Methods and compositions for targeting trans splicing

Abstract

The present disclosure provides compositions for targeting trans-splicing of an mRNA precursor in a cell and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,031, filed on November 30, 2022, the entire contents of which are incorporated herein. Technical Field

[0003] The present disclosure relates to nucleic acid compositions and related methods for targeting trans-splicing of mRNA precursors in cells.

[0004] Submit sequence listing via EFS-WEB

[0005] This application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy, entitled "AMR-014PC / 134241-5014_SequenceListing," were created on November 30, 2023 and are 513,000 bytes in size, the contents of which are incorporated herein by reference in their entirety. Background Art

[0006] Gene editing is widely considered a promising approach for treating a variety of diseases associated with viral infections, enzyme deficiencies, and inherited myopathies. For example, gene editing using the CRISPR / Cas system can introduce double-strand breaks in the gene of interest, which can be repaired by endogenous DNA repair pathways, thereby introducing gene knockout or mutation correction. Appropriate gene editing can 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 methods remain problematic due to the risk of introducing deleterious off-target edits to the genome and packaging limitations for the delivery of system components. An alternative approach to introducing genetic information into cells that avoids the risk of introducing permanent changes to the genome is through regulating the splicing of endogenous nucleic acids (e.g., RNA transcripts).

[0007] Splicing is a reaction that occurs in the nucleus of eukaryotic cells and is catalyzed by a spliceosome (a large ribonucleoprotein (RNP) complex). Splicing removes non-coding sequences (introns) from RNA transcripts (mRNA precursors) and connects coding sequences (exons) together. Spliceosomes typically mediate the cis-splicing of endogenous RNA transcripts, where a loop is formed in the intron, which is then excised to join two exons in 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). Spliceosomes can also perform trans-splicing, where exons from two different primary RNA transcripts are joined end-to-end and connected (Lasda et al. (2011) Wiley Interdiscip Rev RNA 2: 417-34). Trans-splicing produces a chimeric molecule comprising one or more exon regions from a first RNA molecule and one or more exon regions from a second RNA molecule.

[0008] Trans-splicing using exogenous nucleic acids encoding the required genetic information is a promising approach for therapeutic nucleic acid editing and other biotechnology applications. For example, it has been shown that artificial RNA introduced into cells can trans-splicing with endogenous mRNA precursors (see, for example, Puttaraju et al. (1999) Nat Biotech 17: 246). Such trans-splicing work focuses 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 with target mRNA precursors (Puttaraju, 1999). While some research groups have been able to demonstrate in vitro and in vivo activity of SMaRT technology (Mansfield et al. (2000) Genet Apy 7:1885-1895; Liu et al. (2002) Nat. Biotechnol. 20:47), it is a relatively inefficient process and has not yet entered the clinic (Berger et al. (2016) Wiley Interdisciplinary Reviews: RNA 7:487-98).

[0009] Therefore, new approaches are needed to achieve targeted and efficient trans-splicing to introduce the desired genetic information into cells. Summary of the Invention

[0010] 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 nucleotide sequence comprising

[0011] (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary 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 comprises a sequence motif to guide the one or more binding domains to the pre-mRNA target sequence; (b) a splice acceptor and / or splice donor sequence; and (c) at least one exon sequence.

[0012] In some embodiments, the length of one or more binding domain sequences is 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 some embodiments, the length of one or more binding domain sequences is less than 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 some embodiments, the length of one or more binding domain sequences 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 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.

[0013] 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.

[0014] In some embodiments, when the nucleic acid is introduced into the cell, exons in the mRNA precursor are targeted for trans-splicing. In some embodiments, the target sequence is located in a region of the mRNA precursor that contains exons that are targeted for trans-splicing. 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 a splice acceptor.

[0015] 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 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 snoRNA. In some embodiments, the snoRNA comprises an H / ACA box or a C / D box.

[0016] 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 small nuclear RNP (snRNP), small nucleolar RNP (snoRNP), small Cajal body 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 C / D box snoRNP and H / ACA box snoRNP.

[0017] In some embodiments, the ncRNA comprises an Sm sequence motif. In some embodiments, the Sm sequence motif assembles into an RNP with an Sm or Lsm protein. In some embodiments, the Sm or Lsm protein is selected from the group consisting of B / B', D3, D2, D1, E, F, G, LSm5, LSm7, LSm4, LSm8, LSm2, LSm3, LSm6, and LSm10 proteins.

[0018] 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, one or more splicing signals are selected from exon splicing enhancers (ESEs), intron splicing enhancers (ISEs), exon splicing silencers (ESSs), intron splicing silencers (ISSs), polypyrimidine segments, branch points, and combinations thereof. In some embodiments, the at least one intron sequence comprises a branch point and a polypyrimidine segment. In some embodiments, the nucleic acid comprising the nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 exons.

[0019] 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 nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence, the at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a pre-mRNA target sequence; (ii) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or comprises a sequence motif to guide one or more binding domains to the pre-mRNA target sequence; and (iii) one or more splicing signals; (b) a splicing acceptor; and (c) at least one exon sequence. In some embodiments, when the nucleic acid is introduced into the cell, the exons in the pre-mRNA are targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the exons in the pre-mRNA that are targeted for trans-splicing. In some embodiments, the target sequence is located proximal to a splicing site (e.g., a splicing acceptor or a splicing donor). In some embodiments, the target sequence is located proximal to the splice acceptor. In some embodiments, the target sequence is located proximal to the splice donor. In some embodiments, trans-splicing occurs between the splice donor upstream of the exon in the mRNA precursor and the splice acceptor of the nucleic acid. In some embodiments, trans-splicing results in the 3' end of the exon upstream of the splice donor in the mRNA precursor being connected 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.

[0020] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of an mRNA precursor in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′: (a) at least one exon sequence; (b) a splicing donor; (c) at least one intron sequence, the at least one intron sequence comprising (i) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to the mRNA precursor target sequence, wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domains to the mRNA precursor target sequence. In some embodiments, when the nucleic acid is introduced into the cell, the exons in the mRNA precursor are targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the exons in the mRNA precursor. In some embodiments, the target sequence is located proximal to the splicing site (e.g., splicing donor or splicing acceptor). In some embodiments, the target sequence is located proximal to the splicing donor. In some embodiments, the target sequence is located proximal to a splice acceptor. In some embodiments, trans-splicing occurs between a splice donor of the nucleic acid and a splice acceptor downstream of an exon in the pre-mRNA. In some embodiments, trans-splicing results in the 3′ end of the at least one exon sequence of the nucleic acid being joined to the 5′ end of an exon downstream of a splice acceptor in the pre-mRNA.

[0021] 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, U12snRNA, and U7 snRNA. In some embodiments, the snRNA assembles into snRNPs. In some embodiments, the snRNA is U1snRNA. In some embodiments, U1 snRNA assembles into U1 RNPs. In some embodiments, the snRNA is U11snRNA. In some embodiments, U11 snRNA assembles into U11 RNPs. In some embodiments, the snRNA is U7snRNA. In some embodiments, U7 snRNA assembles into U7 RNPs. 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 shown in SEQ ID NOs: 3 and 4. In some embodiments, the Sm sequence motif assembles with the Sm protein into an RNP. In some embodiments, the Sm protein is selected from the group consisting of B / B', D3, D2, Dl, E, F, and G Sm proteins.

[0022] In some embodiments, the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 9-589 or a portion thereof (e.g., a continuous portion thereof). In some embodiments, the ncRNA sequence comprises a region of about 7 to about 40 nucleotides in length, wherein the region comprises an Sm sequence motif. In some embodiments, the ncRNA sequence comprises a region of about 40 to about 300 nucleotides in length, wherein the region comprises a secondary structure and / or an Sm sequence motif. In some embodiments, the Sm sequence motif comprises a sequence selected from SEQ ID NO: 209-399.

[0023] In some embodiments, the at least one intron sequence comprises a binding domain sequence. In some embodiments, the one binding domain sequence 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.

[0024] In some embodiments, the at least one intron sequence comprises more than one binding domain sequence. In some embodiments, the more than one binding domain sequence is each 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.

[0025] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of an mRNA precursor in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence, the at least one intron 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 binding domain sequence being complementary to the mRNA precursor target sequence; and (ii) one or more splicing signals; (b) a splicing acceptor; and (c) at least one exon sequence. In some embodiments, when the nucleic acid is introduced into the cell, the exon in the mRNA precursor is targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the exon in the mRNA precursor. In some embodiments, the target sequence is located proximal to the splice site. In some embodiments, the target sequence is located proximal to the splice donor or splice acceptor. In some embodiments, trans-splicing occurs between the splice donor upstream of the exon in the mRNA precursor and the splice acceptor of the nucleic acid. In some embodiments, trans-splicing results in the 3' end of an exon upstream of the splice donor in the pre-mRNA being joined to the 5' end of at least one exonic sequence of the nucleic acid. In some embodiments, the one or more splicing signals comprise a branch point and a polypyrimidine tract.

[0026] In some aspects, the present disclosure provides a nucleic acid for targeting trans-splicing of an mRNA precursor in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splicing donor; and (c) at least one intron sequence, the at least one intron sequence comprising an ncRNA sequence containing an H / ACA box or a C / D box and one or more binding domain sequences of about 4 to about 30 nucleotides, each binding domain sequence being complementary to the mRNA precursor target sequence. In some embodiments, when the nucleic acid is introduced into the cell, the exon in the mRNA precursor is targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the exon in the mRNA precursor. In some embodiments, the target sequence is located proximal to the splicing site. In some embodiments, the target sequence is located proximal to the splicing donor or splicing acceptor. In some embodiments, trans-splicing occurs between the splicing donor of the nucleic acid and the splicing acceptor downstream of the exon in the mRNA precursor. In some embodiments, trans-splicing results in the 3' end of the at least one exon sequence of the nucleic acid being joined to the 5' end of an exon downstream of the splice acceptor in the pre-mRNA.

[0027] In some embodiments, the ncRNA sequence comprises an H / ACA box comprising an H consensus sequence and an ACA consensus sequence from 5' to 3'. In some embodiments, the ncRNA sequence comprises at least one binding domain sequence located at: (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).

[0028] 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).

[0029] In some embodiments, the ncRNA sequence comprises a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 590-657, or a portion thereof (e.g., a contiguous portion thereof). In some embodiments, the ncRNA sequence comprises a region that is about 40 to about 300 nucleotides in length and comprises an H consensus sequence and an ACA consensus sequence.

[0030] In some embodiments, the ncRNA sequence comprises one binding domain sequence. In some embodiments, the ncRNA sequence comprises more than one binding domain sequence.

[0031] 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 relative to the pre-mRNA target sequence. In some embodiments, the at least one binding domain sequence has at least 95% complementarity with the pre-mRNA target sequence.

[0032] In some embodiments, the nucleic acid comprises a sequence of up to about 20,000 nucleotides in length. In some embodiments, the nucleic acid comprises 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.

[0033] 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 via a viral vector. In some embodiments, the viral vector is AAV. In some embodiments, the nucleic acid is introduced into the cell via a non-viral vector.

[0034] In some embodiments, the efficiency of trans-splicing achieved by introducing the nucleic acid into a cell is greater than that achieved by introducing the nucleic acid into a cell lacking the ncRNA sequence. In some embodiments, the efficiency of trans-splicing is greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%.

[0035] In some embodiments, the nucleic acid is formulated as a lipid nanoparticle.

[0036] In some aspects, the disclosure provides viral vectors comprising the nucleic acids described herein.

[0037] In some aspects, the present disclosure provides lipid nanoparticles comprising a nucleic acid described herein.

[0038] In some aspects, the disclosure provides cells comprising a nucleic acid described herein, a viral vector described herein, or a lipid nanoparticle described herein.

[0039] In some aspects, the present disclosure provides pharmaceutical compositions comprising a nucleic acid described herein, a viral vector described herein, or a lipid nanoparticle described herein and a pharmaceutically acceptable carrier.

[0040] In some aspects, the present disclosure provides pharmaceutical compositions comprising the cells described herein and a pharmaceutically acceptable carrier.

[0041] In some aspects, the present disclosure provides a method 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 when the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition contacts the cell, one or more binding domain sequences bind to the pre-mRNA, thereby targeting the pre-mRNA for trans-splicing.

[0042] In some aspects, the present disclosure provides a method for correcting 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 when the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition contacts the cell, one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA comprising the mutation, thereby correcting the mutation.

[0043] In some aspects, the present disclosure provides a method for treating a patient suffering from a disease or condition associated with a mutation in a pre-mRNA, the method comprising administering to the patient an effective amount of a nucleic acid as described herein, a viral vector as described herein, a lipid nanoparticle as described herein, or a pharmaceutical composition as described herein, wherein upon administration of the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition, one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA containing the mutation, thereby correcting the mutation. In some embodiments, trans-splicing produces an mRNA that alleviates the disease or does not cause or contribute to the disease.

[0044] In some aspects, the present disclosure provides a nucleic acid of any embodiment disclosed herein, a viral vector of any embodiment disclosed herein, a lipid nanoparticle of any embodiment disclosed herein, or a pharmaceutical composition of any embodiment disclosed herein for use in treating a patient suffering from a disease or condition associated with a mutation in a pre-mRNA, the treatment comprising administering to the patient the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition, wherein when the nucleic acid, viral vector, lipid nanoparticle, or pharmaceutical composition is administered, one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA containing the mutation, thereby correcting the mutation.

[0045] In some aspects, the present disclosure provides a nucleic acid of any embodiment disclosed herein, a viral vector of any embodiment disclosed herein, a lipid nanoparticle of any embodiment disclosed herein, or a pharmaceutical composition of any embodiment disclosed herein, for use in the manufacture of a medicament for treating a patient suffering from a disease or condition associated with a mutation in a pre-mRNA, the treatment comprising administering the agent to the patient, wherein when the agent is administered, one or more binding domain sequences of the nucleic acid bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA containing the mutation, thereby correcting the mutation.

[0046] In some aspects, the disclosure provides kits comprising a container comprising a nucleic acid described herein, a viral vector described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein, and instructions for correcting a mutation in an mRNA precursor. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] I don't want to be bound by theory. Figure 1A and Figure 1B A schematic diagram is provided depicting cis-splicing of pre-mRNA ( Figure 1A ) and trans-splicing between two mRNA precursor molecules ( Figure 1B ). “SD” refers to splice donor. “SA” refers to splice acceptor.

[0048] I don't want to be bound by theory. Figure 1C A schematic diagram is provided that depicts an exemplary splicing editor nucleic acid molecule disclosed herein for targeted trans-splicing of mRNA precursors. The labeling of the splicing editor indicates the segments corresponding to the RNA binding domain, non-coding RNA (ncRNA), SA, and exons. The labeling of the mRNA precursor indicates the mRNA precursor segments corresponding to the 5' exon, SD, SA, and 3' exon.

[0049] I don't want to be bound by theory. Figure 1D Schematic diagrams are provided that depict the predicted secondary structures of exemplary splice editor nucleic acid molecules and mRNA precursors of the present disclosure, as well as the interactions between the splice editor and the mRNA precursor to produce a trans-spliced ​​mRNA product.

[0050] Figure 1E A graph showing the proportion of ncRNAs identified as containing sequence motifs (Sm sequence motif, H / ACA box and / or C / D box) is provided.

[0051] Figure 1F A chart is provided showing the length (in number of nucleotides) of exemplary ncRNAs of the present disclosure.

[0052] I don't want to be bound by theory. Figure 2A 、 Figure 2B and Figure 2C A schematic diagram is provided that depicts the extended 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, SA, and an exon ( Figure 2A ), and the interaction of an exemplary splicing editor with a target pre-mRNA, which triggers a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splicing editor ( Figure 2A 、 Figure 2B ).

[0053] Without wishing to be bound by theory, FIG. 3A , Figure 3B and Figure 3C Schematic diagrams are provided that depict the extended and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure, which has an RNA binding domain, U11 snRNA, intron, SA, and exon from 5′ to 3′ ( FIG. 3A ), and the interaction of the exemplary splice editor with a target pre-mRNA that triggers a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splice editor ( FIG. 3A , FIG. Figure 3B ).

[0054] I don't want to be bound by theory. Figure 4A 、 Figure 4B and Figure 4C A schematic diagram is provided that depicts the extended 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 having an Sm sequence motif and a U7 snRNA, an intron, SA, and an exon ( Figure 4A ), and the interaction of an exemplary splicing editor with a target pre-mRNA, which triggers a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splicing editor ( Figure 4A 、 Figure 4B and Figure 4C ).

