HTT trans-splicing molecule

Abstract

HTT nucleic acid trans-splicing molecules are described, which include a coding domain containing one or more exons of HTT, a splice site, and a binding domain that binds to a target intron of HTT pre-mRNA. The HTT nucleic acid trans-splicing molecules described herein can also be used in combination with, for example, an MSH3-binding domain arranged in tandem with an HTT-binding domain, an MSH3 nucleic acid trans-splicing molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA against either MSH3 or HTT, and an MSH3 or HTT microRNA (miRNA), as well as constructs encoding them. Compositions containing the nucleic acid trans-splicing molecules described herein also encompass compositions containing a combination of the nucleic acid trans-splicing molecule and an additional therapeutic agent (e.g., an MSH3 nucleic acid trans-splicing molecule, an MSH3 splice modulator, an antisense oligonucleotide or antisense RNA against either MSH3 or HTT). Nucleic acid trans-splicing molecules and compositions containing them can be used alone or in combination with additional therapeutic agents in methods for treating Huntington's disease (HD). Also described herein are nucleic acid trans-splicing molecules for use alone or in combination with additional therapeutic agents in the treatment of HD or in the preparation of a medicament for treating HD. Also included herein are MSH3 nucleic acid trans-splicing molecules, MSH3 splice modulators, and MSH3 miRNAs, and constructs encoding them, which may be used alone or in combination and / or in combination with additional therapeutic agents to treat a nucleotide repeat disorder (e.g., HD) or in the preparation of a medicament for treating a nucleotide repeat disorder (e.g., HD).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The said XML copy, created on February 14, 2024, is named 61313-709_601_SL.xml and is 521,958 bytes in size. [Background technology]

[0003] Huntington's disease (HD) is a movement-related progressive neurodegenerative disorder. The disease is associated with the loss of cortical pyramidal neurons, striatal medium spiny neurons, and hypothalamic neurons. The genetic cause of HD is autosomal dominant inheritance of an expanded CAG trinucleotide repeat in exon 1 of the HTT gene; the presence of more than 40 CAG repeats in this region causes the disease.

[0004] The HTT locus is large, spanning 180 kb and consisting of 67 exons, and expression of the HTT gene is required for normal development. Although the HTT protein is widely expressed, the brain is most severely affected by pathological expansions of CAG trinucleotide repeats, with early effects observed in the striatum and motor cortex. The mechanism underlying the development of HD is generally believed to be somatic CAG repeat expansions in HTT that occur in affected brain regions (e.g., the striatum) of HD patients.

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

[0006] Described herein are nucleic acid trans-splicing molecules that include a coding domain containing one or more exons of HTT, a splice site, and a binding domain that binds to a target intron of HTT pre-mRNA. In some embodiments, a construct containing the nucleic acid trans-splicing molecule can further include an additional sequence encoding an antisense RNA that binds to the target pre-mRNA, thereby blocking cis-splicing and thereby facilitating trans-splicing of the nucleic acid trans-splicing molecule to a pre-mRNA target (e.g., HTT pre-mRNA). In some embodiments, the nucleic acid trans-splicing molecule can further comprise a second binding domain that binds to a target intron in a second target pre-mRNA (e.g., MSH3 pre-mRNA), thereby trans-splicing it into the target intron of the second target pre-mRNA to generate a hybrid pre-mRNA containing one or more HTT exons and an exon of the second target pre-mRNA (e.g., MSH3 pre-mRNA), which is processed into a hybrid mRNA that is targeted for degradation, e.g., by nonsense-mediated decay. In some embodiments, a construct comprising the nucleic acid trans-splicing molecule can further comprise an additional sequence encoding a small nuclear RNA (snRNA) that blocks normal processing of the second target pre-mRNA (e.g., MSH3 pre-mRNA), e.g., through the introduction of a premature stop codon into the second target pre-mRNA (e.g., MSH3 pre-mRNA), which targets the second target mRNA (e.g., MSH3 mRNA) to nonsense-mediated decay. In some embodiments, constructs comprising nucleic acid trans-splicing molecules may further comprise elements encoding an antisense RNA that blocks normal processing of a second target pre-mRNA (e.g., MSH3 pre-mRNA) and prevents translation of MSH3, e.g., via blocking an MSH3 splice junction or annealing to the 5' UTR or first coding sequence of MSH3.In some embodiments, constructs containing nucleic acid trans-splicing molecules can further include an additional sequence encoding a microRNA (miRNA) specific to endogenous HTT mRNA or MSH3 mRNA, which promotes cleavage of endogenous HTT mRNA or MSH3 mRNA, respectively, thereby reducing the level of endogenous HTT mRNA or MSH3 mRNA. Compositions containing such nucleic acid trans-splicing molecules are also encompassed, as are compositions containing a combination of a nucleic acid trans-splicing molecule and an additional therapeutic agent (e.g., an antisense oligonucleotide or an antisense RNA-encoding construct). Nucleic acid trans-splicing molecules and compositions containing the same can be used alone or in combination with an additional therapeutic agent in methods for treating Huntington's disease (HD). Nucleic acid trans-splicing molecules and compositions containing the same can also be used alone or in combination with an additional therapeutic agent in the treatment of HD or in the preparation of a medicament for treating HD.

[0007] Disclosed herein are HTT nucleic acid trans-splicing molecules 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 pre-mRNA, wherein the target intron comprises intron 2. In some embodiments, the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 60-81, or a sequence having at least 90% identity to any one of SEQ ID NOs: 60-81.

[0008] Also disclosed herein is an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exons 1-3, (b) a splicing domain, and (c) a binding domain that binds to a target intron of HTT pre-mRNA, wherein the target intron comprises intron 3. In some embodiments, the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 158-174, or a sequence having at least 90% identity to any one of SEQ ID NOs: 158-174.

[0009] Also disclosed herein is an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exon 1; (b) a splicing domain; and (c) a binding domain that binds to a target intron of HTT pre-mRNA, wherein the target intron comprises intron 1, and the binding domain comprises any one of SEQ ID NOs: 8 to 21.

[0010] In some embodiments, in the HTT nucleic acid trans-splicing molecules described above, the coding domain comprises, consists essentially of, or consists of HTT exon 1; HTT exon 1 and HTT exon 2; or HTT exons 1-3. In some embodiments, the coding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 3, 59, 157, or 349-353, or a sequence having at least 90% identity to SEQ ID NOs: 3, 59, 157, or 349-353. In some embodiments, the coding domain, splicing domain, and binding domain are operably linked in a 5' to 3' direction.

[0011] In some embodiments, the HTT nucleic acid trans-splicing molecule further comprises a linker, wherein the coding domain, splicing domain, linker, and binding domain are operably linked in a 5' to 3' direction. In some embodiments, the linker is a sequence ranging from 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine; a sequence ranging from 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine. or a sequence ranging from 22 to 42 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine. In some embodiments, the linker comprises, consists essentially of, or consists of any one of SEQ ID NO:38 or a sequence having at least 90% identity to SEQ ID NO:38, SEQ ID NO:39 or a sequence having at least 90% identity to SEQ ID NO:39, SEQ ID NO:40 or a sequence having at least 90% identity to SEQ ID NO:40, or SEQ ID NO:41 or a sequence having at least 90% identity to SEQ ID NO:41.In some embodiments, the linker is selected from the group consisting of SEQ ID NO:37 or a sequence having at least 90% identity to SEQ ID NO:37, SEQ ID NO:42 or a sequence having at least 90% identity to SEQ ID NO:42, SEQ ID NO:43 or a sequence having at least 90% identity to SEQ ID NO:43, SEQ ID NO:44 or a sequence having at least 90% identity to SEQ ID NO:44, SEQ ID NO:45 or a sequence having at least 90% identity to SEQ ID NO:45, SEQ ID NO:46 or a sequence having at least 90% identity to SEQ ID NO:46, SEQ ID NO:106 or a sequence having at least 90% identity to SEQ ID NO:106, SEQ ID NO:107 or a sequence having at least 90% identity to SEQ ID NO:107. the sequence of SEQ ID NO: 112 or a sequence having at least 90% identity to SEQ ID NO: 112, SEQ ID NO: 197 or a sequence having at least 90% identity to SEQ ID NO: 197, or SEQ ID NO: 198 or a sequence having at least 90% identity to SEQ ID NO: 198.

