comprising one or more binding domain sequences of about 4 to about 300 nucleotides, each having complementarity to a pre-mRNA target sequence. a nucleotide sequence comprising The nucleic acid, wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to direct the one or more binding domains to the pre-mRNA target sequence.

43. 43. The nucleic acid of claim 42, wherein the exon of the pre-mRNA is targeted for trans-splicing when the nucleic acid is introduced into the cell.

44. 44. The nucleic acid of claim 43, wherein the target sequence is located downstream of the exon of the pre-mRNA.

45. 45. The nucleic acid of claim 44, wherein the target sequence is located proximal to a splice site.

46. 46. ​​The nucleic acid of claim 44 or 45, wherein the target sequence is located proximal to a splice donor or splice acceptor.

47. 47. The nucleic acid of any one of claims 43 to 46, wherein trans-splicing occurs between the splice donor of the nucleic acid and a splice acceptor downstream of the exon of the pre-mRNA.

48. 48. The nucleic acid of any one of claims 43 to 47, wherein trans-splicing results in ligation of the 3' end of the at least one exon sequence of the nucleic acid to the 5' end of the exon downstream of the splice acceptor of the pre-mRNA.

49. The nucleic acid of any one of claims 34 to 48, wherein the ncRNA sequence is snRNA.

50. 50. The nucleic acid of claim 49, wherein the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atac snRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

51. 51. The nucleic acid of claim 49 or 50, wherein the snRNA is assembled into a snRNP.

52. 51. The nucleic acid of claim 49 or 50, wherein the snRNA is U1 snRNA.

53. 53. The nucleic acid of claim 52, wherein the U1 snRNA is assembled into a U1 RNP.

54. 51. The nucleic acid of claim 49 or 50, wherein the snRNA is U11 snRNA.

55. 55. The nucleic acid of claim 54, wherein the U11 snRNA is assembled into a U11 RNP.

56. 51. The nucleic acid of claim 49 or 50, wherein the snRNA is U7 snRNA.

57. 57. The nucleic acid of claim 56, wherein the U7 snRNA is assembled into a U7 RNP.

58. 51. The nucleic acid of claim 49 or 50, wherein the ncRNA sequence comprises an Sm sequence motif.

59. 59. The nucleic acid of claim 58, wherein the ncRNA sequence comprises an Sm sequence motif and a U7 snRNA.

60. 60. The nucleic acid of claim 58 or 59, wherein the Sm sequence motif comprises the sequence set forth in SEQ ID NOs: 3 and 4.

61. 61. The nucleic acid of any one of claims 58 to 60, wherein the Sm sequence motif is assembled into an RNP with an Sm protein.

62. 62. The nucleic acid of claim 61, wherein the Sm protein is selected from B / B', D3, D2, D1, E, F, and G Sm proteins.

63. 49. The nucleic acid of any one of claims 34 to 48, wherein the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 9 to 589 or a portion thereof.

64. 64. The nucleic acid of claim 63, wherein the ncRNA sequence comprises a region of about 7 to about 40 nucleotides in length, said region comprising an Sm sequence motif.

65. 64. The nucleic acid of claim 63, wherein the ncRNA sequence comprises a region of about 40 to about 300 nucleotides in length, the region comprising a secondary structure and / or an Sm sequence motif.

66. 66. The nucleic acid of any one of claims 34 to 65, comprising one binding domain sequence.

67. 66. The nucleic acid of any one of claims 34 to 65, comprising multiple binding domain sequences.

68. 66. The nucleic acid of any one of claims 34-65, wherein the one or more binding domain sequences are about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 10 to about 50, about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 50 to about 150, about 50 to about 200, about 50 to about 250, about 100 to about 150, about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides in length.

69. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, comprising, 5' to 3': (a) (i) an ncRNA sequence comprising an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides, each having complementarity to a pre-mRNA target sequence, and (ii) at least one intron sequence comprising one or more splicing signals; (b) a splice acceptor, and (c) at least one exon sequence The nucleic acid comprising a nucleotide sequence comprising:

70. 70. The nucleic acid of claim 69, wherein the exon of the pre-mRNA is targeted for trans-splicing when the nucleic acid is introduced into the cell.

71. 71. The nucleic acid of claim 69 or 70, wherein the target sequence is located upstream of the exon of the pre-mRNA.

72. 72. The nucleic acid of claim 71, wherein the target sequence is located proximal to a splice site.

73. 73. The nucleic acid of claim 71 or 72, wherein the target sequence is located proximal to a splice donor or splice acceptor.

