HTT trans-splice molecules

Abstract

An HTT nucleic acid trans-splice molecule is described that includes a coding domain comprising one or more HTT exons, a splice site, and a binding domain that binds to a target intron of an HTT precursor mRNA. The HTT nucleic acid trans-splice molecules described herein may also be used in combination with, for example, an MSH3 binding domain aligned in series with an HTT binding domain, an MSH3 nucleic acid trans-splice molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA for either of MSH3 or HTT, and an MSH3 or HTT microRNA (miRNA) and constructs encoding them. Also encompassed are compositions comprising the nucleic acid trans-splice molecules described herein, as well as compositions comprising a nucleic acid trans-splice molecule in combination with an additional therapeutic agent (e.g., an MSH3 nucleic acid trans-splice molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA for any one of MSH3 or HTT). The nucleic acid trans-splicing molecules may be used alone or in combination with additional therapeutic agents in methods of treating Huntington's disease (HD). Also described herein are nucleic acid trans-splice molecules for use in the treatment of HD or in the preparation of medicaments for the treatment of HD, alone or in combination with additional therapeutic agents. Also encompassed herein are MSH3 nucleic acid trans-splice molecules, MSH3 splice modulators, and MSH3 miRNAs, as well as constructs encoding them, which can be used, alone or in combination and / or in combination with additional therapeutic agents, for the treatment of nucleotide repeat disorders (e.g., HD) or for the preparation of medicaments for the treatment of nucleotide repeat disorders (e.g., HD).

Description

Cross-referencing

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 485,142, filed February 15, 2023, and U.S. Provisional Patent Application No. 63 / 485,146, filed February 15, 2023, each of which is incorporated herein by reference in its entirety.

[0002] sequence list This application contains a sequence list, which has been submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy was created on February 14, 2024, named 61313-709_601_SL.xml, and has a size of 521,958 bytes. Background Technology

[0003] Huntington's disease (HD) is a motor-related progressive neurodegenerative disorder. The disease is associated with the loss of cortical pyramidal neurons, the loss of medium-spinous neurons in the striatum, and the loss of neurons in the hypothalamus. The genetic cause of HD is... HTT The autosomal dominant inheritance of CAG trinucleotide repeat amplification in exon 1 of the gene, with more than 40 CAG repeats in this region being a pathogenic factor.

[0004] HTT The locus is large, spanning 180 kb and consisting of 67 exons, and HTT Gene expression is essential for normal development. Although HTT protein is widely expressed, pathological amplification of CAG trinucleotide repeats has the most severe impact on the brain, with early effects noted in the striatum and motor cortex. It is generally believed that the underlying mechanism of HD pathogenesis involves events occurring in the affected brain regions (e.g., the striatum) of HD patients. HTT Somatic cell CAG repeat expansion.

[0005] HD patients typically develop progressive neurodegeneration within 10 to 20 years after disease onset, leading to death. Currently, there are no disease-modifying treatments for HD. Current treatments are limited to providing symptom relief. Summary of the Invention

[0006] This article describes nucleic acid trans-splicing molecules, which include one or more... HTT The coding domains, splice sites, and binding sites of exons HTT The binding domain of the target intron of the precursor mRNA. In some embodiments, the construct containing the nucleic acid trans-splicing molecule may further include an additional sequence encoding antisense RNA, which binds to the target precursor mRNA, thereby blocking cis-splicing and thus promoting trans-splicing of the nucleic acid trans-splicing molecule to the precursor mRNA target (e.g., HTT Precursor mRNA). In some implementations, the nucleic acid trans-splicing molecule may also contain a precursor mRNA with a second target (e.g., MSH3 The second binding domain of the target intron in the precursor mRNA binds to the target intron, thereby trans-splicing it into the target intron of the second target precursor mRNA, resulting in a structure containing one or more target introns. HTT Exons and second target precursor mRNAs (e.g., MSH3 A hybrid precursor mRNA containing the exons of a precursor mRNA, wherein the hybrid precursor mRNA is processed into a hybrid mRNA, and the hybrid mRNA is targeted for degradation via, for example, nonsense-mediated degradation. In some embodiments, the construct containing the nucleic acid trans-splicing molecule may further include an additional sequence encoding a small nuclear RNA (snRNA), which is introduced into a second target precursor mRNA (e.g., by introducing a premature stop codon) MSH3 To block the second target precursor mRNA (e.g.) in precursor mRNA) MSH3 Normal processing of precursor mRNA, the second target precursor mRNA targets the second target mRNA (e.g., MSH3 mRNA) for nonsense-mediated degradation. In some embodiments, the construct containing nucleic acid trans-splicing molecules may further include elements encoding antisense RNA, which, for example, blocks the MSH3 splice junction or interacts with... MSH3 Annealing the 5'UTR or initial coding sequence to block MSH3 Translation to block the second target precursor mRNA (e.g. MSH3 Normal processing of precursor mRNA. In some implementations, the construct containing nucleic acid trans-splicing molecules may also include encoding for endogenous... HTT mRNA is specific or has a specific effect on MSH3 mRNA contains specific microRNA (miRNA) sequences that promote endogenous [products / functions]. HTT mRNA or MSH3 mRNA cleavage, thereby reducing endogenous mRNA cleavage. HTT mRNA or MSH3 The level of mRNA is also covered. Compositions comprising such nucleic acid trans-splicing molecules, as well as compositions comprising a combination of nucleic acid trans-splicing molecules with an additional therapeutic agent (e.g., an antisense oligonucleotide or an antisense RNA-encoding construct), are also included. The nucleic acid trans-splicing molecules and compositions comprising said molecules can be used alone or in combination with an additional therapeutic agent in methods of treating Huntington's disease (HD). The nucleic acid trans-splicing molecules and compositions comprising said molecules are also used alone or in combination with an additional therapeutic agent for treating HD or for preparing medicaments for treating HD.

[0007] This document discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exon 1 and HTT exon 2; (b) a splicing domain; and (c) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron comprises intron 2. In some embodiments, the binding domain comprises, substantially comprises, or comprises any of the sequences in SEQ ID NO: 60-81, or a sequence having at least 90% identity with any of the sequences in SEQ ID NO: 60-81.

[0008] This document also discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain containing HTT exons 1-3; (b) a splicing domain; and (c) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron contains intron 3. In some embodiments, the binding domain comprises, substantially comprises, or comprises any of the sequences in SEQ ID NO: 158-174, or a sequence having at least 90% identity with any of the sequences in SEQ ID NO: 158-174.

[0009] This article also discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain containing HTT exon 1; (b) a splicing domain; and (c) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron contains intron 1, and wherein the binding domain comprises any one of SEQ ID NO:8-21.

[0010] In some embodiments, in any of the HTT nucleic acid trans-splicing molecules described above, the coding domain comprises, is substantially composed of, or is composed of: HTT exon 1; HTT exon 1 and HTT exon 2; or HTT exons 1-3. In some embodiments, the coding domain comprises, is substantially composed of, or is composed of: any of SEQ ID NO: 3, 59, 157, or 349-353, or a sequence having at least 90% identity with SEQ ID NO: 3, 59, 157, or 349-353. In some embodiments, the coding domain, splicing domain, and binding domain are operatively connected in a 5' to 3' orientation.

[0011] In some implementations, the aforementioned HTT nucleic acid trans-splicing molecule also includes a adapter, wherein the coding domain, splicing domain, adapter, and binding domain are operatively connected in a 5' to 3' orientation. In some embodiments, the linker comprises, is substantially composed of, or consists of: a sequence in the range of 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine; a sequence in the range of 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine; or a sequence in the range of 22 to 42 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine. In some embodiments, the connector comprises, is substantially composed of, or is composed of any of the following: SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; or SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO: 41.In some embodiments, the connector comprises, substantially comprises, or comprises any of the following: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 ... SEQ ID NO: 108 is a sequence with at least 90% identity; SEQ ID NO: 109 or a sequence with at least 90% identity; SEQ ID NO: 110 or a sequence with at least 90% identity; SEQ ID NO: 111 or a sequence with at least 90% identity; SEQ ID NO: 112 or a sequence with at least 90% identity; SEQ ID NO: 197 or a sequence with at least 90% identity; or SEQ ID NO: 198 or a sequence with at least 90% identity.

[0012] In some embodiments, the above-described HTT nucleic acid trans-splicing molecule further comprises a triple-helix terminator, wherein the coding domain, splicing domain, adapter (when present), binding domain, and triple-helix terminator are operatively connected in a 5' to 3' orientation. In some embodiments, the triple-helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 5 or a sequence having at least 90% identity with SEQ ID NO: 5. In some embodiments, the triple-helix terminator comprises, substantially comprises, or comprises SEQ ID NO: 6.

[0013] In some embodiments, the aforementioned HTT nucleic acid trans-splicing molecule further comprises a 5' untranslated region (5' UTR), wherein the 5' UTR, coding domain, splicing domain, adapter (if present), binding domain, and triple-helix terminator (if present) are operatively linked in a 5' to 3' orientation. In some embodiments, the 5' UTR is HTT 5' UTR. In some implementations, HTT The 5' UTR comprises, is substantially composed of, or consists of any of SEQ ID NO: 136 or 192, or a sequence having at least 90% identity with any of SEQ ID NO: 136 or 192.

[0014] In some embodiments, the HTT nucleic acid trans-splicing molecule further comprises a sequence encoding an epitope tag, wherein the 5' UTR (when present), the epitope tag, the coding domain, the splicing domain, the adapter (when present), the binding domain, and the triple-helix terminator (when present) are operatively linked in a 5' to 3' orientation. In some embodiments, the sequence encoding the epitope tag comprises, substantially comprises, or comprises: SEQ ID NO: 4.

[0015] This document also discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain containing HTT exon 1 and HTT exon 2; (b) a splicing domain; (c) a linker; and (d) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron comprises intron 2. In some embodiments, the binding domain comprises, substantially comprises, or comprises any of the sequences in SEQ ID NO: 60-81, or a sequence having at least 90% identity with any of the sequences in SEQ ID NO: 60-81.

[0016] This document also discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain containing HTT exons 1-3; (b) a splicing domain; (c) a linker; and (d) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron contains intron 3. In some embodiments, the binding domain comprises, substantially comprises, or comprises any of the sequences in SEQ ID NO: 158-174, or a sequence having at least 90% identity with any of the sequences in SEQ ID NO: 158-174.

[0017] This document also discloses an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain containing HTT exon 1; (b) a splicing domain; (c) a linker; and (d) a binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron contains intron 1. In some embodiments, the binding domain comprises, substantially comprises, or comprises any of the sequences in SEQ ID NO: 8-21, or a sequence having at least 90% identity with any of the sequences in SEQ ID NO: 8-21.In some embodiments, the connector comprises any of the following SEQ ID NO:, substantially consists of any of the following SEQ ID NO:, or consists of any of the following SEQ ID NO:: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO: 41; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 47 or a sequence having at least 90% identity with SEQ ID NO: 47; SEQ ID NO: 48 or a sequence having at least 90% identity with SEQ ID NO: 49; SEQ ID NO: 49 or a sequence having at least 90% identity with SEQ ID NO: 49; SEQ ID NO: 40 ... SEQ ID NO: 45, a sequence having at least 90% identity; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 or a sequence having at least 90% identity with SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity with SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity with SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity with SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity with SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity with SEQ ID NO: 197. 197 is a sequence with at least 90% identity; or SEQ ID NO: 198 or a sequence with at least 90% identity to SEQ ID NO: 198.In some embodiments, the HTT nucleic acid trans-splicing molecule further comprises a triple-helix terminator, wherein the coding domain, splicing domain, adapter, binding domain, and triple-helix terminator are operatively connected in a 5' to 3' orientation; and optionally, it further comprises a 5' UTR, wherein the 5' UTR (when present), coding domain, splicing domain, adapter, binding domain, and triple-helix terminator (when present) are operatively connected in a 5' to 3' orientation.

[0018] This document also discloses a nucleic acid trans-splicing molecule comprising a linker, wherein the linker comprises, is substantially composed of, or is composed of: a sequence in the range of 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine. In some embodiments, the linker comprises, is substantially composed of, or is composed of: a sequence in the range of 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine. In some embodiments, the adapter comprises, is substantially composed of, or consists of a sequence in the range of 22 to 42 nucleotides in length, wherein the adapter comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine. In some embodiments, the adapter comprises, is substantially composed of, or consists of: SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; or SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO: 41.

[0019] This document also discloses a nucleic acid trans-splicing molecule comprising a linker, wherein the linker comprises, substantially comprises, or comprises the following: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 ... SEQ ID NO: 108 is a sequence with at least 90% identity; SEQ ID NO: 109 or a sequence with at least 90% identity; SEQ ID NO: 110 or a sequence with at least 90% identity; SEQ ID NO: 111 or a sequence with at least 90% identity; SEQ ID NO: 112 or a sequence with at least 90% identity; SEQ ID NO: 197 or a sequence with at least 90% identity; or SEQ ID NO: 198 or a sequence with at least 90% identity.

[0020] In some embodiments of the aforementioned HTT nucleic acid trans-splicing molecule, the HTT nucleic acid trans-splicing molecule further comprises binding... MSH3 The target intron binding domain of the precursor mRNA. In some implementations, MSH3 Target introns contain MSH3 Either intron 5 or intron 15. In some embodiments, combined with MSH3The target intron binding domain of the precursor mRNA comprises, is substantially composed of, or is composed of any one of SEQ ID NO: 140, 142, 144, 146, 209, or 210, or a sequence having at least 90% identity with any one of SEQ ID NO: 140, 142, 144, 146, 209, or 210. In some embodiments, the nucleic acid trans-splicing molecule comprises any one of SEQ ID NO: 149-154 or SEQ ID NO: 212-223, or a sequence having at least 90% identity with any one of SEQ ID NO: 149-154 or SEQ ID NO: 212-223.

[0021] In some embodiments, any of the HTT nucleic acid trans-splicing molecules described above further comprises a nucleic acid sequence encoding a primary miRNA, said primary miRNA comprising a microRNA (miRNA) sequence specific to exon 1 of endogenous HTT mRNA, wherein exon 1 of said nucleic acid trans-splicing molecule comprises a nucleotide sequence variation that weakens the binding of the miRNA to at least a portion of the mRNA encoded by said nucleic acid trans-splicing molecule. In some embodiments, the miRNA sequence comprises any one of SEQ ID NO: 339 or 342, or a nucleic acid sequence having at least 90% identity with any one of SEQ ID NO: 339 or 342. In some embodiments, the nucleic acid sequence encoding the primary miRNA comprises any one of SEQ ID NO: 341 or 344. In some embodiments, the primary miRNA comprises a mir-33 scaffold sequence. In some embodiments, the primary miRNA comprises a mir-30a scaffold sequence, a mir-30a loop sequence, a mir-155 scaffold sequence, a mir-155 loop sequence, a mir-33 scaffold sequence, or a mir-33 loop sequence. In some embodiments, the mir-30a stent sequence comprises the 5' stent sequence shown in SEQ ID NO: 227 or the 3' stent sequence shown in SEQ ID NO: 228; wherein the mir-30a loop sequence comprises SEQ ID NO: 229; wherein the mir-155 stent sequence comprises the 5' stent sequence shown in SEQ ID NO: 230 or the 3' stent sequence shown in SEQ ID NO: 231; wherein the mir-155 loop sequence comprises SEQ ID NO: 232; wherein the mir-33 stent sequence comprises the 5' stent sequence shown in SEQ ID NO: 259 or the 3' stent sequence shown in SEQ ID NO: 260; or wherein the mir-33 loop sequence comprises SEQ ID NO: 261.

[0022] This document also discloses an MSH3 exon-jumping nucleic acid construct comprising operatively linked: (a) a sequence encoding an antisense RNA that promotes exon jumping of a target exon of an MSH3 precursor mRNA, wherein the target exon is any one of MSH3 exons 2-4, 6-8, or 15, wherein the target exon contains a 5' exon-intron junction and a 3' exon-intron junction sequence; and (b) a sequence encoding a small nuclear RNA (snRNA) sequence. In some embodiments, the MSH3 exon-jumping nucleic acid construct further comprises a U1 promoter and a U1 terminator operatively linked to (a) and (b). In some embodiments, the snRNA is a modified snRNA. In some embodiments, the modified snRNA comprises a U7 Sm OPT sequence or a U2 snRNA sequence. In some embodiments, the antisense RNA targets the 5' exon-intron junction or the 3' exon-intron junction of the target exon. In some embodiments, the antisense RNA comprises, is substantially composed of, or is composed of any of the following: SEQ ID NO: 274, 275, 276, 277, 278, 279, 280, 300, 302, 301, 303, 281, 282, 306, 308, 305, 307, 311, 313, 310, 312, 316, 318, 315, 317, 321, 323, 320, or 322, or is combined with SEQ ID NO: The sequence has at least 90% identity with any one of SEQ ID NO: 274, 275, 276, 277, 278, 279, 280, 300, 302, 301, 303, 281, 282, 306, 308, 305, 307, 311, 313, 310, 312, 316, 318, 315, 317, 321, 323, 320, or 322. In some embodiments, the above-described MSH3 exon skipping nucleic acid construct comprises any one of SEQ ID NO: 284, 285, 286, 287, 288, 289, 290, 325, 326, 291, 292, 328, 329, 331, 332, 334, 335, 337, and 338.

[0023] In some embodiments, the antisense RNA targets both the 5' exon-intron junction and the 3' exon-intron junction. In some embodiments, the antisense RNA contains a sequence that is at least 80% complementary to the entire sequence of the target exon.

[0024] In some embodiments, the antisense RNA further comprises: (a) a sequence at least 80% complementary to a 5-nucleotide sequence upstream of the 5' exon-intron junction; and (b) a sequence at least 80% complementary to a 5-nucleotide sequence downstream of the 3' exon-intron junction. In some embodiments, the antisense RNA comprises any one of SEQ ID NO: 299, 304, 309, 314, or 319, or a sequence having at least 90% identity with any one of SEQ ID NO: 299, 304, 309, 314, or 319. In some embodiments, the above-described MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 324, 327, 330, 333, or 336.

[0025] In some embodiments, the antisense RNA comprises: (a) a sequence targeting the 3' exon-intron junction, operably linked in a 5' to 3' orientation; (b) a linker sequence of at least 15 nucleotides that does not anneal to the target exon; and (c) a sequence targeting the 5' exon-intron junction. In some embodiments, the complementarity of the linker sequence with all sequences of the target exon of the same length as the linker is less than 50%. In some embodiments, the antisense RNA comprises any one of SEQ ID NO: 300, 301, 302, 303, 305, 306, 307, 308, 310, 311, 312, 313, 315, 316, 317, 318, 320, 321, 322, or 323, or any combination thereof. In some embodiments, the MSH3 exon skipping nucleic acid construct comprises any one of SEQ ID NO: 325, 326, 328, 329, 331, 332, 334, 335, 337 or 338.

[0026] In some embodiments, the antisense RNA targets exon 7 of MSH3. In some embodiments, the MSH3 exon-jumping nucleic acid construct comprises SEQ ID NO: 309. In some embodiments, the MSH3 exon-jumping nucleic acid construct comprises, from 5' to 3': (a) SEQ ID NO: 310 (In7 / Ex7 asRNA), SEQ ID NO: 298 (linker), and SEQ ID NO: 311 (In7 / Ex7 asRNA); or (b) SEQ ID NO: 312 (In7 / Ex7 asRNA), SEQ ID NO: 298 (linker), and SEQ ID NO: 313 (In7 / Ex7 asRNA). In some embodiments, the antisense RNA comprises any one of SEQ ID NO: 309, 310, 311, 312, or 313, or any combination thereof, or a sequence having at least 90% identity with any one of SEQ ID NO: 309, 310, 311, 312, or 313. In some embodiments, the MSH3 exon skipping nucleic acid construct comprises at least one of SEQ ID NO: 330-332, or any combination thereof.

[0027] This paper also discloses an MSH3 miRNA nucleic acid construct comprising a sequence encoding a primary miRNA, wherein the primary miRNA comprises a scaffold sequence, a loop sequence, and a miRNA sequence targeting endogenous MSH3 mRNA, wherein: (a) the scaffold sequence is derived from mir-30a, mir-33, or mir-155; (b) the loop sequence is derived from mir-22, mir-30a, mir-33, or mir-155; and (c) the miRNA sequence comprises any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256, or 257, or a sequence having at least 90% identity with any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256, or 257. In some embodiments, the scaffold sequence comprises any one of SEQ ID NO: 227, 228, 230, 231, 259, or 260. In some embodiments, the loop sequence comprises any one of SEQ ID NO: 229, 232, or 261. In some embodiments, the primary miRNA sequence comprises any one of SEQ ID NO: 234, 235, 238-241, or 262-269. In some embodiments, the sequence encoding the primary miRNA is operatively linked to a U6 promoter or a CMV promoter.

[0028] This document also discloses an MSH3 nucleic acid trans-splicing molecule comprising: (a) a coding domain sequence; (b) a splicing domain; and (c) a binding domain that binds to a target intron of MSH3 precursor mRNA; wherein the coding domain sequence is not an MSH3 coding domain sequence. In some embodiments, the coding domain sequence includes a sequence that causes frameshifting of mature MSH3 mRNA when trans-spliced ​​into MSH3 precursor mRNA. In some embodiments, the coding domain sequence includes one or more of exons 1, 2, and 3 of HTT. In some embodiments, the target intron of the MSH3 precursor mRNA is intron 5 or intron 15. In some embodiments, the binding domain comprises any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210, or a sequence having at least 90% identity with any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210.

[0029] This document also discloses an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprising: (a) any of the above-described HTT nucleic acid trans-splicing molecules; and (b) any of the above-described MSH3 exon-jumping nucleic acid constructs. In some embodiments, (a) and (b) are contained on a single vector. In some embodiments, the single vector is an AAV vector. In some embodiments, the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 356, 357, 363, or 364. In some embodiments, the AAV vector is a scAAV or ssAAV vector. In some embodiments, the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 369, 370, and 371.

[0030] This document also discloses an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct comprising: (a) any of the above-described HTT nucleic acid trans-splicing molecules; and (b) any of the above-described MSH3 exon-jumping nucleic acid constructs. In some embodiments, (a) and (b) are contained on a single vector. In some embodiments, the HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 358 or 359. In some embodiments, the single vector is an AAV vector.

[0031] This document also discloses an HTT trans-splicing and MSH3 miRNA nucleic acid construct comprising: (a) any of the above-described HTT nucleic acid trans-splicing molecules; and (b) any of the above-described MSH3 miRNA nucleic acid constructs. In some embodiments, (a) and (b) are contained on a single vector. In some embodiments, the HTT trans-splicing and MSH3 miRNA nucleic acid construct comprises any one of SEQ ID NO: 354 or 355. In some embodiments, the vector is an AAV vector.

[0032] An AAV vector is also disclosed, which comprises any of the HTT nucleic acid trans-splicing molecules described above. In some embodiments, the AAV vector comprises any one of SEQ ID NO: 356, 357, 363, or 364.

[0033] An AAV vector is also disclosed, which contains any of the above-mentioned HTT nucleic acid trans-splicing molecules or any of the above-mentioned nucleic acid trans-splicing molecules.

[0034] Also disclosed is a ribonucleic acid trans-splicing molecule, which comprises any one of SEQ ID NO: 23-36, 47-56, 83-105, 113-125, 175-191 or 199-206.

[0035] A ribonucleic acid trans-splicing molecule is also disclosed, which is transcribed from any of the above-mentioned HTT nucleic acid trans-splicing molecules or any of the above-mentioned nucleic acid trans-splicing molecules.

[0036] In some embodiments of the aforementioned HTT nucleic acid trans-splicing molecule, the HTT precursor mRNA contains at least one mutation associated with Huntington's disease (HD). In some embodiments, at least one HD-associated mutation contains HTT Amplification of CAG repeats in gene alleles. In some implementations, HTT The amplification of CAG repeats in the gene alleles contains more than 35 CAG repeats. In some embodiments, the at least one HD-associated mutation is autosomal dominant. In some embodiments, the at least one HD-associated mutation is expressed in at least one of: cortical pyramidal neurons, striatal polyspinous neurons, or hypothalamic neurons.

[0037] A vector is also disclosed, the vector comprising any of the above-mentioned HTT nucleic acid trans-splicing molecules; any of the above-mentioned nucleic acid trans-splicing molecules; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecules; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; or any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs.

[0038] A vector comprising any of the HTT nucleic acid trans-splicing molecules described above is also disclosed. In some embodiments, the vector includes a 5' regulatory domain operatively linked to the coding domain. In some embodiments, the 5' regulatory domain is operatively linked to a 5' untranslated region. In some embodiments, the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter. In some embodiments, the constitutive promoter is a CMV promoter or a CAGGS promoter.

[0039] A proviral plasmid is also disclosed, wherein the proviral plasmid comprises any of the above-mentioned HTT nucleic acid trans-splicing molecules; any of the above-mentioned nucleic acid trans-splicing molecules; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecules; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; or any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs.

[0040] Also disclosed is an adeno-associated virus (AAV) comprising any of the above-mentioned HTT nucleic acid trans-splicing molecules; any of the above-mentioned nucleic acid trans-splicing molecules; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecules; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; or any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs.

[0041] Also disclosed is an adeno-associated virus (AAV) comprising any of the HTT nucleic acid trans-splicing molecules described above, wherein the AAV optionally includes a 5' operably linked 5' regulatory domain in the nucleic acid trans-splicing molecule. In some embodiments, the AAV includes a 5' operably linked 5' regulatory domain in the coding domain. In some embodiments, the 5' regulatory domain is operably linked to a 5' untranslated region. In some embodiments, the 5' regulatory domain includes a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter or a CAGGS promoter. In some embodiments, the AAV exhibits neurotropic characteristics. In some embodiments, the AAV is AAV9, AAV8, AAV5, AAV2, AAV7, or AAV2.7m8, AAV-retro, AAV1, AAV4, or AAV-PHP.eB.

[0042] A composition is also disclosed comprising any of the above-described HTT nucleic acid trans-splicing molecules; any of the above-described nucleic acid trans-splicing molecules; any of the above-described MSH3 exon-jumping nucleic acid constructs; any of the above-described MSH3 miRNA nucleic acid constructs; any of the above-described MSH3 nucleic acid trans-splicing molecules; any of the above-described HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-described vectors; any of the above-described proviral plasmids; or any of the above-described AAVs. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition further comprises at least one antisense oligonucleotide or a construct encoding at least one antisense RNA that inhibits cis-splicing of HTT precursor mRNA. In some embodiments, the at least one antisense oligonucleotide comprises any one of SEQ ID NO: 126-135, or the construct encoding at least one antisense RNA binds to a target sequence bound to any one of SEQ ID NO: 126-135. In some embodiments, the at least one antisense oligonucleotide comprises SEQ ID NO: 131, or the construct encoding at least one antisense RNA binds to a target sequence bound to SEQ ID NO: 131.

[0043] A method for expressing bioactive HTT in target cells to restore the functional level of HTT protein in the target cells is also disclosed, the method comprising transducing the target cells with any of the following: any HTT nucleic acid trans-splicing molecule; any of the above-mentioned nucleic acid trans-splicing molecule; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecule; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned vectors; any of the above-mentioned proviral plasmids; any of the above-mentioned AAVs; or any of the above-mentioned combinations. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of at least HD-associated mutated HTT precursor mRNA is replaced in the target cells. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the target cells containing at least one HD-associated mutated HTT precursor mRNA are replaced. In some embodiments, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the target cells containing at least one HD-associated mutated HTT precursor mRNA are replaced. In some embodiments, the functional level of HTT in the target cells is restored by expressing a biologically functional HTT protein, and / or a mutant HTT protein. HTT RNA and related transcripts (e.g.) HTT1a () has decreased.

[0044] A method for reducing HTT expression in subjects is also disclosed, wherein the HTT comprises a polyglutamine repeat of more than 35 consecutive glutamine residues, the method comprising transfecting or transducing target cells, more particularly neurons, in subjects with any of the following: any HTT nucleic acid trans-splicing molecule; any of the above-mentioned nucleic acid trans-splicing molecule; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecule; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-mentioned vectors; any of the above-mentioned proviral plasmids; any of the above-mentioned AAVs; or any of the above-mentioned combinations.

[0045] A method for correcting at least one mutation in the HTT exon sequence of HTT precursor mRNA in target cells of a subject is also disclosed, the method comprising administering to the subject any of the following: any of the above-described HTT nucleic acid trans-splicing molecules; any of the above-described nucleic acid trans-splicing molecules; any of the above-described MSH3 exon-jumping nucleic acid constructs; any of the above-described MSH3 miRNA nucleic acid constructs; any of the above-described MSH3 nucleic acid trans-splicing molecules; any of the above-described HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-described vectors; any of the above-described proviral plasmids; any of the above-described AAVs; or any of the above-described combinations.

[0046] Also disclosed is a method for treating Huntington's disease (HD) in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any of the following: any of the above-described HTT nucleic acid trans-splicing molecules; any of the above-described nucleic acid trans-splicing molecules; any of the above-described MSH3 exon-jumping nucleic acid constructs; any of the above-described MSH3 miRNA nucleic acid constructs; any of the above-described MSH3 nucleic acid trans-splicing molecules; any of the above-described HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-described vectors; any of the above-described proviral plasmids; any of the above-described AAVs; or any of the above-described combinations.

[0047] In some embodiments of the above method, the method includes administering the following to the brain of the subject: any of the above-described HTT nucleic acid trans-splicing molecules; any of the above-described nucleic acid trans-splicing molecules; any of the above-described MSH3 exon-jumping nucleic acid constructs; any of the above-described MSH3 miRNA nucleic acid constructs; any of the above-described MSH3 nucleic acid trans-splicing molecules; any of the above-described HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid constructs; any of the above-described HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-described vectors; any of the above-described proviral plasmids; any of the above-described AAVs; or any of the above-described combinations. In some embodiments, the subject is a mammal, preferably a rodent, a non-human primate, or a human. In some embodiments, the subject is genetically susceptible to HD or has been diagnosed with HD.

[0048] It also discloses any of the above-mentioned HTT nucleic acid trans-splicing molecules; any of the above-mentioned nucleic acid trans-splicing molecules; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecules; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-mentioned vectors; any of the above-mentioned proviral plasmids; any of the above-mentioned AAVs; or any of the above-mentioned combinations, for the prevention or treatment of HD in subjects in need.

[0049] This document also discloses any of the above-mentioned HTT nucleic acid trans-splicing molecules; any of the above-mentioned nucleic acid trans-splicing molecules; any of the above-mentioned MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned MSH3 miRNA nucleic acid constructs; any of the above-mentioned MSH3 nucleic acid trans-splicing molecules; any of the above-mentioned HTT trans-splicing and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA and MSH3 exon-jumping nucleic acid constructs; any of the above-mentioned HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above-mentioned vectors; any of the above-mentioned proviral plasmids; any of the above-mentioned AAVs; or any of the above-mentioned combinations, for the preparation of a medicament for the treatment or prevention of HD in subjects of need.

[0050] A method is also disclosed, comprising introducing a nucleic acid trans-splicing molecule into a cell, the nucleic acid trans-splicing molecule being configured to splice to both a first target precursor mRNA and a second target precursor mRNA, wherein splicing to the first target precursor mRNA corrects a defect in the first target precursor mRNA, and wherein splicing to the second target precursor mRNA introduces a defect in the second target precursor mRNA. In some embodiments, the nucleic acid trans-splicing molecule comprises a first binding domain configured to target an intron of the first target precursor mRNA and a second binding domain configured to target an intron of the second target precursor mRNA. In some embodiments, the nucleic acid trans-splicing molecule further comprises a coding domain sequence comprising a functional sequence of one or more exons of the first target precursor mRNA, the functional sequence correcting a defect in the first target precursor mRNA. In some embodiments, the defect in the second target precursor mRNA comprises a frameshift in the coding sequence of the second target precursor mRNA. In some embodiments, the frameshift produces a premature stop codon in the second target precursor mRNA. In some embodiments, the defect involves the elimination of the endogenous start codon of the second target precursor mRNA. In some embodiments, the defect involves an inserted 5' UTR that prevents the translation of the protein encoded by the second target precursor mRNA. In some embodiments, the defect involves an inserted 3' UTR that destabilizes the precursor mRNA or prevents the second target precursor mRNA from being exported from the nucleus. In some embodiments, the defect involves the removal of a 5' cap or a 3' polyA tail from the second target precursor mRNA. In some embodiments, the defect leads to nonsense-mediated degradation of the second target precursor mRNA. In some embodiments, the introduction results in a decrease in the abundance of the gene product of the second target precursor mRNA in the cell compared to the abundance of the gene product before the introduction.

[0051] A method for reducing protein abundance in cells is also disclosed, the method comprising introducing a nucleic acid trans-splicing molecule into the cells, the nucleic acid trans-splicing molecule introducing a defect into a precursor mRNA encoding the protein. In some embodiments, the defect comprises one or more of the following: (a) introducing a frameshift in the coding sequence of the precursor mRNA; (b) removing an endogenous start codon from the precursor mRNA; (c) introducing a premature stop codon into the coding sequence of the precursor mRNA; (d) replacing the endogenous coding sequence of the precursor mRNA with an alternative coding sequence; (e) inserting a 5' UTR that prevents the translation of the endogenous coding sequence of the precursor mRNA; (f) inserting a 3' UTR that destabilizes the precursor mRNA; (g) inserting a 3' UTR that prevents the precursor mRNA from being exported from the nucleus; (h) removing a 5' cap from the precursor mRNA; or (i) removing a 3' polyA tail from the precursor mRNA. In some embodiments, the protein is MSH3. In some embodiments, the nucleic acid trans-splicing molecule includes a binding domain that binds to an intron of the precursor mRNA. In some embodiments, the nucleic acid trans-splicing molecule includes a heterologous coding domain sequence. Attached Figure Description

[0052] Figure 1 Depicting HTT The range of CAG trinucleotide repeats found in exon 1 of the gene. As shown, 8–35 CAG trinucleotide repeats reflect the presence of repeats in genes that do not exhibit similar characteristics. HTT Phenotypic normal (wild-type) range found in humans with signs of gene-related disease. 35–39 CAG trinucleotide repeats are associated with incomplete penetrance of HD. HTT The presence of more than 40 CAG trinucleotide repeats in this region of exon 1 of the gene is a pathogenic factor for HD.

[0053] Figure 2 Depicting HTT Somatic CAG repeat amplification in exon 1 of the gene, which is a potential mechanism in the pathogenesis of HD.

[0054] Figure 3 Demonstrated through HTT Precursor mRNA trans-splicing for exon editing is used as a treatment for HD. Intron 1-targeting, intron 2-targeting, or intron 3-targeting exon editors were designed and their efficiency in replacing mutant HTT exon 1 was tested.