[0012] In some embodiments, the HTT nucleic acid trans-splicing molecule further comprises a triple helix terminator, wherein the coding domain, splicing domain, linker (if present), binding domain, and triple helix terminator are operably linked in a 5' to 3' direction. In some embodiments, the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO:5 or a sequence having at least 90% identity to SEQ ID NO:5. In some embodiments, the triple helix terminator comprises, consists essentially of, or consists of SEQ ID NO:6.

[0013] In some embodiments, the HTT nucleic acid trans-splicing molecule further comprises a 5' untranslated region (5'UTR), wherein the 5'UTR, coding domain, splicing domain, linker (if present), binding domain, and triple helix terminator (if present) are operably linked in a 5' to 3' direction. In some embodiments, the 5'UTR is an HTT 5'UTR. In some embodiments, the HTT 5'UTR comprises, consists essentially of, or consists of any one of SEQ ID NOs: 136 or 192, or a sequence having at least 90% identity to any one of SEQ ID NOs: 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 (if present), epitope tag, coding domain, splicing domain, linker (if present), binding domain, and triple helix terminator (if present) are operably linked in a 5' to 3' direction. In some embodiments, the sequence encoding the epitope tag comprises, consists essentially of, or consists of SEQ ID NO:4.

[0015] Also disclosed herein is an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising 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 pre-mRNA, wherein the target intron comprises intron 2. In some embodiments, the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 60-81, or a sequence having at least 90% identity to any one of SEQ ID NOs: 60-81.

[0016] Also disclosed herein is an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exons 1-3, (b) a splicing domain, (c) a linker, and (d) a binding domain that binds to a target intron of HTT pre-mRNA, wherein the target intron comprises intron 3. In some embodiments, the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 158-174, or a sequence having at least 90% identity to any one of SEQ ID NOs: 158-174.

[0017] Also disclosed herein is an HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exon 1, (b) a splicing domain, (c) a linker, and (d) a binding domain that binds to a target intron of HTT pre-mRNA, wherein the target intron comprises intron 1. In some embodiments, the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 8-21, or a sequence having at least 90% identity to any one of SEQ ID NOs: 8-21.In some embodiments, the linker is selected from the group consisting of SEQ ID NO:37 or a sequence having at least 90% identity to SEQ ID NO:37, SEQ ID NO:38 or a sequence having at least 90% identity to SEQ ID NO:38, SEQ ID NO:39 or a sequence having at least 90% identity to SEQ ID NO:39, SEQ ID NO:40 or a sequence having at least 90% identity to SEQ ID NO:40, SEQ ID NO:41 or a sequence having at least 90% identity to SEQ ID NO:41, SEQ ID NO:42 or a sequence having at least 90% identity to SEQ ID NO:42, SEQ ID NO:43 or a sequence having at least 90% identity to SEQ ID NO:43, SEQ ID NO:44 or a sequence having at least 90% identity to SEQ ID NO:44, SEQ ID NO:45 or a sequence having at least 90% identity to SEQ ID NO:45, SEQ ID NO:46 or a sequence having at least 90% identity to SEQ ID NO:46, SEQ ID NO:47 or a sequence having at least 90% identity to SEQ ID NO:47, The nucleic acid sequence of any one of the following SEQ ID NOs: 106 or a sequence having at least 90% identity to SEQ ID NO: 106, SEQ ID NO: 107 or a sequence having at least 90% identity to SEQ ID NO: 107, SEQ ID NO: 108 or a sequence having at least 90% identity to SEQ ID NO: 108, SEQ ID NO: 109 or a sequence having at least 90% identity to SEQ ID NO: 109, SEQ ID NO: 110 or a sequence having at least 90% identity to SEQ ID NO: 110, SEQ ID NO: 111 or a sequence having at least 90% identity to SEQ ID NO: 111, SEQ ID NO: 112 or a sequence having at least 90% identity to SEQ ID NO: 112, SEQ ID NO: 197 or a sequence having at least 90% identity to SEQ ID NO: 197, or SEQ ID NO: 198 or a sequence having 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, linker, binding domain, and triple helix terminator are operably linked in a 5' to 3' direction; optionally, the molecule further comprises a 5' UTR, wherein the 5' UTR (if present), coding domain, splicing domain, linker, binding domain, and triple helix terminator (if present) are operably linked in a 5' to 3' direction.

[0018] Also disclosed herein are nucleic acid trans-splicing molecules comprising a linker, wherein the linker comprises, consists essentially of, or consists of a sequence ranging from 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine. In some embodiments, the linker comprises, consists essentially of, or consists of a sequence ranging from 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine. In some embodiments, the linker comprises, consists essentially of, or consists of a sequence ranging from 22 to 42 nucleotides in length, the linker comprising 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine. In some embodiments, the linker comprises, consists essentially of, or consists of SEQ ID NO:38 or a sequence having at least 90% identity to SEQ ID NO:38, SEQ ID NO:39 or a sequence having at least 90% identity to SEQ ID NO:39, SEQ ID NO:40 or a sequence having at least 90% identity to SEQ ID NO:40, or SEQ ID NO:41 or a sequence having at least 90% identity to SEQ ID NO:41.

[0019] Also disclosed herein are nucleic acid trans-splicing molecules comprising a linker, wherein the linker is selected from the group consisting of SEQ ID NO:37 or a sequence having at least 90% identity to SEQ ID NO:37, SEQ ID NO:42 or a sequence having at least 90% identity to SEQ ID NO:42, SEQ ID NO:43 or a sequence having at least 90% identity to SEQ ID NO:43, SEQ ID NO:44 or a sequence having at least 90% identity to SEQ ID NO:44, SEQ ID NO:45 or a sequence having at least 90% identity to SEQ ID NO:45, SEQ ID NO:46 or a sequence having at least 90% identity to SEQ ID NO:46, SEQ ID NO:106 or a sequence having at least 90% identity to SEQ ID NO:106, SEQ ID NO:107 or a sequence having at least 90% identity to SEQ ID NO:107. The nucleic acid sequence of the present invention may comprise, consist essentially of, or consist of: SEQ ID NO: 108 or a sequence having at least 90% identity to SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity to SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity to SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity to SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity to SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity to SEQ ID NO: 197; or SEQ ID NO: 198 or a sequence having at least 90% identity to SEQ ID NO: 198.

[0020] In some embodiments of the above-described HTT nucleic acid trans-splicing molecule, the HTT nucleic acid trans-splicing molecule further comprises a binding domain that binds to a target intron of MSH3 pre-mRNA. In some embodiments, the MSH3 target intron comprises any one of intron 5 or intron 15 of MSH3. In some embodiments, the binding domain that binds to the target intron of MSH3 pre-mRNA comprises, consists essentially of, or consists of any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210, or a sequence having at least 90% identity to any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210. In some embodiments, the nucleic acid trans-splicing molecule comprises any one of SEQ ID NOs: 149-154 or SEQ ID NOs: 212-223, or a sequence having at least 90% identity to any one of SEQ ID NOs: 149-154 or SEQ ID NOs: 212-223.

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

[0022] Also disclosed herein is an MSH3 exon skipping nucleic acid construct comprising: (a) a sequence encoding an antisense RNA that promotes exon skipping of a target exon in an MSH3 pre-mRNA, wherein the target exon is any one of MSH3 exons 2-4, 6-8, or 15, and the target exon comprises a 5' exon-intron junction sequence and a 3' exon-intron junction sequence; and (b) a sequence encoding a small nuclear RNA (snRNA) sequence, operably linked to the sequences. In some embodiments, the MSH3 exon skipping nucleic acid construct further comprises a U1 promoter and a U1 terminator operably 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 either the 5' exon-intron junction or the 3' exon-intron junction of the target exon. In some embodiments, the antisense RNA is any one of SEQ ID NOs: 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 ... , 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 MSH3 exon skipping nucleic acid construct comprises any one of SEQ ID NOs: 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, hi some embodiments, the antisense RNA comprises a sequence that is at least 80% complementary to the entire sequence of the targeted exon.

[0024] In some embodiments, the antisense RNA further comprises (a) a sequence that is at least 80% complementary to a 5-nucleotide sequence upstream of the 5' exon-intron junction, and (b) a sequence that is 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 NOs: 299, 304, 309, 314, or 319, or a sequence having at least 90% identity to any one of SEQ ID NOs: 299, 304, 309, 314, or 319. In some embodiments, the MSH3 exon skipping nucleic acid construct described above comprises any one of SEQ ID NOs: 324, 327, 330, 333, or 336.