74. 74. The nucleic acid of any one of claims 70 to 73, wherein trans-splicing occurs between a splice donor upstream of the exon of the pre-mRNA and the splice acceptor of the nucleic acid.

75. 74. The nucleic acid of any one of claims 70 to 73, wherein trans-splicing results in ligation of the 3' end of the exon upstream of the splice donor of the pre-mRNA to the 5' end of the at least one exon sequence of the nucleic acid.

76. 76. The nucleic acid of any one of claims 69 to 75, wherein the one or more splicing signals comprise a branch point and a polypyrimidine tract.

77. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, comprising, 5' to 3': (a) at least one exon sequence; (b) a splice donor, and (c) at least one intron sequence, including an ncRNA sequence including an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides, each having complementarity to a pre-mRNA target sequence; The nucleic acid comprising a nucleotide sequence comprising:

78. 78. The nucleic acid of claim 77, wherein an exon of the pre-mRNA is targeted for trans-splicing when the nucleic acid is introduced into the cell.

79. 79. The nucleic acid of claim 78, wherein the target sequence is located downstream of the exon of the pre-mRNA.

80. 80. The nucleic acid of claim 79, wherein the target sequence is located proximal to a splice site.

81. 81. The nucleic acid of claim 79 or 80, wherein the target sequence is located proximal to a splice donor or splice acceptor.

82. 82. The nucleic acid of any one of claims 78 to 81, wherein trans-splicing occurs between the splice donor of the nucleic acid and a splice acceptor downstream of the exon of the pre-mRNA.

83. 82. The nucleic acid of any one of claims 78 to 81, wherein trans-splicing results in ligation of the 3' end of the at least one exon sequence of the nucleic acid to the 5' end of the exon downstream of the splice acceptor of the pre-mRNA.

84. 84. The nucleic acid of any one of claims 69 to 83, wherein the ncRNA sequence comprises, from 5' to 3', an H / ACA box comprising an H consensus sequence and an ACA consensus sequence.

85. 85. The nucleic acid of claim 84, (i) upstream of the H consensus sequence; (ii) downstream of the ACA consensus sequence; (iii) between the H consensus sequence and the ACA consensus sequence; or (iv) A combination of (i) to (iii) The nucleic acid comprising at least one binding domain sequence located at

86. 84. The nucleic acid of any one of claims 69 to 83, wherein the ncRNA sequence comprises a C / D box comprising, from 5' to 3', a C consensus sequence, a D' consensus sequence, a C' consensus sequence, and a D consensus sequence.

87. 87. The nucleic acid of claim 86, (i) upstream of the C consensus sequence; (ii) between the C consensus sequence and the D′ consensus sequence; (iii) between the C' consensus sequence and the D consensus sequence; (iv) downstream of the D consensus sequence; or (iv) A combination of (i) to (iii) The nucleic acid comprising at least one binding domain located at

88. 84. The nucleic acid of any one of claims 78 to 83, wherein the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 590 to 657, or a portion thereof.

89. 89. The nucleic acid of claim 88, wherein the ncRNA sequence comprises a region of about 40 to about 300 nucleotides in length that includes the H consensus sequence and the ACA consensus sequence.

90. The nucleic acid of any one of claims 69 to 89, wherein the ncRNA sequence comprises one binding domain sequence.

91. 90. The nucleic acid of any one of claims 69 to 89, wherein the ncRNA sequence comprises multiple binding domain sequences.

92. 92. The nucleic acid of any one of claims 1 to 91, comprising at least one binding domain sequence having perfect complementarity to the pre-mRNA target sequence.

93. 93. The nucleic acid of any one of claims 1 to 92, comprising at least one binding domain sequence having partial complementarity to the pre-mRNA target sequence.

94. 94. The nucleic acid of Claim 93, wherein said at least one binding domain sequence comprises one or more mismatches to said pre-mRNA target sequence.

95. 95. The nucleic acid of claim 93 or 94, wherein the at least one binding domain sequence has at least 95% complementarity to the pre-mRNA target sequence.

96. 96. The nucleic acid of any one of claims 1 to 95, comprising a sequence of up to about 20,000 nucleotides in length.

97. 97. The nucleic acid of any one of claims 1 to 96, comprising a sequence of about 50 to about 500, about 50 to about 1000, about 100 to about 500, about 100 to about 1000, about 500 to about 1000, about 500 to about 2000, about 500 to about 3,000, about 500 to about 4,000, about 500 to about 5,000, about 1,000 to about 5,000, about 1,000 to about 10,000, about 5,000 to about 15,000, or about 5,000 to about 20,000 nucleotides in length.