[0055] Figure 4 Depicting coding targets HTTAn exemplary general structure of a construct for an RNA exon editor for intron 1. The exemplary exon editor generated from the depicted construct includes a 5' UTR, an exon 1 coding sequence, a splicing donor site, a linker, a binding domain, and a terminator sequence. The binding domain changes to follow the... HTT Intron 1 targets different locations. In some implementations, the promoter is a CMV promoter; in other implementations, the 5' UTR is... HTT 5' UTR; In some embodiments, the connector is a 40 polymer connector; In some embodiments, HTT The 5' UTR is combined with a 40-mer adapter; in some embodiments, an epitope tag is included, an example of which is an N-terminal 3X FLAG tag for mid-target protein detection. The above embodiments can be combined, wherein at least one of these embodiments is included in an RNA exon editor, and any and all combinations thereof, including a combination of all these embodiments in an RNA exon editor.

[0056] Figure 5 An illustration depicts an HTT intron 1-targeting RNA exon editor exhibiting varying levels of trans-splicing efficiency (replacement %), depending on the targeting location of the binding domain within the intron. This is demonstrated by targeting various regions of intron 1. HTT HEK293 cells were transfected with an intron-1-targeting RNA exon editor. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron at the 5' start position of the binding domain, according to SEQ ID NO: 1)_(length of the binding domain (nt)). For example, 701_150 indicates that the binding domain is the inverse complement sequence of bases 701-850 (according to SEQ ID NO: 1) in intron 1. The results shown are compared with exemplary models containing the indicated binding domain targets. HTT It is related to the intron 1-targeting RNA exon editor, where the 5' UTR contains HTT 5' UTR (SEQ ID NO:136) and the connector contains a 40-mer connector (SEQ ID NO: 37). NBD_150 is a control editor in which the target HTT The binding domain is not targeted HTT Replacement of the combined structural domain.

[0057] Figure 6The location and trans-splicing efficiency (replacement %) of the HTT intron 1-targeting RNA exon editor were graphically depicted. Different levels of trans-splicing efficiency (replacement %) were determined, reflecting the targeting location of the binding domain within the intron. Various regions of intron 1 were targeted. HTT HEK293 cells were transfected with an intron-1-targeting RNA exon editor. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in the intron at the 5' start position of the binding domain, according to SEQ ID NO: 1)_(length of the binding domain (nt)). For example, 701_150 indicates that the binding domain is the inverse complement sequence of bases 701-850 (according to SEQ ID NO: 1) in intron 1. The results shown are compared with exemplary models containing the indicated binding domain targets. HTT It is related to the intron 1-targeting RNA exon editor, where the 5' UTR contains HTT 5' UTR and the connector includes a 40 polymer connector.

[0058] Figure 7 The general structure of an exemplary construct encoding an exon editor for HTT intron 1-targeting RNA is depicted. An exemplary exon editor is shown, wherein expression is driven by a CMV promoter. The depicted exemplary exon editor includes an HTT 5' UTR, an N-terminal 3X FLAG tag, an exon 1 coding sequence, a splicing donor site, a linker, a binding domain (HTT_intron1_11704_100), and a terminator sequence.

[0059] Figure 8 The activity of an exemplary HTT intron 1-targeting RNA exon editor including various adapters is illustrated. As shown therein, in the HTT intron 1 (HTT_intron1_11704_100) exon editor, some adapters increase trans-splicing efficiency relative to the 40-mer adapter. HEK293 cells were transfected with the HTT intron 1-targeting RNA exon editor containing the indicated adapters. Cells were harvested 48 hours post-transfection and trans-splicing efficiency was determined by RT-qPCR.

[0060] Figure 9 Depicting coding targets HTT The general structure of an exemplary construct of an RNA exon editor for intron 2. The depicted exemplary exon editor includes a 5' UTR, exon 1-2 coding sequences, a splicing donor site, a linker, a binding domain, and a terminator sequence. The binding domain changes to follow the... HTT Intron 2 targets different locations. In some implementations, the 5' UTR is... HTT 5' UTR; In some embodiments, the connector is a 40 polymer connector; In some embodiments, HTT The 5' UTR is combined with a 40-mer connector. In some embodiments, the promoter is a CMV promoter; in some embodiments, the 5' UTR is... HTT 5' UTR; in some embodiments, the connector is a 40-mer connector; in some embodiments, the CMV promoter is with HTT 5' UTR combination; in some implementations, the CMV promoter is combined with HTT The 5' UTR and 40-mer adapter combination; in some embodiments, an epitope tag, an example of which is an N-terminal 3X FLAG tag for mid-target protein detection. The above embodiments can be combined, wherein at least one of these embodiments is included in an RNA exon editor, and any and all combinations thereof, including a combination of all these embodiments in an RNA exon editor.

[0061] Figure 10 Various examples are shown HTT The activity of an intron 2-targeting RNA exon editor, which exhibited varying levels of trans-splicing efficiency (replacement %), was assessed, depending on the targeting position of the binding domain within the intron. HEK293 cells were transfected with an exemplary HTT intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domain was: (nucleotide base position in the intron at the 5' start position of the binding domain, according to SEQ ID NO: 57) - (length of the binding domain (nt)). The results shown are compared with exemplary models containing the indicated binding domain targets. HTT It is related to the intron 2-targeting RNA exon editor, where the 5' UTR contains HTT 5' UTR and the connector contains a 40-mer connector. NBD is a contrast editor where targeting... HTT The binding domain is not targeted HTT The binding domain is replaced. Splice mutants are control editors for those lacking functional splice donor sites.

[0062] Figure 11The location and trans-splicing efficiency (replacement %) of the HTT intron 2-targeting RNA exon editor were graphically depicted. Different levels of trans-splicing efficiency (replacement %) were determined, reflecting the targeting location of the binding domain within the intron. HEK293 cells were transfected with the HTT intron 2-targeting RNA exon editor targeting various regions of intron 2. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature of the binding domain is: (nucleotide base position in the intron at the 5' start position of the binding domain, according to SEQ ID NO: 57) _ (length of the binding domain (nt)). The results shown are compared with exemplary models containing the indicated binding domain targets. HTT It is related to the intron 2-targeting RNA exon editor, where the 5' UTR contains HTT 5' UTR and the connector includes a 40 polymer connector.

[0063] Figure 12A and Figure 12B An exemplary HTT intron 2-targeting RNA exon editor targeting the upstream region of the branch point is depicted. A) The RNA exon editor is designed to target the upstream region of the intron 2 branch point, and the length of the binding domain varies. Exon editor expression is driven by the CMV promoter. The exon editor contains an HTT 5' UTR, an N-terminal 3X FLAG tag for mid-target protein detection, exon 1-2 coding sequences, a splice donor site, a 41-mer_2 linker, the indicated binding domain, and a terminator sequence. The binding domain is named as: (nucleotide base position in the intron at the 5' start position of the binding domain, according to SEQ ID NO: 57)_(length of the binding domain (nt)). The binding domain varies to target different lengths upstream of the branch point in intron 2. B) The length of the binding domain affects targeting efficiency based on substitution percentage calculations. HEK293 cells were transfected with an HTT intron 2-targeting RNA exon editor with varying binding domain lengths. Cells were harvested 48 hours after transfection and trans-splicing efficiency was determined by RT-qPCR.

[0064] Figure 13 The general structure of an exemplary construct encoding an exon editor for HTT intron 2-targeting RNA is described. Exon editor expression is driven by a CMV promoter. The exemplary exon editor includes an HTT 5' UTR, an N-terminal 3XFLAG tag, exon 1-2 coding sequences, a splicing donor site, a linker, a binding domain (HTT_intron2_12061_150), and a terminator sequence.

[0065] Figure 14The results showed that the exon editor containing the indicated adapter did not exhibit significantly different trans-splicing efficiency compared to the 40-mer adapter in HTT intron 2 (HTT_intron2_12061_150). HEK293 cells were transfected with the HTT intron 2-targeting RNA exon editor, in which the adapters included were altered. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR.

[0066] Figure 15 A cartoon illustration depicts the trans-splicing reaction and its competition with cis-splicing. Abbreviations used include: MALAT1 term; binding domain (BD); linker (L); splice site (SS). Cis-splicing molecules produced by cis-splicing contain amplified CAG repeats. Chimeric trans-splicing molecules produced by HTT 5' RTM-mediated 5' trans-splicing contain HTT exon 1, which contains a normal number of CAG repeats (8-35). Abbreviations used include: MALAT1 term; binding domain (BD); linker (L); splice site (SS); codon optimization (C / O).

[0067] Figure 16 A cartoon illustration shows antisense oligonucleotides (ASOs) designed to block competitive cis-splicing sites (ASO8-10) and upstream exon cis-splicing sites (ASO2-7). Each of these ASOs was co-transfected with the indicated HTT intron 2-targeting exon editor (HTT_intron2_12061_150), and in vitro trans-splicing efficiency was measured in HEK293 cells. Abbreviations used: MALAT1 terminator; binding domain (BD); linker (L); splicing site (SS).

[0068] Figure 17 The percentage of substitution activity is shown in the combination of the exemplary HTT intron 2-targeting exon editor (HTT_intron2_12061_150) and the indicated ASO. ASO6 is designed to block cis-splicing of the upstream intron, resulting in improved in vitro trans-splicing efficiency. HEK293 cells were co-transfected with the exemplary HTT intron 2-targeting RNA exon editor construct (REEC) and ASOs designed to block competitive cis-splicing sites or ASOs designed to block splicing of the upstream intron. Cells were harvested 48 hours after transfection and trans-splicing efficiency was determined by RT-qPCR.

[0069] Figure 18 The diagram shows the use of the indicated elements. HTTRepresentative RT-qPCR and Western blot images of whole-cell lysates from HEK293 cells transfected with an RNA exon editor (detecting N-terminal FLAG epitopes). Notably, the detection level of the target protein is related to that of... HTT RNA substitution % represents the trans-splicing efficiency. (Used with N-terminal FLAG markers) HTT HEK293 cells were transfected with an exon editor, and the exon editor exhibited a range of activities based on RT-qPCR (top panel). Anti-FLAG antibody was used to detect proteins generated after successful trans-splicing in whole-cell lysates (bottom panel). Anti-huntington protein antibody was used to detect native and ONT proteins generated after successful trans-splicing in whole-cell lysates (bottom panel). The intensity of the FLAG and ONT bands was proportional to the relative performance of the qPCR-based exon editor.

[0070] Figure 19 A schematic diagram is shown, illustrating the potential mechanism of a hybrid treatment approach designed to treat HD. This hybrid approach combines an agent that inhibits somatic CAG expansion (e.g., reduction via MSH3) with a targeted... HTT RNA exon editor for precursor mRNA. Targeting HTT The RNA exon editor of the precursor mRNA is used to replace any mutants that may result from DNA that "escapes" the inhibition of somatic cell expansion. HTT RNA.

[0071] Figure 20 A schematic diagram is shown depicting the targeting of tandem-binding domain RNA exon editors. HTT and MSH3 Potential mechanisms of action of precursor mRNA. In one exemplary implementation, the expression of the exon editor is driven by the CMV promoter. Such an exemplary exon editor may contain... HTT 5' UTR, N-end 3X FLAG tag, HTT Exon 1 coding sequence, splice donor site, adapter, MSH3 Combined with structural domains (targeted, for example) MSH3 Intron 5 or intron 15 of the precursor mRNA HTT Combine structural fields (e.g., HTT_intron1_11704_100) with terminating subsequences. HTT The structural domain will be combined with a targeted exon editor to generate corrections after successful trans-splicing. HTT RNA, and MSH3 Combining structural domains will target the exon editor to produce a chimera with premature stop codons. HTT Exon 1- MSH3RNA molecules, which will undergo nonsense-mediated degradation (NMD) and lead to subsequent... MSH3 The reduction in expression.

[0072] Figures 21A-21C The results were presented, showing the targeting. HTT and MSH3 The tandem-binding domain RNA exon editor demonstrated successful trans-splicing to two precursor mRNAs. RT-qPCR was performed on HEK293 cells: A) HTT Inverse splicing efficiency of the target (ONT) (through) HTT (Combined with structural domains), B) HTT-MSH3 Interlocking reverse shearing efficiency (through) MSH3 Combining structural domains, and C) MSH3 RNA transcript expression levels, the cells used for targeted therapy HTT Intron 1 and MSH3 The RNA exon editor of the tandem binding domain of intron 5 was transfected.

[0073] Figures 22A-22C The results were presented, showing the targeting. HTT and MSH3 The tandem-binding domain RNA exon editor demonstrated successful trans-splicing to two precursor mRNAs. RT-qPCR was performed on HEK293 cells: A) HTT Inverse splicing efficiency of the target (ONT) (through) HTT (Combined with structural domains), B) HTT-MSH3 Interlocking reverse shearing efficiency (through) MSH3 Combining structural domains, and C) MSH3 RNA transcript expression levels, the cells used for targeted therapy HTT Intron 1 and MSH3 The RNA exon editor of the tandem binding domain of intron 15 was transfected.

[0074] Figure 23 The general structure of an exemplary construct encoding an exon editor for HTT intron 3-targeting RNA is depicted. The depicted exemplary exon editor comprises a 5' UTR, exon 1-3 coding sequences, a splicing donor site, a linker, a binding domain, and a terminator sequence. The binding domain changes to follow the... HTT Intron 3 targets different locations. In some implementations, the 5' UTR is... HTT 5' UTR; in some embodiments, the connector is a 40-mer connector. In some embodiments, the promoter is a CMV promoter; in some embodiments, the 5' UTR is HTT5' UTR; in some embodiments, the connector is a 40-mer connector; in some embodiments, the CMV promoter is with HTT 5' UTR combination; in some implementations, the CMV promoter is combined with HTT The 5' UTR and 40-mer adapter combination; in some embodiments, an epitope tag, an example of which is an N-terminal 3X FLAG tag for mid-target protein detection. The above embodiments can be combined, wherein at least one of these embodiments is included in an RNA exon editor, and any and all combinations thereof, including a combination of all these embodiments in an RNA exon editor.

[0075] Figure 24 Various examples are shown HTT The activity of an intron 3-targeting RNA exon editor, which exhibited varying levels of trans-splicing efficiency (replacement %), was assessed based on the binding sites of the binding domains within the introns. HEK293 cells were transfected with HTT intron 3-targeting RNA exon editors targeting different regions of intron 3. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature of the binding domains was: (nucleotide base position in intron 3 at the 5' start position of the binding domain, according to SEQ ID NO: 155) - (length of the binding domain (nt)).

[0076] Figure 25 A direct comparison between the HTT intron 2-targeting exon editor and the HTT intron 3-targeting exon editor is shown. HEK293 cells were transfected in parallel with both HTT intron 2-targeting and intron 3-targeting RNA exon editors. Cells were harvested 48 hours after transfection, and trans-splicing efficiency was determined by RT-qPCR. The nomenclature for the binding domain is: (nucleotide base position in intron 3 at the 5' start position of the binding domain) _ (length of the binding domain (nt)).

[0077] Figure 26 This demonstrates that reduced self-splicing does not affect the trans-splicing efficiency of the HTT exon editor. Table 1 shows the cryptic splicing sites identified in the original exon editor and the sequence changes made in the self-splicing-reduced exon editor. HEK293 cells were transfected with HTT intron 2-targeting (HTT_intron2_12061_150) RNA exon editors with or without reduced self-splicing. Cells were harvested 48 hours after transfection, and trans-splicing efficiency was determined by RT-qPCR.

[0078] Figure 27Representative RT-qPCR and Western blot images of lysate from HEK293 cells transfected with an HTT RNA exon editor are shown, testing two promoters and different 5' UTR combinations. HEK293 cells were transfected with an N-terminal FLAG-labeled HTT exon editor (with or without an HTT 5' UTR) driven by a CMV or CAGGS promoter, and testing was performed for wild-type (GTAAGT) splicing sites targeting intron 2 (HTT_intron2_12061_150), splicing mutants targeting intron 2 (HTT_intron2_12061_150), or wild-type splicing sites with a non-target-binding domain (NBD). RT-qPCR was performed on RNA from these cells (top panel). α-FLAG antibody was used to detect proteins (containing N-terminal FLAG epitopes) generated after successful trans-splicing in whole-cell lysate (bottom panel). ONT: The target HTT protein that has successfully undergone trans-splicing. NSP: Non-splicing protein.

[0079] Figure 28 This illustrates how, in an exemplary 5' HTT intron 1-targeting exon editor, non-splicing proteins (NSPs) are reduced in a combined manner by three tandem repeats (3X UBS; SEQ ID NO: 345) containing a U1 snRNA binding site and one AU-rich element (ARE; SEQ ID NO: 346). HEK293 cells were transfected with an HTT intron 1-targeting (HTT_intron1_11704_100) RNA exon editor, which was modified at its linker region to include the indicated NSP-reducing element. Cells were harvested 48 hours post-transfection for Western blot analysis. ONT: Mid-target HTT protein that successfully underwent trans-splicing. NSP: Non-splicing protein.

[0080] Figure 29 This illustrates how non-splicing proteins (NSPs) are reduced in combination within a 5' HTT intron 2-targeting exon editor via three tandem repeats (3X UBS; SEQ ID NO: 345) containing a U1 snRNA binding site and an AU-rich element (ARE; SEQ ID NO: 346). HEK293 cells were transfected with an HTT intron 2-targeting (HTT_intron2_12061_150) exon editor, which undergoes alterations at its linker region to include the indicated NSP-reducing element. Cells were harvested 48 hours post-transfection, and trans-splicing efficiency was determined by RT-qPCR (top panel) or Western blot analysis was performed (bottom panel).

[0081] Figure 30This demonstrates the targeting of tandem-binding domain RNA exon editors. HTT Intron 2 and MSH3 Mechanism of action of precursor mRNA. Exon editor expression is driven by the CMV promoter and contains the HTT 5' UTR, N-terminal 3X FLAG tag, HTT exon 1 and exon 2 coding sequences, splice donor site, adapter, and MSH3 binding domain (targeting). MSH3 The precursor mRNA contains intron 5 or intron 15, an HTT-binding domain (HTT_intron2_12061_150), and a terminator sequence. The HTT-binding domain targets the exon editor to produce corrected HTT RNA after successful trans-splicing, while the MSH3-binding domain targets the exon editor to produce a chimeric HTT exon 1+2-MSH3 RNA molecule with a premature stop codon. This molecule undergoes nonsense-mediated degradation (NMD) and leads to a reduction in subsequent MSH3 expression.

[0082] Figure 31A and Figure 31B This presentation showcases an overview of RT-qPCR for HTT trans-splicing and HTT-MSH3 chimera generation (via MSH3 trans-splicing) in a tandem-binding domain exon editor. RT-qPCR in HEK293 cells: A) HTT target (ONT) trans-splicing efficiency (via MSH3 trans-splicing). HTT Intron 2 targeting binding domain), and B) HTT-MSH3 chimeric trans-splicing efficiency (via MSH3 Intron 5 targeting binding domain), the cell is used for targeting HTT Intron 2 and MSH3 The RNA exon editor containing the tandem-binding domain of intron 5 was transfected. For all exon editors tested here, targeting... HTT The binding domain of intron 2 is HTT_intron2_12061_150, while MSH3_intron5_213_100 and MSH3_intron5_188_150 are targeted at... MSH3 Intron 5 targeting binding domains were tested. The binding domains were tandemly located, and the order of the binding domains is shown. Each MSH3 binding domain of the exon editor was also tested, with the MALAT1 triple helix positioned between two tandem binding domains.

[0083] Figure 32A and Figure 32BThis presentation showcases an overview of RT-qPCR for HTT trans-splicing and HTT-MSH3 chimera generation (via MSH3 trans-splicing) in a tandem-binding domain exon editor. RT-qPCR in HEK293 cells: A) HTT target (ONT) trans-splicing efficiency (via MSH3 trans-splicing). HTT Intron 2 targeting binding domain), and B) HTT-MSH3 chimeric trans-splicing efficiency (via MSH3 Intron 15 targeting binding domain), the cell is used for targeting HTT Intron 2 and MSH3 The RNA exon editor containing the tandem-binding domain of intron 15 was transfected. For all exon editors tested here, targeting... HTT The binding domain of intron 2 is HTT_intron2_12061_150, while MSH3_intron15_6523_120 and MSH3_intron15_6498_150 target... MSH3 Intron 15 targeting binding domains were tested. The binding domains were tandemly located, and the order of the binding domains is shown. Each MSH3 binding domain of the exon editor was also tested, with the MALAT1 triple helix positioned between two tandem binding domains.

[0084] Figure 33 A cartoon was displayed, depicting the process of targeting... MSH3 MSH3 knockdown is achieved through miRNA targeting of mRNA. MSH3 miRNA knockdown that degrades mRNA and transcripts.

[0085] Figure 34 Western blot analysis of the MSH3 exon 23-targeting RNAi constructs is shown. Constructs (SEQ ID NO: 234, 235, 238-241) containing the MSH3 exon 23-targeting miRNA active sequence TTAATCCATAACTCCTTGC (SEQ ID NO: 224) and control constructs (SEQ ID NO: 236, 237, 242, and 243) were analyzed. ImageJ analysis of the Western blots was performed to analyze MSH3 protein knockdown (top panel). U6 promoter-driven shRNA and CMV promoter-driven primary miRNA mimics were designed and tested. Variations included strand placement of the guide strand (5' arm or 3' arm), including protrusions in the stem structure, and alterations to the miRNA scaffold. Negative controls included constructs containing the non-targeting sequence or without hairpin loops.

[0086] Figure 35RT-qPCR and Western blot analysis of the MSH3-targeting RNAi construct are shown. The coding target was analyzed. MSH3 Constructs of miRNAs from different regions of the transcript were developed. ImageJ analysis of Western blots was performed to analyze MSH3 protein knockdown. Primary miRNA mimics driven by the CMV promoter targeting different exon sequences of MSH3 were designed and tested.

[0087] Figure 36 A cartoon illustration depicts MSH3 knockdown achieved via antisense RNA (asRNA) based on small nuclear RNA (snRNA). MSH3 can be inactivated by antisense RNA encoded in a snRNA scaffold, which... MSH3 Splice junction annealing prevents exon inclusion. This leads to exon skipping and premature termination codon generation, ultimately causing NMD in the MSH3 transcript. The diagram here illustrates an example of an MSH3 splicing regulator targeting exon 2 skipping.

[0088] Figure 37 The relative MSH3 RNA expression levels at exon 1-exon 2 and exon 2-exon 3 junctions in the MSH3 splicing regulator targeting exon 2 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcript is SEQ ID NO: 284-287. SEQ ID NO: 284: U7SmOPT containing MSH3 In1 / Ex2 asRNA (SEQ ID NO: 274); SEQ ID NO: 285: U7SmOPT containing MSH3 Ex2 / In2 asRNA (SEQ ID NO: 275); SEQ ID NO: 286: U7SmOPT containing MSH3 In1 / Ex2 + Ex2 / In2 asRNA (SEQ ID NO: 276); SEQ ID NO: 287: MSH3 In1 / Ex2 + Ex2 / In2 long (160nt) asRNA (SEQ ID NO: 277).

[0089] Figure 38The relative MSH3 RNA expression levels at exon 2-exon 3 and exon 3-exon 4 junctions in the MSH3 splicing regulator targeting exon 3 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcripts are SEQ ID NO: 288-290. SEQ ID NO: 288: U7SmOPT containing MSH3 In2 / Ex3 asRNA (SEQ ID NO: 278); SEQ ID NO: 289: U7SmOPT containing MSH3 Ex3 / In3 asRNA (SEQ ID NO: 279); SEQ ID NO: 290: U7SmOPT containing MSH3 In2 / Ex3+Ex3 / In3 asRNA (SEQ ID NO: 280).

[0090] Figure 39 The relative MSH3 RNA expression levels at exon 3-exon 4 and exon 4-exon 5 junctions in the MSH3 splicing regulator targeting exon 4 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcripts are SEQ ID NO: 291-293. SEQ ID NO: 291: U7SmOPT containing MSH3 In3 / Ex4 asRNA (SEQ ID NO: 281); SEQ ID NO: 292: U7SmOPT containing MSH3 Ex4 / In4 asRNA (SEQ ID NO: 282); SEQ ID NO: 293: U7SmOPT containing MSH3 In3 / Ex4 + Ex4 / In4 asRNA (SEQ ID NO: 283).

[0091] Figure 40The relative MSH3 RNA expression levels at exon 2-exon 3 and exon 3-exon 4 junctions in the MSH3 splicing regulator targeting exon 3 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcripts are SEQ ID NO: 290, 324-326. SEQ ID NO: 290: U7SmOPT containing MSH3 In2 / Ex3 + Ex3 / In3 asRNA (SEQ ID NO: 280); SEQ ID NO: 324: MSH3 U7 SmOPT splicing regulator containing In3 / Ex3 / In2 asRNA (SEQ ID NO: 299); SEQ ID NO: 325: MSH3 U7SmOPT splicing regulator containing In3 / Ex3-1 + adapter + Ex3 / In2-1 asRNA (SEQ ID NO: 300 + 298 + 301); SEQ ID NO: 326: MSH3 U2 splicing regulator containing In3 / Ex3-2 + adapter + Ex3 / In2-2 asRNA (SEQ ID NO: 302 + 298 + 303).

[0092] Figure 41 The relative MSH3 RNA expression levels at exon 5-exon 6 and exon 6-exon 7 junctions in the MSH3 splicing regulator targeting exon 6 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcript is SEQ ID NO: 327-329. SEQ ID NO: 327: MSH3 U7 SmOPT splicing regulator containing In6 / Ex6 / In5 asRNA (SEQ ID NO: 304); SEQ ID NO: 328: MSH3 U7SmOPT splicing regulator containing In6 / Ex6-1 + adapter + Ex6 / In5-1 asRNA (SEQ ID NO: 305 + 298 + 306); SEQ ID NO: 329: MSH3 U2 splicing regulator containing In6 / Ex6-2 + adapter + Ex6 / In5-2 asRNA (SEQ ID NO: 307 + 298 + 308).

[0093] Figure 42 The relative positions of exon 6-exon 7 and exon 7-exon 8 junctions in the MSH3 splicing modulator targeting exon 7 skipping are depicted. MSH3 RNA expression levels (bottom), and explanation MSH3 Cartoon illustration of the asRNA target region in the precursor mRNA (top). The splicing regulator transcript is SEQ ID NO: 330-332. The antisense RNA (asRNA) contained in SEQ ID NO: 330 is MSH3 In7 / Ex7 / In6 asRNA (asRNA region SEQ ID NO: 309); the asRNA contained in SEQ ID NO: 331 is In7 / Ex7-1 (asRNA region SEQ ID NO: 310) + adapter (SEQ ID NO: 298) + Ex7 / In6-1 (asRNA region SEQ ID NO: 311); the asRNA contained in SEQ ID NO: 332 is In7 / Ex7-2 (asRNA region SEQ ID NO: 312) + adapter (SEQ ID NO: 298) + Ex7 / In6-2 (asRNA region SEQ ID NO: 313).

[0094] Figure 43 The relative MSH3 RNA expression levels at exon 7-exon 8 and exon 8-exon 9 junctions in the MSH3 splicing regulator targeting exon 8 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcript is SEQ ID NO: 333-335. SEQ ID NO: 333: MSH3 U7 SmOPT splicing regulator containing In8 / Ex8 / In7 asRNA (SEQ ID NO: 314); SEQ ID NO: 334: MSH3 U7SmOPT splicing regulator containing In8 / Ex8-1 + adapter + Ex8 / In7-1 asRNA (SEQ ID NO: 315 + 298 + 316); SEQ ID NO: 335: MSH3 U2 splicing regulator containing In8 / Ex8-2 + adapter + Ex8 / In7-2 asRNA (SEQ ID NO: 317 + 298 + 318).

[0095] Figure 44The relative MSH3 RNA expression levels at the exon 14-exon 15 and exon 15-exon 16 junctions in the MSH3 splicing regulator targeting exon 15 skipping are depicted (bottom), along with a cartoon illustrating the asRNA target region in the MSH3 precursor mRNA (top). The splicing regulator transcript is SEQ ID NO: 336-338. SEQ ID NO:336: MSH3 U7 SmOPT splicing regulator containing In15 / Ex15 / In14 asRNA (SEQ ID NO: 319); SEQ ID NO: 337: MSH3 U7 SmOPT splicing regulator containing In15 / Ex15-1 + adapter + Ex15 / In14-1 asRNA (SEQ ID NO: 320 + 298 + 321); SEQ ID NO: 338: MSH3 U2 splicing regulator containing In15 / Ex15-2 + adapter + Ex15 / In14-2 asRNA (SEQ ID NO: 322 + 298 + 323).

[0096] Figure 45A , Figure 45B and Figure 45C The results showed that the MSH3 exon 7 splicing regulator resulted in a decrease in MSH3 RNA and protein levels. HEK293 cells were transfected with a snRNA-based splicing regulator designed to skip MSH3 exon 7. Cells were harvested 48 hours after transfection, and MSH3 knockdown was measured by RT-qPCR. Figure 45B Or perform Western blot analysis. Figure 45C The splicing regulator transcripts are SEQ ID NO: 330-332. SEQ ID NO: 330: MSH3 U7 SmOPT splicing regulator containing In7 / Ex7 / In6 asRNA (SEQ ID NO: 309); SEQ ID NO: 331: MSH3 U7 SmOPT splicing regulator containing In7 / Ex7-1 + adapter + Ex7 / In6-1 asRNA (SEQ ID NO: 310 + 298 + 311); SEQ ID NO: 332: MSH3U2 splicing regulator containing In7 / Ex7-2 + adapter + Ex7 / In6-2 asRNA (SEQ ID NO: 312 + 298 + 313).

[0097] Figure 46 This demonstrates how trans-splicing and knockdown of unedited microRNAs (miRNAs) can be achieved. HTTCombination strategies for species (including HTT1a) to correct mutant HTT.

[0098] Figure 47 A vectorized hybrid molecule is shown, which combines an HTT exon editor with a target unedited exon. HTT miRNAs of mRNA. Short hairpin RNAs (shRNAs) or microRNAs (miRNAs) designed to reduce HTT gene expression can be added to exon editors within the same cistron (e.g., within introns of exon editors) or as standalone cistrons with their own regulatory sequences. RNAi can reduce unedited targets (e.g., mutants). HTT The expression of ) is to selectively reduce unedited ) using RNAi. HTT The exon editor contains HTT CDS, which contains the sequence-modified portion, makes the edited... HTT It is resistant to shRNA or miRNA.

[0099] Figure 48A and Figure 48B This section presents a profile of trans-splicing and HTT knockdown of HTT exon editor, HTT miRNA-1, and a dual-hybrid molecule of HTT exon editor and HTT miRNA-1. A) All HTT Trans-splicing (editing) in transcripts HTT Transcript percentage, and B) unedited and trans-splicing (edited) in each treatment. HTT Transcript copy number. HEK293 cells were transfected with a double hybrid molecule consisting of an HTT intron 2-targeting RNA exon editor, HTT miRNA-1, and an HTT intron 2-targeting exon editor (SEQ ID NO: 204) + HTT miRNA-1 (HTT miRNA-1 coding sequence SEQ ID NO: 341, containing the HTT miRNA-1 active sequence SEQ ID NO: 339). Cells were harvested 48 hours after transfection and RNA was subjected to RT-qPCR. A double hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), wherein the double hybrid comprises SEQ ID NO: 341 and SEQ ID NO: 204.

[0100] Figures 49A-49CThis study presents the in vitro trans-splicing and HTT knockdown profiles of molecules containing HTT miRNA-1 and HTT miRNA-2. HEK293 cells were transfected with an HTT intron 2-targeting RNA exon editor and a double hybrid molecule consisting of an HTT intron 2-targeting exon editor and either HTT miRNA-1 or HTT miRNA-2. Cells were harvested 48 hours post-transfection, and RNA was analyzed by RT-qPCR: A) Trans-splicing profile, B) HTT knockdown profile, and C) HTT Copy number analysis. A double-hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), the double hybrid comprising SEQ ID NO: 341 and SEQ ID NO: 204. A double-hybrid molecule of HTT exon editor and HTT miRNA-2 (SEQ ID NO: 355), the double hybrid comprising SEQ ID NO: 344 and SEQ ID NO: 204.

[0101] Figure 50 The results show that HTT miRNA-1 successfully knocked down the unedited [protein / protein]. HTT Transcripts, with exon editors and edited HTT Transcriptional interactions were minimal. HEK293 cells were transfected with a double hybrid molecule of HTT exon editor + / - HTT miRNA-1. Cells were harvested 48 hours after transfection, and HTT knockdown and trans-splicing efficiency were determined by RT-qPCR (top image) or Western blot analysis was performed (bottom image). The double hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), wherein the double hybrid comprises SEQ ID NO: 341 and SEQ ID NO: 204.

[0102] Figure 51 A cartoon illustration was shown depicting the reduction of MSH3 through a combination of splice modulation and HTT reverse splicing.

[0103] Figure 52A and Figure 52BResults are presented demonstrating the performance of the HTT exon editor + MSH3 splicing regulator dual hybrid molecule. HEK293 cells were transfected with the HTT exon editor + / - MSH3 splicing regulator. Cells were harvested 48 hours post-transfection and assayed by RT-qPCR to determine: A) trans-splicing profile and B) MSH3 knockdown profile. The HTT exon editor + MSH3 splicing regulator dual hybrid molecule is: MSH3 splicing regulator + HTT exon editor (SEQ ID NO: 356), the dual hybrid comprising SEQ ID NO: 331 and SEQ ID NO: 204.

[0104] Figure 53 AD demonstrates a comparison of self-complementary AAV (scAAV) versus single-stranded AAV (ssAAV) using a dual hybrid molecule of HTT exon editor + MSH3 splicing regulator. HEK293 cells were transduced with scAAV or ssAAV expressing a dual hybrid molecule of HTT exon editor + MSH3 exon 7 skipping splicing regulator. AAV2 serotype was used. Cells were harvested 48 hours after transduction and subjected to RT-qPCR and Western blot analysis. The indicated sequence was inserted between the ITRs of AAV2, whether ssAAV or scAAV. The dual hybrid molecule of HTT exon editor + MSH3 splicing regulator (SEQ ID NO: 357), comprising SEQ ID NO: 331 and SEQ ID NO: 204, is head-to-head oriented.