[0025] In some embodiments, the antisense RNA comprises, operably linked in the 5' to 3' direction, (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) a sequence targeting the 5' exon-intron junction. In some embodiments, the linker sequence is less than 50% complementary to any sequence in the target exon of the same length as the linker. In some embodiments, the antisense RNA comprises any one of SEQ ID NOs: 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 NOs: 325, 326, 328, 329, 331, 332, 334, 335, 337, or 338.

[0026] In some embodiments, the antisense RNA targets MSH3 exon 7. In some embodiments, the MSH3 exon skipping nucleic acid construct comprises SEQ ID NO: 309. In some embodiments, the MSH3 exon skipping 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 NOs: 309, 310, 311, 312, or 313, or any combination thereof, or a sequence having at least 90% identity to any one of SEQ ID NOs: 309, 310, 311, 312, or 313. In some embodiments, the MSH3 exon skipping nucleic acid construct comprises at least one of SEQ ID NOs: 330-332, or any combination thereof.

[0027] Also disclosed herein is an MSH3 miRNA nucleic acid construct comprising a sequence encoding a pri-miRNA comprising a scaffold sequence, a loop sequence, and a miRNA sequence that targets an 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 NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257, or a sequence having at least 90% identity to any one of SEQ ID NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257. In some embodiments, the scaffold sequence comprises any one of SEQ ID NOs: 227, 228, 230, 231, 259, or 260. In some embodiments, the loop sequence comprises any one of SEQ ID NOs: 229, 232, or 261. In some embodiments, the pri-miRNA sequence comprises any one of SEQ ID NOs: 234, 235, 238-241, or 262-269. In some embodiments, the sequence encoding the pri-miRNA is operably linked to a U6 promoter or a CMV promoter.

[0028] Also disclosed herein are MSH3 nucleic acid trans-splicing molecules comprising (a) a coding domain sequence, (b) a splicing domain, and (c) a binding domain that binds to a target intron of MSH3 pre-mRNA, wherein the coding domain sequence is not the MSH3 coding domain sequence. In some embodiments, the coding domain sequence comprises a sequence that, when trans-spliced ​​into the MSH3 pre-mRNA, causes a frameshift in the mature MSH3 mRNA. In some embodiments, the coding domain sequence comprises one or more of exons 1, 2, and 3 of HTT. In some embodiments, the target intron of the MSH3 pre-mRNA is intron 5 or intron 15. In some embodiments, the binding domain comprises any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210, or a sequence having at least 90% identity to any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210.

[0029] Also disclosed herein are HTT trans-splicing and MSH3 exon skipping nucleic acid constructs comprising (a) any of the above-described HTT nucleic acid trans-splicing molecules and (b) any of the above-described MSH3 exon skipping 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 skipping nucleic acid construct comprises any one of SEQ ID NOs: 356, 357, 363, or 364. In some embodiments, the AAV vector is an scAAV or ssAAV vector. In some embodiments, the HTT trans-splicing and MSH3 exon skipping nucleic acid construct comprises any one of SEQ ID NOs: 369, 370, and 371.

[0030] Also disclosed herein are HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid constructs comprising (a) any of the above-described HTT nucleic acid trans-splicing molecules, and (b) any of the above-described MSH3 exon skipping 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 skipping nucleic acid construct comprises any one of SEQ ID NOs: 358 or 359. In some embodiments, the single vector is an AAV vector.

[0031] Also disclosed herein are HTT trans-splicing and MSH3 miRNA nucleic acid constructs 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 NOs: 354 or 355. In some embodiments, the vector is an AAV vector.

[0032] Also disclosed are AAV vectors comprising any of the above-mentioned HTT nucleic acid trans-splicing molecules. In some embodiments, the AAV vector comprises any one of SEQ ID NOs: 356, 357, 363, or 364.

[0033] Also disclosed are any of the above HTT nucleic acid trans-splicing molecules or AAV vectors comprising any of the above nucleic acid trans-splicing molecules.

[0034] Also disclosed are ribonucleic acid trans-splicing molecules comprising any one of SEQ ID NOs: 23-36, 47-56, 83-105, 113-125, 175-191, or 199-206.

[0035] Also disclosed are ribonucleic acid trans-splicing molecules transcribed from any of the above HTT nucleic acid trans-splicing molecules or from any of the above nucleic acid trans-splicing molecules.

[0036] In some embodiments of the above-mentioned HTT nucleic acid trans-splicing molecule, the HTT pre-mRNA comprises at least one mutation associated with Huntington's disease (HD). In some embodiments, the at least one mutation associated with HD comprises a CAG repeat expansion in an HTT gene allele. In some embodiments, the CAG repeat expansion in an HTT gene allele comprises more than 35 CAG repeats. In some embodiments, the at least one mutation associated with HD is autosomal dominant. In some embodiments, the at least one mutation associated with HD is expressed in at least one of cortical pyramidal neurons, striatal medium spiny neurons, or hypothalamic neurons.

[0037] Also disclosed are vectors comprising any of the above HTT nucleic acid trans-splicing molecules; any of the above nucleic acid trans-splicing molecules; any of the above MSH3 exon skipping nucleic acid constructs; any of the above MSH3 miRNA nucleic acid constructs; any of the above MSH3 nucleic acid trans-splicing molecules; any of the above HTT trans-splicing and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid constructs; or any of the above HTT trans-splicing and MSH3 miRNA nucleic acid constructs.

[0038] Also disclosed are vectors comprising any of the above-described HTT nucleic acid trans-splicing molecules. In some embodiments, the vector comprises a 5' regulatory domain operably linked to the 5' side of 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 comprises a constitutive promoter or a tissue-specific promoter. In some embodiments, the constitutive promoter is a CMV promoter or a CAGGS promoter.

[0039] Also disclosed are proviral plasmids comprising any of the above HTT nucleic acid trans-splicing molecules; any of the above nucleic acid trans-splicing molecules; any of the above MSH3 exon skipping nucleic acid constructs; any of the above MSH3 miRNA nucleic acid constructs; any of the above MSH3 nucleic acid trans-splicing molecules; any of the above HTT trans-splicing and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid constructs; or any of the above HTT trans-splicing and MSH3 miRNA nucleic acid constructs.

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

[0041] Also disclosed are adeno-associated viruses (AAV) comprising any of the above-described HTT nucleic acid trans-splicing molecules, wherein the AAV optionally comprises a 5' regulatory domain operably linked to the 5' side of the nucleic acid trans-splicing molecule. In some embodiments, the AAV comprises a 5' regulatory domain operably linked to the 5' side of 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 comprises a constitutive promoter. In some embodiments, the constitutive promoter is a CMV promoter or a CAGGS promoter. In some embodiments, the AAV exhibits neuronal tropism. In some embodiments, the AAV is AAV9, AAV8, AAV5, AAV2, AAV7, or AAV2.7m8, AAV-retro, AAV1, AAV4, or AAV-PHP.eB.

[0042] Also disclosed are compositions comprising any of the above HTT nucleic acid trans-splicing molecules; any of the above nucleic acid trans-splicing molecules; any of the above MSH3 exon skipping nucleic acid constructs; any of the above MSH3 miRNA nucleic acid constructs; any of the above MSH3 nucleic acid trans-splicing molecules; any of the above HTT trans-splicing and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above vectors; any of the above proviral plasmids; or any of the above 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 pre-mRNA. In some embodiments, at least one antisense oligonucleotide comprises any one of SEQ ID NOs: 126-135, or a construct encoding at least one antisense RNA binds to a target sequence bound by any one of SEQ ID NOs: 126-135. In some embodiments, at least one antisense oligonucleotide comprises SEQ ID NO: 131, or a construct encoding at least one antisense RNA binds to a target sequence bound by SEQ ID NO: 131.

[0043] Also disclosed are methods for expressing biologically active HTT in a target cell and restoring functional levels of HTT protein in the target cell, comprising transducing a target cell with 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 skipping 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 skipping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions. 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 the HTT pre-mRNA containing at least one mutation associated with HD in the target cell is replaced. 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 HTT pre-mRNA containing at least one mutation associated with HD in the target cell is 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 HTT pre-mRNA containing at least one mutation associated with HD in the target cell is replaced. In some embodiments, functional levels of HTT are restored in the target cell by expressing biologically functional HTT protein and / or mutant HTT RNA, resulting in a decrease in related transcripts (e.g., HTT1a).

[0044] Also disclosed are methods for reducing expression of HTT containing a polyglutamine repeat of more than 35 consecutive glutamine residues in a subject, the method comprising transfecting or transducing a target cell, more particularly a neuron, in the subject with 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 skipping 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 skipping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions.