[0055] I don't want to be bound by theory. Figure 5A 、 Figure 5B and Figure 5C A schematic diagram is provided that depicts the extended 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 having an Sm sequence motif, an intron, SA, and an exon ( Figure 5A), and the interaction of an exemplary splicing editor with a target pre-mRNA, which triggers a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splicing editor ( Figure 5B and Figure 5C ).

[0056] I don't want to be bound by theory. Figure 6A 、 Figure 6B and Figure 6C A schematic diagram is provided that depicts the extended and folded secondary structure of an exemplary splice editor nucleic acid molecule of the present disclosure having, from 5′ to 3′, a snoRNA inserted with two RNA binding domains, an intron, SA, and an exon ( Figure 6A ), and the interaction of an exemplary splicing editor with a target pre-mRNA, which triggers a trans-splicing event between the SD of the pre-mRNA and the SA of the exemplary splicing editor ( Figure 6B and Figure 6C ).

[0057] I don't want to be bound by theory. Figure 7A is an image showing the design of a snoRNA-guided construct for exon skipping.

[0058] Figure 7B is a graph showing the results of exon skipping for four splice acceptor-targeted snoRNAs (labeled as "snord45a_11_receptor", "snord45a_7_receptor", "aca46_receptor_29" and "aca44_2_receptor_35", sequences as shown in Table 4).

[0059] Figure 8 is a graph showing GFP production as a result of exon skipping for four different splice acceptor-targeting snoRNAs (labeled "V5_snord45a_11_receptor", "V4_snord45a_7_receptor", "V1_aca46_receptor_29" and "V2_aca44_2_receptor_35", sequences shown in Table 4).

[0060] Figure 9 is a graph showing the relationship between four splice acceptor-targeted snoRNAs (labeled as "snord45a_11_receptor", "snord45a_7_receptor", "aca46_receptor_29" and "aca44_2_receptor_35") and Figure 8 Exon skipping results of four randomized boot comparisons.

[0061] I don't want to be bound by theory. Figure 10 is an image showing the design of the U7 leader construct for trans-splicing.

[0062] Figure 11 is a graph showing the binding of U7 leader constructs to different target elements and the effect of trans-splicing.

[0063] FIG12 is a graph showing the Figure 11 The first four bars on the left show U7 constructs with specific targeting elements for trans-splicing, and the remaining bars on the graph show U7 constructs with non-targeting elements for trans-splicing.

[0064] 13 is a graph showing a comparison of U7 of USH2A targeted for piggybac integration (773; hybridization region and hairpin intact) with non-targeting guides (774 and 776) and a mutated U7 hairpin (775; hybridization region intact, but hairpin region mutated). DETAILED DESCRIPTION

[0065] The present disclosure provides nucleic acid molecules for trans-splicing of target RNA (e.g., mRNA precursor) in targeting cells. In some embodiments, nucleic acid molecules are engineered to include a nucleotide sequence, the nucleotide sequence including (i) at least one non-coding sequence (intron sequence), the non-coding sequence including an RNA guide domain bound to one or more target sequences in a target RNA (e.g., mRNA precursor), (ii) a splicing acceptor and / or a splicing donor, and (iii) at least one coding sequence (exon sequence). The nucleic acid molecules disclosed herein are referred to herein as "splicing editor nucleic acid" or "splicing editor nucleic acid molecules". Without being bound by theory, the binding event causes the splicing editor nucleic acid to be close to the region of the selection for trans-splicing of the target RNA (e.g., mRNA precursor), and the spliceosome is recruited to the target RNA (e.g., mRNA precursor), so that effective trans-splicing occurs. In some embodiments, the target RNA is an mRNA precursor. In some embodiments, the mRNA precursor includes a nucleotide sequence containing a pathogenic mutation. In some embodiments, trans-splicing produces an mRNA comprising a desired change compared to the mRNA produced by cis-splicing of the pre-mRNA. For example, in some embodiments, the desired change is correction of a disease-causing mutation in the pre-mRNA.

[0066] In some embodiments, the RNA guide domain comprises (i) one or more binding domains, each of which is complementary to a target sequence in a 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, the assembly of ncRNA to form RNPs confers one or more desired properties to the trans-splicing nucleic acid molecule that enable efficient trans-splicing. For example, in some embodiments, RNPs play the following roles: (i) stabilizing RNA secondary structures present in splicing editor nucleic acid molecules, mRNA precursors, or both; (ii) stabilizing RNA-RNA interactions formed between splicing editor nucleic acid molecules and mRNA precursors; (iii) protecting splicing editor nucleic acid molecules and / or mRNA precursors from degradation; (iv) localizing splicing editor nucleic acid molecules to subcellular compartments where mRNA precursors are present; and (v) combinations of (i)-(iv).

[0067] In some embodiments, the present disclosure provides a method for trans-splicing of a target RNA (e.g., an mRNA precursor) in a targeted cell, comprising introducing a splicing editor nucleic acid molecule as described herein into the cell. In some embodiments, the present disclosure provides a method for correcting a mutation in a target RNA (e.g., an mRNA precursor) in a cell, comprising introducing a splicing editor nucleic acid molecule as described herein into the cell. In some aspects, the introduction is performed in vivo. In some embodiments, the introduction is performed in vitro. In some embodiments, the methods described herein are used to introduce the desired editing into the target nucleic acid editing in a manner that avoids certain shortcomings of gene editing (e.g., gene editing performed using a CRISPR / Cas system). Although gene editing has the risk of introducing permanent and pathogenic off-target editing into the genome, the present disclosure provides a trans-splicing method that avoids changing genomic DNA and achieves transient editing. Therefore, without being bound by theory, the method of the present disclosure is used to introduce editing of nucleic acids in cells in a safer manner than gene editing. In addition, in some embodiments, the method of the present disclosure is used to inactivate the bad off-target gene editing introduced into the genome, thereby preventing or improving the harmful phenotype associated with the gene editing method.

[0068] In some embodiments, the present disclosure provides a method for treating a disease or condition in a subject in need thereof, the disease or condition being associated with (i) one or more gene mutations, and / or (ii) abnormal expression levels and / or activity of a gene or its transcription or translation product, the method comprising administering to the subject one or more splicing editor nucleic acid molecules described herein.

[0069] In some embodiments, the present disclosure provides methods and compositions for delivering splicing editor nucleic acid molecules to cells or subjects. In some embodiments, splicing editor nucleic acid molecules are delivered as DNA. In some embodiments, splicing editor nucleic acid molecules are delivered as RNA. In some aspects, delivery includes administering a recombinant expression vector (e.g., a viral vector, such as AAV) comprising a splicing editor nucleic acid molecule. In some aspects, delivery includes administering a non-viral vector (e.g., a lipid particle) comprising a splicing editor nucleic acid molecule.

[0070] Splice editor nucleic acid molecules for targeted trans-splicing

[0071] Accurate pre-mRNA splicing is crucial for correct protein expression. Nuclear pre-mRNA splicing is catalyzed by the spliceosome. The gene structure of vertebrates is usually composed of relatively long introns and relatively short internal exons. The exon-intron boundary is defined by the splice donor (5' splice site or splice site at the 3' end of the exon) and the splice acceptor (3' splice site or splice site at the 5' end of the exon). In addition to recognizing splice sites, the spliceosome also relies on various splicing signals to mediate splicing events, including branch point sequences and polypyrimidine segments. Typically, the branch point sequence contains an adenosine within the consensus sequence and is located approximately 18-40 nucleotides upstream of the 3' splice site. The polypyrimidine segment contains uracil repeats and is located proximal to the 3' splice site. Alternative signals can enhance or reduce splicing activity and include exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs), and intronic splicing silencers (ISSs). Cis-splicing occurs when the 2'OH group of an intronic branched adenosine makes a nucleophilic attack on the 5' splice site (splice donor). This results in cleavage of the site and joins the 5' end of the intron to the branched adenosine, forming a noose structure. The 3' splice site (splice acceptor) is attacked by the 3'OH of the 5' exon, resulting in the ligation of the 5' exon and the 3' exon to form the mRNA and release the intron noose (see, e.g. Figure 1A ).

[0072] In contrast, trans-splicing occurs between two different RNA molecules, in which the 3′ splice site (splice acceptor) of the second RNA is attacked by the 3′ OH of the 5′ exon of the first RNA, resulting in the ligation of the 5′ exon of the first RNA to the 3′ exon of the second RNA, thereby forming a chimeric RNA (see, e.g., Figure 1B ).

[0073] The present disclosure provides a splicing editor nucleic acid molecule for trans-splicing of a target RNA (e.g., an mRNA precursor) in a targeted cell, the splicing editor nucleic acid molecule comprising a nucleotide sequence comprising (i) at least one intron sequence comprising an RNA guide 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 guide domain is designed to bind to a specific region of the target RNA (e.g., an mRNA precursor) so that splicing can be performed between one or more splice sites of the splicing editor nucleic acid molecule and one or more splice sites of the target RNA (e.g., an mRNA precursor). In some embodiments, trans-splicing produces a chimeric mRNA comprising at least one exon sequence of the splicing editor nucleic acid and one or more exons of the target RNA (e.g., an mRNA precursor).

[0074] In humans, the boundaries of exons are determined by the splice sites that span the exon pairing (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., branch point sequences, polypyrimidine stretches, exon (or intron) splicing enhancers, and silencers) contribute to the correct splicing of exons together to form mature mRNA. During pre-mRNA splicing, the spliceosome looks for a pair of closely spaced splice sites. Without being bound by theory, the splice editor nucleic acid molecules described herein mediate efficient trans-splicing by bringing the splice site of the target RNA (e.g., an mRNA precursor, e.g., a splice acceptor or splice donor of a target mRNA precursor) into close proximity with the splice site of the splice editor nucleic acid molecule (e.g., a splice acceptor or splice donor of a splice editor nucleic acid molecule) so that the spliceosome mediates splicing between the splice site of the target mRNA precursor and the splice site of the splice editor nucleic acid molecule.

[0075] 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 guide domain; (ii) a splice acceptor; and (iii) at least one exon sequence.

[0076] In some embodiments, a splice editor nucleic acid molecule comprises a nucleotide sequence comprising, from 5′ to 3′, (i) at least one exonic sequence; (ii) a splice donor; and (iii) at least one intronic sequence comprising an RNA guide domain.

[0077] In some embodiments, the at least one intron sequence comprises one or more splicing signals (e.g., a branch point 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).

[0078] RNA guide domain

[0079] In some embodiments, the RNA guide domain comprises a nucleotide sequence comprising (i) one or more binding domains, each of which is complementary to a target sequence in a target RNA (e.g., a pre-mRNA); and (ii) an ncRNA. In some embodiments, the one or more binding domains mediate binding of a trans-splicing nucleic acid molecule to a target RNA (e.g., a pre-mRNA) in a cell. In some embodiments, the ncRNA mediates assembly into an RNP.

[0080] In some embodiments, the RNA guide domain comprises a nucleotide sequence having, from 5′ to 3′, (i) one or more binding domains, each binding domain being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); and (ii) an ncRNA.

[0081] In some embodiments, the RNA guide domain comprises a nucleotide sequence having, from 5′ to 3′, (i) an ncRNA; and (ii) one or more binding domains, each binding domain being complementary to a target sequence in a target RNA (e.g., a pre-mRNA).

[0082] In some embodiments, the RNA guide domain comprises a nucleotide sequence having a ncRNA, wherein one or more binding domains are inserted into the ncRNA or exchanged with consecutive nucleotides of the ncRNA.

[0083] Target sequence

[0084] In some embodiments, the one or more binding domains of the RNA guide domain are each complementary to a target sequence in a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing.

[0085] As used herein, the term "target sequence" refers to a continuous nucleotide sequence that is present in a target RNA (e.g., an mRNA precursor) and is targeted for trans-splicing. As used herein, the term "continuous nucleotides" refers to a string of nucleotides that are covalently linked and adjacent to each other. In some embodiments, the length of 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 some embodiments, the length of the target sequence is less than about 300, 250, 200, 100, 150, or 50 nucleotides. In some embodiments, the target sequence is about 5-10, about 5-15, about 5-20, about 10-20, about 10-30, about 10-40, about 10-50, about 10-60, about 10-70, about 10-80, about 10-90, about 10-100, about 50-100, about 50-150, about 50-200, about 50-250, about 50-300, about 100-200, about 100-300, or about 200-300 nucleotides in length.

[0086] In some embodiments, the target sequence is located in a region comprising a splice site targeted for trans-splicing in a target RNA (e.g., a precursor mRNA). As used herein, "a splice site targeted for trans-splicing in a target RNA (e.g., a precursor mRNA)" refers to a splice site selected for trans-splicing in a target RNA (e.g., a precursor mRNA), wherein after a splicing editor nucleic acid molecule as described herein is introduced into a cell comprising a target RNA (e.g., a precursor mRNA), a trans-splicing event mediates the connection between the splice site of the target RNA (e.g., a precursor mRNA) and the splice site of the splicing editor nucleic acid molecule. In some embodiments, the target sequence is located upstream of a splice site targeted for trans-splicing in a target RNA (e.g., a precursor mRNA). In some embodiments, the target sequence is located downstream of a splice site targeted for trans-splicing in a target RNA (e.g., a precursor mRNA). In some embodiments, the target sequence is located in a region comprising a splice site in a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing, wherein the region spans 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, about 5,000 nucleotides.

[0087] In some embodiments, the target sequence is located proximal to a splice site in a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing. As used herein, the term "proximal to a splice site" refers to a region extending less than about 500 nucleotides upstream and / or downstream of a splice site in a target RNA (e.g., a pre-mRNA) that is targeted for trans-splicing.

[0088] In some embodiments, the target sequence is located proximal to a splice acceptor that is targeted for trans-splicing. In some embodiments, the target sequence is located upstream of a splice acceptor that is targeted for trans-splicing. In some embodiments, the target sequence is located downstream of a splice acceptor that is targeted for trans-splicing. In some embodiments, the target sequence overlaps with a splice acceptor that is targeted for trans-splicing.

[0089] In some embodiments, the target sequence is located proximal to the splice donor that is targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the splice donor that is targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the splice donor that is targeted for trans-splicing. In some embodiments, the target sequence overlaps with the splice donor that is targeted for trans-splicing.

[0090] In some embodiments, the target sequence is located in a region of a target RNA (e.g., a pre-mRNA) that contains an exon that is targeted for trans-splicing. As used herein, "an exon targeted for trans-splicing" refers to an exon in a target RNA that is selected for removal following trans-splicing between the target RNA and a splicing editor nucleic acid as described herein, wherein trans-splicing results in one or more exons of the target RNA (e.g., a pre-mRNA) being joined to the at least one exon sequence of the splicing editor nucleic acid to form a chimeric RNA molecule, and wherein the exon targeted for trans-splicing is present in the target RNA but not in the chimeric RNA molecule formed by the trans-splicing event.

[0091] In some embodiments, the target sequence is located upstream of the exon targeted for trans-splicing. In some embodiments, the target sequence is located downstream of the exon targeted for trans-splicing. In some embodiments, the target sequence is within the exon targeted for trans-splicing.

[0092] In some embodiments, the target sequence is located proximal to the splice acceptor of the exon targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the splice acceptor of the exon targeted for trans-splicing. In some embodiments, the target sequence is located 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.

[0093] In some embodiments, the target sequence is located proximal to the splice donor of the exon targeted for trans-splicing. In some embodiments, the target sequence is located upstream of the splice donor of the exon targeted for trans-splicing. In some embodiments, the target sequence is located 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.

[0094] RNA-binding domain

[0095] In some embodiments, the binding domain that is complementary to a target sequence in a target RNA (e.g., a precursor 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, 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. 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.

[0096] 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 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.

[0097] In some embodiments, the binding domain is 10-50 nucleotides in length, for example, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 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. 47, 48, 49, or 50 nucleotides.

[0098] In some embodiments, the RNA guide domain comprises one binding domain. In some embodiments, the RNA guide domain comprises more than one binding domain. In some embodiments, the RNA guide domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 binding domains. In some embodiments, the more than one binding domains are immediately adjacent to each other. In some embodiments, the more than one binding domains are connected by an intervening nucleotide spacer sequence.

[0099] In some embodiments, one or more binding domains each comprise a sequence sufficiently complementary to its target sequence so that the splicing editor nucleic acid molecule can specifically bind to the target sequence by forming a base pair. As used herein, the term "base pair" refers to two nucleobases on relatively complementary nucleic acid chains that interact by forming specific hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds). In some embodiments, the base pair is formed by Watson-Crick base pairing. As understood by those skilled in the art, Watson-Crick base pairing refers to a set of base pairing rules in which purine nucleobases bind to pyrimidine nucleobases to form complementary base pairs. The nature of the hydrogen bond depends on the specific base pair. For example, guanosine-cytosine base pairs are formed by three hydrogen bonds, while adenine-thymine or adenine-uracil base pairs are formed by two hydrogen bonds. It is understood that analogs or derivatives of canonical nucleobases will form base pair interactions by Watson-Crick base pairing or non-canonical base pairing.