[0105] Figure 54Results are presented, showing the performance of the triple hybrid molecule of HTT exon editor + HTT miRNA + MSH3 splicing regulator compared to its control. HEK293 cells were transfected with either HTT exon editor + / - HTT miRNA-1 or HTT miRNA-2 + / - MSH3 splicing regulator. Cells were harvested 48 hours post-transfection and assayed by RT-qPCR: Top panel) % of trans-spliced ​​HTT transcripts, Middle panel) HTT knockdown profile, and Bottom panel) MSH3 knockdown profile. The control hybrid molecule was also tested, which contained an exon editor with a splicing donor mutation, a splicing regulator containing a scrambled asRNA sequence, or a miRNA containing a scrambled asRNA. "1" for HTT miRNA indicates HTT miRNA-1, and "2" for HTT miRNA indicates the use of HTT miRNA-2. SM, splicing mutant. Scr, scrambled control. miR-33 was used for the miRNA scaffold. HTT exon editor (SEQ ID NO: 204); a double hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), the double hybrid comprising SEQ ID NO: 341 and SEQ ID NO: 204; a double hybrid molecule of HTT exon editor + MSH3 splicing regulator: MSH3 splicing regulator + HTT exon editor (SEQ ID NO: 356), the double hybrid comprising SEQ ID NO: 331 and SEQ ID NO: 204; a triple hybrid molecule of HTT exon editor + HTTmiRNA-1 + MSH3 splicing regulator (SEQ ID NO: 358), the triple hybrid comprising SEQ ID NO: 331, SEQ ID NO: 341 and SEQ ID NO: 204; a triple hybrid molecule of HTT exon editor + HTT miRNA-2 + MSH3 splicing regulator (SEQ ID NO: 356). 359), the triple hybrid comprising SEQ ID NO: 331, SEQ ID NO: 344 and SEQ ID NO: 204; and a double hybrid molecule of HTT exon editor and HTT miRNA-2 (SEQ ID NO: 355), the double hybrid comprising SEQ ID NO: 344 and SEQ ID NO: 204.

[0106] Figure 55The trans-splicing profile of HD molecules in iCell GlutaNeurons is shown, as measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced using either an HTT exon editor + HTT miRNA double hybrid molecule or an HTT exon editor + MSH3 splicing regulator double hybrid molecule, both packaged in AAV2.7m8 cells. Cells were harvested after 18–21 days for RT-ddPCR and Western blotting. The indicated sequence was inserted between the ITRs of AAV2.7m8 cells. A double hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), the double hybrid comprising SEQ ID NO: 341 and SEQ ID NO: 204; a double hybrid molecule of HTT exon editor + MSH3 splicing regulator (SEQ ID NO: 357), the double hybrid comprising SEQ ID NO: 331 and SEQ ID NO: 204, in a head-to-head orientation; a triple hybrid molecule of HTT exon editor + HTT miRNA-1 + MSH3 splicing regulator (SEQ ID NO: 358), the triple hybrid comprising SEQ ID NO: 331, SEQ ID NO: 341 and SEQ ID NO: 204.

[0107] Figure 56 The knockdown profile of HTT miRNA in iCell GlutaNeurons is shown, as measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced with either an HTT exon editor + HTT miRNA double hybrid molecule or an HTT exon editor + MSH3 splicing regulator double hybrid molecule, both packaged in AAV2.7m8. Cells were harvested after 18 days for RT-ddPCR and Western blotting. The indicated sequence was inserted between the ITRs of AAV2.7m8. A double hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), comprising SEQ ID NO: 341 and SEQ ID NO: 204; and a double hybrid molecule of HTT exon editor + MSH3 splicing regulator (SEQ ID NO: 357), comprising SEQ ID NO: 331 and SEQ ID NO: 204, were shown head-to-head.

[0108] Figure 57The MSH3 knockdown profile of the MSH3 splicing regulator in iCell GlutaNeurons is shown, as measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced with either an HTT exon editor + HTT miRNA-1 double hybrid molecule or an HTT exon editor + MSH3 splicing regulator double hybrid molecule, both packaged in AAV2.7m8 cells. Cells were harvested after 18 days for RT-ddPCR and Western blotting. The indicated sequence was inserted between the ITRs of the AAV2.7m8 cells. A double-hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), the double hybrid comprising SEQ ID NO: 341 and SEQ ID NO: 204; and a double-hybrid molecule of HTT exon editor + MSH3 splicing regulator (SEQ ID NO: 357), the double hybrid comprising SEQ ID NO: 331 and SEQ ID NO: 204, in a head-to-head orientation.

[0109] Figure 58 This study presents the profile of HTT trans-splicing in the brains of BAC-CAG mice. BAC-CAG mice were neonatally injected with the indicated HD molecule packaged in AAV9 (1E+11 or 3E+11 vg / animal). Cortical and striatal tissues were harvested 4 weeks post-injection, and the efficiency of HTT exon substitutions was analyzed by trans-splicing morphology analysis using RT-ddPCR and Western blotting. SEQ ID NO: 369: scAAV, mouse Msh3 splicing regulator + CMVp::exon editor (double heterozygote of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 370: ssAAV, mouse Msh3 splicing regulator + CMVp::exon editor (double heterozygote of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 371: ssAAV, mouse Msh3 splicing regulator + CAGGSp::exon editor (double heterozygote of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation).

[0110] Figure 59The relationship between exon editor RNA copy number and trans-splicing efficiency (HTT substitution %) observed in the brains of BAC-CAG mice was depicted. SEQ ID NO: 369: scAAV, mouse Msh3 splicing regulator + CMVp::exon editor (double heterozygous for SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 370: ssAAV, mouse Msh3 splicing regulator + CMVp::exon editor (double heterozygous for SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 371: ssAAV, mouse Msh3 splicing regulator + CAGGSp::exon editor (double heterozygous for SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation). Detailed Implementation

[0111] The following examples are provided to illustrate some embodiments of this disclosure, but are not intended to limit the scope of this disclosure; it should be understood that other procedures, methods or techniques known to those skilled in the art may be used alternatively by way of their exemplary nature.

[0112] The compositions and methods described herein relate to the treatment of [a condition caused by] [a specific condition]. HTT Trans-splicing molecules (e.g., precursor mRNA trans-splicing molecules) cause diseases or conditions due to gene mutations. Such mutations include... HTT The CAG trinucleotide repeat in exon 1 of the gene is amplified. The compositions and methods described herein utilize precursor mRNA trans-splicing molecules for gene therapy (e.g., in vivo gene therapy, such as delivery via adeno-associated virus) to treat [the condition caused by...]. HTT Diseases caused by CAG trinucleotide repeat amplification, such as HD. The compositions and methods described herein also employ combinations of precursor mRNA trans-splicing molecules with other therapeutic agents described herein for gene therapy (e.g., in vivo gene therapy) to treat diseases caused by CAG trinucleotide repeat amplification, such as HD. HTT Diseases caused by at least one mutation in the blood, such as HD.

[0113] The compositions and methods described herein also relate to therapeutic molecules that knock down MSH3 expression. MSH3 knockdown constructs can be used alone as a therapeutic agent or in combination with HTT-correcting therapeutic agents and / or HTT-knockdown therapeutic agents. MSH3 knockdown constructs can also be used in combination with other therapeutic agents besides HTT-correcting therapeutic agents, such as other therapeutic agents designed to correct trinucleotide repeat amplification disorders.

[0114] As described in this article, Henoch-Schönlein dysplasia (HD) is a hereditary, progressive neurodegenerative disorder that can only be treated palliatively. These treatments include medication, physical therapy, and counseling, which provide some symptom relief. Although HD manifests in various ways, it is characterized by a progressive loss of motor control, mood regulation, and cognitive function. HD typically occurs in people in their 30s and 40s. The disease is associated with the loss of cortical pyramidal neurons, moderately spinous neurons in the striatum, and hypothalamic neurons. The genetic cause of HD is... HTT An autosomal dominant inheritance of CAG trinucleotide repeat amplification in exon 1 of a gene, where the presence of more than 40 CAG repeats in this region is a pathogenic factor. See also Figure 1 .

[0115] HTT The locus is large, spanning 180 kb and consisting of 67 exons. HTT Gene expression is essential for normal development. Although HTT protein is widely expressed, pathological amplification of CAG trinucleotide repeats has the most severe impact on the brain, with early pathological effects observed in the striatum and motor cortex. A potential mechanism in the pathogenesis of HD involves events occurring in the affected brain regions (e.g., the striatum) of HD patients. HTT Somatic cell CAG repeat expansion. See also Figure 2 Human genetic evidence suggests that genes in DNA mismatch repair pathways (such as...) MSH2, MSH3, FAN1, MLH1) Involved in controlling this process and altering clinical outcomes of HD.

[0116] Despite considerable efforts by numerous biotechnology and pharmaceutical companies to develop therapeutics for HD, there is currently no disease-modifying treatment for HD. The inventors of this invention attempt to address this need using a variety of different modalities, each of which can be implemented individually or in combination, to provide a therapeutic intervention for HD.

[0117] Exon replacement via trans-splicing of HTT precursor mRNA Exon replacement via trans-splicing of precursor mRNA is well-suited as a treatment for HD because it can replace mutant exon replacements. HTT Exon 1, while retaining HTT The wild-type copy. Furthermore, the method can theoretically treat 100% of the HD population because it addresses the entire spectrum of genetic variations in HD patients. In other words, it is not limited to addressing specific HD patient-specific variations (e.g., SNPs), but rather serves as a pan-specific therapeutic agent capable of correcting genetic defects across the entire exon.

[0118] As described herein, the inventors designed a targeted HTTRNA exon editors for introns 1, 2, or 3 of precursor mRNA were selected and tested. Therefore, these RNA exon editors can replace... HTT All exons 1, 1 and 2, or exons 1-3 of the mRNA, thereby correcting any and all pathogenic mutations present in exons 1, 1 and 2, or exons 1-3 (e.g., correcting for amplified CAG trinucleotide repeats in exon 1 for wild-type numbers). Figure 3 As shown, RNA exon editing via trans-splicing enables the use of any of the intron 1-targeted exon editors, intron 2-targeted exon editors, or intron 3-targeted exon editors with wild-type RNA. HTT Exon 1 substitution mutant HTT Exon 1.

[0119] In some implementation schemes, targeting HTT The RNA exon editor of intron 1 of the precursor mRNA contains the CMV promoter (SEQ ID NO: 137) (used here for in vitro experiments in HEK293 cells), 5' UTR (e.g., HTT The 5' UTR (SEQ ID NO: 136 or 192), epitope tags (e.g., 3X FLAG tags (SEQ ID NO: 4)) used to detect the intermediate target (ONT) HTT protein generated after successful trans-splicing, HTT Exon 1 (SEQ ID NO: 3) (which may be codon-modified, followed by the native sequence), splice donor sequence (GTAAGT), adapter (e.g., 40-mer adapter (SEQ ID NO: 37)), target HTT The binding domain of any of the regions in the intron 1 precursor mRNA and the triple-helix terminator (e.g., the MALAT1 terminator (SEQ ID NO:5 or a modified form thereof, such as SEQ ID NO:6)). See, for example Figure 4 In some implementation schemes, targeting HTT The RNA exon editor of intron 1 of the precursor mRNA does not contain one or more of the following: CMV promoter (SEQ ID NO: 137), 5' UTR (e.g., HTTThe promoter may contain a 5'UTR (SEQ ID NO: 136 or 192), an epitope tag (e.g., a 3X FLAG tag (SEQ ID NO: 4)), a splice donor sequence GTAAGT, a 40-mer adapter (SEQ ID NO: 37), and a triple-helix terminator (e.g., the MALAT1 terminator (SEQ ID NO: 5 or a modified form thereof, e.g., SEQ ID NO: 6)). In some embodiments, the CMV promoter may be replaced by a different promoter. A promoter with properties suitable for in vivo studies and therapeutic agents containing an RNA exon editor may be selected. In some embodiments, exemplary RNA exon editors do not contain a FLAG tag or any epitope tag. In such embodiments, therapeutic agents containing RNA exon editors may not contain any epitope tags, which may reduce the likelihood of immunogenicity. Figure 5 As shown, HTT The exon substitution level (trans-splicing efficiency) varies with the targeting position of the binding domain within intron 1. The results presented in this paper indicate that targeting the 3' end of intron 1 near the branch point results in trans-splicing to… HTT Increased efficiency in precursor mRNA is associated with this. See also Figure 6 .

[0120] Next, the inventors attempted to select an exemplary combination domain. HTT _intron1_11704_100, and determined the role of different linkers operably linked to this binding domain in the context of RNA exon editors, further improving HTT Inverse splicing efficiency of intron-1 targeted exon editor. See, for example... Figure 7 It demonstrates the inclusion of combined structural domains. HTT A schematic diagram of the arrangement of an exemplary RNA exon editor for _intron1_11704_100. The results presented herein demonstrate that several adapters identified through extensive trial and error (23-mer GU adapter, 33-mer GU adapter, 34-mer GU adapter, 41-mer GU adapter, 40mer_2, 41mer_2, 68-mer, 84-mer, and 60-mer) confer a higher level of trans-splicing efficiency compared to the 40-mer (SEQ ID NO: 37). See, for example... Figure 8 The sequences of the 23-mer GU adapter (approximately 69-70% guanine content), 33-mer GU adapter (approximately 66-67% guanine content), 34-mer GU adapter (approximately 67-68% guanine content), 41-mer GU adapter (approximately 73-74% guanine content), 40mer_2, 41mer_2, 68-mer, 84-mer, and 60-mer correspond to SEQ ID NO: 38-46, respectively.

[0121] Next, the inventors will target... HTT The RNA exon editor for intron 2 was tested. See also Figure 9 ,about HTT A general schematic diagram of an intron 2-targeted RNA exon editor. Similar to the intron 1-targeted exon editor, a scanning of binding domains is initially performed in intron 2 to identify binding domains that bind to target regions in intron 2, which are associated with and facilitate high levels of trans-splicing efficiency. See, for example... Figure 10 The inventors have determined that the 3' end of the intron (10-20 nucleotides (nt) upstream of branch point A) is targeted for trans-splicing to... HTT A particularly effective region within intron 2. See, for example... Figure 11 Further analysis examined the correlation between the length and function of the binding domain in the upstream subregion of the branch point, revealing that the binding domain length in the range of 125-200 nt has the highest relative inverse splicing efficiency. See, for example, Figure 12.

[0122] The inventors further investigated the combined functionality of the bonding structural domains operably connected to different connectors, such as... Figure 13 As shown graphically, the inventors examined various exemplary connectors for high-level performance of the bonding structure domain pairs. HTT Intron 2 ( HTT The effect of intron2_12061_150) on the trans-splicing activity. For example... Figure 14 As shown, it includes components operably connected to the indicated connector. HTT The trans-splicing activity of the RNA exon editor of _intron2_12061_150 did not change significantly. In fact, when HTT When the _intron2_12061_150 is operably connected to different connectors, only minimal impact is observed, indicating that the use of HTT The reverse splicing of _intron2_12061_150 is highly optimized.

[0123] The results presented in this paper show that none of the tested joints significantly improved the inverse shear efficiency compared to the inverse shear efficiency imparted by the 40-mer joint, indicating that... HTTThe _intron2_12061_150 exon editor achieves high efficiency through recruitment by combining affinity and related spliceosome mechanisms. Results also indicate that different adapter sequences may be included in HTT RNA exon editors that efficiently induce trans-splicing. By designing antisense oligonucleotides (ASOs) and co-expressing each of these ASOs with an RNA exon editor targeting intron 2, the inventors experimentally tested whether inhibition of cis-splicing could promote enhanced trans-splicing, wherein the ASOs specifically block competitive cis-splicing sites (ASO8 (SEQ ID NO: 133), ASO9 (SEQ ID NO: 134), ASO10 (SEQ ID NO: 135)) and cis-splicing sites involved in upstream intron splicing (ASO2-7 (SEQ ID NO: 127-132, respectively)). See also Figure 16 . Figure 17 As shown, HTT Intron 2 Targeted Exon Editor ( HTT The combination of intron2 (12061_150) and ASO6 (SEQ ID NO: 131) blocks the cis-splicing of the upstream intron, resulting in improved efficiency of in vitro trans-splicing. Some embodiments described herein involve the use of... HTT The strategy involves using an RNA exon editor to block these cis-splicing events by expressing antisense RNA in the same plasmid. See also the combined implementation section below.

[0124] To confirm the existence of the truly chimeric HTT protein generated after trans-splicing, the selected... HTT Cell lysate prepared from exon-edited HEK293 cells was subjected to Western blot analysis, using an antibody that specifically binds to the N-terminal FLAG epitope for detection. Figure 18 As shown, Western blotting confirmed the generation of mid-target (ONT) trans-splicing and the resulting mid-target protein after trans-splicing by detecting HTT proteins with FLAG tags of predicted molecular weight. Notably, the intensity of the anti-FLAG ONT protein band correlated with trans-splicing efficiency (RNA substitution %). See also Figure 18 .

[0125] The inventors also designed a target HTT An RNA exon editor for intron 3 was developed and tested. See also Figure 23 ,about HTTA general schematic diagram of the intron 3-targeted RNA exon editor. Similar to the intron 1 and intron 2-targeted exon editors, a binding domain scan is initially performed to identify binding domains that bind to target regions in intron 3, which are associated with and facilitate high levels of trans-splicing efficiency. See, for example... Figure 24 The inventors determined that the 3' end of the intron (5-15 nt upstream of branch point A) is targeted for trans-splicing to... HTT A particularly effective region in intron 3.

[0126] To compare the performance of the intron 2-targeting exon editor versus the intron 3-targeting exon editor, the inventors performed parallel transfections of exon editors containing the highest-performance binding domains in each target intron, and analyzed the RNA by RT-ddPCR. The results presented herein indicate that the HTT intron 3-targeting exon editor exhibits slightly higher trans-splicing efficiency compared to the intron 2-targeting exon editor. See, for example... Figure 25 .

[0127] To investigate hidden self-splicing (via AAV multipliers or intermolecular trans-splicing) in HTT exon editors and constructs containing HTT exon editors, hidden splicing sites were identified using computer prediction of self-splicing sites (see Table 1) to reduce such adverse events. An exemplary HTT intron 2-targeting exon editor with reduced hidden splicing sites (SEQ ID NO: 203) was generated (with four identified self-splicing sites altered) and its trans-splicing efficiency was tested and compared with the original (unaltered) exon editor (SEQ ID NO: 95). The results presented herein confirm that the small sequence changes applied do not affect the trans-splicing activity of the exemplary exon editor with reduced hidden splicing sites. See, for example... Figure 26 .

[0128] The initial rounds of HTT exon editor molecules were expressed by the CMV promoter (SEQ ID NO: 137) and contained the native HTT 5' UTR (SEQ ID NO: 136). The inventors then investigated how the exon editor behaved when expressed by the CAGGS promoter (SEQ ID NO: 196). Since the CAGGS promoter contains a 5' UTR (CBA exon + rabbit β-globulin exon), this study included testing exon editors with or without the native HTT 5' UTR in combination with the CAGGS promoter. It is noteworthy that although the results are presented in the context of HTT intron 2-targeting exon editors, the effects of these modifications are applicable to exon editors targeting other introns.

[0129] In HEK293 cell transfection experiments, the inventors discovered that the exon editor driven by the CAGGS promoter, containing both CAGGS 5' UTR and HTT 5' UTR, has a similar HTT mRNA substitution percentage to the exon editor driven by the CMV promoter. See, for example... Figure 27 When the HTT 5' UTR was removed from the CAGGS promoter-driven exon editor, a decrease in HTT RNA substitution % was observed, and a significant increase in ONT protein expression was observed by Western blotting, indicating the presence of a protein translation activation element in the CAGGS 5' UTR. Simultaneously, the inventors observed an increase in non-splicing protein (NSP) and background (possibly OFT) protein signaling with the removal of the HTT 5' UTR.

[0130] To explore NSP reduction strategies in the context of 5' HTT exon editors, multiple elements were integrated into HTT intron 1-targeting exon editors and HTT intron 2-targeting exon editors, and tested to evaluate their individual and combined effects on exon editor performance. In the context of minimal NSP (i.e., minimizing neoantigens by stopping codons in or immediately following splicing donor sequences in the exon editor), these NSP reduction strategies included: 1) three tandem repeats (3X UBS) at the U1 snRNA binding site (SEQ ID NO: 345), and 2) AU-rich elements (AREs) (SEQ ID NO: 346).

[0131] In the HTT intron 1 targeted exon editor, the inventors demonstrated that inclusion of 3X UBS in the linker domain reduced NSP levels by approximately 75% compared to baseline NSP levels in a 40-component linker control only. Inclusion of ARE in the linker reduced NSP levels by approximately 40% compared to baseline NSP levels in a 40-component linker control only. The combination of 3X UBS and ARE in the exon editor reduced NSP levels by approximately 88% compared to baseline NSP levels in a 40-component linker control only. See, for example... Figure 28 .

[0132] Similarly, in the HTT intron 2-targeted exon editor, the inventors demonstrated that inclusion of 3X UBS in the linker domain reduced NSP levels by approximately 38% relative to the baseline 40-mer linker control. Inclusion of ARE in the linker reduced NSP levels by approximately 33% relative to the baseline 40-mer linker control. The combination of 3X UBS and ARE in the exon editor, when used in combination, reduced NSP levels by approximately 66% compared to the baseline 40-mer linker control. See, for example... Figure 29 .

[0133] Targeted HTT RNA exon editors for introns (e.g., intron 1, intron 2, or intron 3) may include target-specific elements, such as those for... HTT Target introns possess specific binding domains and encoding HTT A sequence of coding domains (e.g., a sequence encoding all or part of exon 1, exon 2, or exon 3, or any combination thereof (e.g., exons 1 and 2 or exons 1-3)). Targeting HTT An RNA exon editor for introns may include one or more target-independent elements that can improve the functionality of the exon editor. Target-independent elements may include, for example, epitope tags, adapters, splice donor sequences, one or more repeats of a U1 snRNA binding site, AU-rich elements, or terminators. Embodiments described herein may include one or more such target-independent elements. Embodiments described herein may exclude one or more such target-independent elements. An RNA exon editor lacking one or more of the target-independent elements described herein may still be able to perform exon editing. For example, in some embodiments, the RNA exon editor described herein does not include a triple-helix terminator, a MALAT-1 terminator, or any terminator sequence. In some embodiments, the RNA exon editor described herein does not include a CMV promoter or a CAGGS promoter. In some embodiments, the RNA exon editor described herein does not include any 5' UTR sequence, or does not include... HTT 5' UTR sequence. In some embodiments, the RNA exon editor described herein does not include the GTAAGT splicing donor sequence. In some embodiments, the RNA exon editor described herein does not include any epitope tags, or does not include 3X FLAG tags. In some embodiments, the RNA exon editor described herein does not include any adapters described herein, including any of SEQ ID NO: 37-46. In some embodiments, the RNA exon editor described herein does not include three tandem repeats (3XUBS) of the U1 snRNA binding site (SEQ ID NO: 345). In some embodiments, the RNA exon editor described herein does not include AU-rich elements (ARE) (SEQ ID NO: 346).

[0134] Inhibit somatic CAG repeat expansion Mounting evidence suggests that a potential mechanism in the pathogenesis of HD is somatic instability in the CAG repeat segment. This supports exploring factors that modify somatic CAG proliferation as viable therapeutic targets. The inventors have devised and tested a strategy to inhibit somatic CAG proliferation. See, for example... Figure 19 Recent genome-wide association studies (GWAS) in HD patients have identified several components of the DNA mismatch repair pathway as key genetic modifiers associated with disease progression rate. For example, in HD patients, single nucleotide variants (SNVs) with reduced expression of the MSH3 gene are associated with delayed disease onset age. MSH3 is presumed to be a good target for HD therapeutics, and there is evidence that MSH3 knockdown inhibits both in vitro and in vivo somatic CAG repeat expansion. Here, the inventors explore several methods for reducing MSH3 levels.

[0135] The inventors have explored a novel approach to using trans-splicing to reduce the expression level of target genes, specifically by trans-splicing into target precursor mRNA to generate RNA that degrades rapidly or fails to produce functional proteins. The inventors envision a 5' exon editor or a 3' exon editor achieving this result. A 5' exon editor can replace one or more 5' exons of the target mRNA to remove the start codon and some or all of the coding sequence, thereby replacing it with an alternative coding or non-coding sequence. A 3' exon editor can replace one or more 3' exons of the target mRNA, thereby replacing it with an alternative coding or non-coding sequence. In the resulting chimeric mRNA, it is possible that no amino acids from the target mRNA are translated. The 5' exon editor can also insert a 5' untranslated region (UTR), thereby preventing translation. The 3' exon editor can insert a 3' UTR, thereby destabilizing the transcript or preventing its export from the nucleus or translation. It is speculated that the remaining portion of the target mRNA after the trans-splicing reaction will degrade rapidly because it lacks a 5' cap or a 3' polyA tail.

[0136] The inventors infer that editing the target (e.g.) HTT The exon editor can be configured to include two different binding domains (one targeting HTT and the other targeting...) MSH3 Simultaneously knock down a second gene (e.g., MSH3). One implementation of this dual effect is that a 5' HTT exon editor containing the 5' portion of HTT can be trans-spliced ​​to... HTT To reconstruct functional wild-type HTT, and also to reverse splice to MSH3 To produce non-functionality MSH3 In one implementation, chimeric nonfunctional mRNA. HTT-MSH3The mRNA encodes the HTT polypeptide, which terminates at the first stop codon in the MSH3 region of the mRNA. Therefore, the normal wild-type... MSH3 Precursor mRNA is converted into chimeric nonfunctional HTT-MSH3 mRNA encodes very little or no code MSH3 The amino acids in the coding sequence are likely to undergo nonsense-mediated degradation. As shown in this paper, this method is applicable to targeting several different amino acids. MSH3 The intron-binding domain, and in fact, will also be applicable to targeting any MSH3 Intron binding domains. Furthermore, although the embodiments provided herein relate to a 5' exon editor, similar approaches can also be implemented using a 3' exon editor with reasonable expectation of success.

[0137] Therefore, the inventors have designed and tested a strategy to treat HD at an earlier stage of pathogenesis by inhibiting somatic CAG amplification. The embodiments described herein (e.g., those for inhibiting somatic CAG amplification) can be used alone or in combination with RNA exon editor-mediated trans-splicing to correct pathogenic mutations. While inhibiting somatic amplification alone is a feasible treatment for HD, the inventors have focused on a strategy designed to inhibit somatic CAG amplification and correct pathogenic mutations. HTT Mixed therapeutic approaches using exon 1 RNA and dual-acting / hybrid molecules were tested. See, for example... Figure 19 and Figure 20 Therefore, the implementation schemes described herein (e.g., those designed to inhibit somatic CAG amplification) can be used alone or in combination with RNA exon editor-mediated trans-splicing to correct pathogenic mutations.

[0138] In addition to the above, in order to suppress the potential mechanism of HD pathogenesis (i.e., somatic CAG amplification), and also replace the mutant... HTT Exon 1, the inventors designed a set of dual-function / hybrid RNA exon editors, the exon editors containing HTT and MSH3 The target sequences of both precursor mRNAs. In some implementations, exon editor expression is driven by a CMV promoter. In some implementations, an exemplary exon editor includes... HTT 5' UTR, N-end 3X FLAG tag, HTT Exon 1 coding sequence, splice domain (splicing donor site), linker, MSH3 Combining structural domains, HTT Combine structural domains and terminating subsequences. HTT Targeting the exon editor by combining structural domains HTTPrecursor mRNA, which, upon successful trans-splicing, leads to the generation of correctional molecules. HTT RNA. MSH3 Targeting the exon editor by combining structural domains MSH3 Precursor mRNA, resulting in the production of a premature stop codon (due to the replacement of the upstream with HTT exon 1). MSH3 Exon-induced frameshift chimerism HTT Exon 1 - MSH3 RNA molecules undergo nonsense-mediated degradation (NMD) and thus lead to a reduction in subsequent MSH3 expression. See, for example... Figure 20 .

[0139] Using an exon editor containing tandem-binding domains, the inventors' experiments demonstrate successful trans-splicing to... HTT and MSH3 Of the two precursor mRNAs, one can be designed to detect chimeric "correction" separately. HTT RNA and chimerism HTT Exon 1- MSH3 RT-qPCR was used to measure RNA molecules. See, for example, Figures 21 and 22. The constructs used in the tests... MSH3 Combining the sequences of the structural domains as shown in SEQ ID NO: 140 and 142 (Figure 21) and SEQ ID NO: 144 and 146 (Figure 22), the tested constructs used HTT The sequences of the combined structural domains are shown in SEQ ID NO: 20, 141, and 145 (Figures 21 and 22), and the tested constructs contain... HTT The sequence of CDS is shown in SEQ ID NO: 3. It is noteworthy that, due to NMD, chimerism... HTT Exon 1 - MSH3 RNA levels may be underestimated. To address this issue, the inventors also measured... MSH3 RNA transcript levels.

[0140] In another embodiment, the inventors also designed a target. HTT Intron 2 (using HTT_intron2_12061_150 binding domain (SEQ ID NO: 72)) and MSH3 The tandem binding domain exon editor of the precursor mRNA was tested. See, for example... Figure 30-3 2. The components used in the tested construct MSH3 The sequences of the combined structural domains are shown in SEQ ID NO: 140, 209, 144 and 210, and the tested constructs contain... HTTThe sequence of CDS is shown in SEQ ID NO: 59.

[0141] Targeted HTT Intron 2 and MSH3 RT-qPCR analysis of the intron 5 tandem-binding domain exon editor showed successful trans-splicing to HTT and MSH3 Of the two, such as those designed to detect chimerism correction HTT RNA and chimerism HTT Exon 2- MSH3 RT-qPCR was used to measure exon 6 RNA molecules. See, for example... Figure 31A and Figure 31B Compared to MSH3_intron5_213_100 (SEQ ID NO: 140), MSH3_intron5_188_150 (SEQ ID NO: 209) exhibits higher MSH3 trans-splicing efficiency. Similarly, targeting... HTT Intron 2 and MSH3 The tandem binding domain exon editor of intron 15 demonstrates successful trans-splicing to HTT and MSH3 Of the two. See, for example. Figure 32A and Figure 32B It is worth noting that, due to NMD, chimerism HTT Exon 2 - MSH3 RNA levels may be underestimated.

[0142] The results presented in this paper demonstrate that tandem-binding domain exon editors are effective and may have therapeutic benefits. Furthermore, these experiments identified [a specific mechanism / effect] for targeting [specific applications]. MSH3 Efficient binding domains for trans-splicing of precursor mRNA. In addition to their use in the context of tandem binding domains as described herein, these MSH3-targeting binding domains can also be used in the context of exon editors with NMD-inducible coding sequences; such exon editors can be used alone to target MSH3 knockdown, or they can be packaged in the same AAV vector with HTT-targeting exon editors and co-delivered to target tissues for a combined approach.

[0143] In some implementations, the MSH3 exon editor can be used to knock down MSH3 expression, wherein the MSH3 exon editor has the targeting described above. MSH3 The intron-binding domain of the precursor mRNA, but excluding the targeting domain. HTT The binding domain of precursor mRNA or any other precursor mRNA. The aforementioned dual-binding domain RNA exon editor can remove the target... HTTThe binding domains of the introns are modified. In some implementations, the following can be applied: MSH3 Exon editors can be used without simultaneously administering treatments that target or correct another gene sequence. In some implementations, such MSH3 exon editors can be used in methods for treating or preventing trinucleotide duplication syndromes.

[0144] MSH3 knockdown via RNAi RNA interference (RNAi) is a natural mechanism that induces gene silencing in a sequence-specific manner by targeting and degrading mRNA, specifically double-stranded RNA (dsRNA). In the initial design, to identify vectorized RNAi constructs that effectively knock down MSH3, a miRNA sequence was used as the basis. MSH3 Various constructs were designed for Ex23 targeting (TTAATCCATAACTCCTTGC; SEQ ID NO: 224). Pol III promoter-driven shRNAs were constructed as potential positive controls. These U6 promoter-driven shRNAs were compared with those expressing Pol II promoter (CMV promoter)-driven targeting... MSH3 The constructs of primary miRNA (pri-miRNA)-like transcripts were tested together. See, for example... Figure 33 .

[0145] For example Figure 34 As shown, Western blot analysis reveals information about this... MSH3 The Ex23-targeting miRNA (TTAATCCATAACTCCTTGC; SEQ ID NO: 224) was observed to knock down approximately 50% of MSH3 protein when expressed in a miR-30a scaffold background. Furthermore, placing the guide RNA on the 5' arm or adding a protrusion to the stem structure did not improve the knockdown efficiency (37% and 23% knockdown, respectively).

[0146] Additional designs focusing on altering the active sequence of miRNAs demonstrate that, in addition to Figure 34 In addition to the exon 23 targeting sequence tested, exon 22 targeting (SEQ ID NO: 246), exon 9 targeting (SEQ ID NO: 248), exon 12 targeting (SEQ ID NO: 256), and exon 15 targeting (SEQ ID NO: 257) miRNA sequences also performed well, with approximately 50% MSH3 protein knockdown observed. See, for example... Figure 35 This MSH3 RNAi modality can be therapeutically relevant to many duplication syndromes on its own, and / or can be combined with exon editors to enhance therapeutic potential.

[0147] In some embodiments, the MSH3-targeting RNAi construct disclosed herein can be used in conjunction with an RNA exon editor that targets HTT or other genes associated with trinucleotide repeat amplification diseases as described above. In other embodiments, the MSH3-targeting RNAi construct disclosed herein can be used independently of HTT-targeting therapies. In some embodiments, the MSH3-targeting RNAi construct disclosed herein can be administered to treat or prevent trinucleotide repeat amplification diseases.

[0148] MSH3 splicing adjustment MSH3 can be inactivated by incorporating a sequence complementary to the MSH3 splice junction into a snRNA sequence (e.g., U7 snRNA). The inventors devised and tested a strategy to block cis-splicing events using antisense RNA expressed on the same plasmid as the HTT-targeting RNA exon editor. The modified snRNA (e.g., U7 SmOPT) was designed as follows: 1) the target sequence (e.g., the histone-binding sequence in the 5′ region of the U7 snRNA) was changed to a sequence complementary to the gene to be modified; and 2) the binding site (AAUUUGUCUAG; SEQ ID NO: 367; U7 SmWT) of the U7 snRNP-specific proteins (Lsm10 and Lsm11) was changed to a shared sequence derived from the major spliceosome uridine-rich small nucleoribonucleoprotein (U snRNP) (AAUUUUUGGAG; SEQ ID NO: 368; U7 Sm OPT), resulting in the formation of a spliceosome-type heptameric protein core encapsulating the U7 Sm OPT. In some implementations, the asRNA molecule described herein consists of a U1 promoter, snRNA (with targeting...) MSH3 It consists of an asRNA sequence (intron-exon boundary and shared Sm binding site) and a U1 terminator. See, for example, Figure 36 .