[0045] Also disclosed are methods for correcting at least one mutation in an HTT exon sequence of an HTT pre-mRNA in a target cell of a subject, comprising administering to a 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 skipping 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 skipping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions.

[0046] Also disclosed are methods of treating Huntington's disease (HD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of 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 skipping 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 skipping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions.

[0047] In some embodiments of the above-mentioned method, the method comprises administering to the brain of a subject 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 skipping 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 skipping nucleic acid constructs; any of the above-mentioned HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions. In some embodiments, the subject is a mammal, preferably a rodent, non-human primate, or human. In some embodiments, the subject has a genetic predisposition to HD or has been diagnosed with HD.

[0048] Also disclosed are any of the above HTT nucleic acid trans-splicing molecules; any of the above nucleic acid trans-splicing molecules; any of the above MSH3 exon skipping nucleic acid constructs; any of the above MSH3 miRNA nucleic acid constructs; any of the above MSH3 nucleic acid trans-splicing molecules; any of the above HTT trans-splicing and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing, HTT miRNA, and MSH3 exon skipping nucleic acid constructs; any of the above HTT trans-splicing and MSH3 miRNA nucleic acid constructs; any of the above vectors; any of the above proviral plasmids; any of the above AAVs; or any of the above compositions for use in preventing or treating HD in a subject in need thereof.

[0049] Also disclosed herein is 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 skipping 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 skipping nucleic acid constructs; any of the above-described HTT trans-splicing, HTT miRNA, and MSH3 exon skipping 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 compositions, for use in the preparation of a medicament for treating or preventing HD in a subject in need thereof.

[0050] Also disclosed are methods comprising introducing into a cell a nucleic acid trans-splicing molecule configured to splice to both a first target pre-mRNA and a second target pre-mRNA, where splicing to the first target pre-mRNA corrects a defect in the first target pre-mRNA and splicing to the second target pre-mRNA introduces a defect in the second target pre-mRNA. In some embodiments, the nucleic acid trans-splicing molecule comprises a first binding domain configured to target an intron of the first target pre-mRNA and a second binding domain configured to target an intron of the second target pre-mRNA. In some embodiments, the nucleic acid trans-splicing molecule further comprises a coding domain sequence comprising functional sequences of one or more exons of the first target pre-mRNA that correct the defect in the first target pre-mRNA. In some embodiments, the defect in the second target pre-mRNA comprises a frameshift in the coding sequence of the second target pre-mRNA. In some embodiments, the frameshift creates a premature stop codon in the second target pre-mRNA. In some embodiments, the defect comprises an endogenous start codon of the second target pre-mRNA that is removed. In some embodiments, the defect comprises an inserted 5' UTR that prevents translation of a protein encoded by the second target pre-mRNA. In some embodiments, the defect comprises an inserted 3' UTR that destabilizes the pre-mRNA or prevents export of the second target pre-mRNA from the nucleus. In some embodiments, the defect comprises removal of a 5' cap or 3' polyA tail from the second target pre-mRNA. In some embodiments, the defect results in nonsense-mediated decay of the second target pre-mRNA. In some embodiments, the introduction reduces the abundance of a gene product of the second target pre-mRNA in the cell compared to the abundance of the gene product before the introduction.

[0051] Also disclosed are methods for reducing the abundance of a protein in a cell, comprising introducing into the cell a nucleic acid trans-splicing molecule that introduces a defect in a pre-mRNA encoding the protein. In some embodiments, the defect comprises one or more of: (a) a frameshift introduced into the coding sequence of the pre-mRNA; (b) removal of the endogenous start codon of the pre-mRNA; (c) introduction of a premature stop codon into the coding sequence of the pre-mRNA; (d) replacement of the endogenous coding sequence of the pre-mRNA with an alternative coding sequence; (e) insertion of a 5' UTR that prevents translation of the endogenous coding sequence of the pre-mRNA; (f) insertion of a 3' UTR that destabilizes the pre-mRNA; (g) insertion of a 3' UTR that prevents the pre-mRNA from being exported from the nucleus; (h) removal of the 5' cap from the pre-mRNA; or (i) removal of the 3' polyA tail from the pre-mRNA. In some embodiments, the protein is MSH3. In some embodiments, the nucleic acid trans-splicing molecule comprises a binding domain that binds to an intron of the pre-mRNA. In some embodiments, the nucleic acid trans-splicing molecule comprises a heterologous coding domain sequence. [Brief explanation of the drawings]