[0100] A binding domain that "specifically binds" a target sequence in a target RNA (e.g., a precursor to an mRNA) refers to a binding domain that does not significantly bind to a reference sequence (e.g., a nucleic acid lacking the target sequence). For example, a splicing editor nucleic acid molecule comprising a binding domain that specifically binds to a target sequence will exhibit a significantly higher binding affinity for a target RNA (e.g., a precursor to an mRNA) comprising a nucleotide sequence containing the target sequence as compared to a target RNA (e.g., a precursor to an mRNA) lacking the target sequence. As understood by those skilled in the art, the binding affinity between a first nucleic acid chain and a second nucleic acid chain is measured as a melting temperature (Tm), which is the temperature at which half of the first nucleic acid chain is duplexed with the second nucleic acid chain.

[0101] In some embodiments, if the binding domain is base-paired with the target sequence under conditions suitable for regulating trans-splicing, the binding domain is complementary to the target sequence in the target RNA (e.g., mRNA precursor). Such conditions can be stringent conditions, such as the target RNA (e.g., mRNA precursor) and the splicing editor nucleic acid molecule are combined at a temperature of 50 ° C -70 ° C in a buffer comprising 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA for 12-16 hours, then washed (see, for example, " Molecular Cloning: A Laboratory Manual ", Sambrook et al. (1989) Cold Spring Harbor Laborator y Press). Other conditions include physiologically relevant conditions that may be encountered in an organism. Those skilled in the art can determine the condition set that is most suitable for testing the complementarity of two sequences according to the final application of the hybridization nucleotide.

[0102] noncoding RNA

[0103] In some embodiments, the RNA guide domain comprises an ncRNA. As used herein, "ncRNA" refers to an RNA sequence that does not encode a protein but plays a role 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 guide 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 RNA and protect it from degradation (Darzacq 2006); and (iii) localize to relevant subcellular compartments (Roithová et al. (2018) Nucleic acid research 46:3774-3790). In some embodiments, ncRNAs are identified according to the methods described herein.

[0104] In some embodiments, the method comprises identifying a 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 annotated with unique identifiers and information about one or more species in which the RNA sequence has been observed.

[0105] In some embodiments, the method includes identifying ncRNA expressed by a cell or organism. Methods for identifying ncRNA are known in the art (see, for example, Hüttenhofer et al. (2006) Nucleic Acids Res 34:635). In some embodiments, cellular RNA is extracted from a cell or organism, separated by PAGE and eluted from the gel, and the ncRNA is identified by sequence analysis (e.g., by 2D RNA fingerprint analysis or enzymatic or chemical RNA sequencing). In some embodiments, ncRNA obtained from a cell or organism through a selection process based on size or antibody binding is reverse transcribed to generate a cDNA library, which is then subjected to sequence analysis. In some embodiments, total RNA is harvested from a cell or organism and ncRNA is detected using microarray hybridization. In some embodiments, genomic SELEX is used to identify ncRNA obtained from a cell or organism. In some embodiments, ncRNA sequences are identified from any known organism. In some embodiments, the organism is a bacterium. In some embodiments, the organism is an archaeon. 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.

[0106] In some embodiments, ncRNA plays a role in modifying, changing, inhibiting or promoting RNP formation and / or standard processing. In some embodiments, ncRNA is assembled into RNP. In some embodiments, RNP plays a role in stabilizing the RNA secondary structure of splicing editor nucleic acid. In some embodiments, RNP plays a role in stabilizing the RNA-RNA interaction between splicing editor nucleic acid and / or splicing editor nucleic acid and target RNA (e.g., mRNA precursor). In some embodiments, RNP plays a role in protecting splicing editor nucleic acid from degradation. In some embodiments, RNP plays a role in localizing splicing editor nucleic acid to a subcellular compartment comprising a target mRNA precursor. Methods for measuring the assembly of one or more nucleic acids (e.g., RNA or DNA) with one or more proteins to form RNPs are known in the art. Such methods include but are not limited to electrophoretic mobility shift assays (EMSA), DNA or RNA pull-down assays, oligonucleotide-targeted RNase H protection assays, fluorescence in situ hybridization co-localization, immunoprecipitation assays, and RNA sequencing and cross-linking methods, such as high-throughput sequencing cross-linked immunoprecipitation (HITS-CLIP).

[0107] In some embodiments, an 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 a splice editor nucleic acid. In some embodiments, a portion of the ncRNA sequence is incorporated into a splice editor nucleic acid.

[0108] In some embodiments, a splice editor of the present disclosure comprises a ncRNA sequence or a portion thereof, wherein the ncRNA is selected from the group consisting of snRNA, snoRNA, lncRNA, rRNA, ribozyme, sRNA, scaRNA, vault RNA, and combinations thereof.

[0109] In some embodiments, the length of ncRNA sequence or its part is less than about 500 nucleotides. In some embodiments, the length of ncRNA sequence or its part is less than about 400 nucleotides. In some embodiments, the length of ncRNA sequence or its part is less than about 300 nucleotides. In some embodiments, the length of ncRNA sequence or its part 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.

[0110] 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.

[0111] 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 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.

[0112] In some embodiments, ncRNA sequence or its portion comprises one or more RNA secondary structures that are assembled into RNP. Methods for determining the secondary structure formed by RNA sequence are known in the art. In some embodiments, the method comprises experimental determination, such as nuclear magnetic resonance, cryogenic electron microscopy or X-ray crystal structure analysis. In some embodiments, the method comprises computational prediction, e.g., based on a thermodynamic model, such as the Turner 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. ACSSymposiu m Series Vol. 682, 246–257; Reuther et al., BMC Bioinform. 11, 129 (2010)); machine learning techniques, such as CONTRAfold (Do et al., Bioinform matics 22, e90–8 (2006); Foo et al., Advances in Neural Informatio n Processing Systems 20, 377–384), ContextFold (Zakov et al., J. Comput. Biol. 18, 1525–1542 (2011)); probabilistic generative models, such as stochastic context-free grammars (Rivas et al., RNA 18, 193–212 (2012)); hybrid models such as 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 methods such as SPOT-RNA (Singh et al., Nat. Commun. 10, 5407 (2019)) or E2Efold (Chen et al., Proceedings of the 8th International Conference on Learning Representations; arXi v: 2002.05810 (2020)). .

[0113] In some embodiments, one or more RNA secondary structures comprise single-stranded RNA sequence, double-stranded RNA sequence or its combination.In some embodiments, one or more RNA secondary structures comprise duplex structure, stem-loop, pseudoknot, inner loop, multi-branched loop, convex loop, outer loop or its combination.In some embodiments, ncRNA sequence or its part comprise the sequence motif that is assembled into RNP.In some embodiments, sequence motif comprises the single-stranded RNA sequence that is assembled into RNP.In some embodiments, ncRNA sequence or its part comprise sequence motif and one or more RNA secondary structures that are assembled into RNP.In some embodiments, one or more proteins in secondary structure and / or sequence motif and human cell are assembled to form RNP.

[0114] In some embodiments, the ncRNA sequence or a portion thereof comprises one or more RNA secondary structures. In some embodiments, the ncRNA sequence or a portion thereof comprises one or more sequence motifs. In some embodiments, the ncRNA sequence or a 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, the H consensus sequence comprising the sequence shown in Table 1 or consisting of the sequence shown in Table 1. In some embodiments, the H consensus sequence comprises SEQ ID NO: 1 or consists of SEQ ID NO: 1. In some embodiments, the sequence motif comprises an ACA consensus sequence, the ACA consensus sequence comprising the sequence shown in Table 1 or consisting of the sequence shown in Table 1. In some embodiments, the ACA consensus sequence comprises SEQ ID NO: 2 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 sequence comprising a sequence shown 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 shown 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 shown 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, wherein the C / D box comprises a C consensus sequence and a D consensus sequence, each comprising a sequence shown 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 shown 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.

[0115] Table 1: ncRNA sequence motifs

[0116]

[0117] In some embodiments, the splice editor of the present disclosure comprises an ncRNA sequence that is 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%, about 99% identical to a sequence selected from SEQ ID NOs: 9-657, or a portion thereof. In some embodiments, the splice editor of the present disclosure comprises an ncRNA sequence selected from SEQ ID NOs: 9-657, or a portion thereof.

[0118] In some embodiments, the ncRNA sequence or a portion thereof comprises a contiguous nucleotide sequence 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 contiguous nucleotide sequence comprises an Sm sequence motif (e.g., an Sm sequence motif shown in Table 1).

[0119] In some embodiments, the ncRNA sequence or a 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 shown in Table 1).

[0120] 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 shown in Table 1).

[0121] 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 shown in Table 1); (ii) an ACA consensus sequence (e.g., an ACA consensus sequence shown in Table 1); or (iii) a combination of (i)-(ii).

[0122] 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 shown in Table 1), (ii) a C′-box motif described herein (e.g., a C′-box motif shown in Table 1), (iii) a D-box motif described herein (e.g., a D-box motif shown in Table 1), (iv) a D′-box motif described herein (e.g., a D′-box motif shown in Table 1), or (v) a combination of (i)-(iv).

[0123] In some embodiments, the ncRNA sequence or a portion thereof comprises a nucleotide sequence that is 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% identical 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 shown in Table 1).

[0124] In some embodiments, the ncRNA sequence or a portion thereof comprises a nucleotide sequence that is 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% identical 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 shown in Table 1).

[0125] In some embodiments, the ncRNA sequence or portion thereof comprises a nucleotide sequence that is 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%, about 99% identical 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 shown in Table 1).

[0126] In some embodiments, the ncRNA sequence or a portion thereof comprises a sequence selected from SEQ ID The sequence of NO:9-657 has a nucleotide sequence that is 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%, about 99% identical, 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 (i) an H consensus sequence (e.g., an H consensus sequence shown in Table 1); (ii) an ACA consensus sequence (e.g., an ACA consensus sequence shown in Table 1); or (iii) a combination of (i)-(ii).

[0127] In some embodiments, the ncRNA sequence or a portion thereof comprises a nucleotide sequence that is 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%, about 99% identical to a sequence selected from SEQ ID NOs: 9-657, wherein the nucleotide sequence comprises a length 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. The invention further comprises a region of about 100 to about 200, about 100 to about 250, or about 100 to about 300 nucleotides, wherein the region comprises (i) a C-box motif described herein (e.g., a C-box motif shown in Table 1), (ii) a C′-box motif described herein (e.g., a C′-box motif shown in Table 1), (iii) a D-box motif described herein (e.g., a D′-box motif shown in Table 1), (iv) a D′-box motif described herein (e.g., a D′-box motif shown in Table 1), or (v) a combination of (i)-(iv).

[0128] In some embodiments, ncRNA is snRNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure comprises a full-length snRNA or a portion thereof, and the full-length snRNA or a portion thereof comprises a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the snRNA sequence described herein or the snRNA sequence identified according to the methods described herein is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length snRNA sequence described herein or the full-length snRNA sequence identified according to the methods described herein is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, a portion of the snRNA sequence described herein or a portion of the snRNA sequence identified according to the methods described herein (e.g., a continuous nucleotide region in the snRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length snRNA or a portion of the snRNA is assembled into a small nuclear RNP (snRNP). In some embodiments, the full-length snRNA or a portion of the snRNA comprises one or more secondary RNA structures assembled to form the 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, wherein (i), (ii), or both assemble to form a snRNP.

[0129] Exemplary metazoan snRNA systems include U1 and U11 snRNAs, which are snRNAs that 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 RNPs in the major and minor spliceosomes (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 responsible for the cleavage and polyadenylation of histone pre-mRNAs (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).

[0130] 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 comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), and wherein the snRNA is selected from the group consisting 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, RNU1-120P, RNU1-121P, RNU1-122P, RNU1-123P, RNU1-124P, RNU1-125P, RNU1-126P, RNU1-127P, RNU1-128P, RNU1-129P, RNU1-130P, RNU1-131P, RNU1-132P, RNU1-133P, RNU1-134P, RNU1-135P, RNU1-136P, RNU1-137P, RNU1-138P, RNU1-139P, RNU1-140P, RNU1-141P, RNU1-142P, RNU1-143P, RNU1-144P, RNU1-145P, RNU1-146P, RNU1-147P, RNU1-148P, RNU1-149P, RNU1-150P, RNU1-151P, RNU1-15 9P, RNU1-11P, RNU1-123P, RNU1-124P, RNU1-125P, RNU1-128P, RNU1-129P, RNU1-130P, RNU1-131P, RNU1-132P, RNU1-133P, RNU1-134P, RNU1-136P, RNU 1-138P, 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, RNU1-24P, RNU1-2 7P, 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、RNU6-1207P、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、R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6-545P、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、<h2 style=";text-align:left;direction:ltr">RNU7-194P、RNU7-195P、RNU7-196P、RNU7-197P、RNU7-19P、RNU7-200 P、RNU7-20P、RNU7-21P、RNU7-22P、RNU7-23P、RNU7-24P、RNU7-25P、RN U7-26P、RNU7-27P、RNU7-28P、RNU7-29P、RNU7-2P、RNU7-30P、RNU7-34 P、RNU7-35P、RNU7-37P、RNU7-38P、RNU7-3P、RNU7-40P、RNU7-41P、RNU 7-43P、RNU7-45P、RNU7-46P、RNU7-47P、RNU7-48P、RNU7-49P、RNU7-4 P、RNU7-50P、RNU7-51P、RNU7-52P、RNU7-53P、RNU7-54P、RNU7-55P、RN U7-56P、RNU7-57P、RNU7-59P、RNU7-60P、RNU7-61P、RNU7-62P、RNU7-6 3P、RNU7-65P、RNU7-66P、RNU7-67P、RNU7-69P、RNU7-6P、RNU7-70P、RN U7-71P、RNU7-73P、RNU7-74P、RNU7-75P、RNU7-77P、RNU7-79P、RNU7- 7P、RNU7-80P、RNU7-81P、RNU7-82P、RNU7-84P、RNU7-85P、RNU7-87P、R NU7-88P、RNU7-8P、RNU7-90P、RNU7-92P、RNU7-93P、RNU7-94P、RNU7-9 5P、RNU7-96P、RNU7-97P、RNU7-99P、RNU7-9P、RNVU1-1、RNVU1-14、RNV U1-15、RNVU1-17、RNVU1-18、RNVU1-19、RNVU1-2、RNVU1-21、RNVU1-22 、RNVU1-23、RNVU1-24、RNVU1-25、RNVU1-26、RNVU1-27、RNVU1-28、RNV U1-29、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。、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0131] 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 comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), and wherein the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atacsnRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0132] In some embodiments, the snRNA is U1 snRNA. In some embodiments, U1 snRNA assembles into U1 RNPs. In some embodiments, the snRNA is U2 snRNA. In some embodiments, U2 snRNA assembles into U2 RNPs. In some embodiments, the snRNA is U4 snRNA. In some embodiments, U1 snRNA assembles into U4 RNPs. In some embodiments, the snRNA is U4atac snRNA. In some embodiments, U1 snRNA assembles into U4atac RNPs. In some embodiments, the snRNA is U5 snRNA. In some embodiments, U1 snRNA assembles into U5 RNPs. In some embodiments, the snRNA is U6 snRNA. In some embodiments, U1 snRNA assembles into U6 RNPs. In some embodiments, the snRNA is U6atac snRNA. In some embodiments, U1 snRNA assembles into U6atac RNPs. In some embodiments, the snRNA is U7 snRNA. In some embodiments, U1 snRNA assembles into U7 RNPs. 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.

[0133] 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, and G Sm protein.

[0134] In some embodiments, the splicing editor nucleic acid molecules of the present disclosure comprise a full-length snoRNA or a portion thereof, the full-length snoRNA or a portion thereof comprising a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the snoRNA sequences described herein or the snoRNA sequences identified according to the methods described herein are incorporated into the splicing editor nucleic acids of the present disclosure. In some embodiments, the full-length snoRNA sequences described herein or the full-length snoRNA sequences identified according to the methods described herein are incorporated into the splicing editor nucleic acids of the present disclosure. In some embodiments, a portion of the snoRNA sequences described herein or a portion of the snoRNA sequences identified according to the methods described herein (e.g., a continuous nucleotide region in the snRNA) is incorporated into the splicing editor nucleic acids of the present disclosure.