[0149] The inventor designed a target MSH3MSH3 splicing regulators for exon 2, 3, and 4 skipping were tested. For each group, asRNA sequences targeting upstream intron-exon boundaries (e.g., intron 1 / exon 2 boundary of exon 2 splicing regulators (SEQ ID NO: 274)), downstream exon-intron boundaries (e.g., exon 2 / intron 2 boundary of exon 2 splicing regulators (SEQ ID NO: 275)), and tandem sequences of two asRNAs (SEQ ID NO: 276 and 277) were tested. Sequences of exemplary MSH3 splicing regulators mediating exon 2 skipping are shown in SEQ ID NO: 284-286, which respectively contain MSH3 splicing regulator elements SEQ ID NO: 274-276. For MSH3 exon 2 skipping molecules, asRNA molecules targeting the intron 1 / exon 2 and exon 2 / intron 2 boundaries without the U7SmOPT snRNA scaffold were also tested. RT-qPCR assays measured splicing... MSH3 The levels of mRNA product show that the U7SmOPT splicing regulator (rather than the asRNA sequence without the U7SmOPT scaffold) exhibits intentional skipping of the target exon. Exemplary MSH3 splicing regulator sequences mediating exon 3 skipping are shown in SEQ ID NO: 288-290, which respectively contain MSH3 splicing regulator elements SEQ ID NO: 278-280. See, for example... Figures 37-39 It is worth noting that, MSH3 Exon 4 skipping does not lead to NMD, but because exon 4 contains 213 nucleotides, it results in a shorter MSH3 product, thus producing an in-frame exon 4 skipped mRNA product. The sequences of exemplary MSH3 splicing regulators mediating exon 4 skipping are shown in SEQ ID NO: 291-293, which respectively contain MSH3 splicing regulator elements SEQ ID NO: 281-283. The inventors observed truncated protein products of exon 4-targeting splicing regulators in Western blots probing MSH3 with antibodies, demonstrating that the splicing regulator skipped the target exon as expected.

[0150] Subsequent design iterations tested MSH3 splice modulators targeting the following jumps: exon 3 (exemplary MSH3 splice modulator sequences shown in SEQ ID NO: 324-326, comprising MSH3 splice modulator elements SEQ ID NO: 299, SEQ ID NO: 300 + 298 + 301, and SEQ ID NO: 302 + 298 + 303, respectively), exon 6 (exemplary MSH3 splice modulator sequences shown in SEQ ID NO: 327-329, comprising MSH3 splice modulator elements SEQ ID NO: 304, SEQ ID NO: 305 + 298 + 306, and SEQ ID NO: 307 + 298 + 308, respectively), and exon 7 (exemplary MSH3 splice modulator sequences shown in SEQ ID NO: 330-332, comprising MSH3 splice modulator elements SEQ ID NO: 304, SEQ ID NO: 305 + 298 + 306, and SEQ ID NO: 307 + 298 + 308, respectively). 309, SEQ ID NO: 310 + 298 + 311 and SEQ ID NO: 312 + 298 + 313), exon 8 (the sequences of exemplary MSH3 splice modulators are shown in SEQ ID NO: 333-335, which respectively contain MSH3 splice modulator elements SEQ ID NO: 314, SEQ ID NO: 315 + 298 + 316 and SEQ ID NO: 317 + 298 + 318) and exon 15 (the sequences of exemplary MSH3 splice modulators are shown in SEQ ID NO: 336-338, which respectively contain MSH3 splice modulator elements SEQ ID NO: 319, SEQ ID NO: 320 + 298 + 321 and SEQ ID NO: 322 + 298 + 323). These exons were chosen because they are well-suited for splicing regulation, taking into account a variety of factors, such as the length of the new epitope (if it produces a protein rather than a transcript for NMD), splicing characteristics (e.g., splicing primarily mediated by the minor spliceosome), alternative splicing, etc.

[0151] The inventor designed a target MSH3MSH3 splicing regulators for skipping exons 3, 6, 7, 8, and 15 were tested. For each group, the following molecules were tested: 1) U7SmOPT molecules containing 12–13 nucleotide antisense sequences across the entire length of the target exon and flanking introns; 2) U7SmOPT molecules containing antisense sequences at both the downstream exon / intron boundary and the upstream intron / exon boundary (separated by unstructured adapters); and 3) U2 snRNA molecules containing antisense sequences at both the downstream exon / intron boundary and the upstream intron / exon boundary (separated by unstructured adapters). The tested... MSH3 The sequences of the splice modulators are as shown in SEQ ID NO: 290 and 324-326 ( Figure 40 ); SEQ ID NO:327-329 ( Figure 41 ); SEQ ID NO: 330-332 ( Figure 42 ); SEQ ID NO: 333-335 ( Figure 43 ); and SEQ IDNO: 336-338 ( Figure 44 As shown in the figure. The corresponding asRNA region SEQ ID NO: and the target region are respectively located in... Figures 40-44 Each of these is shown in the diagram. The inventors identified effective splicing modulators for all target exons tested. Generally, U7SmOPT molecules (e.g., SEQ ID NO: 328, 331, 334, and 337) containing sequences antisense to both the downstream exon / intron boundary and the upstream intron / exon boundary (separated by unstructured linkers) show particularly high degrees of MSH3 expression reduction, but there are some exceptions. See, for example... Figures 40-44 Western blot analysis using antibodies to reveal MSH3 showed that the level of MSH3 protein knockdown corresponded to a profile of RNA exon skipping efficiency. The tested... MSH3 The sequence of the splice modulator is shown in SEQ ID NO: 330-332. See, for example, Figure 45.

[0152] In some embodiments, the MSH3 splicing regulator disclosed herein can be used in combination with RNA exon editors that target HTT or other genes associated with trinucleotide repeat amplification disorders as described above. In some embodiments, the MSH3 splicing regulator disclosed herein can be used independently of HTT-targeted therapies. In some embodiments, the MSH3 splicing regulator disclosed herein can be administered to treat or prevent trinucleotide repeat amplification disorders.

[0153] Knockdown of HTT-toxic species To enhance the efficacy of the HTT exon editor described herein, the inventors conceived of a combined approach to further address toxic species (e.g., HTT1a). To this end, a vectorized hybrid molecule was designed and tested, comprising the HTT exon editor and targeting the unedited exon. HTT The miRNA of exon 1, the element of which is intended to be packaged in a single AAV. See, for example... Figure 46 The sequences of the miRNAs targeting unedited HTT (HTT miRNA-1 and HTT miRNA-2) are shown in SEQ ID NO: 339 and 342. The CDS of the HTT exon editor is codon-modified to prevent interaction between the miRNAs and the exon editor, thereby avoiding degradation of the exon editor itself and the corrected HTT products. See, for example... Figure 47 .

[0154] The inventors first evaluated the efficacy of exon editor alone, HTT miRNA alone, and the combination of both modes with exon editor + HTT miRNA. In this experiment, the HTT intron 2-targeting exon editor (SEQ ID NO: 204) and HTT miRNA-1 (active sequence SEQ ID NO: 339) were tested. SEQ ID NO: 341 corresponds to HTT miRNA-1 (SEQ ID: 339) encoding the primary miRNA sequence in the mir-33 scaffold (SEQ ID: 259, 260, 261). As shown, for example, in Figure 48, in the presence of HTT miRNA, due to the total HTT Transcript copy number decreased, all HTT Trans-splicing (editing) in transcripts HTT The proportion of transcripts increased. However, regardless of the presence or absence of HTTmiRNA-1, the editing of trans-splicing... HTT The copy number of the transcripts remained unchanged, indicating that the interaction between HTT miRNA-1 and the exon editor was minimal or nonexistent.

[0155] Additional comparisons were made with two independent miRNAs targeting HTT exon 1: HTT miRNA-1 (active sequence SEQ ID NO: 339; encoding primary miRNA sequence SEQ ID NO: 341) and HTT miRNA-2 (active sequence SEQ ID NO: 342). SEQ ID NO: 344 corresponds to HTT miRNA-2 (SEQ ID: 342) encoding the primary miRNA sequence in the mir-33 scaffold (SEQ ID: 259, 260, 261). The coding sequence of the HTT intron 2-targeting exon editor was further codon-optimized to confer and ensure resistance to both miRNAs (exemplary variants encoding domain sequences are shown in SEQ ID NO: 59 and 349-351). No negative impact of HTT miRNA-1 or HTT miRNA-2 on trans-splicing was observed, such as through unchanged (or potentially increased) trans-splicing in the presence of the miRNA. HTT Copy number and a higher percentage of trans-splicing generated by the exon editor. HTT As demonstrated by mRNA. See, for example, Figure 49. The inventors have demonstrated that HTTmiRNA-1 and HTT miRNA-2 have similar HTT knockdown efficiencies, and that neither miRNA negatively impacts exon editor performance, such as through trans-splicing that remains unchanged regardless of whether each miRNA is used. HTT The transcript copy number is used as evidence. See, for example, Figure 49.

[0156] The inventors then evaluated the HTT exon editor + miRNA dual molecule at the protein level. Tests on the HTT intron 2-targeting exon editor + HTT miRNA dual hybrid molecule showed that, in the presence of HTT miRNA (HTT miRNA-1; SEQ ID NO: 339), unedited [proteins / exons] were achieved. HTT Approximately 50% of the transcripts were knocked down. Figure 50 (Left figure). Analysis of exon editor trans-splicing activity showed that in the presence of HTT miRNA, unedited and therefore total exon editing occurred due to the miRNA. HTT The number of copies is reduced, and the editing... HTT RNA% increased ( Figure 50 (See right image).

[0157] Construction of hybrid molecules As described herein, the inventors have explored a hybrid approach targeting multiple mechanisms and / or pathogenic species. As detailed herein, these approaches may include replacing mutant HTT with an exon editor, knocking down mutant HTT and associated transcripts (e.g., HTT1a) via RNAi, and reducing MSH3 through trans-splicing or vectorized splicing regulation. These approaches represent a multimodal mechanism of action when combined, yet can still be delivered in a single AAV. A subset of designs aimed at knocking down MSH3 are achieved by designing hybrid molecules (e.g., tandem-binding domain exon editors). Similarly, the inventors have designed HTT-targeting miRNAs and considered combining them with HTT exon editors; therefore, additional consideration was given to codon modification of the miRNA target sites in the exon editor to minimize the interaction between the HTT-targeting miRNA and the exon editor. Such combined approaches can be used to achieve high therapeutic efficacy in some (if not all) HD patients. In fact, such approaches can confer significant disease improvement.

[0158] HTT Exon Editor + MSH3 Splicing Modulator While inhibiting somatic cell proliferation alone may have a positive therapeutic effect on HD, inhibiting somatic CAG proliferation and correcting HTT Hybrid molecules of exon 1 RNA may hold significant therapeutic promise because this approach can simultaneously combat the disease on multiple fronts. Any HTT-targeted RNA exon editor described herein can be used in combination with any one or more MSH3 splicing modulators described herein. Any HTT-targeted RNA exon editor described herein can be used without the simultaneous use of any MSH3 modulator described herein, and any MSH3 splicing modulator described herein can also be used without the simultaneous use of any HTT-targeted RNA exon editor.

[0159] To achieve this combined effect, a hybrid vector encoding an HTT exon editor and an MSH3 splicing modulator was designed. See, for example... Figure 51 Preliminary examination of these double-hybrid molecules used HTT Intron 2 targeting (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) and exemplary MSH3Exon 7 skipped splicing modulator (SEQ ID NO: 331). However, the combined effect will also apply to any combination of HTT exon editor and MSH3 splicing modulator (exemplary MSH3 splicing modulator sequences are shown in, for example, SEQ ID NO: 330-332, which respectively contain MSH3 splicing modulator elements SEQ ID NO: 309, SEQ ID NO: 310 + 298 + 311 and SEQ ID NO: 312 + 298 + 313; and SEQ ID NO: 284-293, 324-329 and 333-338). It should also be understood that the combinatorial effects observed with the exemplary HTT intron 2-targeting (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein will also apply to other HTT intron 2-targeting (HTT_intron2_12061_150) exon editors, including those containing any of SEQ ID NO: 59, 350, or 351 corresponding to the modified coding domain sequence. Therefore, HTT intron 2-targeting exon editors may contain any of the variant coding domain sequences shown in SEQ ID NO: 59 and 349-351. It should also be understood that the combined effects observed with the exemplary HTT intron 2-targeting (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein will also apply to HTT intron 3-targeting (containing, for example, HTT_intron3_4223_150 or HTT_intron3_4233_150) exon editors or HTT intron 1-targeting (containing, for example, HTT_intron1_11704_100 or HTT_intron1_11724_100) exon editors. As shown herein, the inventors examined the trans-splicing profile and MSH3 knockdown efficiency of exon editors alone, MSH3 splicing modulators alone, and exon editors + MSH3 splicing modulator dual-hybrid molecules. RT-qPCR analysis showed that exon editor performance was unchanged regardless of the use of MSH3 splicing modulator, as indicated by similar levels of HTT substitution %. See, for example Figure 52A Similarly, the performance of the MSH3 splicing modulator remained unchanged regardless of whether an exon editor was used. See, for example... Figure 52B This experiment demonstrates that there is no interaction between the exon editor and the MSH3 splicing regulator when they are co-transmitted within the same DNA fragment.

[0160] Effective trans-splicing requires sufficient levels of AAV delivery to the target tissue and high levels of exon editor expression. To achieve high levels of vector expression, self-complementary AAV (scAAV) was tested. To compare the performance of conventional single-stranded AAV (ssAAV) with scAAV, exemplary [examples will be provided]. HTT Intron 2 Targeted Exon Editor+ MSH3 A dual-hybrid molecule of exon 7 skipping splicing regulator was inserted into both the ssAAV compatible plasmid (containing wild-type ITR) and the scAAV compatible plasmid (ITR / ITR-Δtrs), packaged into AAV2 cells, and used for transduction of HEK293 cells. Characterization of MSH3 expression at the RNA and protein levels showed that the dual-hybrid molecule performed better for both modes when packaged in scAAV (see, for example...). Figure 53 (Left figure). Trans-splicing characterization also supports the superior performance of molecules packaged in scAAV compared to ssAAV, as detected by Western blotting. HTT The higher percentage of transcript replacements and the correspondingly increased full-length trans-splicing of HTT proteins are evidenced by (see, for example) Figure 53 (See right figure). The inventors also determined the expression level of the exon editor and observed higher transgene expression in cells transduced with scAAV. One consideration when using the scAAV vector is the limited cargo space (approximately half the size compared to ssAAV), which limits its use when exon editor expression is driven by a larger promoter (e.g., the CAGGS promoter).

[0161] HTT exon editor + HTT miRNA + MSH3 splicing regulator The inventors have also designed, constructed, and tested triple hybrid molecules containing multiple modalities targeting multiple mechanisms and / or pathogenic species. These methods include replacing mutant HTT with an exon editor, knocking down mutant HTT and related transcripts (e.g., HTT1a) via RNAi, and reducing MSH3 through splicing regulation. These methods represent a multimodal mechanism of action when combined, but can still be delivered in a single AAV. Such combined approaches can be used to achieve high therapeutic efficacy for some (if not all) HD patients. In fact, such approaches can confer significant disease improvement. As mentioned above, each of these modalities is designed to minimize interactions with each other (e.g., codon modifications by an exon editor resistant to HTT miRNAs).

[0162] To explore this method, triple hybrid molecules containing the CAGGS promoter (SEQ ID NO: 196) were analyzed. HTTIntron 2-targeted exon editor (SEQ ID NO: 204), exemplary MSH3 The exon 7 skipping splicing regulator (SEQ ID NO: 331) and SEQ ID NO: 341 [HTT miRNA-1 (SEQ ID: 339), encoding the primary miRNA sequence in the mir-33 scaffold (SEQ ID: 259, 260, 261)] or SEQ ID NO: 344 [HTT miRNA-2 (SEQ ID: 342), encoding the primary miRNA sequence in the mir-33 scaffold (SEQ ID: 259, 260, 261)]. The activity of the triplet hybrid was compared with a control hybrid molecule containing a splicing mutant exon editor, a splicing regulator containing a scrambled control sequence, and a miRNA containing a scrambled control sequence. The combined effect will also apply to any combination of HTT exon editor, MSH3 splicing regulator (exemplary MSH3 splicing regulator sequences are shown, for example, in SEQ ID NO: 330-332, which respectively contain MSH3 splicing regulator elements SEQ ID NO: 309, SEQ ID NO: 310 + 298 + 311 and SEQ ID NO: 312 + 298 + 313; and SEQ ID NO: 284-293, 324-329 and 333-338) and HTT miRNA-1 or HTT miRNA-2. It should also be understood that the combinatorial effects observed with the exemplary HTT intron 2-targeting (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein will also apply to other HTT intron 2-targeting (HTT_intron2_12061_150) exon editors, including those containing any of SEQ ID NO: 59, 350, or 351 corresponding to the modified coding domain sequence. Therefore, HTT intron 2-targeting exon editors may contain any of the variant coding domain sequences shown in SEQ ID NO: 59 and 349-351. It should also be understood that the combined effects observed using the exemplary HTT intron 2 targeting (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein will also apply to HTT intron 3 targeting (containing, for example, HTT_intron3_4223_150 or HTT_intron3_4233_150) exon editors or HTT intron 1 targeting (containing, for example, HTT_intron1_11704_100 or HTT_intron1_11724_100) exon editors.

[0163] As previously seen in Figure 52, the inverse splicing efficiency of the HTT exon editor is similar regardless of whether the MSH3 splice modulator is used (see example). Figure 54 (Top image). In the presence of HTT-miRNA 1 or HTT-miRNA 2, unedited HTT transcripts are produced due to the HTT miRNA (and therefore the total). HTT Transcripts reduced, edited HTT Transcription percentage increased (see example) Figure 54 (Top image). Figure 54 The intermediate plot evaluated the performance of HTT miRNAs, and similar efficiencies were observed with HTT miRNA-1 and HTT miRNA-2 in both double-hybrid (exon editor + HTT miRNA) and triple-hybrid (exon editor + HTT miRNA + MSH3 splicing regulator) backgrounds. Evaluation of MSH3 knockdown efficiency showed that the knockdown efficiency induced by the splicing regulator remained unchanged in the presence of either an exon editor or HTT miRNA. See example. Figure 54 (Bottom figure) These results indicate that there is no interaction between the exon editor, HTT miRNA, and MSH3 splicing regulator when they are co-delivered within the same DNA fragment. Notably, the combined effect described will also apply to any combination of the HTT exon editor, HTT miRNA, and MSH3 splicing regulator.

[0164] Mechanism verification in iCell GlutaNeurons To confirm and validate the in vitro efficacy of the aforementioned molecules in additional cellular systems, the inventors utilized iCellGlutaNeurons (FujiFilm), human glutamatergic cortical neurons derived from induced pluripotent stem (iPS) cells. Exemplary molecules were packaged in AAV2.7m8 and used for transduction to express the molecules of interest in iCell GlutaNeurons.

[0165] The inventors tested two different AAV2.7m8 molecules to conduct mechanism verification experiments for three different modes: 1) HTT exon substitution via trans-splicing, 2) via targeted... HTT HTT knockdown of exon 1 miRNA, and 3) via MSH3 Exon 7 splicing regulation results in MSH3 reduction. Notably, one of the tested AAVs (SEQ ID NO: 354) packaged a double-hybrid molecule that expressed... HTTThe test included both an intron 2-targeting exon editor (SEQ ID NO: 204) and HTT miRNA-1 (active sequence SEQ ID NO: 339; encoding primary miRNA sequence SEQ ID NO: 341), while another tested AAV (SEQ ID NO: 357) packaged a dual hybrid molecule expressing both the intron 2-targeting exon editor (SEQ ID NO: 204) and HTT miRNA-1 (active sequence SEQ ID NO: 339; encoding primary miRNA sequence SEQ ID NO: 341). MSH3 Exon 7 skipped splicing modulators (SEQ ID NO: 331), each of which is packaged in AAV2.7m8.

[0166] RT-ddPCR analysis measured the editing (trans-splicing) HTT RNA% levels indicate that, without the use of HTT miRNA, the dual-hybrid molecule HTT exon editor + MSH3 splicing regulator resulted in approximately 37% substitution via trans-splicing in iCell GlutaNeurons. In the dual-hybrid molecule exon editor + HTT miRNA, the inventors observed an edited RNA% of approximately 50%, partly due to the HTT miRNA causing native RNA to be edited. HTT Copy number decreased. Western blot analysis of the N-terminal FLAG tag confirmed the presence of the trans-spliced ​​full-length HTT protein. See, for example... Figure 55 .

[0167] For the unedited HTT Further analysis at the transcriptional level revealed that, without the use of HTT miRNA, the dual heterozygous molecule HTT exon editor + MSH3 splicing regulator confirmed the unedited RNA in iCell GlutaNeurons due to trans-splicing (replacing the native RNA with the corrected RNA). HTT RNA was reduced by 47%. In the presence of HTT miRNA mode, the dual heterozygous molecular exon editor + HTT miRNA uses miRNA to remove unedited RNA. HTT Transcript knockdown by 50% resulted in even more favorable editing: unedited HTT Transcription ratio. Total HTT (trans-spliced ​​and unedited HTT proteins) was visualized by Western blot analysis, confirming that unedited HTT was achieved using additional HTT miRNA patterns. HTT Knock down. See example Figure 56 .

[0168] The inventors also tested the MSH3 splicing regulation mechanism in iCell GlutaNeurons. Western blot analysis of the MSH3 protein showed that a dual heterozygous exon editor + MSH3 exon 7 splicing regulator (SEQ ID NO: 331) resulted in a >40% knockdown of the MSH3 protein in these cells. See, for example... Figure 57 Overall, the inventors confirmed similar activity of each of the three HD modes (HTT exon editor, HTT miRNA, and MSH3 splicing regulator) in HEK293 cells and iCell GlutaNeurons.

[0169] Additional combined implementation schemes and their related details: Improvement passed HTT Exon replacement via trans-splicing of precursor mRNA The results presented in this paper (e.g.) Figure 17 The results show that blocking HTT Forward splicing events can lead to enhanced inverse splicing efficiency and HTT Exon 1 substitution. Therefore, embodiments of this disclosure include strategies to block these cis-splicing events via antisense RNA (asRNA). In order to... HTT Plasmids expressing cis-splicing blockers with the same targeting RNA exon editor can use modified snRNAs previously shown to act as splicing regulators, such as modified U7 snRNA (U7 SmOPT). As detailed herein, snRNAs (e.g., U7 Sm OPT) are designed as follows: 1) the targeting sequence (e.g., the histone-binding sequence in the 5′ region of the U7 snRNA) is changed to a complementary sequence to the gene to be modified, and 2) the binding sites (AAUUUGUCUAG; SEQ ID NO: 367; U7 Sm WT) of U7 snRNP-specific proteins (Lsm10 and Lsm11) are changed to a common binding sequence derived from the major spliceosome U snRNP (AAUUUUUGGAG; SEQ ID NO: 368; U7 Sm OPT), resulting in the binding of the Sm protein found in the spliceosome snRNP to U7 Sm OPT. In some embodiments, the asRNA molecule may contain a U1 promoter, a snRNA (with targeting...) HTT The asRNA sequence includes the intron-exon boundary and the shared Sm binding site, and the U1 terminator. Exemplary asRNA sequences include (SEQ ID NO: 127-135). Among them, HTT Intron 2 Targeted Exon Editor ( HTTThe combination of intron2 (12061_150) and ASO6 (SEQ ID NO: 131) blocks the cis-splicing of upstream introns, resulting in a particularly significant improvement in the efficiency of in vitro trans-splicing. See, for example... Figure 16 and Figure 17 .

[0170] Implementation plan for inhibiting the repetitive expansion of somatic CAG To avoid being bound by theory, the underlying pathogenesis for HD and many other duplicative aberration disorders is considered to be somatic expansion of the duplicative segments. As detailed in this article, human genetic evidence strongly supports the view that clinical outcomes of HD can be explained, at least in part, by somatic instability resulting from somatic expansion of the CAG duplicative segments. Therefore, inhibiting the underlying pathogenesis mechanism caused by somatic instability should alter the disease course and may be a potential therapeutic approach for HD and many other duplicative disorders.

[0171] Based on the goal of targeting the pathology of HD, the inventors designed and tested a strategy to treat HD at an earlier stage of pathogenesis by inhibiting somatic CAG amplification. The embodiments described herein (e.g., those for inhibiting somatic CAG amplification) can be used alone or in combination with RNA exon editor-mediated trans-splicing to correct pathogenic mutations. While inhibiting somatic amplification alone may have a positive therapeutic effect on HD (see the combined embodiments section above), the inventors have focused on strategies designed to inhibit somatic CAG amplification and correct pathogenic mutations. HTT Mixed therapeutic approaches using exon 1 RNA and dual-acting / hybrid molecules were tested. See, for example... Figure 19 and Figure 20 The inventors have also demonstrated that this hybrid treatment method uses cells designed to inhibit somatic CAG expansion and correct [therapeutic effects]. HTT Dual-acting / hybrid molecules of exon 2 RNA are effective. See, for example... Figure 30-3 2. Figure 52 and Figure 53 Therefore, the implementation schemes described herein (e.g., those designed to inhibit somatic CAG amplification) can be used alone or in combination with RNA exon editor-mediated trans-splicing to correct pathogenic mutations.

[0172] The mechanisms controlling this process involve cellular mechanisms, such as DNA mismatch repair enzymes, including MSH2 and MSH3 . MSH3 It is presumed to be a good target for inhibiting the recurrent proliferation of somatic CAG. Here, the inventors outline several ongoing methods designed to reduce... MSH3 Levels and inhibition of somatic CAG repeat expansion.

[0173] Through reverse splicing MSH3 Knockdown of other genes: As described in this article, trans-splicing can be used alone or in combination with other applications of trans-splicing to reduce MSH3 The expression of other genes.

[0174] Implementation plan A1.1. Used for MSH3 Trans-splicing for knockdown of other genes: As described herein, trans-splicing can be used for mRNA editing, but this paper also describes trans-splicing to reduce the expression level of target genes. As described herein, reducing the expression level of target genes can be achieved by trans-splicing into target precursor mRNA or mature mRNA to produce RNA that degrades rapidly or cannot produce functional proteins. See, for example... Figure 20-2 2 and Figure 30-3 2. Both 5' and 3' exon editors can achieve this result. For example, a 5' exon editor can replace one or more 5' exons of the target mRNA to remove the start codon and some or all of the coding sequence, thus replacing it with an alternative coding or non-coding sequence. For example, a 3' exon editor can replace one or more 3' exons of the target mRNA, thus replacing it with an alternative coding or non-coding sequence. In the resulting chimeric mRNA, no amino acids from the target mRNA may be translated. For example, a 5' exon editor can also insert a 5' untranslated region (UTR), thereby preventing translation. For example, a 3' exon editor can also insert a 3' UTR, thereby destabilizing the transcript or preventing its export from the nucleus or translation. It is speculated that the remaining portion of the target mRNA after trans-splicing will degrade rapidly because it lacks a 5' cap (5' exon editor) or a 3' polyA tail (3' exon editor).

[0175] Implementation Scheme A1.2. Tandem Binding Exon Editor: Editing Targets (e.g.) HTT The exon editor can be configured to contain two distinct binding domains (one target). HTT And another target MSH3 Simultaneously knock down the second gene (e.g.) MSH3 ). Figure 19-2 2 and Figure 30-3 2 provides an embodiment of how this dual effect can be achieved. Simply put, it contains... HTT 5' of the 5' HTT Exon editors can reverse splice to HTT To reconstruct functional wild type HTT mRNA can also be trans-spliced ​​to MSH3 To produce non-functionality MSH3 mRNA. In this implementation, chimeric nonfunctional mRNA is incorporated. HTT - MSH3 mRNA will encode HTT polypeptide, the polypeptide in mRNA MSH3 The process terminates at the stop codon within the first box of the section. Therefore, this type of reverse splicing process... MSH3 The precursor mRNA will be altered to become mRNA, which encodes very little or no code. MSH3 The amino acids in the coding sequence will likely undergo nonsense-mediated degradation. This method is applicable to targeting any... MSH3 Intron-binding domains. Although the embodiments provided herein ( Figure 19-2 2 and Figure 30-3 2) A 5' exon editor was used, but a similar approach can be taken with a 3' exon editor.

[0176] Implementation Scheme A.1.3. Tandem RNA Exon Editors: Tandem exon editors are two distinct exon editors inserted into the same plasmid, AAV, or other delivery medium. They can be arranged head-to-head or head-to-tail. Each exon editor can have its own regulatory sequence (including a promoter) or can be generated by the cleavage event of a single bicistronic transcript. One exon editor can target a single gene or intron, and the second exon editor can target a different gene or intron. In some implementations, one exon editor can be used with wild-type RNA. HTT Replacement mutant HTT Furthermore, the second exon editor can reduce MSH3 Expression. In some implementations, an exon editor can target... HTT One intron, and the second editor can target HTT The second intron, thus increasing HTT Overall efficiency of exon editing.

[0177] Implementation Plan A.2. Through RNAi and HTT Combination of reverse splicing to reduce MSH3 Or other genes: Short hairpin RNAs (shRNAs) or microRNAs (miRNAs) designed to reduce gene expression can be added to exon editors within the same cistron (e.g., within introns of exon editors) or as standalone cistrons with their own regulatory sequences. RNAi can reduce unedited targets (e.g., mutants). HTT ) or another gene (e.g. MSH3 The expression of ) was reduced. To selectively reduce mutant expression using RNAi. HTT The exon editor will replace the mutant with a sequence that is resistant to shRNA or miRNA. HTT Part of a transcript. See, for example... Figure 33-35 , Figures 46-50 .

[0178] Implementation plan A.3. Reduce by vectorized splicing regulation or translational blocking MSH3 In some implementation schemes, MSH3 By blocking one or more MSH3 Exon inclusion is used for inactivation, while the deletion of said exon prevents the translation of functional proteins by causing premature translation termination. In some embodiments, MSH3 By with MSH3 The splice junction complementary sequence is incorporated into a small nuclear RNA (snRNA) sequence (e.g., U7 snRNA) to inactivate it, thereby preventing exon inclusion. Here, splice regulation can be vectorized, i.e., delivered by an AAV, which may also include an exon editor. See, for example... Figure 36-4 5. Figures 51-54 .

[0179] Implementation Plan A.3.1. Targeting MSH3 small kernel-based RNA ( For example U7SmOPT) of asRNA: In some implementation schemes, MSH3 By with MSH3 Inactivation is achieved by incorporating sequences complementary to the splice junction into the snRNA sequence (e.g., U7 snRNA). See example. Figure 36-4 5.

[0180] Implementation Scheme A.3.2. Combination of MSH3 Reduction with HTT Trans-splicing: In some implementation schemes, strategy A.3.1 and other strategies described herein are combined into the same DNA fragment, or combined with an exon editor into the same fragment, or co-packaged into a single AAV for co-delivery to patient tissue. See, for example... Figure 33-54 .

[0181] Reduced potential HTT toxicity in species: Short hairpin RNAs (shRNAs) or microRNAs (miRNAs) engineered to reduce gene expression can be added as individual cistrons with their own regulatory sequences to the exon editor. RNAi can reduce unedited targets (e.g., mutants). HTT The expression of ) was reduced. To selectively reduce mutant expression using RNAi. HTT The exon editor will replace the mutant with a sequence that is resistant to shRNA or miRNA. HTT Part of the transcript. See also Figures 46-50 and Figures 54-56 .

[0182] To address the potential for enhanced HD treatment through combination therapies, the inventors have devised a hybrid, multi-pronged approach that targets multiple mechanisms and / or pathogenic species. In some embodiments, these methods include replacing mutants via exon editors. HTT Knockdown of mutants via RNAi HTT and related transcripts (e.g.) HTT1a ), and through the following reduction MSH3 Examples include trans-splicing, antisense RNA, RNAi, vectorized splicing regulation or translational blockade, or any combination thereof. When combined with the same DNA fragment or with an exon editor, these methods represent a multimodal mechanism of action, yet can still be delivered in a single AAV for co-delivery to patient tissues. Implementations utilizing such combined approaches can be used to achieve improved therapeutic efficacy for some (if not all) HD patients. Such approaches can confer even more disease-improving effects.

[0183] animal models BACHD Mouse Model: This animal model is a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) expressing full-length human mutant huntingtin protein (fl-mhtt). It was developed to express fl-mhtt containing 97 glutamine repeats under the control of an endogenous htt regulatory mechanism on BAC. The glutamine repeats are encoded by 97 mixed CAG-CAA repeats. BACHD mice reproduce human HD disease and disease progression by exhibiting progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, including severe cortical and striatal atrophy and striatal dark neuronal degeneration. Based on the robustness of the documented behavioral and neuropathological phenotypes, BACHD mice are considered suitable fl-mhtt mouse models for preclinical studies. BACHD mice are described in detail in Gray et al. (2008, JNeuroscience 28:6182; the entire contents are incorporated herein by reference) and are commercially available.

[0184] In some implementations, the therapeutic efficacy of the nucleic acid trans-splicing molecule / RNA exon editor described herein in the context of a BACHD mouse model can be measured by at least one of the following: pathogenicity. HTT An increase in the percentage of RNA replacement, a decrease in pathogenic HTT, a decrease in pathogenic HTT aggregates, an improvement in motor coordination and balance (as measured by, for example, a rotarod test), a decrease in forebrain atrophy, or any combination thereof.

[0185] BAC-CAG Mouse Model: This animal model is a human genome BAC transgenic mouse model of HD that expresses a human mutant huntingtin protein (mHTT) containing long, uninterrupted, and somatically unstable CAG repeats (120–130 pure CAG repeats) and exhibits a progressive disease-associated phenotype. Unlike other transgenic models with stable, CAA-disrupted, polyglutamine-encoded repeats of mHTT, BAC-CAG mice exhibit strong striatal-selective intranuclear inclusions and transcriptional dysregulation, which have also been observed in HD patients and huntingtin knock-in models. BAC-CAG is described in detail by Gu et al. (2022, Neuron 110:1173; the entire contents are incorporated herein by reference) and is available for purchase. As described therein, striatal transcriptional abnormalities in HD models are associated with the length of their uninterrupted CAG repeats, rather than the length of the polyglutamine repeats. As mentioned above, somatic CAG repeat instability and nuclear mHTT aggregation are most associated with early-onset striatal selective molecular pathogenesis and motor and sleep deficits, while repeat RNA-related pathology and repeat-related non-AUG (RAN) translation may respectively affect less selective or late pathogenicity.

[0186] In some implementations, the therapeutic efficacy of the nucleic acid trans-splicing molecule / RNA exon editor described herein in the context of the BAC-CAG mouse model can be measured by at least one of the following: pathogenicity. HTT An increase in the percentage of RNA replacement, a decrease in pathogenic HTT, a decrease in pathogenic HTT aggregates, an improvement in motor coordination and balance (as measured by, for example, a rotarod test), a decrease in forebrain atrophy, or a decrease in striatal-specific transcriptional lesions, or any combination thereof.