[0052] [Figure 1] The range of CAG trinucleotide repeats found in exon 1 of the HTT gene is shown. As shown, 8 to 35 CAG trinucleotide repeats reflect the phenotypically normal (wild-type) range found in humans who show no signs of disease associated with the HTT gene. 35 to 39 CAG trinucleotide repeats are associated with incomplete penetrance of HD. The presence of more than 40 CAG trinucleotide repeats in this region of exon 1 of the HTT gene is responsible for HD disease. [Figure 2] Figure 1 shows the somatic CAG repeat expansion of an expanded CAG trinucleotide repeat in exon 1 of the HTT gene, the mechanism underlying the development of HD. [Figure 3]We demonstrate exon editing by HTT pre-mRNA trans-splicing as a therapeutic approach for HD. Intron 1-directed, intron 2-directed, or intron 3-directed exon editors were designed and tested for their efficiency in replacing mutant HTT exon 1. [Figure 4] 1 shows an exemplary general structure of a construct encoding an RNA exon editor targeting HTT intron 1. The exemplary exon editor produced by the depicted construct includes a 5'UTR, exon 1 coding sequence, a splice donor site, a linker, a binding domain, and a terminator sequence. The binding domain was varied to target different locations along intron 1 of HTT. In some embodiments, the promoter is a CMV promoter, in some embodiments, the 5'UTR is an HTT 5'UTR, in some embodiments, the linker is a 40-mer linker, in some embodiments, an HTT 5'UTR is combined with a 40-mer linker, and in some embodiments, an epitope tag is included, one example of which is an N-terminal 3X FLAG tag for on-target protein detection. The foregoing embodiments may be combined, such that at least one of these embodiments is included within an RNA exon editor, and any combination thereof, for example, a combination of all of these embodiments, is included in a single RNA exon editor. [Figure 5]Figure 1 shows a graph depicting HTT intron 1-directed RNA exon editors, showing different levels of trans-splicing efficiency (% substitution) depending on where the binding domain is targeted within the intron. HEK293 cells were transfected with HTT intron 1-directed RNA exon editors targeting various regions of intron 1. 48 hours after transfection, cells were harvested and assayed for trans-splicing efficiency by RT-qPCR. Binding domains are designated as follows: (nucleotide base position in the intron at the 5' end start position of the binding domain, numbered according to SEQ ID NO: 1)_(length of binding domain (nt)). For example, 701_150 indicates that the binding domain is the reverse complement of bases 701 to 850 within intron 1 (numbered according to SEQ ID NO: 1). The results shown are for an exemplary HTT intron 1-directed RNA exon editor containing the indicated binding domain target, where the 5'UTR comprises the HTT 5'UTR (SEQ ID NO: 136) and the linker comprises a 40-mer linker (SEQ ID NO: 37). NBD_150 is a control editor in which the binding domain that targets HTT has been replaced with a binding domain that does not target HTT. [Figure 6]The location and trans-splicing efficiency (% substitution) of HTT intron 1-directed RNA exon editors are shown. Various levels of trans-splicing efficiency (% substitution) were determined, reflecting the location within the intron to which the binding domain is targeted. HEK293 cells were transfected with HTT intron 1-directed RNA exon editors targeting various regions of intron 1. Forty-eight hours after transfection, cells were harvested and assayed for trans-splicing efficiency by RT-qPCR. The binding domains are designated as follows: (nucleotide base position in the intron at the 5' end start position of the binding domain, numbered according to SEQ ID NO: 1)_(length of the binding domain (nt)). For example, 701_150 indicates that the binding domain is the reverse complement of bases 701 to 850 within intron 1 (numbered according to SEQ ID NO: 1). The results shown are for an exemplary HTT intron 1-directed RNA exon editor containing the indicated binding domain target, where the 5'UTR comprises the HTT 5'UTR and the linker comprises a 40-mer linker. [Figure 7] 1 shows the general structure of an exemplary construct encoding an HTT intron 1-directed RNA exon editor. An exemplary exon editor is shown, whose expression is driven by a CMV promoter. The exemplary exon editor shown includes an HTT 5'UTR, an N-terminal 3X FLAG tag, exon 1 coding sequence, a splice donor site, a linker, a binding domain (HTT_intron1_11704_100), and a terminator sequence. [Figure 8] Figure 1 shows the activity of exemplary HTT intron 1-directed RNA exon editors containing various linkers. As shown, some linkers increased trans-splicing efficiency in the HTT intron 1 (HTT_intron1_11704_100) exon editor compared to a 40-mer linker. HEK293 cells were transfected with HTT intron 1-directed RNA exon editors containing the indicated linkers. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR. [Figure 9] 1 shows the general structure of an exemplary construct encoding an RNA exon editor that targets HTT intron 2. The exemplary exon editor shown includes a 5'UTR, exon 1-2 coding sequence, a splice donor site, a linker, a binding domain, and a terminator sequence. The binding domain was varied to target different locations along intron 2 of HTT. In some embodiments, the 5'UTR is an HTT 5'UTR, and in some embodiments, the linker is a 40-mer linker, and in some embodiments, the HTT 5'UTR is combined with a 40-mer linker. In some embodiments, the promoter is a CMV promoter, in some embodiments, the 5'UTR is an HTT 5'UTR, in some embodiments, the linker is a 40mer linker, in some embodiments, a CMV promoter is combined with an HTT 5'UTR, in some embodiments, a CMV promoter is combined with an HTT 5'UTR and a 40mer linker, and in some embodiments, an epitope tag is included, one example of which is an N-terminal 3X FLAG tag for on-target protein detection. The foregoing embodiments may be combined, such that at least one of these embodiments is included within an RNA exon editor, and any combination thereof, for example, a combination of all of these embodiments, is included in one RNA exon editor. [Figure 10]Figure 1 shows the activity of various exemplary HTT intron 2-directed RNA exon editors, which exhibit different levels of trans-splicing efficiency (% replacement) depending on where the binding domain is targeted within the intron. HEK293 cells were transfected with exemplary HTT intron 2-directed RNA exon editors targeting various regions of intron 2. 48 hours after transfection, cells were harvested and assayed for trans-splicing efficiency by RT-qPCR. Binding domains are designated as follows: (nucleotide base position in the intron at the 5'-end start position of the binding domain, numbered according to SEQ ID NO: 57)_(length of binding domain (nt)). Results shown are for exemplary HTT intron 2-directed RNA exon editors containing the indicated binding domain targets, where the 5'UTR comprises the HTT 5'UTR and the linker comprises a 40-mer linker. NBD is a control editor in which the HTT-targeting binding domain is replaced with a binding domain that does not target HTT. Splice variants are control editors that lack a functional splice donor site. [Figure 11] The location and trans-splicing efficiency (% replacement) of HTT intron 2-directed RNA exon editors are illustrated. Various levels of trans-splicing efficiency (% replacement) were determined, reflecting where the binding domain targets the intron. HEK293 cells were transfected with HTT intron 2-directed RNA exon editors targeting various regions of intron 2. Forty-eight hours after transfection, cells were harvested and assayed for trans-splicing efficiency by RT-qPCR. The binding domains are designated as follows: (nucleotide base position in the intron at the 5'-end start position of the binding domain, numbered according to SEQ ID NO: 57)_(length of the binding domain (nt)). The results shown are for an exemplary HTT intron 2-directed RNA exon editor containing the indicated binding domain target, where the 5'UTR comprises the HTT 5'UTR and the linker comprises a 40-mer linker. [Figure 12A]Figure 1 shows an exemplary HTT intron 2-directed RNA exon editor that targets the region upstream of the branch site. The RNA exon editors were designed to target the region upstream of the intron 2 branch site and have different binding domain lengths. Expression of the exon editor is driven by a CMV promoter. The exon editor contains the HTT 5'UTR, an N-terminal 3X FLAG tag for on-target protein detection, exon 1-2 coding sequence, a splice donor site, a 41mer_2 linker, the indicated binding domain, and a terminator sequence. The binding domains are named as follows: (nucleotide base position in the intron at the 5' end start position of the binding domain, numbered according to SEQ ID NO: 57)_(length of the binding domain (nt)). The binding domains were varied to target different lengths upstream of the branch site in intron 2. [Figure 12B] Figure 1 shows an exemplary HTT intron 2-directed RNA exon editor targeting the upstream region of the branch site. The length of the binding domain affects targeting efficiency, based on calculation of % replacement. HEK293 cells were transfected with HTT intron 2-directed RNA exon editors with different binding domain lengths. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR. [Figure 13]

[0023] Figure 1 shows the general structure of an exemplary construct encoding an HTT intron 2-directed RNA exon editor. Expression of the exon editor is driven by a CMV promoter. The exemplary exon editor includes the HTT 5'UTR, an N-terminal 3X FLAG tag, exon 1-2 coding sequence, a splice donor site, a linker, a binding domain (HTT_intron2_12061_150), and a terminator sequence. [Figure 14]This shows that exon editors containing the indicated linkers do not exhibit significantly different trans-splicing efficiencies compared to a 40-mer linker in HTT intron 2 (HTT_intron2_12061_150). HEK293 cells were transfected with HTT intron 2-directed RNA exon editors that differed in the linker they contained. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR. [Figure 15] A schematic diagram showing the trans-splicing reaction and its competition with cis-splicing is shown. Abbreviations used in the figure: MALAT1 terminator (term); binding domain (BD); linker (L); splice site (SS). The cis-spliced ​​molecule resulting from cis-splicing contains a CAG repeat expansion. The trans-spliced ​​chimeric molecule resulting from 5' trans-splicing mediated by HTT5'RTM contains an HTT exon 1 with a normal number of CAG repeats (8-35). Abbreviations used in the figure: MALAT1 terminator (term); binding domain (BD); linker (L); splice site (SS); codon optimized (C / O). [Figure 16]A schematic diagram showing antisense oligonucleotides (ASOs) designed to block competing cis-splice sites (ASOs 8–10) and cis-splice sites for upstream exons (ASOs 2–7) is shown. Each of these ASOs was cotransfected with the indicated HTT intron 2-directed exon editor (HTT_intron2_12061_150) and assayed for trans-splicing efficiency in vitro in HEK293 cells. Abbreviations used in the diagram: MALAT1 terminator (term); binding domain (BD); linker (L); splice site (SS). [Figure 17] Figure 1 shows the percent replacement activity of an exemplary HTT intron 2-directed exon editor (HTT_intron2_12061_150) in combination with the indicated ASO. ASO6, designed to block cis-splicing of the upstream intron, resulted in improved trans-splicing efficiency in vitro. HEK293 cells were co-transfected with an exemplary HTT intron 2-directed RNA exon editor construct (REEC) and an ASO designed to block a competing cis-splice site or an ASO designed to block splicing of the upstream intron. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR. [Figure 18]Representative RT-qPCR and Western blot images (probed for the N-terminal FLAG epitope) of whole-cell lysates from HEK293 cells transfected with HTT RNA exon editors containing the indicated elements are shown. It is noteworthy that the level of on-target protein detection correlates with trans-splicing efficiency, expressed as the percent displacement of HTT RNA. HEK293 cells were transfected with N-terminally FLAG-tagged HTT exon editors with a range of activities based on RT-qPCR assays (top panel). An anti-FLAG antibody was used to detect proteins generated after successful trans-splicing in whole-cell lysates (bottom panel). An anti-huntingtin 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 ONT band corresponds to the relative performance of the exon editor based on qPCR. [Figure 19] A schematic illustrating the potential mechanism of a hybrid therapeutic approach designed to treat HD is shown. In this approach, a drug that inhibits somatic CAG expansion (e.g., by reducing MSH3) is combined with an RNA exon editor that targets HTT pre-mRNA. The RNA exon editor targets HTT pre-mRNA and functions to replace any mutant HTT RNA that may be generated from DNA that "escapes" the inhibition of the somatic expansion process. [Figure 20]