[0135] In some embodiments, the full-length snoRNA or a portion thereof is assembled into a small nucleolar RNP (snoRNP). snoRNAs are responsible for RNA methylation and RNA pseudouridylation (Bachellerie 2002, Kiss 2004). There are two types of snoRNAs: (i) H / ACA box snoRNAs responsible for pseudouridylation and (ii) C / D box snoRNAs responsible for 2'-O-ribose methylation (Jorjani 2016, Kufel 2019). snoRNAs can also form RNPs called snoRNPs (Khanna 2006) and hybridize to their RNA targets through Watson-Crick base pairing (Jin 2007).

[0136] 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 shown in SEQ ID NO: 1) and an ACA consensus sequence (e.g., an ACA consensus sequence comprising the sequence shown 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 C consensus sequence (e.g., a C consensus sequence comprising the sequence shown in SEQ ID NO: 5), a D′ consensus sequence (e.g., a D′ consensus sequence comprising the sequence shown in SEQ ID NO: 8), a C′ consensus sequence (e.g., a C′ consensus sequence comprising the sequence shown in SEQ ID NO: 7), and a D consensus sequence (e.g., a D consensus sequence comprising the sequence shown in SEQ ID NO: 6). In some embodiments, C / D box snoRNPs assemble to form C / D snoRNPs.

[0137] In some embodiments, the splice editor nucleic acid molecules of the present disclosure comprise a full-length snoRNA or a portion thereof, the full-length snoRNA or the portion thereof comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), and wherein the snoRNA is selected from SCARNA18, SCARNA18B, SNOR A1, SNORA10, SNORA108, SNORA10B, SNORA11, SNORA11B, SNORA11C, SNORA11D, SNORA11E, SNORA11F, SNORA11G, SNORA12, SNORA13, SNORA14A, SNORA14B, SNORA15, SNOR A16, SNORA17, SNORA18B, SNORA19, SNORA20, SNORA210, SNORA220, SNORA23, SNORA24, SNORA25, SNORA26, SNORA27, SNORA28, SNORA29, SNORA30, SNORA31, SNORA32, SNORA33, SNORA34, SNORA35, SNORA36, SNORA37, SNORA38, SNORA39, SNORA40, SNORA41 ORA15B-1, SNORA15B-2, SNORA16A, SNORA16B, SNORA17A, SNORA17B, SNORA18, SNORA19, SNORA1B, SNORA20, SNO RA20B, SNORA21, SNORA21B, SNORA22, SNORA22B, SNOR A22C, SNORA24, SNORA24B, SNORA25, SNORA25B, SNORA 26, SNORA27, SNORA28, SNORA29, SNORA2A, SNORA2B, SNORA2C, SNORA30, SNORA30B, SNORA31, SNORA31B, SNO RA32, SNORA33, SNORA35, SNORA35B, SNORA36A, SNORA 36B, SNORA36C, SNORA37, SNORA38, SNORA38B, SNORA3A, SNORA3B, SNORA3C, SNORA4, SNORA40, SNORA40B, SNORA40C, SNORA41, SNORA41B, SNORA44, SNORA46, SNO RA47, SNORA48, SNORA48B, SNORA49, SNORA50A, SNORA 50B, SNORA50C, SNORA50D, SNORA51, SNORA52, SNORA54, SNORA55, SNORA56, SNORA57, SNORA58, SNORA58B, SNORA59A, SNORA5A, SNORA5B, SNORA5C, SNORA6, SNOR A60, SNORA61, SNORA62, SNORA63, SNORA63B, SNORA63C, SNORA63D, SNORA63E, SNORA64, SNORA65, SNORA66, SNORA67, SNORA68,SNORA68B、SNORA69、SNORA70、SNO RA70B、SNORA70C、SNORA70D、SNORA70E、SNORA70F、SNORA70G、SNORA70H、SNORA70I、SNORA70J、SNORA71、SNO RA71A、SNORA71C、SNORA71D、SNORA71E、SNORA72、SNORA73、SNORA74、SNORA74D、SNORA75、SNORA75B、SNORA 77、SNORA77B、SNORA78、SNORA79、SNORA79B、SNORA7A、SNORA7B、SNORA8、SNORA80A、SNORA80B、SNORA80C、SNORA80D、SNORA80E、SNORA81、SNORA84、SNORA9、SNORA 9B、SNORD10、SNORD100、SNORD101、SNORD102、SNORD104、SNORD105、SNORD105B、SNORD107、SNORD108、SNORD109A、SNORD109B、SNORD11、SNORD110、SNORD111、SNORD 111B、SNORD112、SNORD113-1、SNORD113-2、SNORD113-3、SNORD113-4、SNORD113-5、SNORD113-6、SNORD113-7、SNORD113-8、SNORD113-9、SNORD114-1、SNORD1 14-10、SNORD114-11、SNORD114-12、SNORD114-13、SNORD114-14、SNORD114-15、SNORD114-16、SNORD114-17、SNORD114-18、SNORD114-19、SNORD114-2、SNORD114-2 114-20、SNORD114-21、SNORD114-22、SNORD114-23、SNORD114-24、SNORD114-25、SNORD114-26、SNORD114-27、SNORD114-28、SNORD114-29、SNORD114-3、SNORD D114-30、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、SNORD 116-23、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、SNORD 36B、SNORD36C、SNORD37、SNORD38A、SNORD38B、SNORD 38C、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、SNORD 65、SNORD65B、SNORD65C、SNORD66、SNORD67、SNORD68、SNORD69、SNORD7、SNORD70、SNORD70B、SNORD71、SNORD72、SNORD73A、SNORD73B、SNORD74B、SNORD77B、SNORD79、SNORD8、SNORD81、SNORD82、SNORD83、SNORD83A、SNORD83B、SNORD84、SNORD86、SNORD87, SNORD88A, SNORD88B, SNORD88C, SNORD89, SNORD9, SNORD90, SNORD 92, SNORD93, SNORD94, SNORD95, SNORD96A, SNORD96B, SNORD97, SNORD98, SNORD99, U8, snoZ196. ,

[0138] In some embodiments, ncRNA is scaRNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure includes full-length scaRNA or a portion thereof, and the full-length scaRNA or a portion thereof includes a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the scaRNA sequence described herein or the scaRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length scaRNA sequence described herein or the full-length scaRNA sequence identified according to the methods described herein are incorporated into the splicing 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 continuous nucleotide region in scaRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length scaRNA or a portion thereof is assembled into small Cajal body RNP (scaRNP). In some embodiments, the full-length scaRNA or a portion thereof includes one or more secondary RNA structures assembled to form scaRNP. In some embodiments, the full-length scaRNA or a portion thereof comprises one or more sequence motifs that assemble to form a scaRNP. In some embodiments, the full-length scaRNA or a portion thereof comprises (i) one or more secondary RNA structures, and (ii) one or more sequence motifs, wherein (i), (ii) or both assemble to form a scaRNP. In some embodiments, the full-length scaRNA or a portion thereof comprises an H / ACA box, wherein the H / ACA box comprises a nucleotide sequence that comprises, from 5′ to 3′, an H consensus sequence (e.g., an H consensus sequence comprising the sequence shown in SEQ ID NO: 1) and an ACA consensus sequence (e.g., an ACA consensus sequence comprising the sequence shown in SEQ ID NO: 2).

[0139] 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 comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), wherein the scaRNA is selected from SCARNA1, SCARNA11, SCARNA14, SCARNA15, SCARNA17, SCARNA20, SCARNA21, SCARNA21B, SCARNA22, SCARNA23, SCARNA3, SCARNA4, SCARNA8.

[0140] In some embodiments, ncRNA is lncRNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure includes a full-length lncRNA or a portion thereof, and the full-length lncRNA or a portion thereof includes a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the lncRNA sequence described herein or the lncRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length lncRNA sequence described herein or the full-length lncRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, a portion of the lncRNA sequence described herein or a portion of the lncRNA sequence identified according to the methods described herein (e.g., a continuous nucleotide region in the lncRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length lncRNA or a portion thereof is assembled into RNP. In some embodiments, the full-length lncRNA or a portion thereof includes one or more secondary RNA structures assembled to form RNP. In some embodiments, the full-length lncRNA or a portion thereof includes one or more sequence motifs assembled to form RNP. In some embodiments, the full-length lncRNA or a portion thereof comprises (i) one or more secondary RNA structures, and (ii) one or more sequence motifs, wherein (i), (ii), or both assemble to form an RNP.

[0141] In some embodiments, the splicing editor nucleic acid molecules of the present disclosure comprise a full-length lncRNA or a portion thereof, wherein the full-length lncRNA or the portion thereof comprises a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), wherein the lncRNA is selected from 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, LINC0 0511, LINC00824, LINC01060, LINC01358, LINC01378, LINC01409, LINC01606, LINC01676, LI NC01943, 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.

[0142] In some embodiments, ncRNA is miscRNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure includes full-length miscRNA or a portion thereof, and the full-length miscRNA or a portion thereof includes a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the miscRNA sequence described herein or the miscRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length miscRNA sequence described herein or the full-length miscRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, a portion of the miscRNA sequence described herein or a portion of the miscRNA sequence identified according to the methods described herein (e.g., a continuous nucleotide region in miscRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length miscRNA or a portion thereof is assembled into RNP. In some embodiments, the full-length miscRNA or a portion thereof includes one or more secondary RNA structures assembled to form RNP. In some embodiments, the full-length miscRNA or a portion thereof includes one or more sequence motifs assembled to form 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, wherein (i), (ii), or both assemble to form an RNP.

[0143] In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length mis cRNA or a portion thereof, wherein the full-length mis cRNA or the portion thereof comprises a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), wherein the miscRNA is selected from RN7SKP12, RN7SKP223, RN7SKP233, RN7SKP260, RN7SKP295, RN7SKP298, RN7SKP35, RN7SK P83, RN7SKP98, RNY1, RNY1P1, RNY1P10, RNY1P11, RNY1P12, RNY1P13, RNY1P14, RNY1P15, RNY1P16, RNY1P2, RNY1P3 1P3, RNY1P4, RNY1P5, RNY1P6, RNY1P7, RNY1P8, RNY1P9, RNY3, RNY3P1, RNY3P10, RNY3P11, RNY3P12, RNY3P13, RNY3P14, R NY3P15, RNY3P16, RNY3P2, RNY3P3, RNY3P4, RNY3P5, RNY3P7, RNY3P8, RNY3P9, RNY4, RNY4P10, RNY4P13, RNY4P14, RNY4P16 , RNY4P17, RNY4P18, RNY4P19, RNY4P20, RNY4P23, RNY4P24, RNY4P25, RNY4P27, RNY4P28, RNY4P29, RNY4P3, RNY4P30, RNY4 P34, RNY4P36, RNY4P37, RNY4P6, RNY4P7, RNY4P9, VTRNA1-1, VTRNA1-2, VTRNA1-3, VTRNA2-2P, VTRNA3-1P and Vault,Y_RNA.

[0144] In some embodiments, ncRNA is Mt tRNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure comprises a full-length Mt tRNA or a portion thereof, and the full-length Mt tRNA or a portion thereof comprises a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the Mt tRNA sequence described herein or the MttRNA sequence identified according to the methods described herein is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length Mt tRNA sequence described herein or the full-length Mt tRNA sequence identified according to the methods described herein is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, a portion of the Mt tRNA sequence described herein or a portion of the Mt tRNA sequence identified according to the methods described herein (e.g., a continuous nucleotide region in the MttRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length MttRNA or a portion thereof is assembled into RNP. In some embodiments, the full-length Mt tRNA or a portion thereof comprises one or more secondary RNA structures that assemble to form RNP. In some embodiments, the full-length Mt tRNA or a portion thereof comprises one or more sequence motifs that assemble to form an RNP. In some embodiments, the full-length Mt tRNA or a portion thereof comprises (i) one or more secondary RNA structures, and (ii) one or more sequence motifs, wherein (i), (ii), or both assemble to form an RNP.

[0145] In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length Mt RNA, or a portion thereof, comprising a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), wherein the Mt tRNA is selected from the group consisting of 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.

[0146] In some embodiments, ncRNA is rRNA.In some embodiments, the splicing editor nucleic acid molecule of the present disclosure includes full-length rRNA or a portion thereof, and the full-length rRNA or a portion thereof includes a nucleotide sequence containing one or more sequence motifs as described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the rRNA sequence described herein or the rRNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length rRNA sequence described herein or the full-length rRNA sequence identified according to the methods described herein are incorporated into the splicing 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 continuous nucleotide region in rRNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, full-length rRNA or a portion thereof is assembled into RNP. In some embodiments, full-length rRNA or a portion thereof includes one or more secondary RNA structures assembled to form RNP. In some embodiments, full-length rRNA or a portion thereof includes one or more sequence motifs assembled to form 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, wherein (i), (ii), or both assemble to form an RNP.

[0147] In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a full-length rRNA or a portion thereof, wherein the full-length rRNA or the portion thereof comprises a nucleotide sequence comprising one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1), wherein the rRNA is selected from RNA5S1, RNA5S2, RNA5S3, RNA5S4, RNA5S5, RNA5S6, RNA5S7, RNA5S8, RNA5S9, RNA5S10, RNA5S11, RNA5S12, RNA5S13, RNA5S14, RNA5S15, RNA5S16, RNA5S17, RNR1, R NR2, 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.

[0148] In some embodiments, ncRNA is vault RNA. In some embodiments, the splicing editor nucleic acid molecule of the present disclosure comprises a full-length vault RNA or a portion thereof, and the full-length vault RNA or a portion thereof comprises a nucleotide sequence containing one or more sequence motifs described herein (e.g., one or more sequence motifs shown in Table 1). In some embodiments, the vault RNA sequence described herein or the vault RNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length vault RNA sequence described herein or the full-length vault RNA sequence identified according to the methods described herein are incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, a portion of the vault RNA sequence described herein or a portion of the vault RNA sequence identified according to the methods described herein (e.g., a continuous nucleotide region in the vault RNA) is incorporated into the splicing editor nucleic acid of the present disclosure. In some embodiments, the full-length vault RNA or a portion thereof is assembled into RNP. In some embodiments, the full-length vault RNA or a portion thereof comprises one or more secondary RNA structures assembled to form RNP. In some embodiments, the full-length vault RNA or a portion thereof comprises one or more sequence motifs assembled to form 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, wherein (i), (ii), or both assemble to form an RNP.

[0149] In some embodiments, a splice editor nucleic acid molecule of the present disclosure comprises a vault RNA or a portion thereof, wherein the vault RNA is VTRNA2-1.

[0150] Methods of Engineering the Disclosed Splice Editor Nucleic Acids

[0151] The present disclosure provides a method for engineering a splicing editor nucleic acid as described herein. In some embodiments, the method includes (A) identifying one or more candidate ncRNAs; (B) obtaining an ncRNA sequence from one or more candidate ncRNAs; and (C) producing a splicing editor nucleic acid comprising a nucleotide sequence comprising (i) an intron sequence, the intron sequence comprising (a) an ncRNA sequence, and (b) one or more binding domains described herein; (ii) a splicing acceptor and / or a splicing donor; and (iii) one or more exon sequences, thereby providing a splicing editor nucleic acid for trans-splicing of a target RNA (e.g., a target mRNA precursor). In some embodiments, the method includes introducing a splicing editor nucleic acid into a cell or a cell population and determining the trans-splicing efficiency of a target RNA (e.g., a target mRNA precursor) according to the methods described herein. In some embodiments, the trans-splicing efficiency of the splicing editor nucleic acid is compared with the trans-splicing efficiency of a control nucleic acid. In some embodiments, the control nucleic acid comprises (i), (ii), and (iii) and lacks an ncRNA sequence.

[0152] Methods for identifying candidate ncRNA sequences

[0153] In some embodiments, identifying one or more candidate ncRNA sequences includes (i) obtaining one or more ncRNA sequences from a database and / or by experimentally analyzing RNA expressed by a cell or organism as described herein; (ii) predicting a secondary structure formed by one or more ncRNA sequences according to the methods described herein; (iii) comparing the predicted secondary structure of (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 is substantially similar to the reference secondary structure. Computational methods for predicting the secondary structure formed by an ncRNA sequence are known in the art (see, e.g., Lorenz et al., Vienna RNA Package 2.0 Algorithms for Molecular Biology, 6:126, 2011). Methods for RNA sequence analysis by comparing a predicted secondary structure with a reference secondary structure are also known in the art (see, e.g., Eddy et al. (1994) Nucleic Acids Res 22:2079).