[0187] To demonstrate the in vivo mechanisms of the brain, the inventors utilized the BAC-CAG mouse model (Gu et al., 2022). As described above, this transgenic mouse strain was engineered to contain the human mutant HTT genomic locus (>120 unbroken CAG repeats in HTT exon 1) and exhibit abnormal features resembling a disease phenotype, including... Nuclear HTT aggregates and transcriptional dysregulation appeared at 12 months of age. The inventors conducted a mechanism verification study on HTT trans-splicing in the brain of BAC-CAG mice using an exemplary molecule packaged in AAV9. In this study, three different AAV test objects were tested: a CMV promoter-driven HTT intron 2-targeting exon editor (SEQ ID NO: 204) packaged in a self-complementary AAV (scAAV) (SEQ ID NO: 363, which contains SEQ ID NO: 204); a CMV promoter-driven HTT intron 2-targeting exon editor (SEQ ID NO: 204) packaged in a single-stranded AAV (ssAAV) (SEQ ID NO: 363, which contains SEQ ID NO: 204); and a CAGGS promoter-driven HTT intron 2-targeting exon editor (SEQ ID NO: 204) packaged in ssAAV (SEQ ID NO: 364, which contains SEQ ID NO: 204). All three test objects also contained a mouse-targeting alternative MSH3 exon 7 splicing regulator (SEQ ID NO: 362), which were co-packaged as hybrid molecules. The complete sequences of the test objects are as follows: a CMV promoter-driven HTT intron 2-targeting exon editor packaged in scAAV (SEQ ID NO: 369), a CMV promoter-driven HTT intron 2-targeting exon editor packaged in ssAAV (SEQ ID NO: 370), and a CAGGS promoter-driven HTT intron 2-targeting exon editor packaged in ssAAV (SEQ ID NO: 371). To test the dose-response of the exon editors, two different doses were selected for injection – bilateral injection of 1E+11 vector genomes (vg) and 3E+11 vg per animal. Two independent administration routes were chosen: intraventricular (ICV) injection at neonatal P0 (Table 2) and intrastriatal injection at 8 weeks of age. Four weeks after injection, mouse cortex and striatum were harvested, and the morphological efficiency of HTT exon substitution via trans-splicing and MSH3 knockdown via splicing regulation was analyzed by RT-ddPCR and Western blotting.

[0188] Table 2. Study design for in vivo mechanism validation studies using ICV injection in the brains of BAC-CAG mice. For each treatment, the age at final collection was 4 weeks, and the brain region collected was the corticostratus.

[0189] like Figure 58As shown, analysis of the HTT trans-splicing profile revealed that over 30% HTT replacement was achieved in the mouse brain via ICV injection in newborn mice. The CAGGS promoter-driven exon editor outperformed its CMV promoter equivalent, as indicated by the higher HTT replacement percentage in both the cortex and striatum. A significant dose-response was observed in the CMV promoter-driven exon editor, resulting in animals receiving 3E+11 vg having a higher HTT replacement percentage than those receiving 1E+11 vg. Notably, trans-spliced ​​full-length HTT protein was detected by Western blotting targeting the N-terminal FLAG tag. This experiment also demonstrated that higher levels of trans-splicing were achieved with increasing exon editor transcript levels. Figure 59 This suggests that identifying a stronger promoter or administering a higher dose may lead to even higher exon editor activity.

[0190] I. Definition As used herein, "trans-splicing" refers to the linking of a first RNA molecule containing one or more exons (e.g., exogenous exons or exons that are part of the coding domain of a trans-splicing molecule) to a second RNA molecule (e.g., a precursor mRNA molecule, such as an endogenous mRNA precursor molecule) via a spliceosome-mediated mechanism, replacing a portion of the second RNA molecule with a portion of the first RNA molecule. For example, Figure 3 This explains the general mechanism of RNA trans-splicing.

[0191] A “nucleic acid trans-splicing molecule” or “trans-splicing molecule” has three main components: (a) a binding domain that confers specificity by tethering the trans-splicing molecule to its target gene (e.g., a precursor mRNA); (b) a splicing domain (e.g., a splicing domain having a 3' or 5' splicing site); and (c) a coding domain configured to trans-splice onto the target nucleic acid, said coding domain being capable of replacing one or more exons in the target nucleic acid (e.g., one or more mutant exons). A “precursor mRNA trans-splicing molecule” or “RTM” refers to a nucleic acid trans-splicing molecule that targets precursor mRNA. The terms “nucleic acid trans-splicing molecule” and “trans-splicing molecule” refer to (1) DNA encoding RNA, wherein the RNA transcript is an effector molecule that physically binds to the target precursor mRNA; and (2) the RNA transcript itself. For clarity, this document uses the term "coding sequence" (e.g., trans-splicing molecule coding sequence) to specify a subject-encoded effector (e.g., the coding sequence is DNA and the effector is RNA). In some embodiments, the trans-splicing molecule coding sequence may include cDNA, for example, as a functional exon (e.g., functional...). HTT A part of the exon, used to replace the mutant. HTT Exons.

[0192] As used herein, the term “exon editor” may refer to trans-splicing molecules or vectors containing trans-splicing molecules (e.g., AAV vectors containing DNA encoding RNA transcripts as trans-splicing molecules).

[0193] As used herein, “trans-splicing efficiency” refers to the ratio of the detected expression level of the desired trans-spliced ​​RNA product (i.e., a chimeric RNA molecule generated via RNA trans-splicing, comprising a functional exon of the trans-splicing molecule operatively linked to an endogenous target precursor mRNA) to the amount of DNA or RNA (or a reference molecule) introduced for the trans-splicing molecule. In some cases, the expression level of the trans-spliced ​​RNA product is detected in RNA isolated from cells or tissues using RNA-seq.

[0194] As used in this article, “RNA replacement %” refers to the portion of the total target mRNA population that has been successfully trans-spliced ​​(TS), and is calculated using the following equation: Target (ONT) TS% = 100 * (ONT copy number / (ONT copy number + native copy number)).

[0195] As used herein, "relative trans-splicing efficiency" refers to the ratio of the tested trans-splicing efficiency to a reference trans-splicing efficiency, where the tested trans-splicing efficiency is the trans-splicing efficiency of the trans-splicing molecule (e.g., the nucleic acid trans-splicing molecule described herein), and the reference trans-splicing efficiency is the trans-splicing efficiency of a reference molecule (e.g., a reference molecule having the same elements as the nucleic acid trans-splicing molecule, but with the binding domain replaced by an out-of-order binding domain or a non-targeted binding domain (e.g., containing or consisting of a binding domain of SEQ ID NO: 7)). The relative trans-splicing efficiency of a trans-splicing molecule can be expressed as the ratio (also known as a fold increase) of the tested trans-splicing RNA efficiency tested under similar conditions to the reference trans-splicing efficiency.

[0196] As used herein, the term "operably linked" refers to an arrangement of elements in which the described components are configured to perform their usual function. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked" to the other nucleic acid sequence. Elements do not need to be adjacent to be operably linked. Thus, for example, an intervening sequence may exist between operably linked sequences (e.g., a binding domain and a coding sequence may be separated by an intervening sequence, and the binding domain is still considered "operably linked" to the coding sequence).

[0197] As used herein, the term "coding domain" refers to a nucleic acid sequence (e.g., an RNA sequence, a DNA sequence, or a combination of RNA and DNA) that encodes a portion of a protein (e.g., a target protein in which a mutation is being corrected). Therefore, a coding domain may include one or more functional exons (e.g., sequences of functional exons). In some cases, one or more functional exons of a coding domain are not separated by introns (e.g., as in endogenous precursor mRNA) but are adjacent to each other (e.g., as in cDNA). In some cases, a coding domain may include one or more introns (e.g., native introns), or untranslated regions (UTRs, such as native UTRs) located between exons or, in other cases, adjacent to exons (e.g., upstream or downstream).

[0198] As used in this article, "native 5'" HTT "Non-translated area" or "native 5'" HTT "UTR" refers to a sequence longer than 20 nucleotides, which is related to the original sequence. HTT Genes (e.g., human genes) HTT The gene has at least 90% sequence identity in the region of the ATG start codon 5'. HTT An example of an untranslated region is given by the DNA sequence of SEQ ID NO: 136. 5' HTT An example of a modified form of the untranslated region is given by the DNA sequence of SEQ ID NO: 192.

[0199] As used in this article, "5' HTT "Functional sequence of exons" refers to the sequence of exons containing HTT The nucleic acid sequence of one or more of exons 1-3 (e.g., exon 1; or exon 1 and exon 2; or exon 1, exon 2, and exon 3), wherein the exon encodes HTT The functional (biologically active) portion of a protein. In some implementations, "5' HTT "Functional sequence of exons" refers to the sequence of exons containing HTT Exon 1 or HTT Exon 1-2 or HTT The nucleic acid sequences of exons 1-3, wherein the exons encode HTT The functional (biologically active) part of a protein. When the 3' trans-splicing at the binding site is transferred to the endogenous protein... HTT Exon timing, 5' HTT The functional sequence of exons provides functionality HTT Proteins (e.g., unmutated) HTT Expression of proteins. In some cases, 5' HTTThe functional sequence of exons includes the exon sequence adjacent to the exon that is undergoing trans-splicing by the trans-splicing molecule (e.g., binding). HTT Intron 2 and endogenous HTT Trans-splicing molecules that undergo trans-splicing of exon 3 may include 5' cells containing exons 1 and 2. HTT Functional sequence of exons, or binding HTT Intron 3 and endogenous HTT Trans-splicing molecules that undergo trans-splicing of exon 4 may include 5' cells containing exons 1-3. HTT (Functional sequence of exons).

[0200] As used herein, the term "functional" in a protein context refers to a biologically active protein. The term "functional" can also refer to the amount of protein activity necessary to support normal cellular function. Regarding... HTT The term "functional" can be used to refer to recovery in the context of, for example, cortical pyramidal neurons, striatal polyspinous neurons, and / or hypothalamic neurons. HTT Activity levels necessary to support normal cell function HTTThe amount of protein activity. Furthermore, reducing the amount of mutant HTT and / or distorting the mutant:wild-type ratio will significantly contribute to reducing neurodegeneration of cortical pyramidal neurons, striatal polyspinous neurons, and / or hypothalamic neurons. In the context of treating disorders associated with pathogenic HTT activity (e.g., HD) or using therapeutic agents containing nucleic acid trans-splicing molecules as described herein, "functional" refers to reducing the amount of defective (non-functional) HTT proteins, such as those containing polyglutamine segments with more than 35 or 40 glutamine repeats, to eliminate one or more symptoms of disorders associated with pathogenic HTT activity (e.g., HD). In some embodiments, such methods or uses result in a reduction in pathogenic HTT activity and an increase in wild-type HTT protein activity. In some embodiments, such a reduction in pathogenic HTT protein activity, relative to control (untreated) cells expressing mutant pathogenic HTT, reduces the pathogenic HTT activity level by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) of HTT activity. In some embodiments, relative to cells where HTT is present at normal wild-type levels (e.g., those present in cells expressing unmutated HTT), such an increase in HTT protein activity restores the HTT activity level to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) of HTT activity.

[0201] As used herein, “cryptic splice site corrected,” “cryptic splice site mitigated,” or “crypticsplice site resistant” refers to a nucleic acid trans-splicing molecule or a portion thereof (e.g., a coding domain sequence therein) that has been modified to alter individual nucleotides therein, thereby reducing the splicing frequency occurring at cryptic splice sites identified in the context of a nucleic acid trans-splicing molecule. In some embodiments, the modification does not result in any change to the amino acid sequence thereby encoded. In some embodiments, the cryptic splice site resistant nucleic acid sequence within the nucleic acid trans-splicing molecule is located within the coding domain sequence (CDS). In some embodiments, crypticsplice site resistance… HTT CDS comprises, is essentially composed of, or consists of: HTTThe gene contains exon 1, or exons 1 and 2, or exons 1, 2 and 3, wherein a hidden splicing site has been identified in the context of a nucleic acid trans-splicing molecule, and wherein at least one of the hidden splicing sites has been modified to reduce the splicing frequency at the at least one site without altering the amino acid encoded therefrom.

[0202] As used herein, a "splicing domain" refers to a nucleic acid sequence having a motif recognized by the spliceosome and mediating trans-splicing. A splicing domain includes a splice site (e.g., a single splice site, i.e., one and only one splice site), which may be a 3' splice site or a 5' splice site. A splicing domain may include other regulatory elements. In some embodiments, the splicing domain contains GUAAGT or GTAAGT. In some embodiments, the splice site consists essentially of GUAAGT or GTAAGT. In some embodiments, the splice site consists of GUAAGT or GTAAGT.

[0203] As used in this article, the “binding domain” of a trans-splicing molecule is a multinucleotide sequence that binds to a target gene at the binding site through hybridization (i.e., complete or partial complementarity with the binding site).

[0204] As used herein, the term "binding site" refers to the target precursor mRNA (e.g., endogenous gene (e.g.)). HTT An endogenous polynucleotide sequence in the precursor mRNA of a nucleic acid trans-splicing molecule that binds to a binding domain. The binding site extends from the 5' end nucleotide to which the binding domain binds to the 3' end nucleotide. In some embodiments, the length of the binding site is the same as that of the binding domain. In other embodiments, the binding site is 1-10 nucleotides longer or shorter than the binding domain (i.e., some nucleotides of the binding site or the binding domain do not hybridize). In embodiments involving a binding domain having at least two non-overlapping sequences that are at least 80% complementary to the binding site, the binding site may be substantially shorter than the binding domain.

[0205] As used in this article, “complementarity” and its grammatical variations refer to the percentage of nucleotide bases in a given sequence that pair with a reference sequence via hydrogen bonding.

[0206] As used herein, if each nucleotide base of a given sequence (e.g., a binding domain sequence) pairs with a reference sequence (e.g., an endogenous precursor mRNA binding site) via hydrogen bonding to form a double-stranded sequence (e.g., via Watson-Crick base pairing, such as each A pairing with T or U, and each C pairing with G), then the given sequence is “100% complementary” to the reference sequence, or has “100% complementarity”. For example, a binding domain oriented antisense to the binding site is complementary to the binding site. RNA pairing includes G-U pairing; therefore, an RNA binding domain that pairs GU with the binding site is 100% complementary to the binding site. Thus, a binding domain that is exactly the anticomplement of the binding site (i.e., the A of the binding domain pairs with the U of the binding site) can be modified to replace any one or more A's with G or C's with T without substantially affecting binding.

[0207] As used herein, if X% of the nucleotide bases of a given sequence (e.g., a binding domain sequence) pair with a reference sequence (e.g., an endogenous precursor mRNA binding site) via hydrogen bonding, for example, by hybridization to form a double-stranded sequence (e.g., via Watson-Crick base pairing, such as A pairing with T or U, and C pairing with G), then the given sequence is “at least X% complementary” to the reference sequence, or has “X% complementarity”. For example, if at least 135 of the 150 residues of a binding domain sequence of length 150 bases pair with a binding site of length 150 bases via hydrogen bonding through Watson-Crick base pairing, and the remaining 15 or fewer mismatched nucleotides, then the binding domain sequence is at least 90% complementary to the binding site.

[0208] For the sequences provided herein and in the accompanying sequence listing, it should be understood that the RNA transcript encoded by the DNA sequence contains uridine (U) at the position corresponding to the thymidine (T) listed in the corresponding DNA sequence. In some cases, the sequences of RNA exon editor components are disclosed herein in DNA sequence form. For any sequence disclosed herein in DNA sequence form, it is also considered to replace the RNA sequence of each T in the sequence with U. Thus, if a given SEQ ID NO is identified as having a sequence that can be included in the RNA exon editor, it is also considered to replace the SEQ ID NO of each T with U.

[0209] As used herein, “binding” between the binding domain and the intron refers to a hydrogen bond (e.g., double helix formation or Watson-Crick pairing) between the binding domain and the target intron, strong enough to mediate trans-splicing by associating the trans-splicing molecule with the target (e.g., precursor mRNA). In some embodiments, the hydrogen bond between the binding domain and the target intron is located between complementary and antisense oriented (e.g., hybridizing) nucleotide bases.

[0210] As used herein, an "artificial intron" refers to a non-coding nucleic acid sequence that links a binding domain (directly or indirectly) to a coding domain. Artificial introns include splicing domains and may also include one or more spacer sequences and / or other regulatory elements.

[0211] As used herein, the term "mutation" can be used to refer to any abnormal nucleic acid sequence encoding a defective RNA or protein product (e.g., a nonfunctional protein product, a biologically inactive protein, a protein product with reduced function, a protein product with pathogenic or abnormal function, and / or a protein product produced in amounts less than or greater than normal). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), duplications, missense mutations, frameshift mutations, deletions, insertions, and splicing mutations. In some embodiments, a mutation refers to a nucleic acid sequence that differs from one or more portions of a corresponding wild-type nucleic acid sequence or a functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence encoding a protein having an amino acid sequence that differs from a corresponding wild-type protein or a functional variant thereof. "Mutant exon" (e.g., mutant exon) HTT An exon is an exon containing a mutation or an exon sequence that reflects a mutation in a different region, such as a hidden exon caused by an intron mutation.

[0212] Unless otherwise stated, the term " HTT "(Huntington protein) refers to any native protein derived from any vertebrate source." HTT The sources include mammals, such as primates (e.g., humans, African green monkeys, and cynomolgus monkeys) and rodents (e.g., mice and rats), as well as functionally equivalent or improved variants (e.g., natural or synthetic variants), mutants, mutant proteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments. Based on known... HTT Signal transduction can identify functionally equivalent and improved variants. HTT Covering the entire length, unprocessed HTT and any form of naturally produced by processing within cells. HTT Exemplary human HTT The sequence is provided as a reference sequence from the National Center for Biotechnology Information (NCBI): NG_009378. In some cases, HTT The fragment is encoded by a therapeutic agent comprising a sequence having at least 95% sequence identity with any of SEQ ID NO: 3, 59, 157, 349, 350, 351, 352 or 353 (e.g., having at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any of SEQ ID NO: 3, 59, 157, 349, 350, 351, 352 or 353), its functional portion and / or variants of its codon modifications.

[0213] As used herein, a “variant” means a polynucleotide that differs from a reference polynucleotide sequence (e.g., a naturally occurring polynucleotide sequence) at at least one nucleic acid residue, or a polypeptide (e.g., an AAV capsid sequence) that differs from a reference polypeptide sequence (e.g., a naturally occurring polypeptide sequence or, for example, any rAAV sequence described herein) at at least one amino acid residue. Differences at at least one residue may include, for example, the substitution, deletion, or insertion of a nucleic acid residue by another nucleic acid, or the substitution of an amino acid residue by another amino acid. A variant may be a homolog, isoform, or transcript variant of a polynucleotide as defined herein, characterized by the degree of identity or homology as defined herein.

[0214] In some cases, variants of polynucleotides or polypeptides include at least one nucleic acid substitution (e.g., 1-100 nucleic acid or amino acid substitutions, 1-50 nucleic acid or amino acid substitutions, 1-20 nucleic acid or amino acid substitutions, 1-10 nucleic acid or amino acid substitutions, such as 1 nucleic acid or amino acid substitution, 2 nucleic acid or amino acid substitutions, 3 nucleic acid or amino acid substitutions, 4 nucleic acid or amino acid substitutions, 5 nucleic acid or amino acid substitutions, 6 nucleic acid or amino acid substitutions, 7 nucleic acid or amino acid substitutions, 8 nucleic acid or amino acid substitutions, 9 nucleic acid or amino acid substitutions, or 10 nucleic acid or amino acid substitutions). Nucleic acid substitutions that give the expressed polypeptide an amino acid exchanged from the same class are referred to herein as conserved substitutions. Specifically, these are amino acids having aliphatic side chains, positively or negatively charged side chains, or aromatic groups in the side chains, wherein the side chains can form hydrogen bridges, such as side chains with hydroxyl functional groups. Through conservative substitution, for example, an amino acid with a polar side chain can be replaced by another amino acid with a corresponding polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain can be replaced by another amino acid with a corresponding hydrophobic side chain (e.g., serine (threonine) is replaced by threonine (serine), or leucine (isoleucine) is replaced by isoleucine (leucine).

[0215] In some cases, the terminology also encompasses insertions, deletions, and / or non-conserved substitutions, such as at locations that do not result in substantial modifications to the protein's three-dimensional structure. Those skilled in the art can readily determine modifications to the three-dimensional structure through insertions or deletions, for example, using CD spectroscopy (circular dichroism spectroscopy).

[0216] The term "homology" refers to the degree of similarity between two nucleic acid sequences. Sequence homology is determined by comparing two sequences aligned under standard conditions in terms of length. The sequences to be compared in this paper may have additions or deletions (e.g., gaps) in the best alignment of the two sequences. In some implementations, sequence homology is calculated by creating alignments using, for example, the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22): 4673 4680). Commonly available sequence analysis software, such as Vector NTI, GENTYX, BLAST, or analysis tools provided by public databases, can also be used.

[0217] As used herein, the term “AAV” or “AAV serotype” refers to dozens of naturally occurring and available adeno-associated viruses, as well as artificially created AAVs. Among the AAVs isolated from or engineered from human or non-human primates (NHPs) and well-characterized, human AAV2 was the first AAV developed as a gene transfer vector; it has been widely used for efficient gene transfer experiments in various target tissues and animal models. For example, AAV9, AAV-retro, AAV1, AAV4, AAV8, AAV5, and AAV-PHP.eB belong to inherently neurotropic AAV serotypes.

[0218] As used herein, with respect to AAV, the term variant means any AAV sequence derived from a known AAV sequence, including those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or greater sequence identity on the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants that may include up to about 10% variation with any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with AAV capsids provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with other AAV capsids. When determining the percentage of identity of the AAV capsid, comparisons can be made with any variable protein (e.g., vp1, vp2, or vp3).

[0219] ITRs or other AAV components can be readily isolated from or engineered from AAVs using techniques available to those skilled in the art. Such AAVs can be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., Manassas, Va.). Alternatively, AAV sequences can be engineered by referencing published sequences (e.g., available in the literature or databases such as GenBank, PubMed, etc.) through synthesis or other suitable methods. AAV viruses can be engineered using conventional molecular biology techniques, making it possible to optimize these particles for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, tuning of stability and particle lifetime, efficient degradation, and / or accurate delivery to the cell nucleus, etc.

[0220] As used herein, the terms “subject,” “individual,” or “patient” include any mammal requiring these treatments or preventative measures, including primates such as humans. Other mammals requiring such treatments or preventative measures include non-human primates (NHPs; e.g., cynomolgus monkeys and African green monkeys), dogs, cats or other domesticated animals, horses, livestock, laboratory animals, etc. Individuals can be male or female. In one implementation, the individual suffers from [a condition caused by…]. HTT Diseases or conditions caused by gene mutations (e.g., HD). In another implementation, an individual is in the process of developing a disease or condition caused by a gene mutation. HTT The risk of diseases or conditions caused by gene mutations. In another implementation, the individual has shown that... HTT Clinical signs of a disease or condition (such as HD) caused by a gene mutation. The individual can be any age at which treatment or preventative therapy may be beneficial. For example, in some implementations, the individual is 0-5 years old, 5-10 years old, 10-20 years old, 20-30 years old, 30-40 years old, 30-50 years old, 40-50 years old, 50-60 years old, 60-70 years old, or over 70 years old.

[0221] As used herein, the terms "mutation-related condition" or "mutation-related condition" refer to the correlation between a condition and a mutation. In some embodiments, a mutation-related condition is known or suspected to be wholly or partially, or directly or indirectly, caused by said mutation. For example, an individual with the mutation may be at risk of developing said condition, and said risk may also depend on other factors, such as other (e.g., independent) mutations (e.g., in the same or different genes) or environmental factors.

[0222] As used herein, the term "treatment" or its grammatical derivatives are defined as reducing disease progression, alleviating the severity of disease symptoms, slowing the progression of disease symptoms, removing disease symptoms, or delaying the onset of disease. In some implementations, the term "treatment" is used to refer to the sustained or durable effect of a therapeutic agent (such as the RNA exon editor described herein).

[0223] As used herein, the term "prevention" of a disease, or its grammatical derivatives, is defined as reducing the risk of disease onset, for example, preventative therapy for individuals at risk of developing a mutation-related disease. An individual can be characterized as "at risk of developing a disease" by identifying mutations associated with the disease, according to any suitable method known in the art or described herein. In some embodiments, an individual at risk of developing a disease has one or more mutations associated with said disease. HTT Mutation. Or, alternatively, if an individual has a family history of the disease, the individual can be characterized as "at risk of developing the disease".

[0224] Conditions in individuals can be treated or prevented by directly administering trans-splicing molecules or RNA exon editors (e.g., within a vector, such as an AAV vector or AAV particle). Alternatively, host cells containing trans-splicing molecules can be administered to an individual.

[0225] As used in the methods described herein, the term "application" or its grammatical derivatives refer to the delivery of trans-splicing molecules or RNA exon editors (e.g., within a vector (e.g., an AAV vector or AAV particles)) or combinations thereof, or ex vivo treated cells, to an individual in need, such as those with... HTT Individuals with mutations or defects. For example, in an embodiment targeting striatal cells (e.g., intermediate polyspinous neurons) or cortical cells (e.g., pyramidal neurons), the method involves delivering trans-splicing molecules or RNA exon editors (e.g., within a vector (e.g., AAV vectors or AAV particles)) or combinations thereof to an individual via intracerebral (IC) delivery (e.g., slow delivery injection or convection-enhanced diffusion injection), intraventricular (ICV) delivery, or intrathecal delivery. In some embodiments, IC injection involves stereotactic implantation of a microinjection-guided cannula to improve delivery to specific sites in the brain. In another embodiment, the composition is administered systemically (e.g., intravenously). In view of this disclosure, those skilled in the art may also choose other methods of administration.

[0226] As used in this article, "regulation" HTT "Expression" refers to reducing endogenous mutants. HTT The expression and / or addition of reverse splicing HTT The expression. Regulation. HTTThe expression of [expression name] can be used to refer to, for example, a reduction in endogenous (e.g., mutant) transcripts or protein products relative to endogenous mutant transcripts or protein products. HTT The expression and / or increase of trans-splicing HTT (e.g., with trans-splicing molecule-mediated corrective mutation sites) HTT Expression of transcripts or protein products. This is achieved through trans-splicing of endogenous proteins containing mutated sites. HTT Following the exon, the functional HTT protein is expressed.

[0227] As used herein, “codon optimization” refers to modifying a nucleic acid sequence to alter individual nucleic acids without altering the encoded amino acids. A sequence modified in this manner is referred to herein as “codon-optimized.” This process can be performed on any sequence described in this specification to enhance performance or stability. Codon optimization can be performed, for example, in the manner described below: for example, U.S. Patent Nos. 7,561,972, 7,561,973, or 7,888,112, each of which is incorporated herein by reference in its entirety. Sequences around translation start sites can be converted to common Kozak sequences according to known methods. See, for example, Kozak et al., 1987. Nucleic Acids Res. 15 (20): 8125-8148, which is incorporated herein by reference in its entirety. The term “pharmaceuticalally acceptable” means safe for administration to mammals (e.g., humans). In some embodiments, pharmaceutically acceptable compositions are approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly for use in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or mediator that is administered together with a therapeutic molecule (e.g., the trans-splicing molecule of the present invention or a carrier or cell comprising the trans-splicing molecule). Examples of suitable drug carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition.

[0228] The term “a (a / an)” means “one (species) or more (species)”. For example, “one gene” should be understood to represent one or more such genes. Therefore, the terms “a (a / an)”, “one or more of a (oran)”, and “at least one of a (or an)” are used interchangeably in this document.

[0229] Unless otherwise stated, as used herein, the term “about” means a value that varies within ±10% relative to a reference value.

[0230] II. Trans-splicing molecules This article provides nucleic acid trans-splicing molecules that, through at least one mutant... HTT Exon replacement with functional HTT Exons [e.g., at the 5' binding site] HTT Exons, for example HTT Exon 1 (SEQ ID NO: 348; HTT 5' UTR + exon 1, which includes 21 CAG repeats (within the normal repeat number range)), exon 2 (SEQ ID NO: 365), and exon 3 (SEQ ID NO: 366) can be used for correction. HTT Mutations in [the sequence]. Note that although SEQ ID NO: 348 shows [the mutation]. HTT The sequence of exon 1 comprises 21 CAG repeats, but the number of CAG repeats in individual genomic sequences may differ (i.e., higher or lower). By taking into account any variation in the number of CAG repeats, the corresponding nucleotide numbering positions in sequences with different numbers of CAG repeats can be readily determined. In some embodiments, the nucleic acid trans-splicing molecule is an RNA trans-splicing molecule (RTM). The design of the trans-splicing molecule allows for the replacement of defective or mutant portions of the precursor mRNA exon with a nucleic acid sequence, such as an exon with a functional (e.g., normal) sequence without mutation. The functional sequence can be wild-type, a naturally occurring sequence, or a corrected sequence with some other modifications (e.g., codon optimization).

[0231] The trans-splicing molecule comprises a binding domain, a splicing domain, and a coding domain. In some embodiments, the nucleic acid trans-splicing molecule has a 5' regulatory domain, which has a native 5'... HTT Untranslated regions (e.g., sequences with at least 80% sequence identity to SEQ ID NO: 136 or 192). In some embodiments, the nucleic acid trans-splicing molecule has a splicing site of GTAAGT. In some embodiments, the nucleic acid trans-splicing molecule has a linker domain of more than 25 nucleotides in length. In some embodiments, the nucleic acid trans-splicing molecule has a linker domain comprising, substantially comprising, or comprising any of SEQ ID NO: 37-46 and 106-112, or sequences having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NO: 37-46 and 106-112.

[0232] In some embodiments, the trans-splicing molecule described herein comprises: a 5' untranslated region, a coding domain sequence (e.g., CDS, e.g., coding structure) operably connected in the 5' to 3' direction; and a coding structure domain sequence (e.g., a coding structure domain sequence). HTT The functional sequence of exons (e.g., at the binding site 5') HTT The functional sequence of exons includes the sequence of exons, splice domains (e.g., splice sites), connector domains, binding domains, 3' downstream regions, and terminator domains.

[0233] In some implementations, the nucleic acid trans-splicing molecules described herein are configured to correct the subject's... HTT The alleles of the gene located in HTT At least one mutation in the 5' region of a gene (e.g., in the 5' region of introns 1, 2, or 3): binding to target introns 1, 2, or 3 and mediating a 5' region. HTT The coding domain of the exon's functional sequence is trans-spliced ​​to the endogenous domain of the target intron 3'. HTT Exons. Thus, this type of trans-splicing repairs defects in the target cells of an individual by replacing defective exons and removing the defective portion of the target precursor mRNA. HTT Genes, thereby producing wild-type HTT proteins capable of being transcribed in cells. HTT mRNA.

[0234] HTT The trans-splicing molecules described in this article target HTT A gene can contain one or more mutations associated with HD. Example: Human HTT The sequence is provided as a reference sequence from the National Center for Biotechnology Information (NCBI): NG_009378. In addition to the published sequences, it includes all subsequently obtained corrections or naturally occurring conserved and non-pathogenic variant sequences present in humans or other mammalian populations. It also includes additional conserved nucleotide substitutions or those leading to codon optimization. The sequences provided by the database accession number can also be used to search for homologous sequences in the same or another mammalian organism.

[0235] expected HTT The nucleic acid sequence and the resulting protein can tolerate certain minor modifications at the nucleic acid level, including, for example, modifications to silencing nucleotide bases related to the encoded amino acid. In other embodiments, nucleic acid base modifications that alter amino acids are envisioned, for example, to improve the expression of the resulting peptide / protein. In some embodiments, modifications based on allelic variations arising from the natural degeneracy of the genetic code are envisioned.

[0236] HTTGene modification also includes analogues or modified forms of the encoded amino acid sequence. Typically, these analogues differ from the specific identified protein by only one to four codon changes. Conserved substitutions occur within amino acid families that are related to side chains and chemical properties.

[0237] Functionality HTT The nucleic acid sequence of a gene can originate from any naturally expressed functional gene. HTT Mammals or their homologs. In other embodiments, for HTT Gene sequences are modified to enhance expression in target cells. Such modifications can include codon optimization.

[0238] As mentioned above, HD is... HTT This is caused by an amplification of more than 35 CAG trinucleotide repeats (incomplete penetrance) or more than 40 CAG repeats (adolescent or adult-onset disease) in exon 1 of a gene, where the repeats are inherited in an autosomal dominant manner. See also Figure 1 Compositions containing the trans-splicing molecule described herein can correct amplified CAG repeats in exon 1, regardless of the number of repeats present, because the trans-splicing molecule replaces... HTT The entire pathogenic exon 1 of the gene.

[0239] Coding domain In some implementations, the coding domain of the 5' trans-splicing molecule is included in the target. HTT Introns (e.g.) HTT Intron 1 [(SEQ ID NO: 348; HTT 5' UTR + exon 1, comprising 21 CAG repeats (within the normal repeat number range) or may contain 35-39 CAG repeats (incomplete penetrance) or 40+ CAG repeats (adult-onset or adolescent-onset HD)], intron 2 (SEQ ID NO: 365) and / or intron 3 (SEQ ID NO: 366)) all of the 5' HTT Exons (e.g., functional) HTT (Exons). For example, in 5' trans-splicing molecules targeting HTT In an implementation of intron 2, the coding structure domain may include functional HTT Exons 1-2. In some implementations, functional HTT Exons 1-2 are encoded by sequences containing any one of SEQ ID NO: 59, 349, 350, or 351. In some embodiments, functionality... HTTExons 1-2 are encoded by sequences comprising any one of SEQ ID NO: 59, 349, 350, or 351, said sequences further comprising an ATG start codon at the 5' end. In some embodiments, the binding domain is bound to intron 2, and the encoding domain includes functional HTT Exons 1-2.

[0240] Targeting 5' trans-splicing molecules HTT In some implementations of intron 3, the coding structure domain may include functional HTT Exons 1-3. In some implementations, functional HTT Exons 1-3 are encoded by sequences containing any one of SEQ ID NO: 157, 352, or 353. In some embodiments, functionality... HTT Exons 1-3 are encoded by sequences comprising SEQ ID NO: 157, 352, or 353, said sequences further comprising an ATG start codon at the 5' end. In some embodiments, the binding domain is bound to intron 3, and the encoded domain includes functional HTT Exons 1-3.

[0241] In some implementations, the 5' trans-splicing molecule targets HTT Intron 1, the coding structure field can include functional HTT Exon 1. In some implementations, functional HTT Exon 1 is encoded by a sequence containing SEQ ID NO: 3. In some embodiments, functionality... HTT Exon 1 is encoded by a sequence comprising SEQ ID NO: 3, which further comprises an ATG start codon at the 5' end. In some embodiments, a binding domain is bound to intron 1, and the encoding domain includes functional... HTT Exon 1.

[0242] In some implementations, the coding domain coding sequence (e.g., for transgenes encoding RTM) includes HTT Exons (e.g., HTT cDNA from exons is used to replace mutants. HTT Exons. For example, one or more functional components within a coding domain. HTT Exons can be cDNA sequences. In some implementations, the entire coding domain is a cDNA sequence. Alternatively, all or part of the coding domain, or one or more of its functional components, may be cDNA sequences. HTT Exons can be naturally occurring sequences (e.g., those with endogenous origins). HTTThe exons have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0243] In some implementations, all or part of the coding structure domain or the coding sequence of the coding structure domain, or one or more of its functionalities... HTT Exons are codon-optimized sequences in which the nucleic acid sequence has been modified, for example, to enhance expression or stability, without causing changes to the encoded amino acids. Codon optimization can be performed, for example, in the manner described below: for example, U.S. Patent Nos. 7,561,972, 7,561,973, or 7,888,112, each of which is incorporated herein by reference in its entirety. For delivery via recombinant AAV, as described herein, in one embodiment, the coding domain can be a nucleic acid sequence up to 4,000 nucleotides in length.