98. 98. The nucleic acid of any one of claims 1 to 97, which is introduced into the cell as RNA.

99. 98. The nucleic acid of any one of claims 1 to 97, which is introduced into the cell as DNA.

100. 100. The nucleic acid of any one of claims 1 to 99, which is introduced into the cell by a viral vector.

101. The nucleic acid of claim 100, wherein the viral vector is AAV.

102. The nucleic acid of any one of claims 1 to 100, which is introduced into the cell by a non-viral vector.

103. 103. The nucleic acid of any one of claims 1 to 102, wherein introduction of said nucleic acid into said cell results in more efficient trans-splicing than a nucleic acid lacking said ncRNA sequence.

104. 104. The nucleic acid of claim 103, wherein the efficiency of trans-splicing is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%.

105. The nucleic acid of any one of claims 1 to 104, formulated as a lipid nanoparticle.

106. A viral vector comprising the nucleic acid of any one of claims 1 to 104.

107. A lipid nanoparticle comprising the nucleic acid according to any one of claims 1 to 105.

108. A cell comprising a nucleic acid according to any one of claims 1 to 105, a viral vector according to claim 106, or a lipid nanoparticle according to claim 107.

109. A pharmaceutical composition comprising the nucleic acid of any one of claims 1 to 105, the viral vector of claim 106, the lipid nanoparticle of claim 107, and a pharmaceutically acceptable carrier.

110. 109. A pharmaceutical composition comprising the cells of claim 108 and a pharmaceutically acceptable carrier.

111. 110. A method for targeting trans-splicing of a pre-mRNA in a cell, comprising contacting the cell with a nucleic acid according to any one of claims 1 to 105, a viral vector according to claim 106, a lipid nanoparticle according to claim 107, or a pharmaceutical composition according to claims 109 to 110, wherein upon contact of the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition with the cell, the one or more binding domain sequences bind to the pre-mRNA, thereby targeting the pre-mRNA for trans-splicing.

112. 110. A method for repairing a mutation in a pre-mRNA in a cell, comprising contacting the cell with a nucleic acid described in any one of claims 1 to 105, a viral vector described in claim 106, a lipid nanoparticle described in claim 107, or a pharmaceutical composition described in claims 109 to 110, wherein upon contacting the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition with the cell, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and one or more exons of the pre-mRNA containing the mutation are replaced by trans-splicing, thereby repairing the mutation.

113. 110. A method for treating a patient having a disease or disorder associated with a mutation in a pre-mRNA, comprising administering to the patient an effective amount of a nucleic acid according to any one of claims 1 to 105, a viral vector according to claim 106, a lipid nanoparticle according to claim 107, or a pharmaceutical composition according to claims 109 to 110, wherein upon administration of the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and one or more exons of the pre-mRNA containing the mutation are replaced by trans-splicing, thereby repairing the mutation.

114. 114. The method of claim 113, wherein said trans-splicing results in an mRNA that alleviates said disease or that does not cause or contribute to said disease.

115. 110. The nucleic acid of any one of claims 1 to 105, the viral vector of claim 106, the lipid nanoparticle of claim 107, or the pharmaceutical composition of claims 109 to 110, for use in treating a patient having a disease or disorder associated with a mutation in a pre-mRNA, wherein the treatment comprises administering the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition to the patient, wherein upon administration of the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation and replace one or more exons of the pre-mRNA containing the mutation by trans-splicing, thereby repairing the mutation.

116. 110. The nucleic acid of any one of claims 1 to 105, the viral vector of claim 106, the lipid nanoparticle of claim 107, or the pharmaceutical composition of claims 109 to 110, for the manufacture of a medicament for use in treating a patient having a disease or disorder associated with a mutation in a pre-mRNA, wherein the treatment comprises administering the medicament to the patient, wherein upon administration of the medicament, the one or more binding domain sequences of the nucleic acid bind to the pre-mRNA at a position proximal to the mutation, and one or more exons of the pre-mRNA containing the mutation are replaced by trans-splicing, thereby repairing the mutation.

117. A kit comprising a container containing the nucleic acid of any one of claims 1 to 105, the viral vector of claim 106, the lipid nanoparticle of claim 107, or the pharmaceutical composition of claims 109 to 110, together with instructions for use in repairing a mutation in pre-mRNA.

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