[0154] In some embodiments, identifying one or more candidate ncRNA sequences comprises (i) obtaining one or more ncRNA sequences from a database and / or by experimental analysis of RNA expressed by a cell or organism as described herein; (ii) predicting a secondary structure formed by the one or more ncRNA sequences according to the methods described herein; (iii) comparing the predicted secondary structure of (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 is substantially similar to the reference secondary structure and comprising a sequence motif as described herein (e.g., a sequence motif comprising one or more sequences shown in Table 1).

[0155] In some embodiments, the one or more candidate ncRNA sequences are selected from any one or any combination of the sequences shown in SEQ ID NOs: 9-657.

[0156] Methods for obtaining ncRNA sequences

[0157] In some embodiments, obtaining an ncRNA sequence for incorporation into a splice editor nucleic acid of the present disclosure comprises (i) identifying one or more candidate ncRNA sequences as described herein; and (ii) selecting the ncRNA sequence from the one or more candidate ncRNA sequences.

[0158] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of sequences shown in SEQ ID NOs: 9-657), wherein the ncRNA sequence is at least about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 nucleotides in length and comprises an Sm motif as described herein (e.g., an Sm motif as shown in Table 1). In some embodiments, the ncRNA sequence is about 7 nucleotides in length and comprises an Sm motif as described herein (e.g., an Sm motif as shown in Table 1). In some embodiments, the ncRNA sequence is about 8 nucleotides in length and comprises an Sm motif as described herein (e.g., an Sm motif as shown in Table 1). In some embodiments, the ncRNA sequence is about 9 nucleotides in length and comprises an Sm motif as described herein (e.g., an Sm motif as shown in Table 1). In some embodiments, the ncRNA sequence is about 10 nucleotides in length and comprises an Sm motif as described herein (e.g., an Sm motif as shown 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 shown 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 shown in Table 1).

[0159] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of sequences shown in SEQ ID NOs: 9-657), wherein the ncRNA sequence has a length of 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 and comprises an Sm motif described herein (e.g., an Sm motif shown in Table 1).

[0160] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence that has 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% similarity to 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 sequences shown 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 comprises an Sm motif described herein (e.g., an Sm motif shown in Table 1).

[0161] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of sequences shown 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 comprises an H-box motif described herein (e.g., an H-box motif shown in Table 1).

[0162] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence that has 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% similarity to 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 sequences shown 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 comprises an H-box motif described herein (e.g., an H-box motif shown in Table 1).

[0163] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of sequences shown 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 comprises an ACA box motif described herein (e.g., the ACA box motif shown in Table 1).

[0164] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence that has 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% similarity to 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 sequences shown 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 comprises an ACA box motif described herein (e.g., the ACA box motif shown in Table 1).

[0165] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of sequences shown 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 comprises an H-box and an ACA box motif described herein (e.g., the H-box and ACA box motifs shown in Table 1).

[0166] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence that has 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% similarity to 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 sequences shown 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 comprises an H-box and an ACA box motif described herein (e.g., the H-box and ACA box motifs shown in Table 1).

[0167] In some embodiments, obtaining the ncRNA sequence comprises selecting the ncRNA sequence from one or more candidate ncRNA sequences (e.g., one or more candidate ncRNA sequences selected from any one or any combination of the sequences shown 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 comprises (i) a C-box motif described herein (e.g., a C-box motif shown in Table 1), (ii) a C′-box motif described herein (e.g., a C′-box motif shown in Table 1), (iii) a D-box motif described herein (e.g., a D-box motif shown in Table 1), (iv) a D′-box motif described herein (e.g., a D′-box motif shown in Table 1), or (v) a combination of (i)-(iv).

[0168] In some embodiments, obtaining the ncRNA sequence comprises selecting an ncRNA sequence that has 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% similarity to 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 sequences shown in SEQ ID NOs: 9-657), wherein the length of the sequence is at least about 40, about 45, about 50, about 55, about 60, about 65, about 70, 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%. about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides and comprises (i) a C-box motif described herein (e.g., a C-box motif shown in Table 1), (ii) a C′-box motif described herein (e.g., a C′-box motif shown in Table 1), (iii) a D-box motif described herein (e.g., a D′-box motif shown in Table 1), (iv) a D′-box motif described herein (e.g., a D′-box motif shown in Table 1), or (v) a combination of (i)-(iv).

[0169] Methods for producing splice editor nucleic acids

[0170] The splice editor nucleic acids provided herein are produced by suitable nucleic acid synthesis methods or means known in the art. In some embodiments, the splice editor nucleic acids are produced as RNA. In some embodiments, the splice editor nucleic acids are produced as DNA. The present disclosure also provides a delivery system comprising the splice editor nucleic acid, such as a vector comprising the splice editor nucleic acid, a lipid particle comprising the splice editor nucleic acid.

[0171] Methods for producing splice editor nucleic acids include, but are not limited to, in vitro transcription (IVT), synthesis 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 utilized.

[0172] In some embodiments, the present disclosure provides splice editor nucleic acids produced using IVT enzymatic synthesis methods. Methods for making polynucleotides by IVT are known in the art and are described in International Application PCT / US2013 / 30062.

[0173] In some embodiments, the present disclosure provides a splicing editor nucleic acid chemically synthesized by any means described in the art. In some embodiments, the splicing 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 determined chemical structure (sequence). The method of oligonucleotide synthesis is known in the art (see, for example, Reese (2005) Organic & Biomolecular Chemistry 3 (21): 3851). Although the chemical synthesis procedure is constantly expanding, when the polynucleotide length is significantly increased to more than one hundred nucleotides, it becomes more challenging to purify such nucleic acids by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels, such as PAGE). One method for generating longer nucleic acids is to produce two or more molecules and connect them together.

[0174] Methods for determining trans-splicing efficiency of splice editor nucleic acids

[0175] 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 a splice editor nucleic acid molecule described herein.

[0176] In some embodiments, the method includes using a fluorescence-based splicing reporter assay. In some embodiments, the assay includes contacting a reporter cell or cell population with a splicing editor nucleic acid molecule according to the methods described herein (e.g., by transfection with a viral vector encoding a splicing editor nucleic acid molecule), wherein the splicing editor nucleic acid molecule comprises at least one exon encoding a reporter molecule, wherein the trans-splicing event is indicated by the presence of a fluorescent signal from the reporter molecule, which can be 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 splicing editor nucleic acid molecule comprises a nucleotide sequence encoding a first portion of a fluorescent protein, and the target RNA comprises a nucleotide sequence encoding a second portion of a fluorescent protein, wherein trans-splicing produces an RNA comprising a nucleotide sequence encoding a full-length fluorescent protein, and wherein trans-splicing events are detected using a fluorescence measurement method (e.g., FACS).

[0177] In some embodiments, a splicing editor nucleic acid is introduced into a cell for a period of time using the methods described herein (e.g., by a viral vector or a non-viral vector), followed by extraction of RNA from the cell and detection of trans-splicing products. For example, mRNA spliced ​​from a target RNA (e.g., a target mRNA precursor) is analyzed by a suitable method known in the art (e.g., endpoint or quantitative RT-PCR or RNA sequencing). In some embodiments, a cell or cell population is contacted with a splicing editor nucleic acid molecule, wherein the degree of trans-splicing is determined using next-generation sequencing (NGS) technology. For example, in some embodiments, mRNA extracted from a cell treated with or contacted with a splicing editor nucleic acid provided by the present disclosure is enzymatically converted into cDNA, which is further analyzed by NGS analysis to determine the extent of mRNA molecules comprising exon sequences incorporated from splicing editor nucleic acids.

[0178] In some embodiments, trans-splicing is determined by protein sequence analysis of polypeptides translated from mRNA spliced ​​from mRNA precursors. In some embodiments, RNA-guided molecules correct mutations by incorporating corrected exons, wherein the translation of mRNA produced by trans-splicing of mRNA precursors and splicing editor nucleic acids produces a polypeptide comprising an amino acid sequence encoded by the corrected exons. The techniques used for protein sequence analysis include, but are not limited to, Sanger sequencing, mass spectrometry, functional assays for measuring peptidase activity, or immunoblotting using antibodies that react with the corrected amino acid sequence.

[0179] In some embodiments, trans-splicing is determined by measuring the activity of a protein translated from an mRNA spliced ​​from a pre-mRNA. For example, in some embodiments, the protein is an enzyme, and the method for measuring trans-splicing comprises measuring enzyme activity using a functional ELISA.

[0180] In some embodiments, methods of measuring trans-splicing efficiency using a splice editor nucleic acid of the present disclosure are described in U.S. Application No. 16 / 994,230, which is incorporated herein by reference.

[0181] In some embodiments, the method of measuring the trans-splicing efficiency of a splice editor nucleic acid using the present disclosure is any of the methods 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.

[0182] 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.

[0183] In some embodiments, the splice editor nucleic acid molecules described herein achieve a higher trans-splicing efficiency than a splice editor nucleic acid molecule without an ncRNA, as measured using the methods described herein, wherein the splice editor nucleic acid molecules described herein comprise a nucleotide sequence comprising: (i) at least one intron sequence comprising one or more binding domains and an ncRNA, each binding domain being complementary to a target sequence in a 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. In some embodiments, the trans-splicing efficiency achieved by the splice editor nucleic acid molecules described herein is increased by 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 compared to the trans-splicing efficiency of the splice editor nucleic acid molecule without the ncRNA.

[0184] Exemplary splice editor nucleic acids

[0185] In some embodiments, the present disclosure provides a subgroup I of nucleic acids for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence, the intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in the target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence, the ncRNA sequence forming a secondary structure and / or comprising a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) one or more splicing signals; (b) a splicing acceptor; and (c) at least one exon sequence.

[0186] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) one or more splicing signals, wherein the ncRNA is an 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, U4atacsnRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12snRNA, and U7 snRNA.

[0187] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA comprising a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0188] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure and comprises a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0189] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) a ncRNA sequence that forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA); and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0190] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence that forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA (e.g., a pre-mRNA), wherein the ncRNA is an snRNA; and (iii) 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.

[0191] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) a ncRNA sequence comprising a sequence motif that assembles to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0192] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) a ncRNA sequence, the ncRNA sequence forming a secondary structure and comprising a sequence motif, the secondary structure and the sequence motif assembling to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0193] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a 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 a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA); and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0194] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence forming a secondary structure and / or comprising a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA), wherein the ncRNA is a snRNA; and (iii) 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.

[0195] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence comprising a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0196] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence forming a secondary structure and comprising a sequence motif to guide the one or more binding domain sequences to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0197] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence forming a secondary structure and / or comprising a sequence motif that assembles to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA); and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0198] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence forming a secondary structure and / or comprising a sequence motif that assembles to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA), wherein the ncRNA is a snRNA; and (iii) 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, U6snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0199] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides comprising a sequence motif that assembles to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0200] In some embodiments, the nucleic acids of subgroup I comprise a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA); (ii) an ncRNA sequence of about 7 to about 300 nucleotides in length, the ncRNA sequence forming a secondary structure and comprising a sequence motif, the secondary structure and sequence motif assembling to form an RNP to guide the binding domain to the target RNA (e.g., a pre-mRNA), wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif; and (iii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0201] In some embodiments, the nucleic acids of subgroup I comprise one binding domain. In some embodiments, the nucleic acids of subgroup I comprise two binding domains. In some embodiments, the nucleic acids of subgroup I comprise three binding domains. In some embodiments, the nucleic acids of subgroup I comprise four binding domains. In some embodiments, the nucleic acids of subgroup I comprise five binding domains.

[0202] In some embodiments, the target RNA is an mRNA precursor. In some embodiments, the mRNA precursor comprises from 5' to 3': a 5' exon, a splicing donor, an intron, a splicing 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 nucleic acid of subgroup I is complementary to a target sequence in a target RNA (e.g., an mRNA precursor), wherein the target sequence is located in the 5' exon of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing donor of the mRNA precursor. In some embodiments, the target sequence is located within an intron of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing acceptor of the mRNA precursor. In some embodiments, the target sequence is located in the 3' exon of the mRNA precursor. In some embodiments, trans-splicing occurs between the splicing donor of the mRNA precursor and the splicing acceptor of the nucleic acid of subgroup I. In some embodiments, trans-splicing results in the 3' end of the 5' exon of the pre-mRNA being joined to the 5' end of the at least one exon sequence of the subgroup I nucleic acid.

[0203] In some embodiments, one or more splicing signals of the nucleic acids of subgroup I comprise a branch point. In some embodiments, one or more splicing signals of the nucleic acids of subgroup I comprise a polypyrimidine stretch. In some embodiments, one or more splicing signals of the nucleic acids of subgroup I comprise a branch point and a polypyrimidine stretch. In some embodiments, one or more splicing signals further comprise an ISE. In some embodiments, one or more splicing signals further comprise an ISS.

[0204] In some embodiments, the at least one exonic sequence of the nucleic acids of subset I comprises an ESE. In some embodiments, the at least one exonic sequence of the nucleic acids of subset I comprises an ESS.

[0205] In some embodiments, the present 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 a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, the at least one intron sequence comprising (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domains to the target RNA.

[0206] In some embodiments, the present disclosure provides a nucleic acid of subgroup II for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5' to 3': (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, the at least one intron sequence comprising (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in the target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the ncRNA 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, U12snRNA, and U7 snRNA.

[0207] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0208] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a precursor mRNA), wherein the ncRNA sequence forms a secondary structure and comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0209] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA.

[0210] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5' to 3', (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, the at least one intron sequence comprising (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA, and wherein the ncRNA is a snRNA. In some embodiments, the snRNA is selected from U1 snRNA, U2 snRNA, U4 snRNA, U4atacsnRNA, U5 snRNA, U6 snRNA, U6atac snRNA, U11 snRNA, U12 snRNA, and U7 snRNA.

[0211] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0212] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence, and (ii) one or more binding domain sequences, each binding domain sequence is complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure and comprises a sequence motif, wherein the secondary structure and sequence motif assemble to form an RNP to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0213] In some embodiments, the nucleic acids of subgroup II comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domains to the target RNA.

[0214] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the ncRNA 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.

[0215] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0216] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure and comprises a sequence motif to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0217] In some embodiments, the nucleic acids of subgroup II comprise a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA.

[0218] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, the at least one intron sequence comprising (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA forms a secondary structure and / or comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA, and wherein the ncRNA 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.

[0219] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence comprises a sequence motif that assembles to form an RNP to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0220] In some embodiments, the nucleic acid of subgroup II comprises a nucleotide sequence comprising, from 5′ to 3′, (a) at least one exon sequence; (b) a splice donor; (c) at least one intron sequence, wherein the at least one intron sequence comprises (i) an ncRNA sequence of about 7 to about 300 nucleotides in length, and (ii) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a target sequence in a target RNA (e.g., a pre-mRNA), wherein the ncRNA sequence forms a secondary structure and comprises a sequence motif, wherein the secondary structure and sequence motif assemble to form an RNP to guide the one or more binding domains to the target RNA, and wherein the sequence motif is selected from an Sm sequence motif and an Lsm sequence motif.

[0221] In some embodiments, the nucleic acids of subgroup II comprise one binding domain. In some embodiments, the nucleic acids of subgroup II comprise two binding domains. In some embodiments, the nucleic acids of subgroup II comprise three binding domains. In some embodiments, the nucleic acids of subgroup II comprise four binding domains. In some embodiments, the nucleic acids of subgroup II comprise five binding domains.

[0222] In some embodiments, the target RNA is a mRNA precursor. In some embodiments, the mRNA precursor comprises from 5' to 3': a 5' exon, a splicing donor, an intron, a splicing 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 nucleic acid of subgroup II is complementary to a target sequence in a target RNA (e.g., an mRNA precursor), wherein the target sequence is located in the 5' exon of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing donor of the mRNA precursor. In some embodiments, the target sequence is located within an intron of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing acceptor of the mRNA precursor. In some embodiments, the target sequence is located in the 3' exon of the mRNA precursor. In some embodiments, trans-splicing occurs between the splicing donor of the nucleic acid of subgroup II and the splicing acceptor of the mRNA precursor. In some embodiments, trans-splicing results in the 3' end of the 5' exon of the at least one exon sequence of the nucleic acid of subgroup II being joined to the 5' end of the 3' exon of the mRNA precursor.

[0223] In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup II comprises an ESE. In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup II comprises an ESS.