[0244] In some embodiments, this document describes a nucleic acid trans-splicing molecule comprising, in the 5' to 3' direction: (a) a cDNA coding domain sequence; (b) a splicing donor sequence; and (c) a binding domain sequence configured to bind to an intron of an endogenous RNA molecule; wherein the coding domain sequence contains at least one nucleotide mutation relative to the endogenous RNA molecule sequence, wherein the at least one nucleotide mutation disrupts a cryptic splicing site within the coding domain sequence. In some embodiments, the nucleotide mutation is a synonymous nucleotide mutation. In some embodiments, the cryptic splicing site is identified experimentally. In some embodiments, the cryptic splicing site is predicted based on computer analysis.

[0245] This document also covers a method for modifying RNA molecules in cells, the method comprising providing cells with an exogenous RNA molecule comprising, in the 5' to 3' direction: (a) a cDNA-coding domain sequence containing a nucleotide mutation different from that of an endogenous target RNA molecule in the cell; (b) a splice donor sequence configured to splice to a splice acceptor sequence of the endogenous target RNA molecule; and (c) a binding domain sequence configured to bind to an intron of the endogenous target RNA molecule; wherein the nucleotide mutation disrupts a cryptic splice site within the coding domain sequence of the exogenous RNA molecule. In some embodiments, the nucleotide mutation is a synonymous nucleotide mutation. In some embodiments, the cryptic splice site is identified experimentally. In some embodiments, the cryptic splice site is predicted based on computer analysis.

[0246] This document also covers a method for increasing the trans-splicing efficiency or therapeutic performance of an RNA exon editor, the method comprising introducing a mutation into a coding domain sequence of the RNA exon editor, wherein the mutation disrupts a cryptic splicing site in the coding domain sequence of the RNA exon editor. In some embodiments, the nucleotide mutation is a synonymous nucleotide mutation. In some embodiments, the cryptic splicing site is identified experimentally. In some embodiments, the cryptic splicing site is predicted based on computer analysis.

[0247] Reducing nucleotide changes at cryptic splicing sites can include eliminating or reducing the ability of the cryptic splicing site to be used in splicing reactions. For example, cryptic splicing sites identified in the context of an RNA exon editor can include splice sites, polypyrimidine segments, and branching points. In some embodiments, one or more nucleotide changes can be introduced into at least one or any combination of splice sites, polypyrimidine segments, or branching points of cryptic splicing sites identified in the context of an RNA exon editor. In some embodiments, nucleotide changes are determined to minimize potential effects on the protein thereby encoded. Those skilled in the art will understand that conserved amino acid changes are preferred over non-conserved amino acid changes if the nucleotide changes made to reduce the frequency of cryptic splicing site use also alter the amino acids encoded by the trans-spliced ​​RNA. Furthermore, those skilled in the art can readily analyze protein sequences and structures, focusing on functional domains and important sequences therein, to assess whether such changes can be reasonably expected to alter the function of the protein encoded by the trans-spliced ​​protein. Those skilled in the art can also test proteins containing such amino acid changes using assays known in the art to determine whether altered biological activity has occurred. In some embodiments, more than one nucleotide is changed within the identified cryptic splicing site. In some cases, the number of nucleotides that should change is determined empirically based on computer predictions and / or experimental results. In some embodiments, one or more (also referred to herein as at least one) synonymous mutations may be introduced into at least one or any combination of splicing sites, polypyrimidine segments, or branching points of cryptic splicing sites identified in the context of an RNA exon editor. Synonymous mutations do not alter the amino acid sequence of proteins encoded by trans-spliced ​​RNA. In some embodiments, more than one synonymous mutation may be introduced into at least one or any combination of splicing sites, polypyrimidine segments, or branching points of cryptic splicing sites identified in the context of an RNA exon editor.

[0248] In addition to the above, experimental results and sequence information are typically analyzed as follows: Changes are made by searching for and replacing certain elements of the splice acceptor site to remove experimentally identified cryptic splicing sites. For introducing nucleotide changes, AG sites (and stronger CAG sites) at the ends of the splice acceptor site are preferred. If the AG site is not found or cannot be changed without introducing a non-synonymous mutation, the sequence 42-4 base pairs upstream of the splice site is scanned for branch points (sequences matching YNAH). Any such branch point sequences identified are then analyzed and considered for introducing one or more nucleotide mutations to reduce the cryptic splice site at the experimentally identified site. Furthermore, the sequence is scanned to determine the presence of polypyrimidine segments (multiple Ys immediately upstream of the terminal AG). Typically, such polypyrimidine segments contain at least five pyrimidines within 10 base pairs upstream of the splice site. Once identified, these polypyrimidine segments are then analyzed and considered for introducing nucleotide mutations to reduce the cryptic splice site at the experimentally identified site.

[0249] In some embodiments, the cryptic splicing sites that are modified to reduce off-target splicing, or the off-target splicing sites, are sites that have been empirically identified as off-target splicing sites. For example, such sites can be identified using the techniques described in Example 6 of WO 2023 / 220742, which is hereby incorporated herein by reference in its entirety. In some embodiments, the nucleotide changes to reduce cryptic splicing sites alter all cryptic splicing sites whose frequency of use is above a predetermined threshold.

[0250] In some implementations, the cryptic splice sites that are modified to reduce off-target splicing, or the off-target splice sites that are already predicted to be off-target splice sites, are modified to reduce off-target splicing. Such predictions can be made based on sequence analysis to identify canonical splice sites, polypyrimidine segments, and / or branching points of the presumed cryptic splice sites.

[0251] In some embodiments, reducing the nucleotide change at the cryptic splice site causes a nucleotide sequence that previously matched the canonical splice site common sequence to no longer match the canonical sequence. In some embodiments, reducing the nucleotide change at the cryptic splice site eliminates potential splice site nucleotides. In some embodiments, reducing the nucleotide change at the cryptic splice site eliminates potential polypyrimidine segment nucleotides. In some embodiments, reducing the nucleotide change at the cryptic splice site eliminates potential branching point nucleotides. In some embodiments, reducing the nucleotide change at the cryptic splice site is a synonymous nucleotide change. In some embodiments, reducing the nucleotide change at the cryptic splice site causes a change in the amino acid encoded by the exon editor. In some embodiments, the amino acid change is a conserved amino acid substitution.

[0252] To address the possibility of covert self-splicing (via cis-splicing of AAV multipliers or intermolecular trans-splicing), the inventors used computer prediction of self-splicing sites to reduce such adverse events. See, for example, Table 1.

[0253] Table 1. Reduction of cryptic self-splicing sites identified in the original exon editors.

[0254] Binding domain The article described The trans-splicing molecule provides a binding domain (BD) that is configured as a binding / annealing target. HTT Introns and / or exons. In some cases, the target... HTT Introns are Intron 2. In one embodiment, the binding domain is associated with the target. HTT Intron precursor mRNA (e.g., target mRNA) A nucleic acid sequence that is at least 80% complementary to the intron sequence (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), which can inhibit endogenous target cis-splicing while enhancing trans-splicing molecules and targets. HTT Trans-splicing between precursor mRNAs (e.g., by creating endogenous) A part of mRNA and having one or more functions HTT Chimeric molecules of exon-coding domains, wherein the exons encode wild-type (Amino acid sequence). In one embodiment involving the coding sequence of a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain coding sequence encodes the target. HTT The intron precursor mRNA sequence is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0255] In some cases, the present invention provides a combination Trans-splicing molecules (or vectors thereof) of intron 2, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to an endogenous protein. HTT Exon 3. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to any one or more (e.g., six or more, eight or more, ten or more, or twelve or more) binding sites of nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of SEQ ID NO: 57.

[0256] In some cases, the binding site includes Intron 2 consists of any six or more consecutive nucleotides within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 (e.g., HTT Intron 2 consists of any eight or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. Intron 2 consists of any ten or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. HTT Intron 2 consists of any 12 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. Intron 2 consists of any 20 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. HTT Intron 2 consists of any 30 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. Intron 2 consists of any 40 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. HTT Intron 2 consists of any 50 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. Intron 2 consists of any 100 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. HTT Intron 2 consists of any 150 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. Intron 2 consists of any 200 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251. HTTIntron 2 consists of any 250 or more consecutive nucleic acids within the ranges of 1 to 200, 1,500 to 2,500, or 10,500 to 12,251.

[0257] In some cases, the binding site includes Intron 2 consists of any six or more consecutive nucleotides within a range of 10,000 to 12,251 (e.g., HTT Intron 2 consists of any eight or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. Intron 2 consists of any ten or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. HTT Intron 2 consists of any 12 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. Intron 2 consists of any 20 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. HTT Intron 2 consists of any 30 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. Intron 2 consists of any 40 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. [[ID=6 Intron 2 consists of any 50 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. ​ Intron 2 consists of any 100 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. ​ Intron 2 consists of any 150 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides. ​ Intron 2 consists of any 200 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides or ​ Intron 2 consists of any 250 or more consecutive nucleic acids within a range of 10,000 to 12,251 nucleotides.

[0258] In some cases, the binding site includes ​ Intron 2 consists of any six or more consecutive nucleotides within nucleotides 11,000 to 12,251 (e.g., ​ Intron 2 consists of any eight or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any ten or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 12 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 20 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides.​ Intron 2 consists of any 30 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 40 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 50 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 100 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 150 or more consecutive nucleic acids within a range of 11,000 to 12,251 nucleotides. ​ Intron 2 consists of any 200 or more consecutive nucleic acids within 11,000 to 12,251 nucleotides or ​ Intron 2 consists of any 250 or more consecutive nucleic acids within 11,000 to 12,251 nucleotides.

[0259] In some cases, the binding site includes ​ Any six or more consecutive nucleotides within nucleotides 11,950 to 12,251 of intron 2 (e.g., ​ Intron 2 consists of any eight or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any ten or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 12 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 20 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 30 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 40 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 50 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 100 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 150 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ Intron 2 consists of any 200 or more consecutive nucleic acids within nucleotides 11,950 to 12,251. ​ (Any 250 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of intron 2).

[0260] In some cases, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0261] In some cases, the binding domain comprises a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 60-81 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0262] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 60, 62 or 67-81 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0263] In some cases, the binding domain comprises a nucleic acid sequence having at least 80% identity (at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with any of SEQ ID NO: 60 or 67-81.

[0264] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 67 or 70-81 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0265] In some cases, the binding domain comprises a nucleic acid sequence having at least 80% identity (at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) with any of SEQ ID NO: 71-74 or 77-79.

[0266] In some cases, the target ​ Introns are ​ Intron 3. In one embodiment, the binding domain is associated with the target. ​ Intron precursor mRNA (e.g., target mRNA) ​ A nucleic acid sequence that is at least 80% complementary to the intron sequence (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), which can inhibit endogenous target cis-splicing while enhancing trans-splicing molecules and targets. ​ Trans-splicing between precursor mRNAs (e.g., by creating endogenous) ​ A part of mRNA and having one or more functions ​ Chimeric molecules of exon-coding domains, wherein the exons encode wild-type ​ (Amino acid sequence). In one embodiment involving the coding sequence of a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain coding sequence encodes the target. ​The intron precursor mRNA sequence is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0267] In some cases, the present invention provides a combination ​ Trans-splicing molecules (or vectors thereof) of intron 3, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to an endogenous protein. ​ Exon 4. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to any one or more (e.g., six or more, eight or more, ten or more, or twelve or more) binding sites of nucleotides 3,100 to 4,429 having SEQ ID NO: 155.

[0268] In some cases, the binding site includes ​ Intron 3 contains any six or more consecutive nucleotides from nucleotides 3,100 to 4,429 (e.g., ​ Intron 3 consists of any eight or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any ten or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 12 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 20 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 30 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 40 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 50 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 100 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 150 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​ Intron 3 consists of any 200 or more consecutive nucleic acids within nucleotides 3,100 to 4,429. ​Intron 3 contains any 250 or more consecutive nucleic acids within nucleotides 3,100 to 4,429.

[0269] In some cases, the binding site includes ​ Intron 3 is any six or more consecutive nucleotides from nucleotides 4,100 to 4,429 (e.g., ​ Intron 3 consists of any eight or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any ten or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 12 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 20 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 30 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 40 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 50 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 100 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 150 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ Intron 3 consists of any 200 or more consecutive nucleic acids within nucleotides 4,100 to 4,429. ​ (Any 250 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of intron 3).

[0270] In some cases, the binding site includes ​ Intron 3 is any six or more consecutive nucleotides within the range of 4,100 to 4,388 (e.g., ​ Intron 3 consists of any eight or more consecutive nucleic acids within the range of nucleotides 4,100 to 4,388. ​ Intron 3 consists of any ten or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​ Intron 3 consists of any 12 or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​ Intron 3 consists of any 20 or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​ Intron 3 consists of any 30 or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​Intron 3 consists of any 40 or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​ Intron 3 consists of any 50 or more consecutive nucleic acids within the range of 4,100 to 4,388 nucleotides. ​ Intron 3 consists of any 100 or more consecutive nucleic acids within the range of nucleotides 4,100 to 4,388. ​ Intron 3 consists of any 150 or more consecutive nucleic acids within the range of nucleotides 4,100 to 4,388. ​ Intron 3 consists of any 200 or more consecutive nucleic acids within nucleotides 4,100 to 4,388. ​ (Any 250 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of intron 3).

[0271] In some cases, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0272] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 158-174 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0273] In some cases, the binding domain comprises a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 164-174 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0274] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 170-174 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0275] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 171-172 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0276] The binding domain can be operatively connected to the splice domain 3' (e.g., directly connected to the splice domain or an intervention sequence having a 3' end connecting the splice domain and a 5' end connecting the binding domain).

[0277] In some cases, the target ​ Introns are ​ Intron 1. In one embodiment, the binding domain is associated with the target. ​ Intron precursor mRNA (e.g., target mRNA) ​ A nucleic acid sequence that is at least 80% complementary to the intron sequence (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), which can inhibit endogenous target cis-splicing while enhancing trans-splicing molecules and targets. ​ Trans-splicing between precursor mRNAs (e.g., by creating endogenous) ​ A part of mRNA and having one or more functions ​ Chimeric molecules of exon-coding domains, wherein the exons encode wild-type​ (Amino acid sequence). In one embodiment involving the coding sequence of a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain coding sequence encodes the target. ​ The intron precursor mRNA sequence is a nucleic acid sequence that is at least 80% complementary (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary).

[0278] In some cases, the present invention provides a combination ​ Trans-splicing molecules (or vectors thereof) of intron 1, for example, wherein the nucleic acid trans-splicing molecule is configured to trans-splice the coding domain to an endogenous protein. ​ Exon 2. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to any one or more (e.g., six or more, eight or more, ten or more, or twelve or more) binding sites of nucleotides 1 to 1000 or 11,500 to 11,850 of SEQ ID NO: 1.

[0279] In some cases, the binding site includes ​ Intron 1 consists of any six or more consecutive nucleotides within the range of 1 to 1000 or 11,500 to 11,850 (e.g., ​ Intron 1 consists of any eight or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any ten or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 12 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 20 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 30 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 40 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 50 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​Intron 1 consists of any 100 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 150 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850. ​ Intron 1 consists of any 200 or more consecutive nucleic acids within the range of 1 to 1000 or 11,500 to 11,850. ​ Intron 1 consists of any 250 or more consecutive nucleic acids ranging from 1 to 1000 or from 11,500 to 11,850.

[0280] In some cases, the binding site includes ​ Intron 1 consists of any six or more consecutive nucleotides within the range of 11,500 to 11,850 (e.g., ​ Intron 1 consists of any eight or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any ten or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 12 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 20 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 30 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 40 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 50 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 100 or more consecutive nucleic acids within the range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 150 or more consecutive nucleic acids within the range of 11,500 to 11,850 nucleotides. ​ Intron 1 consists of any 200 or more consecutive nucleic acids within a range of 11,500 to 11,850 nucleotides or ​ Intron 1 consists of any 250 or more consecutive nucleic acids within the range of 11,500 to 11,850 nucleotides.

[0281] In some cases, the binding site includes ​ Any six or more consecutive nucleotides within nucleotides 11,650 to 11,850 of intron 1 (e.g., ​Intron 1 consists of any eight or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any ten or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 12 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 20 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 30 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 40 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 50 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 100 or more consecutive nucleic acids within the range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 150 or more consecutive nucleic acids within the range of 11,650 to 11,850 nucleotides. ​ Intron 1 consists of any 200 or more consecutive nucleic acids within a range of 11,650 to 11,850 nucleotides or ​ (Any 250 or more consecutive nucleic acids within 11,650 to 11,850 nucleotides of intron 1).

[0282] In some cases, the binding domain has at least two non-overlapping sequences that are at least 80% complementary to the binding site.

[0283] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 8 or 14-21 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0284] In some cases, the binding domain comprises a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 16-21 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0285] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 17, 18, 20 or 21 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity).

[0286] In some embodiments, the binding domain is a DNA sequence having at least 80% identity with any of SEQ ID NO: 17 or 18 (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity).

[0287] The binding domain can be operatively connected to the splice domain 3' (e.g., directly connected to the splice domain or an intervention sequence having a 3' end connecting the splice domain and a 5' end connecting the binding domain).

[0288] As detailed in this article, ​ The first stage of exon editor design begins with screening and selecting highly efficient BD sequences that are complementary to the target precursor mRNA introns. As shown in this paper, the target... ​ Intron 2 and ​Intron 3 BD exhibited the highest level of trans-splicing and was therefore identified as an exemplary treatment for the HD patient population. ​ Effective components of a targeted exon editor. See also ​ , ​ Figure 12 ​ and ​ .

[0289] ​ For the 5' exon editor, the splicing domain may include a splice donor site (5' splice site) to mediate trans-splicing.

[0290] For 3' exon editors, splice domains may include splice sites, branch points, and / or polypyrimidine segments (PPTs) to mediate trans-splicing. In some embodiments, the splice domain has a single splice site, indicating that, due to the lack of a corresponding splice site, the splice site is designed to preferentially perform trans-splicing rather than cis-splicing.

[0291] Those skilled in the art can select alternative splice domains based on known methods and principles. In one embodiment, the 5' splice site concordant sequence is the nucleic acid sequence AG / GURAGU (where / denotes the splice site). In another embodiment, endogenous splice sites corresponding to exons and introns proximal to the splice site can be used to maintain any splice regulatory signals.

[0292] In one implementation, a suitable 5' splice site includes GTAAGT or GUAAGT.

[0293] The splice structure domain can be operatively connected to the terminating substructure domain 5' (e.g., directly connected to the terminating substructure domain or an intervention sequence having a 3' end connecting the splice structure domain and the 5' end connecting the terminating substructure domain, such as a joint structure domain and / or a binding structure domain and / or a 3' downstream sequence).

[0294] ​ In some cases, the nucleic acid trans-splicing molecule includes a 5' untranslated region. In some embodiments, the 5' untranslated region comprises, is substantially composed of, or consists of: native 5' ​Untranslated region. In some embodiments, the 5' untranslated region contains a sequence having at least 80% sequence identity with SEQ ID NO: 136 (e.g., at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity).

[0295] In some implementations, the 5' untranslated region can be operatively connected to the coding structure domain 5' (e.g., directly connected to the coding structure domain or having an intervention sequence that connects the 3' end of the 5' untranslated region to the 5' end of the coding structure domain).

[0296] In some implementations, the nucleic acid trans-splicing molecule does not include a 5' untranslated region or does not include a native 5' region. ​ Non-translated area.

[0297] ​ In some cases, the nucleic acid trans-splicing molecule is operatively linked to a 5' regulatory domain operatively linked to the coding domain (e.g., directly to the coding domain or via an intermediate domain (e.g., the untranslated region)). The 5' regulatory domain may include a promoter (e.g., a constitutive promoter, such as the CMV promoter or the EF1-α promoter). In some cases, the 5' regulatory domain includes components operatively linked to a native 5'... ​ Untranslated region promoters (e.g., constitutive promoters, such as CMV / CMV promoters). In some implementations, they are operatively linked to the native 5'. ​ The 5' regulatory domain of the untranslated region contains a sequence having at least 80% sequence identity with SEQ ID NO: 137 (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity).

[0298] In some implementations, it is operatively connected to the native 5' ​The 5' regulatory domain of the untranslated region contains a sequence having at least 80% sequence identity with SEQ ID NO: 196 (e.g., at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity).

[0299] In some implementations, the CMV promoter is replaced by the CAGGS promoter, which drives the expression of the RNA exon editor described herein. See, for example... ​ As shown therein, when comparing CAGGS 5' UTRs with and without HTT5' UTR, protein translation appears to be regulated via HTT5' UTR when CAGGS 5' UTR + HTT5' UTR are operatively linked. When HTT5 UTR is removed, leaving only CAGGS 5' UTR, the inventors observed stronger protein expression, which is likely due to the presence of some translation-upregulating element in CAGGS 5' UTR. Transduction experiments presented herein (where the RNA exon editor is introduced into cells via AAV) also demonstrate that the CAGGs promoter drives significant expression of the RNA exon editor, MSH3 splicing regulator, and miRNA-encoding construct. See, for example... ​ , ​ , ​ and ​ .

[0300] In some implementations, the 5' modulatory domain can be operatively connected to the 5' coded domain (e.g., directly connected to the coded domain or an intervention sequence having a connection between the 3' end of the 5' modulatory domain and the 5' end of the coded domain).

[0301] In some implementations, other types of promoters may be used. In some implementations, the nucleic acid trans-splicing molecule is not operatively linked to the CMV promoter or the CAGGS promoter.

[0302] ​ As discussed herein, increasing trans-splicing efficiency remains an important goal for using nucleic acid trans-splicing molecules as therapeutic agents. To engineer nucleic acid trans-splicing molecules with trans-splicing efficiency suitable for use in therapeutic interventions, the inventors tested multiple linker sequences between the splice domain (SD) and binding domain (BD) and identified a series of exemplary linkers that statistically significantly improved performance compared to the 40-mer linker. See, for example... ​ .

[0303] In addition to the above, nucleic acid trans-splicing molecules may include a linker domain at one or more locations within the molecule. In some embodiments, the linker domain is operatively connected to a splice domain or splice site 3' (e.g., directly connected to a splice domain or splice site). The linker domain can be of any suitable size. In some embodiments, the linker domain is longer than 20 nucleotides (e.g., between 20 and 100 nucleotides or between 20 and 85 nucleotides). In some cases, the adapter domain comprises, is substantially composed of, or consists of a nucleic acid sequence having at least 80% identity with any of SEQ ID NO: 37-46 or 106-112 (e.g., at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity).

[0304] Linker sequences are frequently included in trans-splicing molecules, where they are positioned between the splice donor and the binding domain to provide flexibility and accessibility to each element. As described herein, the functional contributions of different elements are evaluated in different combinations in the context of trans-splicing molecules, targeting the activity conferred therefrom.

[0305] In some implementations, the nucleic acid trans-splicing molecule does not include any of SEQ ID NO: 37-46 or 106-112, or the adapter sequence may not be present.

[0306] The connector domains described in this article can also be included in non-targeted applications. ​ or ​The embodiments disclosed herein include an RNA exon editor comprising a binding domain targeting endogenous precursor mRNA, a coding domain sequence encoding a functional amino acid sequence, and a linker domain between the coding domain sequence and the binding domain, wherein the linker domain comprises one or more of SEQ ID NO: 37-46 or 106-112.

[0307] ​ In some cases, the trans-splicing molecule includes a 3' transcription terminator domain. In some embodiments, such a 3' transcription terminator domain forms a triple helix structure that effectively caps the 3' end of the trans-splicing molecule. In some cases, the 3' transcription terminator domain is derived from the human long non-coding RNA MALAT1 (e.g., wild-type MALAT1). In some embodiments, the 3' transcription terminator domain includes a tRNA-like domain. This can be used as a basis for the present invention. ​ A 3' transcription terminator domain, a portion of a trans-splicing molecule, is described in International Patent Publication No. WO 2020 / 214973, which is incorporated herein by reference in its entirety. For example, in some embodiments, the region operatively linked to the 3' end of the binding domain in the RTM includes a terminator domain comprising, substantially comprising, or comprising: a wild-type MALAT1+mascRNA domain, such as SEQ ID NO: 5. In some embodiments, the region operatively linked to the 3' end of the binding domain in the RNA exon editor includes a terminator domain comprising, substantially comprising, or comprising: a mutant MALAT1+mascRNA (anti-Mut1 masc RNA) domain, such as SEQ ID NO: 6.

[0308] In some embodiments, the nucleic acid trans-splicing molecule does not include a 3' transcription terminator domain or does not include a MALAT1-derived transcription terminator.

[0309] ​ In some implementations, the exemplary RNA exon editor described herein includes components that... ​Those intron 1 binding domains, wherein such exemplary intron 1 binding RNA exon editors may comprise any of SEQ ID NO: 23-36 and 47-56, said sequences comprising a coding sequence (e.g., SEQ ID NO: 3), a splicing domain, a adapter domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence also includes an ATG start codon at the 5' end.

[0310] In some implementations, the exemplary RNA exon editor described herein includes components that... ​ Those intron 2 binding binding domains, wherein such exemplary intron 2 binding RNA exon editors may comprise either SEQ ID NO: 83-105 and 113-125, the sequences comprising a coding sequence (e.g., SEQ ID NO: 59, 349, 350, or 351), a splicing domain, a adapter domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence also includes an ATG start codon at the 5' end.

[0311] In some implementations, the exemplary RNA exon editor described herein includes components that... ​ Those intron 3-binding binding domains, wherein such exemplary intron 3-binding RNA exon editors may comprise any of SEQ ID NO: 175-191, the sequences comprising a coding sequence (e.g., any of SEQ ID NO: 157, 352, or 353), a splice domain, a adapter domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence also includes an ATG start codon at the 5' end.

[0312] In some implementations, the exemplary RNA exon editor described herein includes components that... ​ Intron 1 binding binding domain and binding ​ Those intron binding domains, among which such exemplary hybrids / duplexes ​ / ​ An RNA exon editor may comprise any of SEQ ID NO: 149-154, the sequence comprising a coding sequence (e.g., SEQ ID NO: 3), a splice domain, an adapter domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence further comprises an ATG start codon at the 5' end. In some embodiments, exemplary heterozygous / double... ​ / ​ RNA exon editors include the ability to... ​ Introns and ​The binding structural domain of the two introns.

[0313] In some implementations, the exemplary RNA exon editor described herein includes components that... ​ Intron 2 binding binding domain and binding ​ Those intron binding domains, among which such exemplary hybrids / duplexes ​ / ​ An RNA exon editor may comprise any of SEQ ID NO: 212-223, the sequence comprising a coding sequence (e.g., SEQ ID NO: 59), a splice domain, an adapter domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence further comprises an ATG start codon at the 5' end. In some embodiments, exemplary heterozygous / double... ​ / ​ RNA exon editors include the ability to... ​ Introns and ​ The binding structural domain of the two introns.

[0314] In some implementations, an exemplary RNA exon editor includes... ​ The binding domains of introns (e.g., intron 2) and their interactions with... ​ A binding domain for intron (e.g., intron 5 or intron 15). In some embodiments, ​ The combined structural domain includes intron5_213_100 (SEQ ID NO: 140), intron5_188_150 (SEQ ID NO: 209), intron15_6523_120 (SEQ ID NO: 144), or intron15_6498_150 (SEQ ID NO: 210). In some embodiments, ​ The combined structure field contains HTT_intron2_12061_150 (SEQ ID NO: 95). In some implementations, ​ Associative domains are ​ Combined with domain 5'. In some implementations, ​ Associative domains are ​ Combined with structural domain 3'. In some implementations, the MALAT1 terminator is... ​ Combining structural domains and ​ The domains are combined. In some implementations, the MALAT1 terminator is not present. ​ Combining structural domains and ​ The domains are combined. In some implementations, the MALAT1 terminator is... ​Combining structural domains and ​ Combine the two structural domains 3'.

[0315] In some implementations, an exemplary RNA exon editor includes... ​ A binding domain for intron (e.g., intron 5 or intron 15). In some embodiments, ​ The combined structural domain includes intron5_213_100 (SEQ ID NO: 140), intron5_188_150 (SEQ ID NO: 209), intron15_6523_120 (SEQ ID NO: 144), or intron15_6498_150 (SEQ ID NO: 210). In some embodiments, ​ The targeted RNA exon editor also includes any splicing domains disclosed herein, any 3X UBS sequences described herein, any AU-rich elements described herein, any adapter domains described herein, and / or any terminator sequences disclosed herein. In some embodiments, ​ Targeted RNA exon editor and ​ Targeted exon editors are administered in combination. In some implementations, ​ Targeted exon editor does not ​ Targeted exon editors are used in conjunction.

[0316] In some implementation schemes, by targeting ​ miRNA reduction in mRNA ​Expression. In some embodiments, the primary miRNA comprises one or more of the following: mir-30a [scaffold 5' (SEQ ID NO: 227); scaffold 3' (SEQ ID NO: 228); loop (SEQ ID NO: 229)], mir155 [5' scaffold (SEQ ID NO: 230); 3' scaffold (SEQ ID NO: 231); loop (SEQ ID NO: 232)] or mir-33 [scaffold 5' (SEQ ID NO: 259), scaffold 3' (SEQ ID NO: 260); loop (SEQ ID NO: 261). In some embodiments, the primary miRNA comprises one or more of the following: SEQ ID NO: 234, 235, 238-241 or 262-269. In some embodiments, the miRNA active sequence comprises, is substantially composed of, or consists of any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256, or 257, or a sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256, or 257. Targeting ​ miRNAs of mRNA can be used in combination with any RNA exon editor disclosed herein, including, for example, targeting... ​ , ​ Or both of their RNA exon editors. Targeting ​ miRNAs of mRNA can be used independently to reduce MSH3 expression without the use of any RNA exon editors disclosed herein. Targeting ​ miRNAs of mRNA can be used in methods to treat or prevent trinucleotide duplication syndromes.

[0317] In some implementations, snRNA-based antisense RNA reduces ​ Expression, which can induce exon skipping during precursor mRNA processing. In some embodiments, the snRNA construct comprises one or more of the following: SEQ ID NO: 274, which targets ​ The junction between intron 1 and exon 2; SEQ ID NO: 275, its target ​ The junction between exon 2 and intron 2; SEQ ID NO: 278, 301 or 303, targeting ​The junction between intron 2 and exon 3; SEQ ID NO: 279, 300 or 302, its target ​ The junction between exon 3 and intron 3; SEQ ID NO: 281, its target ​ The junction between intron 3 and exon 4; SEQ ID NO: 282, its target ​ The junction between exon 4 and intron 4; SEQ ID NO: 306 or 308, which targets ​ The junction between intron 5 and exon 6; SEQ ID NO: 305 or 307, its target ​ The junction between exon 6 and intron 6; SEQ ID NO: 311 or 313, its target ​ The junction between intron 6 and exon 7; SEQ ID NO: 310 or 312, its target ​ The junction between exon 7 and intron 7; SEQ ID NO: 316 or 318, its target ​ The junction between intron 7 and exon 8; SEQ ID NO: 315 or 317, its target ​ The junction between exon 8 and intron 8; SEQ ID NO: 321 or 323, its target ​ The junction between intron 14 and exon 15; or SEQ ID NO: 320 or 322, which targets ​ The junction between exon 15 and intron 15.

[0318] In some implementations, one, two, three, four, five or more of the above asRNA constructs are used in combination (e.g., administered to a patient). For example, in some implementations, constructs targeting the intron 1-exon 2 junction and constructs targeting the exon 2-intron 2 junction are used in combination; constructs targeting the intron 2-exon 3 junction and constructs targeting the exon 3-intron 3 junction are used in combination; constructs targeting the intron 3-exon 4 junction and constructs targeting the exon 4-intron 4 junction are used in combination; constructs targeting the intron 5-exon 6 junction and constructs targeting the exon 6-intron 6 junction are used in combination; constructs targeting the intron 6-exon 7 junction and constructs targeting the exon 7-intron 7 junction are used in combination; constructs targeting the intron 7-exon 8 junction and constructs targeting the exon 8-intron 8 junction are used in combination; or constructs targeting the intron 14-exon 15 junction and / or constructs targeting the exon 15-intron 15 junction are used in combination.

[0319] In some embodiments, a single asRNA construct targeting two intron-exon junctions is used. In some embodiments, the asRNA construct comprises a sequence at least partially complementary to the entire exon sequence plus a portion of the intron sequence on either side of the exon (e.g., nucleotides 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of the intron sequence on either side of the exon sequence). Such constructs can target, for example, in… ​ The intron-exon junction on either side of any one of exons 2, 3, 4, 5, 6, 7, 8, or 15. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 324 (In3 / Ex3 / In2 sequence; comprising SEQ ID NO: 299) that targets the entire length of exon 3 and the flanking junction on either side. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 327 (In6 / Ex6 / In5 sequence; comprising SEQ ID NO: 304) that targets the entire length of exon 6 and the flanking junction on either side. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 330 (In7 / Ex7 / In6 sequence; comprising SEQ ID NO: 309) that targets the entire length of exon 7 and the flanking junction on either side. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 333 (In8 / Ex8 / In7 sequence; comprising SEQ ID NO: 314) that targets the entire length of exon 8 and flanking junctions on either side. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 336 (In15 / Ex15 / In14 sequence; comprising SEQ ID NO: 319) that targets the entire length of exon 15 and flanking junctions on either side.

[0320] In some implementations, a single asRNA construct targeting two intron-exon junctions is used. In some implementations, the asRNA construct includes a sequence at least partially complementary to the 5' intron-exon junction sequence, a sequence at least partially complementary to the 3' intron-exon junction sequence, and an unstructured adapter connecting the two sequences. Such constructs can target, for example, in… ​The intron-exon junction on either side of exon 2, 3, 4, 5, 6, 7, 8, or 15. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 325 [U7SmOPT In3 / Ex3 (SEQ ID NO: 300) + adapter + Ex3 / In2 (SEQ ID NO: 301)], targeting the junction on either side of exon 3. In some embodiments, the asRNA construct is a U2 snRNA construct comprising SEQ ID NO: 326 [U2 In3 / Ex3 (SEQ ID NO: 302) + adapter + Ex3 / In2 (SEQ ID NO: 303)], targeting the junction on either side of exon 3. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 328 [U7SmOPT In6 / Ex6 (SEQ ID NO: 305) + adapter + Ex6 / In5 (SEQ ID NO: 306)], which targets the junction site on either side of exon 6. In some embodiments, the asRNA construct is a U2 snRNA construct comprising SEQ ID NO: 329 [U2 In6 / Ex6 (SEQ ID NO: 307) + adapter + Ex6 / In5 (SEQ ID NO: 308)], which targets the junction site on either side of exon 6. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 331 [U7SmOPT In7 / Ex7 (SEQ ID NO: 310) + adapter + Ex7 / In6 (SEQ ID NO: 311)], which targets the junction site on either side of exon 7. In some embodiments, the asRNA construct is a U2snRNA construct comprising SEQ ID NO: 332 [U2 In7 / Ex7 (SEQ ID NO: 312) + adapter + Ex7 / In6 (SEQ ID NO: 313)], which targets the linker site on either side of exon 7. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 334 [U7SmOPT In8 / Ex8 (SEQ ID NO: 315) + adapter + Ex8 / In7 (SEQ ID NO: 316)], which targets the linker site on either side of exon 8.In some embodiments, the asRNA construct is a U2 snRNA construct comprising SEQ ID NO: 335 [U2 In8 / Ex8 (SEQ ID NO: 317) + adapter + Ex8 / In7 (SEQ ID NO: 318)], targeting the linker site on either side of exon 8. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 337 [U7SmOPT In15 / Ex15 (SEQ ID NO: 320) + adapter + Ex15 / In14 (SEQ ID NO: 321)], targeting the linker site on either side of exon 15. In some embodiments, the asRNA construct is a U2 snRNA construct comprising SEQ ID NO: 338 [U2 In15 / Ex15 (SEQ ID NO: 322) + adapter + Ex15 / In14 (SEQ ID NO: 323)], targeting the linker site on either side of exon 15.