[0023] Figure 1 shows a schematic diagram illustrating the possible mechanism of action of a tandem-binding domain RNA exon editor targeting HTT and MSH3 pre-mRNA. In one exemplary embodiment, expression of the exon editor is driven by a CMV promoter. Such an exemplary exon editor may include an HTT 5'UTR, an N-terminal 3X FLAG tag, an HTT exon 1 coding sequence, a splice donor site, a linker, an MSH3-binding domain (e.g., targeting intron 5 or intron 15 of the MSH3 pre-mRNA), an HTT-binding domain (e.g., HTT_intron1_11704_100), and a terminator sequence. The HTT-binding domain targets the exon editor to generate a corrected HTT RNA after successful trans-splicing, and the MSH3-binding domain targets the exon editor to generate an HTT exon 1-MSH3 chimeric RNA molecule with a premature stop codon, which is subject to nonsense-mediated decay (NMD) and subsequently reduces MSH3 expression. [Figure 21] Panels A–C show that tandem-binding domain RNA exon editors targeting HTT and MSH3 demonstrate successful trans-splicing of both pre-mRNAs. RT-qPCR was performed in HEK293 cells transfected with tandem-binding domain RNA exon editors targeting HTT intron 1 and MSH3 intron 5 to measure A) HTT on-target (ONT) trans-splicing efficiency (via the HTT-binding domain), B) HTT-MSH3 chimera trans-splicing efficiency (via the MSH3-binding domain), and C) MSH3 RNA transcript expression. [Figure 22]Panels A–C show results demonstrating that tandem-binding domain RNA exon editors targeting HTT and MSH3 demonstrate successful trans-splicing of both pre-mRNAs. RT-qPCR was performed in HEK293 cells transfected with tandem-binding domain RNA exon editors targeting HTT intron 1 and MSH3 intron 15 to measure A) HTT on-target (ONT) trans-splicing efficiency (via the HTT-binding domain), B) HTT-MSH3 chimera trans-splicing efficiency (via the MSH3-binding domain), and C) MSH3 RNA transcript expression. [Figure 23] 1 shows the general structure of an exemplary construct encoding an HTT intron 3-directed RNA exon editor. The exemplary exon editor shown includes a 5' UTR, exon 1-3 coding sequence, a splice donor site, a linker, a binding domain, and a terminator sequence. The binding domain was varied to target different locations along intron 3 of HTT. In some embodiments, the 5' UTR is the HTT 5' UTR, and in some embodiments, the linker is a 40-mer linker. In some embodiments, the promoter is a CMV promoter, in some embodiments, the 5' UTR is the HTT 5' UTR, and in some embodiments, the linker is a 40-mer linker. In some embodiments, the CMV promoter is combined with the HTT 5' UTR, and in some embodiments, the CMV promoter is combined with the HTT 5' UTR and a 40-mer linker. In some embodiments, an epitope tag is included, such as an N-terminal 3X FLAG tag for on-target protein detection. The foregoing embodiments may be combined, such that at least one of these embodiments is included within an RNA exon editor, and any combination thereof, for example, a combination of all of these embodiments, is included in one RNA exon editor. [Figure 24]Figure 1 shows the activity of various exemplary HTT intron 3-directed RNA exon editors, showing different levels of trans-splicing efficiency (% replacement) depending on the binding site within the intron targeted by the binding domain. HEK293 cells were transfected with HTT intron 3-directed RNA exon editors targeting various regions of intron 3. 48 hours after transfection, cells were harvested and assayed for trans-splicing efficiency by RT-qPCR. Binding domains are designated as follows: (nucleotide base position within intron 3 at the 5'-end start position of the binding domain, numbered according to SEQ ID NO: 155)_(length of binding domain (nt)). [Figure 25] A direct comparison of HTT intron 2-directed exon editors and HTT intron 3-directed exon editors is shown. HEK293 cells were transfected in parallel with HTT intron 2-directed and intron 3-directed RNA exon editors. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR. Binding domains are designated as follows: (nucleotide base position within intron 3 at the 5' end start position of the binding domain)_(length of the binding domain (nt)). [Figure 26] This shows that self-splicing reduction does not affect the trans-splicing efficiency of the HTT exon editor. Table 1 shows the cryptic splice sites identified in the original exon editor and the sequence changes made in the self-splicing reduction exon editor. HEK293 cells were transfected with the HTT intron 2-directed (HTT_intron2_12061_150) RNA exon editor with or without self-splicing reduction. Cells were harvested 48 hours after transfection and assayed for trans-splicing efficiency by RT-qPCR. [Figure 27]Representative RT-qPCR and Western blot images of lysates from HEK293 cells transfected with HTT RNA exon editors testing two promoter and different 5'UTR combinations are shown. HEK293 cells were transfected with N-terminally FLAG-tagged HTT exon editors driven by either the CMV or CAGGS promoter, with or without the HTT 5'UTR, and testing the wild-type (GTAAGT) splice site targeting intron 2 (HTT_intron2_12061_150), a splice variant targeting intron 2 (HTT_intron2_12061_150), or the wild-type splice site with a non-targeting binding domain (NBD). RT-qPCR was performed on RNA from these cells (upper panel). The α-FLAG antibody was used to detect proteins generated after successful trans-splicing, which contain an N-terminal FLAG epitope, in whole cell lysates (lower panel). ONT: on-target HTT protein resulting from successful trans-splicing. NSP: unspliced ​​protein. [Figure 28] This figure shows that unspliced ​​protein (NSP) is reduced in a combinatorial manner by including three tandem repeats of the U1 snRNA binding site (3X UBS; SEQ ID NO: 345) and an AU-rich element (ARE; SEQ ID NO: 346) in an exemplary 5' HTT intron 1-directed exon editor. HEK293 cells were transfected with an HTT intron 1-directed (HTT_intron1_11704_100) RNA exon editor in which the respective linker regions were altered to include the indicated NSP-reducing elements. 48 hours after transfection, cells were harvested and subjected to Western blot analysis. ONT: on-target HTT protein due to successful trans-splicing. NSP: unspliced ​​protein. [Figure 29]Figure 1 shows that unspliced ​​protein (NSP) is reduced in a combinatorial manner by including three tandem repeats of the U1 snRNA binding site (3X UBS; SEQ ID NO: 345) and an AU-rich element (ARE; SEQ ID NO: 346) in a 5' HTT intron 2-directed exon editor. HEK293 cells were transfected with an HTT intron 2-directed (HTT_intron2_12061_150) RNA exon editor in which the respective linker regions had been altered to include the indicated NSP-reducing elements. Cells were harvested 48 hours post-transfection and assayed for trans-splicing efficiency by RT-qPCR (upper panel) or Western blot analysis (lower panel). [Figure 30] This study demonstrates the mechanism of action of a tandem-binding domain RNA exon editor targeting HTT intron 2 and MSH3 pre-mRNA. Expression of the exon editor is driven by a CMV promoter and contains the HTT 5'UTR, an N-terminal 3X FLAG tag, coding sequences for HTT exons 1 and 2, a splice donor site, a linker, an MSH3-binding domain (targeting intron 5 or intron 15 of MSH3 pre-mRNA), an HTT-binding domain (HTT_intron2_12061_150), and a terminator sequence. The HTT-binding domain targets the exon editor to generate corrected HTT RNA after successful trans-splicing. The MSH3-binding domain targets the exon editor to generate an HTT exon 1+2-MSH3 chimeric RNA molecule with a premature stop codon, which is subject to nonsense-mediated decay (NMD) and subsequently reduces MSH3 expression. [Figure 31](A) and (B) show RT-qPCR profiles of HTT trans-splicing and HTT-MSH3 chimera generation (via MSH3 trans-splicing) in tandem binding domain exon editors. RT-qPCR analysis of A) HTT on-target (ONT) trans-splicing efficiency (via the HTT intron 2-directed binding domain) and B) HTT-MSH3 chimera trans-splicing efficiency (via the MSH3 intron 5-directed binding domain) in HEK293 cells transfected with tandem binding domain RNA exon editors targeting HTT intron 2 and MSH3 intron 5. The binding domain targeting HTT intron 2 was HTT_intron2_12061_150 for all exon editors tested here, while MSH3 intron 5-directed binding domains were tested: MSH3_intron5_213_100 and MSH3_intron5_188_150. The binding domains were arranged in tandem, with the order of the binding domains as shown. Exon editors were also tested with each MSH3 binding domain having a MALAT1 triple helix arranged between the two tandem binding domains. [Figure 32](A) and (B) show RT-qPCR profiles of HTT trans-splicing and HTT-MSH3 chimera generation (via MSH3 trans-splicing) in tandem binding domain exon editors. RT-qPCR analysis of