[0224] In some embodiments, the present disclosure provides a subgroup III nucleic acid for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence, the at least one intron sequence comprising (i) a snoRNA sequence comprising an H / ACA box or a C / D box and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence; and (ii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0225] In some embodiments, the present disclosure provides a subgroup III nucleic acid for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence, the at least one intron sequence comprising (i) a snoRNA sequence comprising an H / ACA box or a C / D box to guide one or more binding domain sequences to the target RNA (e.g., pre-mRNA), and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence; and (ii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0226] In some embodiments, the nucleic acid of subgroup III comprises a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence, the at least one intron sequence comprising (i) a snoRNA sequence comprising an H / ACA box or a C / D box, which assembles to form an RNP to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence; and (ii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0227] In some embodiments, the nucleic acid of subgroup III comprises a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence comprising (i) a snoRNA sequence comprising an H / ACA box or a C / D box to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences of about 4 to about 30 nucleotides in length, each binding domain sequence being complementary to the pre-mRNA target sequence; and (ii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0228] In some embodiments, the nucleic acid of subgroup III comprises a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence, the at least one intron sequence comprising (i) a snoRNA sequence comprising an H / ACA box or a C / D box, which assembles to form an RNP to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences of about 4 to about 30 nucleotides in length, each binding domain sequence being complementary to the pre-mRNA target sequence; and (ii) one or more splicing signals; (b) a splice acceptor; and (c) at least one exon sequence.

[0229] In some embodiments, the target RNA is a mRNA precursor. In some embodiments, the mRNA precursor comprises from 5' to 3': a 5' exon, a splicing donor, an intron, a splicing 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 nucleic acid of subgroup III is complementary to a target sequence in a target RNA (e.g., an mRNA precursor), wherein the target sequence is located in the 5' exon of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing donor of the mRNA precursor. In some embodiments, the target sequence is located within an intron of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing acceptor of the mRNA precursor. In some embodiments, the target sequence is located in the 3' exon of the mRNA precursor. In some embodiments, trans-splicing occurs between the splicing donor of the mRNA precursor and the splicing acceptor of the nucleic acid of subgroup III. In some embodiments, trans-splicing results in the 3' end of the 5' exon of the pre-mRNA being joined to the 5' end of the at least one exonic sequence of the nucleic acid of subgroup III.

[0230] In some embodiments, one or more splicing signals of a nucleic acid of subgroup III comprise a branch point. In some embodiments, one or more splicing signals of a nucleic acid of subgroup III comprise a polypyrimidine tract. In some embodiments, one or more splicing signals of a nucleic acid of subgroup III comprise a branch point and a polypyrimidine tract. In some embodiments, one or more splicing signals further comprise an ISE. In some embodiments, one or more splicing signals further comprise an ISS.

[0231] In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup III comprises an ESE. In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup III comprises an ESS.

[0232] In some embodiments, the present disclosure provides a subgroup IV nucleic acid for targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell, the nucleic acid comprising 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, the at least one intron sequence comprising a snoRNA sequence containing an H / ACA box or a C / D box and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence.

[0233] In some embodiments, the nucleic acid of subgroup IV comprises 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, wherein the at least one intron sequence comprises a snoRNA sequence comprising an H / ACA box or a C / D box to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence.

[0234] In some embodiments, the nucleic acid of subgroup IV comprises 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, wherein the at least one intron sequence comprises a snoRNA sequence comprising an H / ACA box or a C / D box, which assembles to form an RNP to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences, each binding domain sequence being complementary to the pre-mRNA target sequence.

[0235] In some embodiments, the nucleic acid of subgroup IV comprises 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, wherein the at least one intron sequence comprises a snoRNA sequence comprising an H / ACA box or a C / D box to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences of about 4 to about 30 nucleotides in length, each binding domain sequence being complementary to the pre-mRNA target sequence.

[0236] In some embodiments, the nucleic acid of subgroup IV comprises 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, wherein the at least one intron sequence comprises a snoRNA sequence comprising an H / ACA box or a C / D box, which assembles to form an RNP to guide one or more binding domain sequences to a target RNA (e.g., a pre-mRNA), and one or more binding domain sequences of about 4 to about 30 nucleotides in length, each binding domain sequence being complementary to the pre-mRNA target sequence.

[0237] In some embodiments, the target RNA is a mRNA precursor. In some embodiments, the mRNA precursor comprises from 5' to 3': a 5' exon, a splicing donor, an intron, a splicing 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 nucleic acid of subgroup IV is complementary to a target sequence in a target RNA (e.g., an mRNA precursor), wherein the target sequence is located in the 5' exon of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing donor of the mRNA precursor. In some embodiments, the target sequence is located within an intron of the mRNA precursor. In some embodiments, the target sequence is located at the proximal end of the splicing acceptor of the mRNA precursor. In some embodiments, the target sequence is located in the 3' exon of the mRNA precursor. In some embodiments, trans-splicing occurs between the splicing donor of the nucleic acid of subgroup IV and the splicing acceptor of the mRNA precursor. In some embodiments, trans-splicing results in the 3' end of the 5' exon of the at least one exon sequence of the nucleic acid of subgroup IV being joined to the 5' end of the 3' exon of the mRNA precursor.

[0238] In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup IV comprises an ESE. In some embodiments, the at least one exonic sequence of the nucleic acids of subgroup IV comprises an ESS.

[0239] In some embodiments, the nucleic acids of subgroup III or subgroup IV comprise an H / ACA box, wherein the H / ACA box comprises a nucleotide sequence from 5′ to 3′ having an H consensus sequence and an ACA consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located upstream of the H consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located downstream of the ACA consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located between the H consensus sequence and the ACA consensus sequence.

[0240] In some embodiments, nucleic acids of subgroup III or subgroup IV comprise an H / ACA box comprising a nucleotide sequence from 5' to 3' comprising an H consensus sequence and an ACA consensus sequence; and a binding domain. In some embodiments, the binding domain sequence is located upstream of the H consensus sequence. In some embodiments, the binding domain sequence is located downstream of the ACA consensus sequence. In some embodiments, the binding domain sequence is located between the H consensus sequence and the ACA consensus sequence.

[0241] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box comprising a nucleotide sequence from 5′ to 3′ 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.

[0242] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box and a binding domain, wherein the H / ACA box comprises a nucleotide sequence having an H consensus sequence and an ACA consensus sequence from 5′ to 3′, and wherein 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.

[0243] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box and two binding domains, wherein the H / ACA box comprises a nucleotide sequence having an H consensus sequence and an ACA consensus sequence from 5′ to 3′, and wherein 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.

[0244] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box and three binding domains, wherein the H / ACA box comprises a nucleotide sequence having an H consensus sequence and an ACA consensus sequence from 5′ to 3′, and wherein 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.

[0245] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box and four binding domains, wherein the H / ACA box comprises a nucleotide sequence having an H consensus sequence and an ACA consensus sequence from 5′ to 3′, and wherein 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.

[0246] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises an H / ACA box and five binding domains, wherein the H / ACA box comprises a nucleotide sequence having an H consensus sequence and an ACA consensus sequence from 5′ to 3′, and wherein 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.

[0247] In some embodiments, the nucleic acids of subgroup III or subgroup IV comprise a C / D box, wherein the C / D box comprises a nucleotide sequence from 5′ to 3′ having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located upstream of the C consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located between the C consensus sequence and the D′ consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located between the C′ consensus sequence and the D consensus sequence. In some embodiments, the one or more binding domain sequences of the nucleic acids of subgroup III or subgroup IV are located downstream of the D consensus sequence.

[0248] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises a C / D box and a binding domain, wherein the C / D box comprises a nucleotide sequence from 5′ to 3′ having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence; and wherein the one 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.

[0249] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises a C / D box and two binding domains, wherein the C / D box comprises a nucleotide sequence having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence from 5′ to 3′; and wherein the two binding domains are each independently 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; and / or downstream of the D consensus sequence.

[0250] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises a C / D box and three binding domains, wherein the C / D box comprises a nucleotide sequence having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence from 5′ to 3′; and wherein the three binding domains are each independently 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; and / or downstream of the D consensus sequence.

[0251] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises a C / D box and four binding domains, wherein the C / D box comprises a nucleotide sequence having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence from 5′ to 3′; and wherein the four binding domains are each independently 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; and / or downstream of the D consensus sequence.

[0252] In some embodiments, the nucleic acid of subgroup III or subgroup IV comprises a C / D box and five binding domains, wherein the C / D box comprises a nucleotide sequence having a C consensus sequence, a D′ consensus sequence, a C′ consensus sequence, and a D consensus sequence from 5′ to 3′; and wherein the five binding domains are each independently 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; and / or downstream of the D consensus sequence.

[0253] In some embodiments, the nucleic acids of the present disclosure (e.g., nucleic acids of any of subgroups I-IV) comprise at least one binding domain sequence that is fully complementary to a target sequence. In some embodiments, the nucleic acids comprise at least one binding domain sequence that is partially complementary to a target sequence (e.g., comprises at least 95% complementarity to the target sequence). In some embodiments, the nucleic acids comprise at least one binding domain sequence that is fully complementary to a target sequence and at least one binding domain sequence that is partially complementary to a target sequence (e.g., comprises at least 95% complementarity to the target sequence).

[0254] In some embodiments, the nucleic acids of the present disclosure (e.g., nucleic acids of any one of subgroups I-IV) have a sequence length of up to about 20,000 nucleotides. In some embodiments, the sequence length is up to about 10,000 nucleotides. In some embodiments, the sequence length is up to about 9,000 nucleotides. In some embodiments, the sequence length is up to about 8,000 nucleotides. In some embodiments, the sequence length is up to about 7,000 nucleotides. In some embodiments, the sequence length is up to about 6,000 nucleotides. In some embodiments, the sequence length is up to about 5,000 nucleotides. In some embodiments, the sequence length is from about 50 to about 500 nucleotides. In some embodiments, the sequence length is from about 50 to about 1000 nucleotides. In some embodiments, the sequence length is from about 100 to about 500 nucleotides. In some embodiments, the sequence length is from about 100 to about 1000 nucleotides. In some embodiments, the sequence length is from about 500 to about 1000 nucleotides. In some embodiments, the length of the sequence is about 500 to about 2000 nucleotides. In some embodiments, the length of the sequence is about 500 to about 3,000 nucleotides. In some embodiments, the length of the sequence is about 500 to about 4,000 nucleotides. In some embodiments, the length of the sequence is about 500 to about 5,000 nucleotides. In some embodiments, the length of the sequence is about 1,000 to about 5,000 nucleotides. In some embodiments, the length of the sequence is about 1,000 to about 10,000 nucleotides. In some embodiments, the length of the sequence is about 5,000 to about 15,000 nucleotides. In some embodiments, the length of the sequence is about 5,000 to about 20,000 nucleotides.

[0255] In some embodiments, the splicing editor nucleic acid molecules of the present disclosure comprise a sequence selected from Table 3 or a portion thereof. Table 3 provides exemplary nucleotide sequences of splicing editor nucleic acids of the present disclosure. As shown in the table, the sequence regions are divided by a hyphen (-), and the identification of the 5' to 3' region is Region 1, Region 2, Region 3, and optional Region 4. In some embodiments, the splicing editor nucleic acid molecules of the present disclosure comprise a sequence having the formula 5'-[A]-[B]-3', wherein [A] is a nucleotide sequence selected from Table 3, and [B] is a sequence from 5' to 3' comprising a splicing acceptor and one or more exon sequences. In some embodiments, [A] comprises a nucleotide sequence selected from Table 3, wherein the RNA binding domain is replaced with an RNA binding domain described herein.

[0256] In some embodiments, the splice editor nucleic acid molecules of the present disclosure comprise one or more RNA binding domains described herein, ncRNA sequences, intron sequences, splice acceptors, and one or more exon sequences from 5′ to 3′, wherein the ncRNA sequences are about 90%, 95%, 98%, 99%, or 100% identical to the ncRNA sequences identified in Table 3. In some embodiments, the splice editor nucleic acid molecules of the present disclosure comprise one or more exon sequences, splice donors, intron sequences, ncRNA sequences, and one or more RNA binding domains described herein from 5′ to 3′, wherein the ncRNA sequences are about 90%, 95%, 98%, 99%, or 100% identical to the ncRNA sequences identified in Table 3. In some embodiments, the intron sequences are about 90%, 95%, 98%, 99%, or 100% identical to the intron sequences identified in Table 3.

[0257] carrier

[0258] In some embodiments, the present disclosure provides vectors comprising one or more splicing 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 attached. 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.

[0259] In some embodiments, the vector is an expression vector, wherein the expression vector 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, such as a plasmid, phage, virus, or cosmid, to which another DNA segment (i.e., an "insert") is attached so that the attached segment is replicated in the cell.

[0260] 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.

[0261] In some embodiments, the vector or expression vector is a viral vector in which the additional nucleic acid segment is connected to the viral genome. Non-limiting exemplary viral vectors include those based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, and picornavirus. Non-limiting exemplary viral vectors also include those based on retroviruses, such as murine leukemia virus, spleen necrosis virus, and those 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, vectors are used for eukaryotic target cells and include, but are not limited to, pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).

[0262] In some embodiments, the vector comprises one or more transcription and / or translation control elements. In some embodiments, the one or more transcription and / or translation control elements used depend on the target cell population and the vector system. In some embodiments, any number of suitable transcription and translation control elements are used in the expression vector, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., such as those described further below.

[0263] In some embodiments, a vector comprising a splicing editor nucleic acid of the present disclosure is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, the transcriptional control element is functional in a eukaryotic cell (e.g., a mammalian cell, e.g., a human cell). In some embodiments, the splicing editor nucleic acid sequence is operably linked to one or more control elements that enable expression in a eukaryotic cell (e.g., a mammalian cell, e.g., a human cell).

[0264] In some embodiments, the promoter contained in the expression vector 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-β-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 molecules including, but not limited to, doxycycline; an RNA polymerase, such as T7 RNA polymerase; an estrogen receptor; an estrogen receptor fusion, etc.

[0265] In some embodiments, the promoter is a constitutive promoter (eg, CMV promoter, UBC promoter).

[0266] In some embodiments, the promoter is a promoter that is spatially restricted and / or time-restricted (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.). The promoter of spatial restriction may also be referred to as an enhancer, a transcriptional control element, a control sequence, etc. Any suitable promoter of spatial restriction is suitable for use in the present disclosure, and the selection of suitable promoters (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 germline, a promoter that drives expression in the lung, a promoter that drives expression in muscle, a promoter that drives expression in islet cells of the pancreas, etc.) will depend on the organism. For example, various promoters of spatial restriction are known for plants, flies, worms, mammals, mice, etc. Therefore, the promoter of spatial restriction can be used to regulate the expression of splicing editor nucleic acids in a variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also time-restricted, so that the promoter is in an "on (ON)" state or an "off (OFF)" state during a specific stage of embryonic development or during a specific stage of a biological process. For purposes 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.

[0267] Promoters suitable for use in the present disclosure include those derived from viruses and referred to herein as viral promoters, or they include those derived from organisms, including prokaryotes or eukaryotes. In some embodiments, promoters suitable for use in the present disclosure include any promoter that drives expression by RNA polymerase (e.g., pol I, pol II, pol III).

[0268] 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 such as 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.

[0269] Exemplary eukaryotic promoters (i.e., promoters that are functional in eukaryotic cells) include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the herpes simplex virus (HSV) thymidine kinase promoter, the early and late SV40 promoters, the long terminal repeats (LTRs) of retroviruses, the human elongation factor-1 promoter (EF1), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG), the murine stem cell virus promoter (MSCV), the phosphoglycerate kinase-1 locus promoter (PGK), and mouse metallothionein-I.

[0270] In some embodiments, the present disclosure provides vectors comprising a splicing editor nucleic acid and an RNA polymerase III promoter (e.g., U6 and H1) as described herein. Descriptions and parameters for enhancing the use of such promoters are known in the art, and additional information and methods are regularly described; see, for example, Ma, H. et al., Molecular Therapy-Nucleic Acids 3, e161 (2014).

[0271] In some embodiments, the expression vector comprises a ribosome binding site and a transcription terminator for translation initiation. In some embodiments, the expression vector comprises an appropriate sequence for amplifying expression. In some embodiments, the expression vector comprises a nucleotide sequence encoding a non-natural tag (e.g., a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.), for example, which is operably connected to a splicing editor nucleic acid.

[0272] Methods for introducing nucleic acids into host cells or host cell populations are known in the art, and any known method can be used to introduce nucleic acids (e.g., expression constructs) into cells. In some embodiments, established transfection techniques are used; see, for example, Angel and Yanik (2010) PLoS ONE 5(7):e 11756, and the commercially available TransMessen from Qiagen. Reagents, Stemfect™ RNA transfection kit from Stemgent and - mRNA transfection kit (see also Beumer et al. (2008) PNAS 105(50):19821-19826), providing the splice editor nucleic acid molecule or a vector comprising the splice editor nucleic acid molecule to the cell population.