[0321] ​ Some embodiments of the splicing regulator construct include an operablely linked sequence of: a sequence encoding a small nuclear RNA (snRNA) sequence (e.g., a U7 Sm OPT sequence or a U2 snRNA sequence), and a sequence encoding an antisense RNA, which promotes ​ Exon skipping of target exons in precursor mRNA. Exon skipping can introduce frameshifts and / or premature stop codons, which may induce nonsense-mediated degradation or otherwise impair the production of functional MSH3. Antisense RNAs that promote exon skipping of target exons may target one or both of the following: ​The 5' exon-intron junction and 3' exon-intron junction of the target exon of the precursor mRNA. As used herein, the antisense RNA is said to "target" the exon-intron junction if the sequence surrounding a particular exon-intron junction is sufficiently complementary to it to facilitate hopping of the target exon during precursor mRNA processing. In some embodiments, the antisense RNA sequence comprises a continuous fragment of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides that is 100% complementary to a continuous nucleotide fragment of the same length on the precursor mRNA including the exon-intron junction. In some implementations, the antisense RNA sequence comprises a continuous fragment of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides, which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a continuous nucleotide fragment of the same length on the precursor mRNA, including the exon-intron junction. In some implementations, the antisense RNA sequence comprises a continuous fragment of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides, which is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a continuous nucleotide fragment of the same length on the precursor mRNA including the exon-intron junction, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the exon-intron junction. Skilled personnel should understand that, in the presence of incomplete complementarity, exon jumping function can be enhanced by increasing the length of the continuous fragment of partially complementary nucleotides. Skilled personnel should also understand that the GC content of the sequence surrounding the exon-intron junction can affect the ability of antisense RNA sequences to effectively target the exon-intron junction and induce jumping. Higher GC content increases annealing intensity, which may result in a smaller required complementary fragment.

[0322] In some embodiments, the MSH3 splicing regulator comprises an antisense RNA sequence targeting two exon-intron junctions. In some embodiments, the antisense RNA sequence targeting two exon-intron junctions comprises a majority or all of the entire exon sequence annealed to the precursor mRNA. In some embodiments, the antisense RNA is 100% complementary to the entire precursor mRNA exon sequence. In some embodiments, the antisense RNA is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the precursor mRNA exon sequence. In some embodiments, the antisense RNA further comprises a nucleotide sequence at least partially complementary to the intron sequences upstream and / or downstream of the targeted precursor mRNA exon sequence. In some embodiments, such intron sequences are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long or longer. In some embodiments, the antisense RNA sequence contains a sequence complementary to such intron sequences by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0323] In some embodiments, the antisense RNA sequence targeting the exon-intron junction of the target exon comprises (a) two non-adjacent sequences, each annealed to the sequence surrounding the exon-intron junction of the target exon, and (b) a linker sequence between the two non-adjacent sequences, the linker sequence having low complementarity (e.g., less than 50%) with the target exon. In some embodiments, the linker sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides, or 10–30, 10–50, 15–25, or 18–22 nucleotides. In some embodiments, the linker has less than 70, 60, 50, 40, or 30% complementarity with all consecutive segments of the same length as the linker in the target exon sequence.

[0324] In some embodiments, any MSH3 splicing regulator disclosed herein may be used in combination with any HTT RNA exon editor disclosed herein. In some embodiments, the MSH3 splicing regulator disclosed herein may not be used in combination with any RNA exon editor disclosed herein. Any MSH3 splicing regulator described herein may be used independently in methods for treating trinucleotide repeat amplification syndromes.

[0325] In some embodiments, multiple constructs are encoded on a single vector (e.g., an AAV vector). Any exon editor, miRNA, and asRNA sequences described herein can be combined on a single vector. In some embodiments, two or more of the exon editor, miRNA, and asRNA constructs are encoded on a single vector. In some embodiments, the exon editor, miRNA, and / or asRNA (if applicable) target the same gene. For example, a single vector may encode a gene for generating correction. ​ mRNA ​ Exon editor and ​ Targeted miRNAs and / or ​ Targeted asRNA is used to reduce the amount of defective HTT. In some implementations, a single vector encodes... ​ Exon editors (e.g., exon editors containing any of SEQ ID NO:83-104, SEQ ID NO:113-125, SEQ ID NO:175-191, SEQ ID NO:199-206, or SEQ ID NO:23-36) and ​ Targeting miRNAs (e.g., SEQ ID NO: 339 and / or 342). In some embodiments, the vector comprises any of SEQ ID NO: 354 or 355, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with such sequences. In some embodiments, the exon editor, miRNA, and / or asRNA (if applicable) target different genes. In some embodiments, for example, a single vector encodes a gene for generating a correction. ​ mRNA HTT Exon editor and for knocking down MSH3 Expressed MSH3 Exon editor MSH3 miRNA and / or MSH3 asRNA (e.g., snRNA constructs). In some implementations, a single vector encodes... HTTExon editors (e.g., exon editors containing any of SEQ ID NO: 83-104, SEQ ID NO: 113-125, SEQ ID NO: 175-191, SEQ ID NO: 199-206, or SEQ ID NO: 23-36) and MSH3 Targeting asRNA (e.g., constructs comprising any one or more of SEQ ID NO: 284-293 or 324-338). In some embodiments, the vector comprises a sequence as shown in SEQ ID NO: 356 or 357 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with such sequences. In some embodiments, the vector encodes... HTT Exon editors (e.g., exon editors containing any of SEQ ID NO: 83-104, SEQ ID NO: 113-125, SEQ ID NO: 175-191, SEQ ID NO: 199-206, or SEQ ID NO: 23-36), MSH3 Targeted asRNAs (e.g., constructs containing any one or more of SEQ ID NO: 284-293 or 324-338) and HTT Targeting miRNA (e.g., SEQ ID NO: 339 and / or 342). In some embodiments, the vector comprises a sequence represented by SEQ ID NO: 358 or 359 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity with such sequences.

[0326] The different treatment modalities disclosed herein can be combined in various combinations, whether encoded on the same vector or on different vectors. For example, any HTT exon editor disclosed herein that has a binding domain targeting intron 1 can be combined with any of the methods disclosed herein. MSH3 Targeted asRNA constructs and / or any disclosed herein HTT Targeted miRNA constructs. In some implementations, miRNAs with a binding domain targeting intron 1 are used. HTT Exon editor MSH3 Targeted asRNA and HTT The targeted miRNA is included on the same vector. As another example, any HTT exon editor disclosed herein that has a binding domain targeting intron 2 can be used with any of the miRNAs disclosed herein. MSH3 Targeted asRNA constructs and / or any disclosed herein HTTTargeted miRNA constructs. In some implementations, a binding domain targeting intron 2 is included. HTT Exon editor MSH3 Targeted asRNA and HTT The targeted miRNA is included on the same vector. As another example, any HTT exon editor disclosed herein that has a binding domain targeting intron 3 can be used with any of the miRNAs disclosed herein. MSH3 Targeted asRNA constructs and / or any disclosed herein HTT Targeted miRNA constructs. In some implementations, miRNAs with a binding domain targeting intron 3 are used. HTT Exon editor MSH3 Targeted asRNA and HTT The targeted miRNAs are contained within the same vector.

[0327] In some embodiments, the binding of trans-splicing molecules to target precursor mRNA is mediated by a complementarity percentage (i.e., based on the base-pairing characteristics of nucleic acids), triple helix formation, or protein-nucleic acid interactions (as described in the literature cited herein), or any combination thereof. In one embodiment, the nucleic acid trans-splicing molecule comprises DNA, RNA, or a DNA / RNA hybrid molecule, wherein the DNA or RNA is single-stranded or double-stranded. This document also includes RNA or DNA that can hybridize with one of the aforementioned RNAs or DNAs, preferably under stringent conditions, such as at 60°C in 2.5x SSC buffer, and several washes at 37°C in a lower buffer concentration (e.g., 0.5x SSC buffer). These nucleic acids may encode proteins exhibiting lipid phosphatase activity and / or associating with the plasma membrane. When trans-splicing molecules are synthesized in vitro, such trans-splicing molecules can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization with target mRNA, transport to cells, stability against enzymatic cleavage in cells, etc. For example, modifying trans-splicing molecules to reduce total charge can enhance cellular uptake of the molecules. Furthermore, modifications can be made to reduce susceptibility to nucleases or chemical degradation. The nucleic acid molecule can be synthesized by conjugation to another molecule (e.g., a peptide, a hybridization-triggered cross-linking agent, a transporter, a hybridization-triggered cleavage agent, etc.).

[0328] Various other well-known modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see above for oligonucleotides). Possible modifications are known in the art. Modifications that can be made to the structure of synthetic trans-splicing molecules include backbone modifications.

[0329] Cell line assay In some cases, the trans-splicing molecules described herein have been tested in cultured cell lines. To screen, select, and improve the functionality of RNA exon editors, cultured cell lines can be obtained or engineered to target specific RNA molecules. HTT The precursor mRNA is expressed at a sufficient level.

[0330] RNA exon editor screening platform As described herein, 5' RNA exon editors contain several functional sequence elements, such as binding domains (BD) for precursor mRNA targeting and adapters that allow access to splice donor (SD) sites. When RNA exon editors are engineered for a given gene target, the ability of multiple sequence options for each of these elements to promote high trans-splicing (TS) efficiency is tested. Such tests can be performed by: (A) cloning and transfecting individual RNA exon editor variants and analyzing efficiency via RT-qPCR / ddPCR and Western blotting; and / or (B) cloning and assembling RNA exon editors in a high-throughput (HT) library-based approach that relies on next-generation sequencing (NGS) and computational analysis to assess efficiency. Both approaches are described below.

[0331] RNA exon editor screening in individual form: This approach can be applied to test small-scale variable elements within RNA exon editor sequences prior to initiating library-based multiplex screening, to validate the performance of RNA exon editors identified in multiplex screening, and / or to improve the performance of lead candidates. TS efficiency is assessed at both the RNA and protein levels.

[0332] At the RNA level, TS activity was assessed by isolating total RNA from cells, followed by reverse transcription and real-time quantitative PCR (RT-qPCR), whereby the RT-qPCR measured the copy number of RNA targeting, for example, the following: RNF20 (used for normalized housekeeping genes); native ( HTT mRNA; exon editor RNA; target-edited exon RNA (ONT), which is the product of positive TS; ONT + exon editor + OFT (off-target), a single assay that captures all three targets. OFT indicates that the RNA exon editor may trans-splice incorrect RNA molecules.

[0333] ONT TS efficiency, also known as substitution percentage or benzyl percentage, represents the total HTT The portion of the mRNA population that has successfully undergone TS is calculated using the following equation: ONT TS% = 100*(ONT copy number / (ONT copy number + native copy number)).

[0334] RNA exon editor TS efficiency is the percentage of RNA exon editor transcripts that have been correctly trans-spliced ​​to... HTT The portion of RNA is calculated using the following equation: Exon editor TS% = 100 * (ONT copy number / (ONT copy number + exon editor copy number + OFT copy number)).

[0335] At the protein level, TS activity is measured by applying Western blot analysis to proteins extracted from cell or tissue samples. β-actin (a cytoskeletal protein) or tubulin can be used as loading controls. For constructs that include tags (e.g., N-terminal FLAG tags), Western blots can be probed using antibodies (Abs) specific to said tags (e.g., FLAG-specific Abs) to assess ONT protein levels.

[0336] III. Carrier Various techniques can be used, such as recombinant adeno-associated virus (AAV) vectors or other vector modalities (e.g., non-viral vectors), to deliver trans-splicing molecules to target cells of an individual. Therefore, this document provides vectors containing / encoding trans-splicing molecules (e.g., viral or non-viral vectors containing / encoding trans-splicing molecules, such as DNA vectors containing / encoding trans-splicing molecules). Any suitable nucleic acid vector can be used in conjunction with the compositions and methods of this invention to design and assemble components of the trans-splicing molecules and recombinant AAV. In one embodiment, the vector is a promoter-driven recombinant AAV carrying a trans-splicing molecule, the promoter expressing the trans-splicing molecule in selected cells of an individual. Methods for assembling recombinant vectors are known in the art. See, for example, Ausubel et al. Current Protocols in Molecular Biology , John Wiley&Sons, New York, 1989; Kay, MA et al., Nat. Medic ,2001, 7(l):33-40; and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0337] In some embodiments described herein, the trans-splicing molecule is delivered to selected cells requiring treatment, such as neurons, using an AAV vector. A variety of naturally occurring AAV serotypes are available. AAV capsids exist in a variety of natural variants, allowing for the identification and use of AAVs with properties particularly suitable for neurons. Artificial AAV vectors can be engineered using conventional molecular biology techniques, making it possible to optimize these particles to specifically deliver the trans-splicing molecule nucleic acid sequence to cells, minimize immunogenicity, adjust stability and particle lifetime, achieve efficient degradation, and accurately deliver to the cell nucleus, etc. For example, such artificial capsids can be generated using any suitable technique, using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and a heterologous sequence that can be obtained from different selected AAVs, discontinuous portions of the same AAV, or non-AAV viral or non-viral sources. Artificial AAVs can be, but are not limited to, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids. A pseudotyped vector in which the capsid of one AAV is replaced by a heterologous capsid protein can be used to deliver the trans-splicing molecule described herein.

[0338] The expression of the trans-splicing molecules described herein can be achieved in selected cells by delivery using recombinantly engineered AAVs or artificial AAVs containing sequences that encode the desired trans-splicing molecules. The use of AAVs is a common method for exogenous DNA delivery because it is relatively non-toxic, provides efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized AAV serotypes isolated from humans or non-human primates, human serotype 2 has been widely used for efficient gene transfer experiments in various target tissues and animal models.

[0339] In some implementations, the AAV is AAV1 or a variant thereof (e.g., SEQ ID NO: 6 or 64 of US20030138772 or SEQ ID NO: 11 or 27 of US20150159173), AAV2 or a variant thereof (e.g., SEQ ID NO: 7 or 70 of US20030138772, SEQ ID NO: 7 or 23 of US20150159173 or SEQ ID NO: 7 of US20150159173), AAV2G9 or a variant thereof, AAV3 or a variant thereof (e.g., SEQ ID NO: 8 or 71 of US20030138772), AAV3a or a variant thereof, AAV3b or a variant thereof (e.g., SEQ ID NO: 1 and 10 of US Patent No. 6,156,303), AAV3-3 or a variant thereof (e.g., SEQ ID NO: WO2005033321). SEQ ID NO: 200 and 217), AAV4 or variants thereof (e.g., SEQ ID NO: 63 of US20030138772), AAV4-4 or variants thereof (e.g., SEQ ID NO: 201 or 218 of WO2005033321), AAV5 or variants thereof (e.g., SEQ ID NO: 114 of US20030138772), AAV6 or variants thereof (e.g., SEQ ID NO: 65 of US20030138772), AAV6.1 or variants thereof (e.g., SEQ ID NO: 29 of US20150159173), AAV6.2 or variants thereof, AAV6.1.2 or variants thereof, AAV7 or variants thereof (e.g., SEQ ID NO: 1-3 of US20030138772), AAV7.2 or variants thereof, AAV8 or variants thereof (e.g., SEQ ID NO: 200 and 217 of US20030138772). 4 and 95 or AAV8(b) (as described in U.S. Patent No. 9,567,376, which is incorporated herein by reference in its entirety)), AAV9 or variants thereof (e.g., SEQ ID NO: 5 and 100 of US20030138772), AAV9.9 or variants thereof, AAV9.11 or variants thereof, AAV9.13 or variants thereof, AAV9.16 or variants thereof, AAV9.24 or variants thereof, AAV9.45 or variants thereof, AAV9.47 or variants thereof, AAV9.61 or variants thereof, AAV9.68 or variants thereof, AAV9.84 or variants thereof (see, for example, N. Pulicherla et al.) Molecular Therapy19(6):1070-1078 (2011), which is incorporated herein by reference in its entirety, AAV10 or variants thereof (e.g., SEQ ID NO: 117 of US20030138772), AAV11 or variants thereof (e.g., SEQ ID NO: 118 of US20030138772), AAV12 or variants thereof (e.g., SEQ ID NO: 118 of US20030138772). 119), AAV16.3 or a variant thereof, AAV24.1 or a variant thereof, AAV27.3 or a variant thereof, AAV42.12 or a variant thereof, AAV42-1b or a variant thereof, AAV42-2 or a variant thereof, AAV42-3a or a variant thereof, AAV42-3b or a variant thereof, AAV42-4 or a variant thereof, AAV42-5a or a variant thereof, AAV42-5b or a variant thereof, AAV42-6b or a variant thereof, AAV42-8 or a variant thereof, AAV42-10 or a variant thereof, AAV42-11 or a variant thereof, AAV42-12 or a variant thereof, AAV42-13 or a variant thereof, AAV42-15 or a variant thereof, AAV42-aa or a variant thereof, AAV43-1 or a variant thereof, AAV43-12 or a variant thereof, AAV43-20 or a variant thereof, AAV43-21 or a variant thereof, AAV43-23 or a variant thereof, AAV43-25 or a variant thereof, AAV43-5 or a variant thereof, A AV44.1 or its variants, AAV44.2 or its variants, AAV44.5 or its variants, AAV223.1 or its variants, AAV223.2 or its variants, AAV223.4 or its variants, AAV223.5 or its variants, AAV223.6 or its variants, AAV223.7 or its variants, AAV1-7 / rh.48 or its variants, AAV1-8 / rh.49 or its variants, AAV2-15 / rh.62 or its variants, AAV AAV2-3 / rh.61 or a variant thereof, AAV2-4 / rh.50 or a variant thereof, AAV2-5 / rh.51 or a variant thereof, AAV3.1 / hu.6 or a variant thereof, AAV3.1 / hu.9 or a variant thereof, AAV3-9 / rh.52 or a variant thereof, AAV3-11 / rh.53 or a variant thereof, AAV4-8 / rh.64 or a variant thereof, AAV4-9 / rh.54 or a variant thereof (e.g., SEQ of WO2005033321) SEQ ID NO: 116), AAV4-19 / rh.55 or its variants (e.g., SEQ ID NO: 117 of WO2005033321), AAV5-3 / rh.57 or its variants, AAV5-22 / rh.58 or its variants, AAV7.3 / hu.7 or its variants, AAV16.8 / hu.10 or its variants, AAV16.12 / hu.11 or its variants, AAV29.3 / bb.1 or its variants, AAV29.5 / bb.2 or its variants, AAV106.1 / hu.37 or its variants, AAV114.3 / hu.40 or its variants, AAV127.2 / hu.41 or its variants, AAV127.5 / hu.42 or its variants, AAV128.3 / hu.44 or its variants, AAV130.4 / hu.48 or its variants, AAV145.1 / hu.53 or its variants, AAV145.5 / hu.54 or its variants, AAV145.6 / hu.55 or its variants, AAV161.10 / hu.60 or its variants, AAV161.6 / h u.61 or a variant thereof, AAV33.12 / hu.17 or a variant thereof, AAV33.4 / hu.15 or a variant thereof, AAV33.8 / hu.16 or a variant thereof, AAV52 / hu.19 or a variant thereof, AAV52.1 / hu.20 or a variant thereof, AAV58.2 / hu.25 or a variant thereof, AAVA3.3 or a variant thereof, AAVA3.4 or a variant thereof, AAVA3.5 or a variant thereof, AAVA3.7 or a variant thereof, AAVC1 or a variant thereof, AAVC2 or a variant thereof, AAVC5 or a variant thereof, AAV-DJ or a variant thereof (e.g., SEQ of US20140359799) SEQ ID NO: 2 or 3), AAV-DJ8 or a variant thereof, AAVF3 or a variant thereof, AAVF5 or a variant thereof, AAVH2 or a variant thereof, AAVH6 or a variant thereof, AAVLK03 or a variant thereof, AAVH-1 / hu.1 or a variant thereof, AAVH-5 / hu.3 or a variant thereof, AAVLG-10 / rh.40 or a variant thereof, AAVLG-4 / rh.38 or a variant thereof, AAVLG-9 / hu.39 or a variant thereof, AAVN721-8 / rh.43 or a variant thereof, AAVCh.5 or a variant thereof (e.g., SEQ ID NO 46 of US20150159173), AAVCh.5R1 or a variant thereof, AAVcy.2 or a variant thereof, AAVcy.3 or a variant thereof, AAVcy.4 or a variant thereof, AAVcy.5 or a variant thereof (e.g., SEQ ID NO: 46 of US20150159173). 8 and 24), AAVcy.5R1 or variants thereof, AAVcy.5R2 or variants thereof, AAVcy.5R3 or variants thereof, AAVcy.5R4 or variants thereof, AAVcy.6 or variants thereof, AAVhu.1 or variants thereof (e.g., SEQ ID NO: 144 of WO2005033321), AAVhu.2 or variants thereof (e.g., SEQ ID NO: 143 of WO2005033321), AAVhu.3 or variants thereof (e.g., SEQ ID NO: 145 of WO2005033321), AAVhu.4 or variants thereof (e.g., SEQ ID NO: 141 of WO2005033321), AAVhu.5 or variants thereof, AAVhu.6 or variants thereof (e.g., SEQ ID NO: 84 of WO2005033321), AAVhu.7 or variants thereof (e.g., SEQ ID NO: 150 of WO2005033321), AAVhu.9 or variants thereof (e.g., SEQ ID NO: 155 of WO2005033321), AAVhu.10 or variants thereof (e.g., SEQ ID NO: 156 of WO2005033321), AAVhu.11 or variants thereof (e.g., SEQ ID NO: 153 of WO2005033321), AAVhu.13 or variants thereof (SEQ ID NO: 153 of US20150159173). 16 and 32), AAVhu.15 or variants thereof (e.g., SEQ ID NO: 147 of WO2005033321), AAVhu.16 or variants thereof (e.g., SEQ ID NO: 148 of WO2005033321), AAVhu.17 or variants thereof (e.g., SEQ ID NO: 83 of WO2005033321), AAVhu.18 or variants thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.19 or variants thereof (e.g., SEQ ID NO: 133 of WO2005033321), AAVhu.20 or variants thereof (e.g., SEQ ID NO: 134 of WO2005033321), AAVhu.21 or variants thereof (e.g., SEQ ID NO: 147 of WO2005033321). 135), AAVhu.22 or variants thereof (e.g., SEQ ID NO: 138 of WO2005033321), AAVhu.23.2 or variants thereof (e.g., SEQ ID NO: 137 of WO2005033321), AAVhu.24 or variants thereof (e.g., SEQ ID NO: 136 of WO2005033321), AAVhu.25 or variants thereof (e.g., SEQ ID NO: 146 of WO2005033321), AAVhu.26 or variants thereof (e.g., SEQ ID NO: 17 and 33 of US20150159173), AAVhu.27 or variants thereof (e.g., SEQ ID NO: 140 of WO2005033321), AAVhu.28 or variants thereof (e.g., SEQ ID NO: 138 of US20150159173). 42) AAVhu.29 or its variants (e.g., SEQ ID NO: 132 of WO2005033321), AAVhu.29R or variants thereof, AAVhu.31 or variants thereof (e.g., SEQ ID NO: 121 of WO2005033321), AAVhu.32 or variants thereof (SEQ ID NO: 122 of WO2005033321), AAVhu.34 or variants thereof (e.g., SEQ ID NO: 125 of WO2005033321), AAVhu.35 or variants thereof (e.g., SEQ ID NO: 164 of WO2005033321), AAVhu.37 or variants thereof (e.g., SEQ ID NO: 18 and 34 of US20150159173), AAVhu.39 or variants thereof (e.g., SEQ ID NO: 102 of WO2005033321), AAVhu.40 or variants thereof (e.g., SEQ ID NO: 102 of WO2005033321). 87), AAVhu.41 or variants thereof (e.g., SEQ ID NO: 91 of WO2005033321), AAVhu.42 or variants thereof (e.g., SEQ ID NO: 85 of WO2005033321), AAVhu.43 or variants thereof (e.g., SEQ ID NO: 160 of WO2005033321), AAVhu.44 or variants thereof (e.g., SEQ ID NO: 45 of US20150159173), AAVhu.44R1 or variants thereof, AAVhu.44R2 or variants thereof, AAVhu.44R3 or variants thereof, AAVhu.45 or variants thereof (e.g., SEQ ID NO: 127 of WO2005033321), AAVhu.46 or variants thereof (e.g., SEQ ID NO: 91 of WO2005033321). AAVhu.47 or variants thereof (e.g., SEQ ID NO: 128 of WO2005033321), AAVhu.48 or variants thereof (e.g., SEQ ID NO: 38 of US20150159173), AAVhu.48R1 or variants thereof, AAVhu.48R2 or variants thereof, AAVhu.48R3 or variants thereof, AAVhu.49 or variants thereof (e.g., SEQ ID NO: 189 of WO2005033321), AAVhu.51 or variants thereof (e.g., SEQ ID NO: 190 of WO2005033321), AAVhu.52 or variants thereof (e.g., SEQ ID NO: 191 of WO2005033321), AAVhu.53 or variants thereof (e.g., SEQ ID NO: 159 of US20150159173). NO:19 and 35), AAVhu.54 or its variants (e.g., SEQ ID NO: 188 of WO2005033321), AAVhu.55 or variants thereof (e.g., SEQ ID NO: 187 of WO2005033321), AAVhu.56 or variants thereof (e.g., SEQ ID NO: 192 of WO2005033321), AAVhu.57 or variants thereof (e.g., SEQ ID NO: 193 of WO2005033321), AAVhu.58 or variants thereof (e.g., SEQ ID NO: 194 of WO2005033321), AAVhu.60 or variants thereof (e.g., SEQ ID NO: 184 of WO2005033321), AAVhu.61 or variants thereof (e.g., SEQ ID NO: 185 of WO2005033321), AAVhu.63 or variants thereof (e.g., SEQ ID NO: 195 of WO2005033321), AAVhu.64 or variants thereof (e.g., SEQ ID NO: 195 of WO2005033321). SEQ ID NO: 196), AAVhu.66 or variants thereof (e.g., SEQ ID NO: 197 of WO2005033321), AAVhu.67 or variants thereof (e.g., SEQ ID NO: 198 of WO2005033321), AAVhu.14 / 9 or variants thereof, AAVhu.t 19 or variants thereof, AAVrh.2 or variants thereof (e.g., SEQ ID NO: 39 of US20150159173), AAVrh.2R or variants thereof, AAVrh.8 or variants thereof (e.g., SEQ ID NO: 41 of US20150159173), AAVrh.8R or variants thereof, AAVrh.10 or variants thereof (e.g., SEQ ID NO: 197 of US20150159173). 9 and 25), AAVrh.12 or variants thereof, AAVrh.13 or variants thereof (e.g., SEQ ID NO: 10 and 26 of US20150159173), AAVrh.13R or variants thereof, AAVrh.14 or variants thereof, AAVrh.17 or variants thereof, AAVrh.18 or variants thereof, AAVrh.19 or variants thereof, AAVrh.20 or variants thereof (e.g., SEQ ID NO: 10 and 26 of US20150159173). 1) AAVrh.21 or variants thereof, AAVrh.22 or variants thereof, AAVrh.23 or variants thereof, AAVrh.24 or variants thereof, AAVrh.25 or variants thereof, AAVrh.31 or variants thereof, AAVrh.32 or variants thereof, AAVrh.33 or variants thereof, AAVrh.34 or variants thereof, AAVrh.35 or variants thereof, AAVrh.36 or variants thereof, AAVrh.37 or variants thereof (e.g., SEQ ID NO: 40 of US20150159173), AAVrh.37R2 or variants thereof, AAVrh.38 or variants thereof (e.g., SEQ ID NO: 86 of WO2005033321), AAVrh.39 or variants thereof (e.g., SEQ ID NO: 3, 20, or 36 of US20150159173), AAVrh.40 or variants thereof (e.g., SEQ ID NO: 92 of WO2005033321), AAVrh.43 or variants thereof (e.g., SEQ ID NO: 21 and 37 of US20150159173), AAVrh.46 or variants thereof (e.g., SEQ ID NO: 4 and 22 of US20150159173), AAVrh.48 or variants thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1 or variants thereof (e.g., SEQ ID NO: 86 of US20150159173). 44) AAVrh.48.1.2 or variants thereof, AAVrh.48.2 or variants thereof, AAVrh.49 or variants thereof (e.g., SEQ ID NO: 103 of WO2005033321), AAVrh.50 or variants thereof (e.g., SEQ ID NO: 108 of WO2005033321), AAVrh.51 or variants thereof (e.g., SEQ ID NO: 104 of WO2005033321), AAVrh.52 or variants thereof (e.g., SEQ ID NO: 96 of WO2005033321), AAVrh.53 or variants thereof (e.g., SEQ ID NO: 97 of WO2005033321), AAVrh.54 or variants thereof (e.g., SEQ ID NO: 97 of US20150159173). 49) AAVrh.56 or variants thereof (e.g., SEQ ID NO: 152 of WO2005033321), AAVrh.57 or variants thereof (e.g., SEQ ID NO: 105 of WO2005033321), AAVrh.58 or variants thereof (e.g., SEQ ID NO: 48 of US20150159173), AAVrh.61 or variants thereof (e.g., SEQ ID NO: 107 of WO2005033321), AAVrh.62 or variants thereof (e.g., SEQ ID NO: 114 of WO2005033321), AAVrh.64 or variants thereof (e.g., SEQ ID NO: 152 of US20150159173). 43) AAVrh.64R1 or variants thereof, AAVrh.64R2 or variants thereof, AAVrh.67 or variants thereof (e.g., SEQ ID NO: 47 of US20150159173), AAVrh.73 or a variant thereof (e.g., SEQ ID NO: 5 of US20150159173) or AAVrh.74 or a variant thereof (e.g., SEQ ID NO: 6 of US2015015917). Non-limiting examples of variations include SEQ ID Nos: 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99 and 101-113 of US20030138772 (the contents of which are incorporated herein by reference in their entirety), and SEQ ID Nos: 1, 2, 4-82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219 and 224-236 of WO2005033321 (the contents of which are incorporated herein by reference in their entirety). In one embodiment, the AAV serotype is any of those described in US 2021 / 0189430 (the contents of which are incorporated herein by reference in their entirety). The amino acid sequence of the AAV may include one or more amino acid substitutions at one or more positions in the AAV capsid protein that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588 (numbered based on the VP1 number of AAV2).

[0340] Unless otherwise stated, the AAV ITR and other selected AAV components described herein can be readily selected from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or other known and unknown AAV serotypes. In one embodiment, the ITR is derived from AAV2. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., Manassas, VA). Alternatively, AAV sequences can be obtained by synthesis or other suitable means referencing published sequences (e.g., available in the literature or databases such as GenBank, PubMed, etc.).

[0341] The AAV fragments required for assembly into a vector include cap proteins (including vp1, vp2, vp3, and hypervariable regions), rep proteins (including rep 78, rep 68, rep 52, and rep 40), and sequences encoding these proteins. These fragments are readily available for use in a variety of vector systems and host cells. Such fragments can be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated using any suitable technique, using a combination of selected AAV sequences (e.g., fragments of the vp1 capsid protein) and heterologous sequences that can be obtained from different selected AAV serotypes, discontinuous portions of the same AAV serotype, or non-AAV viral or non-viral sources. Artificial AAV serotypes can be, but are not limited to, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids. A pseudotype vector, as described herein, is useful for use with an AAV capsid accompanied by an ITR of an AAV having a different capsid protein. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV with an AAV2 ITR and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV with an AAV2 ITR and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises an amino acid sequence found at NCBI reference sequence: YP_077180.1. In another embodiment, the AAV8 capsid comprises a capsid encoded by nt 2121 to 4337 of GenBank accession: AF513852.1.

[0342] In one embodiment, the vector useful in the compositions and methods described herein contains at least a sequence encoding a selected AAV serotype capsid (e.g., AAV2 capsid) or a fragment thereof. In another embodiment, the useful vector contains at least a sequence encoding a selected AAV serotype rep protein (e.g., AAV2 rep protein) or a fragment thereof. Optionally, such vectors may contain both AAV cap and rep protein. In vectors providing both AAV rep and cap, the AAV rep and AAV cap sequences may both be derived from a single serotype source, such as AAV2.

[0343] Alternatively, a vector in which the rep sequence originates from an AAV serotype different from the AAV serotype providing the cap sequence can be used. In one embodiment, the rep and cap sequences are expressed from different sources (e.g., different vectors, or host cells and vectors). In another embodiment, these rep sequences are fused in-frame with cap sequences of different AAV serotypes to form chimeric AAV vectors, such as those described in U.S. Patent No. 7,282,199 (incorporated herein by reference).

[0344] Suitable recombinant AAV (rAAV) is generated by culturing host cells containing a nucleic acid sequence encoding an AAV serotype capsid protein or a fragment thereof as defined herein; a functional rep gene; a small gene consisting of, for example, an AAV ITR and a trans-splicing molecule nucleic acid sequence; and sufficient helper functions to allow the small gene to be packaged into the AAV capsid protein. The components required for culturing in host cells to package the AAV small gene into the AAV capsid may be provided trans-present to the host cells. Alternatively, any one or more of the required components (e.g., the small gene, rep sequence, cap sequence, and / or helper functions) may be provided by stable host cells that have been engineered to contain one or more of the required components using methods known to those skilled in the art.