A) HTT on-target (ONT) trans-splicing efficiency (via the HTT intron 2-directed binding domain) and B) HTT-MSH3 chimera trans-splicing efficiency (via the MSH3 intron 15-directed binding domain) in HEK293 cells transfected with tandem binding domain RNA exon editors targeting HTT intron 2 and MSH3 intron 15. The binding domain targeting HTT intron 2 was HTT_intron2_12061_150 for all exon editors tested here, while MSH3 intron 15-directed binding domains were tested: MSH3_intron15_6523_120 and MSH3_intron15_6498_150. The binding domains were arranged in tandem, with the order of the binding domains as shown. Exon editors were also tested with each MSH3 binding domain having a MALAT1 triple helix arranged between the two tandem binding domains. [Figure 33] 1 shows a schematic diagram illustrating MSH3 knockdown by miRNA targeting MSH3 mRNA. MSH3 can be knocked down by miRNA that targets MSH3 mRNA and degrades the transcript. [Figure 34]Western blot analysis of MSH3 exon 23-directed RNAi constructs is shown. Constructs (SEQ ID NOs: 234, 235, 238-241) containing the MSH3 exon 23-directed miRNA active sequence TTAATCCATAACTCCTTGC (SEQ ID NO: 224) and control constructs (SEQ ID NOs: 236, 237, 242, and 243) were analyzed. Western blots were subjected to ImageJ analysis to analyze MSH3 protein knockdown (top panel). Mimics of U6 promoter-driven shRNAs and CMV promoter-driven pri-miRNAs were designed and tested. Variations included the strand positioning (5' or 3' arm) of the guide strand, such as a bulge in the stem structure, and various miRNA scaffolds. Negative controls included constructs containing non-targeting sequences or a control without a hairpin loop. [Figure 35] RT-qPCR and Western blot analysis of MSH3-directed RNAi constructs are shown. Constructs encoding miRNAs targeting different regions of the MSH3 transcript were analyzed. Western blots were subjected to ImageJ analysis to analyze MSH3 protein knockdown. CMV promoter-driven pri-miRNA mimics targeting different exon sequences of MSH3 were designed and tested. [Figure 36] A schematic diagram showing MSH3 knockdown by small nuclear RNA (snRNA)-based antisense RNA (asRNA) is shown. Antisense RNA encoded in the snRNA scaffold anneals to the MSH3 splice junction, inactivating MSH3 and preventing exon inclusion. This leads to exon skipping and premature stop codon generation, ultimately resulting in NMD of the MSH3 transcript. The diagram shows an example of an MSH3 splice modulator targeting exon 2 skipping. [Figure 37]The relative expression levels of MSH3 RNA at the exon 1-exon 2 junction and exon 2-exon 3 junction for MSH3 splice modulators targeting exon 2 skipping (bottom panel) and a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (top panel) are shown. The splice modulator transcripts are SEQ ID NOS: 284 to 287. SEQ ID NOS: 284: U7SmOPT containing MSH3 In1 / Ex2 asRNA (SEQ ID NOS: 274); SEQ ID NOS: 285: U7SmOPT containing MSH3 Ex2 / In2 asRNA (SEQ ID NOS: 275); SEQ ID NOS: 286: U7SmOPT containing MSH3 In1 / Ex2 + Ex2 / In2 asRNA (SEQ ID NOS: 276); SEQ ID NOS: 287: MSH3 In1 / Ex2 + Ex2 / In2 long (160 nt) asRNA (SEQ ID NOS: 277). [Figure 38] The relative expression levels of MSH3 RNA at the exon 2-exon 3 junction and exon 3-exon 4 junction for MSH3 splice modulators targeting exon 3 skipping (bottom panel) and a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (top panel) are shown. The splice modulator transcripts are SEQ ID NOS: 288-290. SEQ ID NOS: 288: U7SmOPT containing MSH3 In2 / Ex3 asRNA (SEQ ID NOS: 278); SEQ ID NOS: 289: U7SmOPT containing MSH3 Ex3 / In3 asRNA (SEQ ID NOS: 279); SEQ ID NOS: 290: U7SmOPT containing MSH3 In2 / Ex3 + Ex3 / In3 asRNA (SEQ ID NOS: 280). [Figure 39]The relative expression levels of MSH3 RNA at the exon 3-exon 4 junction and exon 4-exon 5 junction for MSH3 splice modulators targeting exon 4 skipping (bottom panel) and a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (top panel) are shown. The splice modulator transcripts are SEQ ID NOS: 291 to 293. SEQ ID NOS: 291: U7SmOPT containing MSH3 In3 / Ex4 asRNA (SEQ ID NOS: 281); SEQ ID NOS: 292: U7SmOPT containing MSH3 Ex4 / In4 asRNA (SEQ ID NOS: 282); SEQ ID NOS: 293: U7SmOPT containing MSH3 In3 / Ex4 + Ex4 / In4 asRNA (SEQ ID NOS: 283). [Figure 40] The relative expression levels of MSH3 RNA at the exon 2-exon 3 junction and exon 3-exon 4 junction (bottom panel) and a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (top panel) are shown for the MSH3 splice modulator targeting exon 3 skipping. The splice modulator transcripts are SEQ ID NOs: 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 splice modulator containing In3 / Ex3 / In2 asRNA (SEQ ID NO: 299); SEQ ID NO: 325: MSH3 U7 SmOPT splice modulator containing In3 / Ex3-1 + linker + Ex3 / In2-1 asRNA (SEQ ID NO: 300 + 298 + 301); SEQ ID NO: 326: MSH3 U2 splice modulator containing In3 / Ex3-2 + linker + Ex3 / In2-2 asRNA (SEQ ID NO: 302 + 298 + 303). [Figure 41]The relative expression levels of MSH3 RNA at the exon 5-exon 6 junction and exon 6-exon 7 junction for MSH3 splice modulators targeting exon 6 skipping are shown (bottom panel), and a schematic diagram (top panel) shows the asRNA target region in MSH3 pre-mRNA. The splice modulator transcripts are SEQ ID NOS: 327-329. SEQ ID NOS: 327: MSH3 U7 SmOPT splice modulator containing In6 / Ex6 / In5 asRNA (SEQ ID NOS: 304); SEQ ID NOS: 328: MSH3 U7 SmOPT splice modulator containing In6 / Ex6-1 + linker + Ex6 / In5-1 asRNA (SEQ ID NOS: 305 + 298 + 306); SEQ ID NOS: 329: MSH3 U2 splice modulator containing In6 / Ex6-2 + linker + Ex6 / In5-2 asRNA (SEQ ID NOS: 307 + 298 + 308). [Figure 42] The relative expression levels of MSH3 RNA at the exon 6-exon 7 junction and exon 7-exon 8 junction for MSH3 splice modulators targeting exon 7 skipping are shown in the figure below, along with a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (figure above). The splice modulator transcripts are represented by SEQ ID NOS: 330 to 332. The antisense RNA (asRNA) contained in SEQ ID NOS: 330 is the MSH3 In7 / Ex7 / In6 asRNA (asRNA region SEQ ID NOS: 309). The asRNA contained in SEQ ID NOS: 331 is the In7 / Ex7-1 (asRNA region SEQ ID NOS: 310) + linker (SEQ ID NOS: 298) + Ex7 / In6-1 (asRNA region SEQ ID NOS: 311). The asRNA contained in SEQ ID NOS: 332 is the In7 / Ex7-2 (asRNA region SEQ ID NOS: 312) + linker (SEQ ID NOS: 298) + Ex7 / In6-2 (asRNA region SEQ ID NOS: 313). [Figure 43]The relative expression levels of MSH3 RNA at the exon 7-exon 8 junction and exon 8-exon 9 junction for MSH3 splice modulators targeting exon 8 skipping are shown (bottom panel), and a schematic diagram (top panel) shows the asRNA target region in MSH3 pre-mRNA. The splice modulator transcripts are SEQ ID NOS: 333 to 335. SEQ ID NOS: 333: MSH3 U7 SmOPT splice modulator containing In8 / Ex8 / In7 asRNA (SEQ ID NOS: 314); SEQ ID NOS: 334: MSH3 U7 SmOPT splice modulator containing In8 / Ex8-1 + linker + Ex8 / In7-1 asRNA (SEQ ID NOS: 315 + 298 + 316); SEQ ID NOS: 335: MSH3 U2 splice modulator containing In8 / Ex8-2 + linker + Ex8 / In7-2 asRNA (SEQ ID NOS: 317 + 298 + 318). [Figure 44] The relative expression levels of MSH3 RNA at the exon 14-exon 15 junction and the exon 15-exon 16 junction (bottom panel) and a schematic diagram showing the asRNA target region in MSH3 pre-mRNA (top panel) are shown for the MSH3 splice modulator targeting exon 15 skipping. The splice modulator transcripts are represented by SEQ ID NOs: 336 to 338. Sequence number 336: MSH3 U7 SmOPT splice modulator containing In15 / Ex15 / In14 asRNA (sequence number 319); sequence number 337: MSH3 U7 SmOPT splice modulator containing In15 / Ex15-1 + linker + Ex15 / In14-1 asRNA (sequence numbers 320 + 298 + 321); sequence number 338: MSH3 U2 splice modulator containing In15 / Ex15-2 + linker + Ex15 / In14-2 asRNA (sequence numbers 322 + 298 + 323). [Figure 45]Figures 45A, 45B, and 45C show that MSH3 exon 7 splice modulators reduce MSH3 RNA and protein levels. HEK293 cells were transfected with snRNA-based splice modulators designed to skip MSH3 exon 7. Cells were harvested 48 hours posttransfection and assayed for MSH3 knockdown by RT-qPCR (Figure 45B) or Western blot analysis (Figure 45C). Splice modulator transcripts are SEQ ID NOs: 330-332. SEQ ID NO: 330: MSH3 U7 SmOPT splice modulator containing In7 / Ex7 / In6 asRNA (SEQ ID NO: 309); SEQ ID NO: 331: MSH3 U7 SmOPT splice modulator containing In7 / Ex7-1 + linker + Ex7 / In6-1 asRNA (SEQ ID NO: 310 + 298 + 311); SEQ ID NO: 332: MSH3 U2 splice modulator containing In7 / Ex7-2 + linker + Ex7 / In6-2 asRNA (SEQ ID NO: 312 + 298 + 313). [Figure 46] We present a combined strategy to correct mutant HTT by trans-splicing and knocking down unedited HTT species (including HTT1a) using microRNAs (miRNAs). [Figure 47]