[0273] In some embodiments, the splicing editor nucleic acid molecule is introduced into a cell or cell population as RNA. In some embodiments, the RNA has chemical properties suitable for delivery, tolerance, and intracellular stability, for example, after administration in vivo or in vitro. In some embodiments, the RNA is modified, for example, comprising a modified sugar moiety, a modified internucleoside bond, a modified nucleoside, a modified nucleotide, and / or a combination thereof. In some embodiments, the modified RNA exhibits one or more of the following properties: no immunostimulatory properties; nuclease resistance; improved cellular uptake; extended half-life; increased translation efficiency; and / or no toxicity to cells or mammals, for example, in vivo or after contact with cells in vitro or in vitro.

[0274] Delivery Agent

[0275] In some embodiments, the delivery of the splicing editor nucleic acid described herein is carried out by one or more methods described herein. In some embodiments, the splicing 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 method described herein is suitable for administering the splicing editor nucleic acid of the present disclosure to a target cell population or target tissue for trans-splicing of mRNA precursors in cells, in vitro, or in vivo targeted target cells or target tissues.

[0276] In some embodiments, delivery comprises administering the splice editor nucleic acid as RNA or DNA. In some embodiments, delivery comprises administering the splice editor nucleic acid as DNA formulated into LNPs or polymer nanoparticles. In some embodiments, delivery comprises administering the splice editor nucleic acid as RNA formulated into LNPs or polymer nanoparticles.

[0277] In some embodiments, delivery includes administering a recombinant expression vector (e.g., plasmid, viral vector) comprising a splicing editor nucleic acid. In some embodiments, the recombinant expression vector is a non-viral vector (e.g., plasmid). In some embodiments, the recombinant expression vector is a viral vector (e.g., AAV). In some embodiments, delivery includes formulating one or more recombinant expression vectors using LNP or polymer nanoparticles. In some embodiments, a combination of viral vectors and non-viral delivery vehicles is used.

[0278] In some embodiments, the splicing editor nucleic acid molecules are delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule-RNA conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. 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, which can also be used to deliver other polynucleotides).

[0279] Viral delivery

[0280] In some embodiments, the splicing 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 splicing editor nucleic acids described herein. In some embodiments, the cloning capacity of the viral vector is sufficient to deliver the splicing editor nucleic acid.

[0281] In some embodiments, recombinant adeno-associated virus (rAAV) vectors are used for delivery. The technology for producing rAAV particles is a standard technology in the art, wherein the AAV genome to be packaged (comprising polynucleotides to be delivered, such as nucleic acids encoding one or more gRNAs and / or site-directed endonucleases), rep and cap genes, and helper virus functions are provided to cells. The production of rAAV generally 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 separated from the rAAV genome (i.e., not in the rAAV genome), and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which recombinant viruses can be derived, and can also be derived from an AAV serotype that differs 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-modified AAV vectors. For example, U.S. Pat. No. 7,056,602 discloses the production of pseudotyped rAAV.

[0282] In some embodiments, the method for generating packaging cells involves creating a cell line that stably expresses all the necessary components required for the production of AAV particles. For example, a plasmid (or multiple plasmids) comprising an rAAV genome lacking AAV rep and cap genes, an AAV rep and cap gene separated from the rAAV genome, and a selectable marker (e.g., a neomycin resistance gene) is integrated into the cell genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79: 2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23: 65-73) or direct blunt end connection (Senapathy and Carter, 1984, J. Biol. Chem., 259: 4661-4666). The packaging cell line can then be infected with a helper virus (e.g., adenovirus). The advantage of this method is that the cell selectivity is good and it is suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0283] 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 methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin 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. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent 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.

[0284] In addition to adeno-associated viral vectors, other viral vectors can also be used. Such viral vectors include, but are not limited to, adenovirus, lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpes virus, Epstein-Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.

[0285] Nanoparticle compositions

[0286] In some embodiments, the splicing editor nucleic acid of the present disclosure or a recombinant expression vector comprising a splicing editor nucleic acid is delivered to a host cell (e.g., in vitro) or a subject via nanoparticles (e.g., lipid nanoparticles). In some embodiments, the nucleic acid or expression vector is formulated in a nanoparticle or other delivery vehicle (e.g., polymer nanoparticles) to promote cellular uptake and / or protect them from degradation when delivered to a subject.

[0287] In some embodiments, the nanoparticle composition comprises lipids. Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any number of lipids may be present, including cationic and / or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, conjugated lipids (e.g., PEGylated lipids), and / or structural lipids. Such lipids may be used alone or in combination.

[0288] Nanoparticles are ultrafine particles with a size generally between 1 and 100 to 500 nanometers (nm), with an interface layer around them, and generally exhibiting size-dependency or size-dependent properties. Nanoparticle compositions are varied and include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, nanoparticle compositions can be liposomes with a lipid bilayer having a diameter of 500 nm or less. In some embodiments, nanoparticle compositions are vesicles comprising one or more lipid bilayers. In certain embodiments, nanoparticle compositions include two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked. The lipid bilayers can include one or more ligands, proteins, or channels.

[0289] In some embodiments, the nanoparticle composition comprises a splice editor nucleic acid and / or a recombinant expression vector comprising a splice editor nucleic acid.

[0290] In some embodiments, the present disclosure provides an LNP composition comprising: (a) a splicing editor nucleic acid molecule as described herein or an expression vector comprising a splicing 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 ionizable lipid, a PEG lipid, a lipidoid, and cholesterol or a cholesterol derivative. In some embodiments, the present disclosure provides an LNP composition comprising: (a) a splicing editor nucleic acid molecule as described herein or an expression vector comprising a splicing editor nucleic acid molecule; and (b) one or more lipid moieties selected from the group consisting of: an ionizable lipid, an amino lipid, an anionic lipid, a neutral lipid, an amphiphilic lipid, a helper lipid, a structural lipid, a PEG lipid, and a lipidoid, and optionally (c) a targeting moiety.

[0291] 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, it is known in the art that certain ionizable lipids can 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.

[0292] 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 online dilution. Other techniques and methods suitable for preparing the LNPs described herein include coacervation, microemulsions, supercritical fluid technology, and phase transition temperature (PIT) technology.

[0293] Pharmaceutical composition

[0294] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a splice editor nucleic acid, recombinant expression vector, or delivery system described herein, in combination with a suitable pharmaceutically acceptable carrier or diluent.

[0295] 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 into 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 one or more splice editor nucleic acids or a recombinant expression vector.

[0296] Depending on the specific mode of administration and dosage form, exemplary pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, etc. Depending on the formulation and route of administration, the contemplated pharmaceutical composition can generally be formulated to achieve a physiologically compatible pH value. In some embodiments, the composition comprises a therapeutically effective amount of one or more splicing editor nucleic acids or recombinant expression vectors, and one or more pharmaceutically acceptable excipients.

[0297] 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 (e.g., oils, water, saline, glycerol, and ethanol), wetting agents or emulsifiers, pH buffer substances, etc.

[0298] The pharmaceutical composition can be formulated into solutions, suppositories, injections, and the like. In some embodiments, the pharmaceutical composition is formulated to result in systemic administration of one or more splicing editor nucleic acids or recombinant expression vectors, for example, following enteral or parenteral administration. In some embodiments, the pharmaceutical composition is formulated to result in local administration of one or more splicing editor nucleic acids or recombinant expression vectors, for example, following regional administration or implantation. In some embodiments, the pharmaceutical composition is formulated for immediate activity or sustained release of one or more splicing editor nucleic acids or recombinant expression vectors.

[0299] Typically, an effective amount of a splicing editor nucleic acid, recombinant expression vector, or delivery system described herein can be provided, for example, as a method for treating a subject suffering from a disease or condition. Methods for calculating an effective amount or effective dose are known to those of ordinary skill in the art. The final amount administered depends on the route of administration and the nature of the condition to be treated. A competent clinician will be able to determine an effective amount of a splicing editor nucleic acid, recombinant expression vector, or delivery system described herein to administer to a patient, thereby stopping or reversing the progression of the condition.

[0300] In some embodiments, based on animal data and other information available for the trans-splicing system, a clinician can determine the maximum safe dose for an individual depending on the route of administration. For example, a dose administered intravenously may be greater than a dose administered intrathecally, as a given therapeutic composition is administered to a greater amount of body fluid. Similarly, a composition that is rapidly cleared from the body may be administered at a higher dose or in repeated doses in order to maintain therapeutic concentrations. Using ordinary techniques, a competent clinician will be able to optimize the dose of a particular therapeutic agent during routine clinical trials.

[0301] For inclusion in a drug, the splicing editor nucleic acid, recombinant expression vector or delivery system described herein can be obtained from a suitable commercial source. In some embodiments, the therapy based on the splicing editor nucleic acid, recombinant expression vector or delivery system described herein to be used for therapeutic administration must be sterile. The therapeutic composition is typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle. In some embodiments, the therapeutic component is stored in a unit or multi-dose container, such as a sealed ampoule or vial, in the form of an aqueous solution or a lyophilized formulation for reconstitution.

[0302] Methods and Usage

[0303] In some embodiments, the present disclosure provides cells, ex vivo and in vivo methods, which include using splicing editor nucleic acids, recombinant expression vectors or delivery systems as described herein to target trans-splicing of target RNA (e.g., mRNA precursors) in cells. In some embodiments, the method includes using splicing editor nucleic acids, recombinant expression vectors or delivery systems as described herein to correct mutations in target RNA (e.g., mRNA precursors). In some embodiments, the present disclosure provides methods for treating patients with diseases or conditions, comprising administering splicing editor nucleic acids, recombinant expression vectors, delivery systems or pharmaceutical compositions as described herein to target trans-splicing of target RNA (e.g., mRNA precursors) in target cell populations and / or target tissues, thereby treating diseases or conditions.

[0304] Cellular RNA editing

[0305] In some embodiments, the method comprises introducing a splicing editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein into a cell or cell population. In some embodiments, the method comprises contacting a cell with a splicing 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.

[0306] The splicing editor nucleic acids, recombinant expression vectors, delivery systems, or pharmaceutical compositions described herein can be introduced into cells by any method known in the art, such as viral or phage 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 penetration, cell contact after fusion with cell penetrating peptides, microinjection, and nanoparticle-mediated delivery. In some embodiments, the vector system can be introduced into cells by viral infection.

[0307] 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 contacting the cell with a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein when the cell is contacted 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 the 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.

[0308] In some embodiments, the present disclosure provides a method for targeting trans-splicing of a pre-mRNA comprising a pathogenic mutation in a cell or a cell population, the method comprising contacting the cell or cell population with a splicing editor nucleic acid, a recombinant expression vector, a delivery system, or a pharmaceutical composition described herein, wherein when the cell is contacted with the splicing editor nucleic acid, the recombinant expression vector, the delivery system, or the pharmaceutical composition, one or more binding domains of the splicing editor nucleic acid bind to the pre-mRNA, and trans-splicing results in the ligation of one or more exons of the pre-mRNA to one or more exons of the splicing editor nucleic acid, thereby producing an mRNA that does not contain the pathogenic mutation.

[0309] In some embodiments, the present disclosure provides a method for targeting trans-splicing of a pre-mRNA in a cell or cell population derived from a patient suffering from a disease or condition, the method comprising contacting the cell or cell population with a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, wherein when the cell or cell population is contacted 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 the 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, wherein the cell or cell population is reintroduced into the patient, thereby treating or ameliorating the disease or condition.

[0310] In vivo RNA editing

[0311] The present disclosure provides methods for treating a patient suffering from a disease or condition using a splicing editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition as described herein. In some embodiments, the disease or condition is associated with one or more mutations in a target RNA, wherein the method targets trans-splicing of the target RNA to remove the one or more mutations.

[0312] In some embodiments, the present disclosure provides methods of treating a patient suffering from a disease or disorder, comprising administering to the patient a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein.

[0313] In some embodiments, the present disclosure provides a method for treating a patient suffering from a disease or condition 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 when the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition is administered, the splice editor nucleic acid binds to the target RNA (e.g., pre-mRNA), and trans-splicing results in the 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, thereby treating or ameliorating the disease or condition.

[0314] In some embodiments, the present disclosure provides a method of treating a patient having a disease or condition 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 when the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition is administered, the splice editor nucleic acid binds to the pre-mRNA, and trans-splicing results in the joining of one or more exons of the pre-mRNA to one or more exons of the splice editor nucleic acid, wherein trans-splicing produces an mRNA that does not contain the disease-causing mutation, thereby treating or ameliorating the disease or condition.

[0315] In some embodiments, the route of administration is any route determined by one of skill in the art to be sufficient to deliver the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein to the target tissue or cell population.

[0316] In some embodiments, administration of a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein results in correction of a mutation in a pre-mRNA in a target tissue or cell population in a patient.

[0317] The term "treatment" refers to the application of one or more methods described herein to improve a disease. In some embodiments, the specific procedure is the administration of a splicing editor nucleic acid, a recombinant expression vector, a delivery system, or a pharmaceutical composition as described herein. "Treatment" of an individual (e.g., a mammal, such as a human) or a cell refers to any type of intervention used to attempt to change the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a splicing editor nucleic acid, a recombinant expression vector, a delivery system, or a pharmaceutical composition as described herein, and may be performed prophylactically or after a pathological event occurs or after exposure to a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or disorder, and may include, for example, a minimal change or improvement in one or more measurable markers of a disease or disorder, and may include, for example, a minimal change or improvement in one or more measurable markers of a disease or disorder being treated.

[0318] The terms "patient," "subject," "individual," and the like are used interchangeably herein to refer to any animal suitable for the methods described herein. In some embodiments, the patient, subject, or individual is a human.

[0319] Reagent test kit

[0320] The present disclosure provides kits for performing the methods described herein. In some embodiments, the kit includes a splice editor nucleic acid, a recombinant expression vector, a delivery system, or a pharmaceutical composition described herein.

[0321] In some embodiments, the kit includes a splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition described herein, and reagents for reconstitution and / or dilution of the splice editor nucleic acid, recombinant expression vector, delivery system, or pharmaceutical composition.

[0322] In some embodiments, the kit includes one or more additional reagents, wherein such additional reagents are selected from a buffer, a buffer for introducing a splice editor nucleic acid, a recombinant expression vector, a delivery system into a cell, a wash buffer, a control reagent, a control vector, a control polynucleotide, a reagent for in vitro production of a recombinant expression vector or a delivery system, an adapter for sequencing, etc. The buffer can be a stabilization buffer, a reconstitution buffer, a dilution buffer, etc. The kit can also include one or more components that can be used to promote or enhance targeted binding or trans-splicing of a splice editor nucleic acid.

[0323] In addition to the components mentioned above, test kit may also include instructions for using the components of test kit to put into practice the method. The instructions for putting into practice the method may be recorded on a suitable recording medium. For example, instructions may be printed on a substrate such as paper or plastic. Instructions may be present in the test kit as a packaging insert, in the label or its component (i.e., associated with packaging or sub-packaging) of the test kit container. Instructions may exist as an electronic storage data file present in a suitable computer-readable storage medium such as a CD-ROM, disk, flash drive, etc. In some cases, actual instructions are not present in the test kit, but may be provided for, for example, obtaining instructions from a remote resource via the Internet. The example of this situation is a test kit comprising a website, in which instructions may be viewed and / or instructions may be downloaded from the website. The same as instructions, this means for obtaining instructions may be recorded on a suitable substrate.

[0324] In some embodiments, the kit includes a container comprising a splice editor nucleic acid, a recombinant expression vector, a delivery system, or a pharmaceutical composition described herein, and instructions for use in targeting trans-splicing of a target RNA (e.g., a pre-mRNA) in a cell or a population of cells.

[0325] In some embodiments, the kit includes a container comprising 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 cell population of the patient.

[0326] definition

[0327] As used herein, the term "pre-mRNA" refers to precursor mRNA, which is RNA containing exons and introns. Pre-mRNA is a primary transcript that is processed into messenger RNA. It is synthesized from a DNA template in the cell nucleus through transcription. In some embodiments, the RNA is derived from mammalian cells. In other embodiments, the RNA is derived from the mitochondria of mammalian cells.

[0328] As used herein, the term "RNA-binding" is used to describe a molecule, protein, nucleic acid, or complex that specifically binds to RNA.

[0329] As used herein, "disease" refers to a state of health in which an animal is unable to maintain homeostasis and, if the disease is not ameliorated, the animal's health will continue to deteriorate. In contrast, a "disorder" in an animal refers to a state of health in which the animal is able to maintain homeostasis, but the animal's health is not as good as it would be without the disorder. A disorder does not necessarily lead to a further decline in the animal's health if left untreated.