[0345] In one embodiment, the AAV includes a promoter (or a functional fragment of a promoter). The promoter to be used in the rAAV can be selected from a large number of constitutive or inducible promoters capable of expressing the selected transgene in desired target cells. See, for example, the list of promoters identified in International Patent Publication No. WO 2014 / 012482 (incorporated herein by reference). In one embodiment, the promoter is cell-specific. The term "cell-specific" means that the specific promoter selected for the recombinant vector can direct the expression of the selected transgene in a specific cell type. In some embodiments, the promoter is specific for the expression of the transgene in neuronal cells. In some embodiments, the promoter is specific for the expression of the transgene in cortical neurons (e.g., cortical pyramidal neurons). In some embodiments, the promoter is specific for the expression of the transgene in striatal neurons (medium-spinous neurons of the striatum). In some embodiments, the promoter is specific for the expression of the transgene in hypothalamic neurons. In some embodiments, the transgene is expressed in at least one cell type or cell.

[0346] In another embodiment, the promoter is the native promoter of t...

Claims

1. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure domain containing HTT exon 1 and HTT exon 2; (b) splice domain; and (c) A binding domain of a target intron that binds to HTT precursor mRNA, wherein the target intron comprises intron 2.

2. The HTT nucleic acid trans-splicing molecule of claim 1, wherein the binding domain comprises, is substantially composed of, or is composed of: Any of SEQ ID NO: 60-81, or a sequence that has at least 90% identity with any of SEQ ID NO: 60-81.

3. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure domain containing HTT exons 1-3; (b) splice domain; and (c) A binding domain of a target intron that binds to HTT precursor mRNA, wherein the target intron comprises intron 3.

4. The HTT nucleic acid trans-splicing molecule of claim 3, wherein the binding domain comprises, substantially consists of, or consists of: Any of SEQ ID NO: 158-174, or a sequence that has at least 90% identity with any of SEQ ID NO: 158-174.

5. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure field containing HTT exon 1; (b) splice domain; and (c) A binding domain that binds to a target intron of HTT precursor mRNA, wherein the target intron includes intron 1, and wherein the binding domain includes any one of SEQ ID NO: 8-21.

6. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, wherein the coding domain comprises, is substantially composed of, or is composed of: HTT exon 1; HTT exon 1 and HTT exon 2; or HTT exon 1-3.

7. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, wherein the coding domain comprises, is substantially composed of, or is composed of: The sequence is any one of SEQ ID NO: 3, 59, 157 or 349-353, or a sequence that has at least 90% identity with SEQ ID NO: 3, 59, 157 or 349-353.

8. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, wherein the coding domain, the splicing domain, and the binding domain are operatively linked in a 5' to 3' orientation.

9. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, the HTT nucleic acid trans-splicing molecule further comprising a adapter, wherein the coding domain, splicing domain, adapter and binding domain are operatively connected in a 5' to 3' orientation.

10. The HTT nucleic acid trans-splicing molecule of claim 9, wherein the linker comprises, substantially consists of, or consists of: A sequence in the range of 20 to 50 nucleotides in length, wherein the linker contains 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine. A sequence ranging from 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine; or A sequence ranging from 22 to 42 nucleotides in length, wherein the linker contains 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine.

11. The HTT nucleic acid trans-splicing molecule of any one of claims 9 or 10, wherein the adapter comprises, is substantially composed of, or is composed of any one of the following: SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; or SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO:

41.

12. The HTT nucleic acid trans-splicing molecule of claim 9, wherein the adapter comprises any of the following, is substantially composed of any of the following, or is composed of any of the following: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 or a sequence having at least 90% identity with SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity with SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity with SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity with SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity with SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity with SEQ ID NO: 197; or SEQ ID NO: 198 or a sequence having at least 90% identity with SEQ ID NO:

198.

13. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, the HTT nucleic acid trans-splicing molecule further comprising a triple-helix terminator, wherein the coding domain, the splicing domain, the linker (if present), the binding domain and the triple-helix terminator are operatively connected in a 5' to 3' orientation.

14. The HTT nucleic acid trans-splicing molecule of claim 13, wherein the triple-helix terminator comprises, substantially comprises, or comprises: SEQ ID NO: 5 or a sequence having at least 90% identity with SEQ ID NO:

5.

15. The HTT nucleic acid trans-splicing molecule of claim 13, wherein the triple-helix terminator comprises, substantially comprises, or comprises: SEQ ID NO:

6.

16. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, the HTT nucleic acid trans-splicing molecule further comprising a 5' untranslated region (5' UTR), wherein the 5' UTR, the coding domain, the splicing domain, the linker (if present), the binding domain, and the triple-helix terminator (if present) are operatively connected in a 5' to 3' orientation.

17. The HTT nucleic acid trans-splicing molecule of claim 16, wherein the 5' UTR is HTT 5' UTR.

18. The HTT nucleic acid trans-splicing molecule of claim 17, wherein... HTT 5' UTR contains the following, is basically composed of the following, or is composed of the following: The sequence is either SEQ ID NO: 136 or 192, or a sequence that is at least 90% identical to either SEQ ID NO: 136 or 192.

19. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, the HTT nucleic acid trans-splicing molecule further comprising a sequence encoding an epitope tag, wherein the 5' UTR (when present), the epitope tag, the coding domain, the splicing domain, the adapter (when present), the binding domain, and the triple-helix terminator (when present) are operatively connected in a 5' to 3' orientation.

20. The HTT nucleic acid trans-splicing molecule of claim 19, wherein the sequence encoding the epitope tag comprises, substantially consists of, or consists of: SEQ ID NO:

4.

21. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure domain containing HTT exon 1 and HTT exon 2; (b) Splice domain; (c) Connector; and (d) A binding domain of a target intron that binds to HTT precursor mRNA, wherein the target intron contains intron 2.

22. The HTT nucleic acid trans-splicing molecule of claim 21, wherein the binding domain comprises, is substantially composed of, or is composed of: Any of SEQ ID NO: 60-81, or a sequence that has at least 90% identity with any of SEQ ID NO: 60-81.

23. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure domain containing HTT exons 1-3; (b) Splice domain; (c) Connector; and (d) A binding domain of a target intron that binds to HTT precursor mRNA, wherein the target intron contains intron 3.

24. The HTT nucleic acid trans-splicing molecule of claim 23, wherein the binding domain comprises, is substantially composed of, or is composed of: Any of SEQ ID NO: 158-174, or a sequence that has at least 90% identity with any of SEQ ID NO: 158-174.

25. An HTT nucleic acid trans-splicing molecule, said HTT nucleic acid trans-splicing molecule comprising: (a) The coding structure field containing HTT exon 1; (b) Splice domain; (c) Connector; and (d) A binding domain of a target intron that binds to HTT precursor mRNA, wherein the target intron contains intron 1.

26. The HTT nucleic acid trans-splicing molecule of claim 25, wherein the binding domain comprises, is substantially composed of, or is composed of: Any of SEQ ID NO: 8-21, or a sequence that has at least 90% identity with any of SEQ ID NO: 8-21.

27. The HTT nucleic acid trans-splicing molecule according to any one of claims 21-26, wherein the adapter comprises any of the following SEQ ID NO:, is substantially composed of any of the following SEQ ID NO:, or is composed of any of the following SEQ ID NO:: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO: 41; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 or a sequence having at least 90% identity with SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity with SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity with SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity with SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity with SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity with SEQ ID NO: 197; or SEQ ID NO: 198 or a sequence having at least 90% identity with SEQ ID NO:

198.

28. The HTT nucleic acid trans-splicing molecule of claim 27, further comprising a triple-helix terminator, wherein the coding domain, the splicing domain, the linker, the binding domain, and the triple-helix terminator are operably connected in a 5' to 3' direction; and optionally, further comprising a 5' UTR, wherein the 5' UTR (when present), the coding domain, the splicing domain, the linker, the binding domain, and the triple-helix terminator (when present) are operably connected in a 5' to 3' direction.

29. A nucleic acid trans-splicing molecule comprising a linker, wherein the linker comprises, is substantially composed of, or is composed of: A sequence ranging from 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine.

30. The nucleic acid trans-splicing molecule of claim 29, wherein the linker comprises, substantially consists of, or consists of: A sequence ranging from 20 to 45 nucleotides in length, wherein the linker contains 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine.

31. The nucleic acid trans-splicing molecule of claim 29 or claim 30, wherein the linker comprises, is substantially composed of, or is composed of: A sequence ranging from 22 to 42 nucleotides in length, wherein the linker contains 60-80% guanine, dispersed with thymidine / uridine; 65-75% guanine, dispersed with thymidine / uridine; or 66-74% guanine, dispersed with thymidine / uridine.

32. The nucleic acid trans-splicing molecule according to any one of claims 29-31, wherein the adapter comprises, is substantially composed of, or is composed of: SEQ ID NO: 38 or a sequence having at least 90% identity with SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity with SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity with SEQ ID NO: 40; or SEQ ID NO: 41 or a sequence having at least 90% identity with SEQ ID NO:

41.

33. A nucleic acid trans-splicing molecule comprising a linker, wherein the linker comprises, substantially comprises, or comprises: SEQ ID NO: 37 or a sequence having at least 90% identity with SEQ ID NO: 37; SEQ ID NO: 42 or a sequence having at least 90% identity with SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity with SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity with SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity with SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity with SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity with SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity with SEQ ID NO: 107; SEQ ID NO: 108 or a sequence having at least 90% identity with SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity with SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity with SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity with SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity with SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity with SEQ ID NO: 197; or SEQ ID NO: 198 or a sequence having at least 90% identity with SEQ ID NO:

198.

34. The HTT nucleic acid trans-splicing molecule according to any one of claims 1-28, wherein the HTT nucleic acid trans-splicing molecule further comprises binding... MSH3 The target intron binding domain of precursor mRNA.

35. The HTT nucleic acid trans-splicing molecule of claim 34, wherein... MSH3 Target introns contain MSH3 Either intron 5 or intron 15.

36. The HTT nucleic acid trans-splicing molecule as described in any one of claims 34 or 35, wherein the binding MSH3 The target intron binding domain of the precursor mRNA contains, is essentially composed of, or is composed of: The sequence is any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210, or has at least 90% identity with any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210.

37. The HTT nucleic acid trans-splicing molecule according to any one of claims 34-36, wherein the nucleic acid trans-splicing molecule comprises any one of SEQ ID NO: 149-154 or SEQ ID NO: 212-223, or a sequence having at least 90% identity with any one of SEQ ID NO: 149-154 or SEQ ID NO: 212-223.

38. The HTT nucleic acid trans-splicing molecule as claimed in any of the preceding claims, the HTT nucleic acid trans-splicing molecule further comprising a nucleic acid sequence encoding a primary miRNA, the primary miRNA comprising a microRNA (miRNA) sequence specific to exon 1 of endogenous HTT mRNA, wherein exon 1 of the nucleic acid trans-splicing molecule comprises a nucleotide sequence variation that weakens the binding of the miRNA to at least a portion of the mRNA encoded by the nucleic acid trans-splicing molecule.

39. The HTT nucleic acid trans-splicing molecule of claim 38, wherein the miRNA sequence comprises any one of SEQ ID NO: 339 or 342, or a nucleic acid sequence having at least 90% identity with any one of SEQ ID NO: 339 or 342.

40. The HTT nucleic acid trans-splicing molecule of claim 39, wherein the nucleic acid sequence encoding the primary miRNA comprises either SEQ ID NO: 341 or 344.

41. The HTT nucleic acid trans-splicing molecule according to any one of claims 38 to 40, wherein the primary miRNA comprises a mir-33 scaffold sequence.

42. The HTT nucleic acid trans-splicing molecule according to any one of claims 38 to 40, wherein the primary miRNA comprises a mir-30a scaffold sequence, a mir-30a loop sequence, a mir-155 scaffold sequence, a mir-155 loop sequence, a mir-33 scaffold sequence, or a mir-33 loop sequence.

43. The HTT nucleic acid trans-splicing molecule of claim 42, wherein the mir-30a scaffold sequence comprises the 5' scaffold sequence shown in SEQ ID NO: 227 or the 3' scaffold sequence shown in SEQ ID NO: 228; wherein the mir-30a loop sequence comprises SEQ ID NO: 229; wherein the mir-155 scaffold sequence comprises the 5' scaffold sequence shown in SEQ ID NO: 230 or the 3' scaffold sequence shown in SEQ ID NO: 231; wherein the mir-155 loop sequence comprises SEQ ID NO: 232; wherein the mir-33 scaffold sequence comprises the 5' scaffold sequence shown in SEQ ID NO: 259 or the 3' scaffold sequence shown in SEQ ID NO: 260; or wherein the mir-33 loop sequence comprises SEQ ID NO:

261.

44. An MSH3 exon-jugating nucleic acid construct, said MSH3 exon-jugating nucleic acid construct comprising operably linked: (a) A sequence encoding an antisense RNA that promotes exon skipping of a target exon of the MSH3 precursor mRNA, wherein the target exon is any one of MSH3 exons 2-4, 6-8, or 15, and wherein the target exon contains a 5' exon-intron junction and a 3' exon-intron junction sequence; and (b) The sequence encoding a small nuclear RNA (snRNA) sequence.

45. The MSH3 exon-jumping nucleic acid construct of claim 44, wherein the MSH3 exon-jumping nucleic acid construct further comprises a U1 promoter and a U1 terminator operatively linked to (a) and (b).

46. ​​The MSH3 exon skipping nucleic acid construct as described in claim 44 or 45, wherein the snRNA is a modified snRNA.

47. The MSH3 exon skipping nucleic acid construct of claim 46, wherein the modified snRNA comprises a U7Sm OPT sequence or a U2 snRNA sequence.

48. The MSH3 exon-jugating nucleic acid construct according to any one of claims 44-47, wherein the antisense RNA targets the 5' exon-intron junction or the 3' exon-intron junction of the target exon.

49. The MSH3 exon skipping nucleic acid construct of claim 44, wherein the antisense RNA comprises, is substantially composed of, or is composed of: SEQ ID NO: any one of 274, 275, 276, 277, 278, 279, 280, 300, 302, 301, 303, 281, 282, 306, 308, 305, 307, 311, 313, 310, 312, 316, 318, 315, 317, 321, 323, 320, or 322, or any one of SEQ ID NO. The sequence NO: 274, 275, 276, 277, 278, 279, 280, 300, 302, 301, 303, 281, 282, 306, 308, 305, 307, 311, 313, 310, 312, 316, 318, 315, 317, 321, 323, 320 or 322 has at least 90% identity.

50. The MSH3 exon-jumping nucleic acid construct of claim 44, wherein the MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 284, 285, 286, 287, 288, 289, 290, 325, 326, 291, 292, 328, 329, 331, 332, 334, 335, 337 and 338.

51. The MSH3 exon-jugating nucleic acid construct according to any one of claims 44-47, wherein the antisense RNA targets both the 5' exon-intron junction and the 3' exon-intron junction.

52. The MSH3 exon skipping nucleic acid construct of claim 51, wherein the antisense RNA comprises a sequence that is at least 80% complementary to the entire sequence of the target exon.

53. The MSH3 exon skipping nucleic acid construct of claim 51, wherein the antisense RNA further comprises: (a) A sequence that is at least 80% complementary to the 5-nucleotide sequence upstream of the 5' exon-intron junction; and (b) A sequence that is at least 80% complementary to the 5-nucleotide sequence downstream of the 3' exon-intron junction.

54. The MSH3 exon skipping nucleic acid construct of claim 52 or 53, wherein the antisense RNA comprises any one of SEQ ID NO: 299, 304, 309, 314 or 319, or a sequence having at least 90% identity with any one of SEQ ID NO: 299, 304, 309, 314 or 319.

55. The MSH3 exon-jumping nucleic acid construct according to any one of claims 51-53, wherein the MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 324, 327, 330, 333 or 336.

56. The MSH3 exon skipping nucleic acid construct of claim 51, wherein the antisense RNA comprises components operably linked in a 5' to 3' orientation: (a) A sequence targeting the 3' exon-intron junction; (b) A linker sequence of at least 15 nucleotides that does not anneal to the target exon; and (c) Sequences targeting the 5' exon-intron junction.

57. The MSH3 exon skipping nucleic acid construct of claim 56, wherein the complementarity of the adapter sequence with all sequences of the target exon of the same length as the adapter is less than 50%.

58. The MSH3 exon skipping nucleic acid construct of claim 56 or 57, wherein the antisense RNA comprises any one of SEQ ID NO: 300, 301, 302, 303, 305, 306, 307, 308, 310, 311, 312, 313, 315, 316, 317, 318, 320, 321, 322 or 323, or any combination thereof.

59. The MSH3 exon-jumping nucleic acid construct according to any one of claims 56-58, wherein the MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 325, 326, 328, 329, 331, 332, 334, 335, 337 or 338.

60. The MSH3 exon skipping nucleic acid construct according to any one of claims 44-59, wherein the antisense RNA targets MSH3 exon 7.

61. The MSH3 exon-jumping nucleic acid construct of claim 60, wherein the MSH3 exon-jumping nucleic acid construct comprises SEQ ID NO:

309.

62. The MSH3 exon-jumping nucleic acid construct of claim 60, wherein the MSH3 exon-jumping nucleic acid construct comprises, from 5' to 3': (a) SEQ ID NO: 310 (In7 / Ex7 asRNA), SEQ ID NO: 298 (linker), and SEQ ID NO: 311 (In7 / Ex7 asRNA); or (b) SEQ ID NO: 312 (In7 / Ex7 asRNA), SEQ ID NO: 298 (linker) and SEQ ID NO: 313 (In7 / Ex7 asRNA).

63. The MSH3 exon skipping nucleic acid construct of claim 60, wherein the antisense RNA comprises any one of SEQ ID NO: 309, 310, 311, 312 or 313, or any combination thereof, or a sequence having at least 90% identity with any one of SEQ ID NO: 309, 310, 311, 312 or 313.

64. The MSH3 exon-jumping nucleic acid construct of claim 60, wherein the MSH3 exon-jumping nucleic acid construct comprises at least one of SEQ ID NO: 330-332, or any combination thereof.

65. An MSH3 miRNA nucleic acid construct, said MSH3 miRNA nucleic acid construct comprising a sequence encoding a primary miRNA, said primary miRNA comprising a scaffold sequence, a loop sequence, and a miRNA sequence targeting endogenous MSH3 mRNA, wherein: (a) The stent sequence is derived from mir-30a, mir-33 or mir-155; (b) The ring sequence is derived from mir-22, mir-30a, mir-33, or mir-155; and (c) The miRNA sequence comprises any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256 or 257, or a sequence having at least 90% identity with any one of SEQ ID NO: 224, 244, 246, 248, 250, 252, 254, 256 or 257.

66. The MSH3 miRNA nucleic acid construct of claim 65, wherein the scaffold sequence comprises any one of SEQ ID NO: 227, 228, 230, 231, 259 or 260.

67. The MSH3 miRNA nucleic acid construct of claim 65 or 66, wherein the loop sequence comprises any one of SEQ ID NO: 229, 232 or 261.

68. The MSH3 miRNA nucleic acid construct according to any one of claims 65-67, wherein the primary miRNA sequence comprises any one of SEQ ID NO: 234, 235, 238-241 or 262-269.

69. The MSH3 miRNA nucleic acid construct according to any one of claims 65-68, wherein the sequence encoding the primary miRNA is operatively linked to the U6 promoter or the CMV promoter.

70. An MSH3 nucleic acid trans-splicing molecule, said MSH3 nucleic acid trans-splicing molecule comprising: (a) Encoding structure field sequence; (b) splice domain; and (c) Binding domain of the target intron that binds to MSH3 precursor mRNA; The sequence of encoded structure fields mentioned therein is not an MSH3 encoded structure field sequence.

71. The nucleic acid trans-splicing molecule of claim 70, wherein the coding domain sequence comprises a sequence that causes frameshifting of mature MSH3 mRNA when trans-spliced ​​into the MSH3 precursor mRNA.

72. The nucleic acid trans-splicing molecule of claim 70 or 71, wherein the coding domain sequence comprises one or more of exons 1, 2 and 3 of HTT.

73. The nucleic acid trans-splicing molecule according to any one of claims 70-72, wherein the target intron of the MSH3 precursor mRNA is intron 5 or intron 15.

74. The nucleic acid trans-splicing molecule of any one of claims 70 to 73, wherein the binding domain comprises any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210, or a sequence having at least 90% identity with any one of SEQ ID NO: 140, 142, 144, 146, 209 or 210.

75. An HTT trans-splicing and MSH3 exon-jumping nucleic acid construct, wherein the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprises: (a) The HTT nucleic acid trans-splicing molecule as described in any one of claims 1-28; and (b) The MSH3 exon skipping nucleic acid construct as described in any one of claims 44-64.

76. The HTT trans-splicing and MSH3 exon skipping nucleic acid construct of claim 75, wherein (a) and (b) are contained on a single vector.

77. The HTT trans-splicing and MSH3 exon skipping nucleic acid construct of claim 76, wherein the single vector is an AAV vector.

78. The HTT trans-splicing and MSH3 exon-jumping nucleic acid construct of claim 77, wherein the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 356, 357, 363 or 364.

79. The HTT trans-splicing and MSH3 exon skipping nucleic acid construct of claim 77, wherein the AAV vector is scAAV or ssAAV vector.

80. The HTT trans-splicing and MSH3 exon-jumping nucleic acid construct of claim 79, wherein the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 369, 370 and 371.

81. A HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct, wherein the HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct comprises: (a) The HTT nucleic acid trans-splicing molecule as described in any one of claims 38-43; and (b) The MSH3 exon skipping nucleic acid construct as described in any one of claims 44-64.

82. The HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid construct as described in claim 81, wherein (a) and (b) are contained on a single vector.

83. The HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct of claim 82, wherein the HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct comprises any one of SEQ ID NO: 358 or 359.

84. The HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid construct as described in claim 82 or 83, wherein the single vector is an AAV vector.

85. An HTT trans-splicing and MSH3 miRNA nucleic acid construct, wherein the HTT trans-splicing and MSH3 miRNA nucleic acid construct comprises: (a) The HTT nucleic acid trans-splicing molecule as described in any one of claims 1-28; and (b) The MSH3 miRNA nucleic acid construct as described in any one of claims 65-69.

86. The HTT trans-splicing and MSH3 miRNA nucleic acid construct of claim 85, wherein (a) and (b) are contained on a single vector.

87. The HTT trans-splicing and MSH3 miRNA nucleic acid construct of claim 86, wherein the HTT trans-splicing and MSH3 miRNA nucleic acid construct comprises any one of SEQ ID NO: 354 or 355.

88. The HTT trans-splicing and MSH3 miRNA nucleic acid construct as described in claim 86 or 87, wherein the vector is an AAV vector.

89. An AAV vector comprising the HTT nucleic acid trans-splicing molecule as described in any one of claims 38-43.

90. The AAV carrier of claim 89, wherein the AAV carrier comprises any one of SEQ ID NO: 356, 357, 363 or 364.

91. An AAV vector comprising an HTT nucleic acid trans-splicing molecule as described in any one of claims 1-28 and 34-43 or a nucleic acid trans-splicing molecule as described in any one of claims 29-33.

92. A ribonucleic acid trans-splicing molecule, said ribonucleic acid trans-splicing molecule comprising any one of SEQ ID NO: 23-36, 47-56, 83-105, 113-125, 175-191 or 199-206.

93. A ribonucleic acid trans-splicing molecule, said ribonucleic acid trans-splicing molecule being transcribed from an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 and 39-43 or a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33.

94. The HTT nucleic acid trans-splicing molecule according to any one of claims 1-28 or 34-43, wherein the HTT precursor mRNA comprises at least one mutation associated with Huntington's disease (HD).

95. The HTT nucleic acid trans-splicing molecule of claim 94, wherein the at least one HD-related mutation comprises HTT Amplification of CAG repeats in gene alleles.

96. The HTT nucleic acid trans-splicing molecule as described in claim 95, wherein... HTT The amplification of CAG repeats in the gene alleles contains more than 35 CAG repeats.

97. The HTT nucleic acid trans-splicing molecule according to any one of claims 94-96, wherein the at least one HD-related mutation is autosomal dominant.

98. The HTT nucleic acid trans-splicing molecule according to any one of claims 94-97, wherein the at least one HD-related mutation is expressed in at least one of: cortical pyramidal neurons, striatal medium spiny neurons, or hypothalamic neurons.

99. A vector comprising: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; or an HTT trans-splicing and MSH3 miRNA nucleic acid construct as claimed in any one of claims 85-88.

100. A vector comprising an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43.

101. The carrier of claim 100, wherein the carrier includes a 5' adjustment structure domain operatively connected to the 5' of the coding structure domain.

102. The carrier of claim 101, wherein the 5' adjustment domain is operatively connected to the 5' untranslated region.

103. The vector of claim 101 or 102, wherein the 5' regulatory domain comprises a constitutive promoter or a tissue-specific promoter.

104. The carrier of claim 103, wherein the constitutive promoter is a CMV promoter or a CAGGS promoter.

105. A proviral plasmid comprising: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; or an HTT trans-splicing and MSH3 miRNA nucleic acid construct as claimed in any one of claims 85-88.

106. An adeno-associated virus (AAV) comprising: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; or an HTT trans-splicing and MSH3 miRNA nucleic acid construct as claimed in any one of claims 85-88.

107. An adeno-associated virus (AAV) comprising an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43, wherein the AAV optionally comprises a 5' operatively linked 5' regulatory domain in the nucleic acid trans-splicing molecule.

108. The AAV of claim 107, wherein the AAV includes a 5' adjustment structure domain operatively connected to the 5' of the coding structure domain.

109. The AAV of any one of claims 107 or 108, wherein the 5' adjustment domain is operatively connected to the 5' untranslated region.

110. The AAV of any one of claims 107-109, wherein the 5' regulation domain comprises a constitutive promoter.

111. The AAV of claim 110, wherein the constitutive promoter is a CMV promoter or a CAGGS promoter.

112. The AAV of any one of claims 107-111, wherein the AAV exhibits neuronal orientation.

113. The AAV as claimed in any one of claims 107-111, wherein the AAV is AAV9, AAV8, AAV5, AAV2, AAV7 or AAV2.7m8, AAV-retro, AAV1, AAV4 or AAV-PHP.eB.

114. A composition comprising: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; an HTT trans-splicing and MSH3 miRNA nucleic acid construct as claimed in any one of claims 85-88; a vector as claimed in any one of claims 99-104; a proviral plasmid as claimed in claim 105; or an AAV as claimed in any one of claims 89-91 or 106-113.

115. The composition of claim 114, wherein the composition comprises a pharmaceutically acceptable excipient.

116. The composition of any one of claims 114 or 115, further comprising at least one antisense oligonucleotide or a construct encoding at least one antisense RNA, said antisense RNA inhibiting cis-splicing of the HTT precursor mRNA.

117. The composition of claim 116, wherein the at least one antisense oligonucleotide comprises any one of SEQ ID NO: 126-135, or the construct encoding the at least one antisense RNA binds to a target sequence bound to any one of SEQ ID NO: 126-135.

118. The composition of claim 116 or 117, wherein the at least one antisense oligonucleotide comprises SEQ ID NO: 131, or the construct encoding the at least one antisense RNA binds to the target sequence bound to SEQ ID NO:

131.

119. A method for expressing bioactive HTT in target cells to restore the functional level of HTT protein in said target cells, the method comprising transducing said target cells with the following: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; or an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 85-88. miRNA nucleic acid construct; the vector as described in any one of claims 99-104; the proviral plasmid as described in claim 105; the AAV as described in any one of claims 89-91 or 106-113; or the composition as described in any one of claims 114-118.

120. The method of claim 119, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of the HTT precursor mRNA containing at least one HD-related mutation in the target cell is replaced.

121. The method of claim 120, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the HTT precursor mRNA containing at least one HD-related mutation in the target cell is replaced.

122. The method of claim 121, wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the HTT precursor mRNA containing at least one HD-related mutation is replaced in the target cell.

123. The method of any one of claims 119-122, wherein the functional level of HTT in the target cells is restored by expressing a biologically functional HTT protein, and / or a mutant HTT protein. HTT RNA and related transcripts (e.g.) HTT1a () has decreased.

124. A method for reducing HTT expression in a subject, said HTT comprising a polyglutamine repeat of more than 35 consecutive glutamine residues, said method comprising transfecting or transducing target cells, more particularly neurons, in the subject with the following: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; an HTT trans-splicing and MSH3... miRNA nucleic acid construct; vector as described in any one of claims 99-104; proviral plasmid as described in claim 105; AAV as described in any one of claims 89-91 or 106-113; or composition as described in any one of claims 114-118.

125. A method for correcting at least one mutation in the HTT exon sequence of HTT precursor mRNA in target cells of a subject, the method comprising administering to the subject the following: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 85-88. miRNA nucleic acid construct; vector as described in any one of claims 99-104; proviral plasmid as described in claim 105; AAV as described in any one of claims 89-91 or 106-113; or composition as described in any one of claims 114-118.

126. A method of treating a subject with Huntington's disease in need, the method comprising administering to the subject a therapeutically effective amount of the following: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; and an HTT trans-splicing and MSH3... miRNA nucleic acid construct; vector as described in any one of claims 99-104; proviral plasmid as described in claim 105; AAV as described in any one of claims 89-91 or 106-113; or composition as described in any one of claims 114-118.

127. The method of any one of claims 119-126, the method comprising administering to the brain of the subject the following: an HTT nucleic acid trans-splicing molecule as claimed in any one of claims 1-28 or 34-43; a nucleic acid trans-splicing molecule as claimed in any one of claims 29-33; an MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 44-64; an MSH3 miRNA nucleic acid construct as claimed in any one of claims 65-69; an MSH3 nucleic acid trans-splicing molecule as claimed in any one of claims 70-74; an HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 75-80; an HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as claimed in any one of claims 81-84; an HTT trans-splicing and MSH3 miRNA nucleic acid construct as claimed in any one of claims 85-88; a vector as claimed in any one of claims 99-104; a proviral plasmid as claimed in claim 105; an AAV as claimed in any one of claims 89-91 or 106-113; or a composition as claimed in any one of claims 114-118.

128. The method of any one of claims 119-127, wherein the subject is a mammal, preferably a rodent, a non-human primate, or a human.

129. The method of any one of claims 124-128, wherein the subject is genetically predisposed to HD or has been diagnosed with HD.

130. The HTT nucleic acid trans-splicing molecule as described in any one of claims 1-28 or 34-43; the nucleic acid trans-splicing molecule as described in any one of claims 29-33; the MSH3 exon-jumping nucleic acid construct as described in any one of claims 44-64; the MSH3 miRNA nucleic acid construct as described in any one of claims 65-69; the MSH3 nucleic acid trans-splicing molecule as described in any one of claims 70-74; the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as described in any one of claims 75-80; the HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as described in any one of claims 81-84; the HTT trans-splicing and MSH3 miRNA nucleic acid construct as described in any one of claims 85-88; the vector as described in any one of claims 99-104; the proviral plasmid as described in claim 105; the AAV as described in any one of claims 89-91 or 106-113; or the composition as described in any one of claims 114-118, for the prevention or treatment of HD in a subject of need.

131. The HTT nucleic acid trans-splicing molecule as described in any one of claims 1-28 or 34-43; the nucleic acid trans-splicing molecule as described in any one of claims 29-33; the MSH3 exon-jumping nucleic acid construct as described in any one of claims 44-64; the MSH3 miRNA nucleic acid construct as described in any one of claims 65-69; the MSH3 nucleic acid trans-splicing molecule as described in any one of claims 70-74; the HTT trans-splicing and MSH3 exon-jumping nucleic acid construct as described in any one of claims 75-80; the HTT trans-splicing, HTT miRNA, and MSH3 exon-jumping nucleic acid construct as described in any one of claims 81-84; the HTT trans-splicing and MSH3 miRNA nucleic acid construct as described in any one of claims 85-88; the vector as described in any one of claims 99-104; the proviral plasmid as described in claim 105; the AAV as described in any one of claims 89-91 or 106-113; or the composition as described in any one of claims 114-118, for preparing a medicament for treating or preventing HD in a subject of need.

132. A method comprising introducing a nucleic acid trans-splicing molecule into a cell, the nucleic acid trans-splicing molecule being configured to splice to both a first target precursor mRNA and a second target precursor mRNA, wherein splicing to the first target precursor mRNA corrects a defect in the first target precursor mRNA, and wherein splicing to the second target precursor mRNA introduces a defect in the second target precursor mRNA.

133. The method of claim 132, wherein the nucleic acid trans-splicing molecule comprises a first binding domain configured to target an intron of the first target precursor mRNA and a second binding domain configured to target an intron of the second target precursor mRNA.

134. The method of claim 132 or 133, wherein the nucleic acid trans-splicing molecule further comprises a coding domain sequence, the coding domain sequence comprising a functional sequence of one or more exons of the first target precursor mRNA, the functional sequence correcting for defects in the first target precursor mRNA.

135. The method of any one of claims 132-134, wherein the defect in the second target precursor mRNA comprises a frameshift in the coding sequence of the second target precursor mRNA.

136. The method of claim 135, wherein the frameshift generates a premature stop codon in the second target precursor mRNA.

137. The method of any one of claims 132-136, wherein the defect comprises the elimination of the endogenous start codon of the second target precursor mRNA.

138. The method of any one of claims 132-137, wherein the defect comprises an inserted 5' UTR that prevents the translation of a protein encoded by the second target precursor mRNA.

139. The method of any one of claims 132-138, wherein the defect comprises an inserted 3' UTR that destabilizes the precursor mRNA or prevents the second target precursor mRNA from being exported from the cell nucleus.

140. The method of any one of claims 132-139, wherein the defect comprises removing the 5' cap or 3' polyA tail from the second target precursor mRNA.

141. The method of any one of claims 132-140, wherein the defect leads to nonsense-mediated degradation of the second target precursor mRNA.

142. The method of any one of claims 132-141, wherein the introduction results in a decrease in the abundance of the gene product of the second target precursor mRNA in the cell compared with the abundance of the gene product prior to the introduction.

143. A method for reducing protein abundance in a cell, the method comprising introducing a nucleic acid trans-splicing molecule into the cell, the nucleic acid trans-splicing molecule introducing a defect into a precursor mRNA encoding the protein.

144. The method of claim 143, wherein the defect comprises one or more of the following: (a) Introducing a frameshift into the coding sequence of the precursor mRNA; (b) Eliminate the endogenous start codon of the precursor mRNA; (c) Introducing a premature stop codon into the coding sequence of the precursor mRNA; (d) Replace the endogenous coding sequence of the precursor mRNA with an alternative coding sequence; (e) Inserting a 5' UTR that prevents the translation of the endogenous coding sequence of the precursor mRNA; (f) Insertion of a 3' UTR that destabilizes the precursor mRNA; (g) Insertion of a 3' UTR to prevent the precursor mRNA from being exported from the nucleus; (h) Remove the 5' cap from the precursor mRNA; or (i) Eliminate the 3' polyA tail from the precursor mRNA.

145. The method of claim 143 or 144, wherein the protein is MSH3.

146. The method of claim 145, wherein the nucleic acid trans-splicing molecule comprises a binding domain that binds to an intron of the precursor mRNA.

147. The method of claim 146, wherein the nucleic acid trans-splicing molecule comprises a heterologous coding domain sequence.

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