[0330] Example

[0331] Example 1: Selection of ncRNA

[0332] This example describes a method for identifying ncRNAs for inclusion in splice editors capable of targeting pre-mRNAs and generating trans-splicing events. Figure 1E As shown, it has been determined that almost all ncRNA sequences mined from public databases contain the sequence motifs identified in Table 1. The secondary structures of ncRNA sequences identified in the public domain, including metazoan U1 snRNA sequences, U11 snRNA sequences, U7 snRNA sequences, Sm sequences and H / ACA snoRNA sequences, were predicted using RNAlib-2.5.1 software. The predicted secondary structures were compared with known secondary structures using the RNA covariance model (see, for example, Eddy et al. (1994) Nucleic Acids Research 22: 2079-2088). ncRNA sequences with predicted secondary structures that are similar to known secondary structures were selected for further evaluation. This method provided more than 120,000 candidate ncRNA sequences. Figure 1F As shown, the length of candidate ncRNA sequences ranges from about 7 nucleotides to more than 300 nucleotides. Exemplary candidate ncRNA sequences identified by this computational analysis are shown in SEQ ID NOs: 9-657.

[0333] Example 2: Design and testing of splice editors for trans-splicing

[0334] A splice editor nucleic acid molecule is designed for targeted trans-splicing. The nucleic acid molecule comprises a nucleotide sequence having the following features: (a) an intron sequence having: (i) at least one binding domain sequence complementary to a target sequence in a pre-mRNA, and (ii) an ncRNA sequence; (b) a splice site; and (c) at least one exon sequence. The ncRNA sequence is selected from the candidate ncRNAs identified as described in Example 1. The splice editor nucleic acid molecule is engineered to incorporate the entire candidate ncRNA sequence or a portion thereof comprising the secondary structure and / or sequence motifs identified in Table 1.

[0335] The first group of nucleic acid molecules is designed to have an ncRNA sequence derived from snRNA and undergo trans-splicing at a splice donor in the mRNA precursor (to correct mutations at the 5' end of the exon). The nucleic acid molecules have a nucleotide sequence arranged from 5' to 3' as follows: (a) an intron having (i) a binding domain sequence, (ii) an snRNA sequence (U1 snRNA; U11 snRNA; Sm sequence motif and U7 snRNA; or Sm sequence motif), (iii) a branch point and (iv) a polypyrimidine tract; (b) a splice acceptor; and (c) an exon. Figure 2A 、 Figure 2B and Figure 2C Schematic diagrams of exemplary U1-based splice editor nucleic acid molecules are shown, and the sequences are provided in the table of Table 3 (in each row of Table 3, the column titled "Region 2" is assigned the label "U1_X_Y", where X and Y are integers). FIG3A, Figure 3B and Figure 3C A schematic diagram of an exemplary U11-based splice editor nucleic acid molecule is shown, and the sequence is provided in the table of Table 3 (in each row of Table 3, the column titled "Region 2" is assigned the label "U11_X_Y", where X and Y are integers). Figure 4A 、 Figure 4B and Figure 4C A schematic diagram of an exemplary U7-based splice editor nucleic acid molecule is shown, and the sequences are provided in Table 3 (in each row of 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 "U7_X", where X is an integer). Figure 5A 、 Figure 5B and Figure 5C Schematics of exemplary Sm-based splice editor nucleic acid molecules are shown, and the sequences are provided in Table 3 (in each row of 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 the label "adeno_intron").

[0336] The second set of nucleic acid molecules was designed to have an ncRNA sequence derived from an H / ACA snoRNA and undergo trans-splicing at a splice donor in the pre-mRNA (to correct mutations at the 5' end of the exon). The nucleic acid molecules had a nucleotide sequence arranged from 5' to 3' as follows: (a) an intron having (i) first and second binding domains inserted into the H / ACA box snoRNA sequence, (ii) a branch point, and (iii) a polypyrimidine tract; (b) a splice acceptor; and (c) an exon. Figure 6A 、 Figure 6B and Figure 6C Schematics of exemplary snoRNA-based splicing editor nucleic acid molecules are shown, and the sequences are provided in Table 3 (in each row of Table 3, the column titled "Region 1" is assigned a label beginning with "sno," "SNO," or "SCARNA").

[0337] The nucleic acid molecules are evaluated for trans-splicing by using reporter cells, where correct RNA editing generates mRNA that produces fluorescent protein. The splicing editor is introduced into the reporter cells by 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 splicing donor reporter construct that encodes an mRNA precursor under the control of a CMV promoter, which contains blue fluorescent protein (BFP), a self-cleaving p2A linker, a truncated GFP (5'GFP) and a splicing donor. BFP is used to confirm the stable expression of the reporter construct. The reporter construct has matrix metallopeptidase 9 (MMP9) intron 1 and exon 2 downstream of the splicing donor to ensure that splicing events can occur, followed by a bovine growth hormone polyadenylation signal (bGHpA) to allow stable expression of the construct. The splicing editor nucleic acid has an exon that is the second half of the truncated GFP (3'GFP), and trans-splicing results in the expression of full-length GFP. Correctly edited reporter cells produce a signal from a fluorescent reporter gene and are sorted by FACS. Sorted cells are sequenced to identify active splice editors.

[0338] Example 3: Design and testing of snoRNA for exon skipping

[0339] In the experiments of this example, a snoRNA guide construct was developed in which the hybridization region was replaced to allow the snoRNA to act as a guide for the RNP complex. In this example, the snoRNA guide construct was tested for exon skipping.

[0340] In these experiments, a snoRNA guide construct was engineered on a plasmid along with a sequence complementary to the target sequence to generate a snoRNP that blocks the splice site, thereby allowing exon skipping. To test exon skipping, a snoRNA guide construct was engineered on a plasmid (i.e., pA0077, Figure 7A , Table 2) was designed on the plasmid, which also included the intron and exon sites of the MMP9 gene and two GFP sites ( Figure 7A The element was then introduced into a separate plasmid target (i.e., pA0016) after the U6 promoter and before the poly-T terminator ( Figure 7A ). These constructs and plasmids were designed so that the snoRNA could bind to the MMP9 gene splice site, thereby blocking the splice site and leading to the production of GFP, as shown in Figure 7B Volcano map and Figure 8 as shown in the chart.

[0341] Figure 7B Four snoRNA candidates targeting splice acceptors (i.e., labeled "snord45a_11_receptor", "snord45a_7_receptor", "aca46_receptor_29" and "aca44_2_receptor_35", which were obtained through high-throughput screening of exon skipping and whose sequences are shown in Table 4) were synthesized as IDT eBlocks and cloned into the pA0016 vector. The vectors were transfected into a HEK293FT cell line stably expressing the PiggyBAC-integrated MMP9 exon skipping reporter gene (pA0077). This cell line was a high-expressing single-cell clone established by FACS sorting for reporter gene BFP expression. 100 ng of the element vector was transfected into HEK293FT cells seeded in 96-well plates using Lipofectamine 2000, allowing the cells to reach 90% confluence at the time of transfection. 48 hours after transfection, cells were analyzed by flow cytometry for GFP% and compared to non-targeting control (ntc) or cells treated with pUC19. Figure 8is a graph showing GFP production as a result of exon skipping for four different snoRNAs targeting splice acceptors (i.e., C / D snoRNA for snord, H / ACA for snora or aca, labeled "V5_snord45a_11_receptor", "V4_snord45a_7_receptor", "V1_aca46_receptor_29", and "V2_aca44_2_receptor_35", sequences shown in Table 4) compared to four randomized guides (i.e., "V10ntc_random_9", "V11ntc_aca42_1_receptor_106", "V12ntc_snord51_123_receptor", pU C19 (separate plasmid)). Figure 7A 、 Figure 7B 、 Figure 8 and Figure 9 The results demonstrate that the snoRNA disclosed herein guides exon skipping.

[0342] Table 4: snoRNAs targeting splice acceptor sequences

[0343]

[0344]

[0345] Example 4: Design of U7 snRNA and trans-splicing test

[0346] In the experiments of this example, U7 snRNA guide constructs were developed and tested for trans-splicing. The U7 snRNA guide constructs were designed on a plasmid along with a sequence complementary to the target sequence to generate a U7 snRNA construct that blocked the splicing site, thereby allowing trans-splicing. To test trans-splicing, a U7 snRNA guide construct was constructed on a plasmid (i.e., pA0120, Figure 10 , Table 2 ), and the U7 snRNA guide was designed on the plasmid, which also included the intron and exon sites of the USH2A gene and two GFP sites ( Figure 10 ).

[0347] The elements were then introduced onto a separate plasmid target (i.e., pA0177) after the pCMV promoter and before the bGHpA ( Figure 10 ) and included an additional GFP site. In these experiments, trans-splicing was observed by expression of GFP because GFP

[0348] The elements were then introduced onto a separate plasmid target (i.e., pA0177) after the pCMV promoter and before the bGHpA ( Figure 10) and include an additional GFP site. In these experiments, trans-splicing is observed by expression of GFP because the GFP site of the plasmid target (i.e., pA0177) will replace and restore the GFP site of the pA0120 plasmid, thereby demonstrating trans-splicing by GFP expression. Figure 11 Figure 12 is a graph showing the binding of U7 snRNA guide constructs to different target elements and the effect of trans-splicing. Figure 11 Independent validation of the results of . The first four bars on the left show U7 snRNA constructs with specific targeting elements for trans-splicing, and the remaining bars on the chart show U7 snRNA constructs with non-targeting elements for trans-splicing. These experiments show, in particular, that targeting close to the splice donor enhances trans-splicing. Figure 13 is a chart showing a comparison of U7 snRNA targeting piggybac-integrated USH2A (773; hybridization region and hairpin intact) with non-targeting guides (774 and 776) and mutated U7 snRNA hairpins (775; hybridization region intact, but hairpin region mutated). These experiments further show, in particular, that targeting the complementary region with a U7 snRNA construct having a hairpin and hybridization region results in significantly higher trans-splicing compared to a non-targeted control.

[0349] Table 2:

[0350]

[0351]

[0352] Table 3: Sequence Listing

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

Claims

1. A nucleic acid for targeting trans-splicing of mRNA precursors in cells, the nucleic acid comprising a nucleotide sequence comprising (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary 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 comprises a sequence motif to direct the one or more binding domains to the pre-mRNA target sequence; (b) splice acceptor and / or splice donor sequences; and (c) at least one exon sequence.

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. The nucleic acid of claim 1 or 2, wherein the one or more binding domain sequences are less than 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.

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 of any one of claims 1 to 4, comprising a binding domain sequence.

6. The nucleic acid of 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 when the nucleic acid is introduced into the cell, exons in the mRNA precursor are targeted for trans-splicing.

9. The nucleic acid of claim 8, wherein the target sequence is located in a region of the pre-mRNA comprising 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. The nucleic acid of claim 9 or 10, wherein the target sequence is located proximal to a splice donor or a splice acceptor.

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 a snRNA.

14. The nucleic acid of claim 13, wherein the snRNA is selected from the group consisting of U7 snRNA, U1 snRNA, U2 snRNA, U4snRNA, 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 assembles into RNP.

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

19. The nucleic acid of any one of claims 1 to 17, wherein the ncRNA sequence comprises a secondary structure that is assembled into 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. The nucleic acid of claim 19 or 20, wherein the secondary structure comprises one or more stem-loops.

22. The nucleic acid of any one of claims 17 to 21, wherein the RNP is selected from the group consisting of small nuclear RNP (snRNP), small nucleolar RNP (snoRNP), small Cajal body RNP (scaRNP), and combinations thereof.

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

24. The nucleic acid of claim 22, wherein the RNP is selected from the group consisting of a C / D box sno RNP and a H / ACA box sno RNP.

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

26. The nucleic acid of claim 25, wherein the Sm sequence motif assembles into RNPs with the Sm or Lsm protein.

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

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

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

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. The nucleic acid of claim 30, wherein the one or more splicing signals are selected from the group consisting of exonic splicing enhancers (ESEs), intronic splicing enhancers (ISEs), exonic splicing silencers (ESSs), intronic splicing silencers (ISSs), polypyrimidine tracts, branch points, and combinations thereof.

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 stretch.

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, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron sequence comprising (i) one or more binding domain sequences of about 4 to about 300 nucleotides, each binding domain sequence being complementary to a pre-mRNA target sequence; (ii) a non-coding RNA (ncRNA) sequence of about 7 to about 300 nucleotides in length that forms a secondary structure and / or comprises a sequence motif to direct the one or more binding domains to the pre-mRNA target sequence; and (iii) one or more splicing signals; (b) splice acceptors; and (c) at least one exon sequence.

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

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

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

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

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

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

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 stretch.

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

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

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

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

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

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 in the pre-mRNA.

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

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

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

51. The nucleic acid of claim 49 or 50, wherein the snRNA assembles into snRNPs.

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

53. The nucleic acid of claim 52, wherein the U1 snRNA assembles into U1 RNP.

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

55. The nucleic acid of claim 54, wherein the U11 snRNA assembles into U11 RNP.

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

57. The nucleic acid of claim 56, wherein the U7 snRNA assembles into U7 RNP.

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

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

60. The nucleic acid of claim 58 or 59, wherein the Sm sequence motif comprises the sequence shown in SEQ ID NO: 3 and 4.

61. A nucleic acid as described in any one in claims 58 to 60, wherein the Sm sequence motif and Sm protein are assembled into RNP.

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

63. 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: 9-589 or a portion thereof.

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

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

66. A nucleic acid as described in any one of claims 34 to 65, comprising a binding domain sequence.

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

68. The nucleic acid of any one of claims 34 to 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, the nucleic acid comprising a nucleotide sequence comprising, from 5′ to 3′: (a) at least one intron 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 binding domain sequence being complementary to a pre-mRNA target sequence; and (ii) one or more splicing signals; (b) splice acceptors; and (c) at least one exon sequence.

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

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

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

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

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

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

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 stretch.

77. A nucleic acid for targeting trans-splicing of a pre-mRNA in a cell, the nucleic acid comprising 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 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 binding domain sequence being complementary to a pre-mRNA target sequence.

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

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

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

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

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 in the pre-mRNA.

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

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

85. The nucleic acid of claim 84, comprising at least one binding domain sequence located at: (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).

86. 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. The nucleic acid of claim 86, comprising at least one binding domain located at: (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).

88. 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: 590-657, or a portion thereof.

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

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

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

92. The nucleic acid of any one of claims 1 to 91, comprising at least one binding domain sequence that is fully complementary to the pre-mRNA target sequence.

93. The nucleic acid of any one of claims 1 to 92, comprising at least one binding domain sequence that is partially complementary to the pre-mRNA target sequence.

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

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

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

97. The nucleic acid of any one of claims 1 to 96, wherein the nucleic acid comprises 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. The nucleic acid of any one of claims 1 to 97, wherein the nucleic acid is introduced into the cell as RNA.

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

100. The nucleic acid of any one of claims 1 to 99, wherein the nucleic acid is introduced into the cell via 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, wherein the nucleic acid is introduced into the cell via a non-viral vector.

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

104. The nucleic acid of claim 103, wherein the trans-splicing efficiency is greater than 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, wherein the nucleic acid is 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 of any one of claims 1 to 105.

108. A cell comprising the nucleic acid of any one of claims 1 to 105, the viral vector of claim 106, or the lipid nanoparticle of 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. A pharmaceutical composition comprising the cells of claim 108 and a pharmaceutically acceptable carrier.

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

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

113. A method of treating a patient having a disease or condition associated with a mutation in a pre-mRNA, the method comprising administering to the patient an effective amount of 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, wherein when the nucleic acid, the viral vector, the lipid nanoparticle, or the pharmaceutical composition is administered, the one or more binding domain sequences bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA containing the mutation, thereby correcting the mutation.

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

115. 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 for use in treating a patient suffering from a disease or condition 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 wherein trans-splicing replaces one or more exons in the pre-mRNA comprising the mutation, thereby correcting the mutation.

116. A nucleic acid as described in any one of claims 1 to 105, a viral vector as described in claim 106, a lipid nanoparticle as described in claim 107, or a pharmaceutical composition as described in claims 109 to 110, for use in the manufacture of a medicament for treating a patient suffering from a disease or condition associated with a mutation in a pre-mRNA, the treatment comprising administering the medicament to the patient, wherein when the medicament is administered, the one or more binding domain sequences of the nucleic acid bind to the pre-mRNA at a position proximal to the mutation, and wherein trans-splicing replaces one or more exons in the pre-mRNA containing the mutation, thereby correcting the mutation.

117. A kit comprising a container comprising 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, and instructions for correcting a mutation in an mRNA precursor.

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