[0013] Figure 1 illustrates a vectorized hybrid molecule that combines an HTT exon editor with an miRNA that targets unedited HTT mRNA. A short hairpin RNA (shRNA) or microRNA (miRNA) designed to reduce HTT gene expression can be added to the exon editor, either within the same cistron (e.g., within an intron of the exon editor) or as a separate cistron with its own regulatory sequence. RNAi can reduce the expression of the unedited target (e.g., mutant HTT). For the purpose of selectively reducing unedited HTT using RNAi, the exon editor contains an HTT CDS that contains a sequence modification that renders the edited HTT resistant to shRNA or miRNA. [Figure 48]Figures A and B show the trans-splicing and HTT knockdown profiles of an HTT exon editor, HTT miRNA-1, and a dual hybrid molecule of an HTT exon editor and HTT miRNA-1. A) The percentage of trans-spliced ​​(edited) HTT transcripts among all HTT transcripts, and B) the copy numbers of unedited and trans-spliced ​​(edited) HTT transcripts in each treatment. HEK293 cells were transfected with an HTT intron 2-directed RNA exon editor, HTT miRNA-1, and a dual hybrid molecule of an HTT intron 2-directed exon editor (SEQ ID NO: 204) plus HTT miRNA-1 (SEQ ID NO: 341, the HTT miRNA-1 coding sequence, containing SEQ ID NO: 339, the HTT miRNA-1 active sequence). 48 hours after transfection, cells were harvested and RNA was subjected to RT-qPCR. A dual hybrid molecule of an HTT exon editor and an HTT miRNA-1 (SEQ ID NO: 354), the dual hybrid comprising SEQ ID NO: 341 and SEQ ID NO: 204. [Figure 49] Figures A-C show 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-directed RNA exon editor and dual hybrid molecules of an HTT intron 2-directed exon editor plus HTT miRNA-1 or HTT miRNA-2. Forty-eight hours after transfection, cells were harvested, and RNA was subjected to RT-qPCR analysis for A) trans-splicing profile, B) HTT knockdown profile, and C) HTT copy number analysis. A dual hybrid molecule of an HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354), comprising SEQ ID NO: 341 and SEQ ID NO: 204. A dual hybrid molecule of an HTT exon editor and HTT miRNA-2 (SEQ ID NO: 355), comprising SEQ ID NO: 344 and SEQ ID NO: 204. [Figure 50] This shows that HTT miRNA-1 successfully knocks down unedited HTT transcripts and has minimal interaction with the exon editor and edited HTT transcripts. HEK293 cells were transfected with a dual hybrid molecule of HTT exon editor + / - HTT miRNA-1. 48 hours after transfection, cells were harvested and assayed for HTT knockdown and trans-splicing efficiency by RT-qPCR (upper panel) or Western blot analysis (lower panel). A dual hybrid molecule of HTT exon editor and HTT miRNA-1 (SEQ ID NO: 354) was obtained, where the dual hybrid comprises SEQ ID NO: 341 and SEQ ID NO: 204. [Figure 51] FIG. 1 shows a schematic diagram illustrating reduction of MSH3 by splice modulation in combination with HTT trans-splicing. [Figure 52] Figures 1A and 1B show results demonstrating the performance of the HTT exon editor + MSH3 splice modulator dual hybrid molecule. HEK293 cells were transfected with the HTT exon editor + / - MSH3 splice modulator. 48 hours post-transfection, cells were harvested and assayed for A) trans-splicing profile and B) MSH3 knockdown profile by RT-qPCR. HTT exon editor + MSH3 splice modulator dual hybrid molecule: MSH3 splice modulator + HTT exon editor (SEQ ID NO: 356), this dual hybrid comprising SEQ ID NO: 331 and SEQ ID NO: 204. [Figure 53]Figures A-D show a comparison of self-complementary AAV (scAAV) and single-stranded AAV (ssAAV) using the HTT exon editor + MSH3 splice modulator dual hybrid molecule. HEK293 cells were transduced with scAAV or ssAAV expressing the HTT exon editor + MSH3 exon 7-skipping splice modulator dual hybrid molecule. The AAV2 serotype was used. Cells were harvested 48 hours after transduction and subjected to RT-qPCR and Western blot analysis. The indicated sequences were inserted between the ITRs of AAV2, regardless of whether it was ssAAV or scAAV. The HTT exon editor + MSH3 splice modulator dual hybrid molecule (SEQ ID NO: 357) contains SEQ ID NO: 331 and SEQ ID NO: 204 in a head-to-head orientation. [Figure 54]Results are shown showing the performance of triple hybrid molecules (compared to their controls) of an HTT exon editor, HTT miRNA, and MSH3 splice modulator. HEK293 cells were transfected with an HTT exon editor, HTT miRNA-1, or HTT miRNA-2, MSH3 splice modulator. Cells were harvested 48 hours posttransfection and assayed by RT-qPCR for the following: (top panel) % trans-spliced ​​HTT transcript, (middle panel) HTT knockdown profile, and (bottom panel) MSH3 knockdown profile. Control hybrid molecules containing an exon editor with a splice donor mutation, a splice modulator containing a scrambled asRNA sequence, or a miRNA containing a scrambled asRNA were also tested. "1" for HTT miRNA indicates that HTT miRNA-1 was used, and "2" for HTT miRNA indicates that HTT miRNA-2 was used. SM, splice variant. Scr, scrambled control. miR-33 was used as the miRNA scaffold.a dual hybrid molecule of an HTT exon editor and an HTT miRNA-1 (SEQ ID NO:354), the dual hybrid comprising SEQ ID NO:341 and SEQ ID NO:204; a dual hybrid molecule of an HTT exon editor and an MSH3 splice modulator; an MSH3 splice modulator and an HTT exon editor (SEQ ID NO:356), the dual hybrid comprising SEQ ID NO:331 and SEQ ID NO:204; a triple hybrid molecule of an HTT exon editor and an HTT miRNA-1 + MSH3 splice modulator (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 an HTT exon editor and an HTT miRNA-2 + MSH3 splice modulator (SEQ ID NO:359), the triple hybrid comprising SEQ ID NO:331, SEQ ID NO:344, and SEQ ID NO:204; A dual hybrid molecule with miRNA-2 (SEQ ID NO: 355), which dual hybrid comprises SEQ ID NO: 344 and SEQ ID NO: 204. [Figure 55]The trans-splicing profile of HD molecules in iCell GlutaNeurons was measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced with either an HTT exon editor + HTT miRNA dual hybrid molecule or an HTT exon editor + MSH3 splice modulator dual hybrid molecule (both packaged in AAV2.7m8). Cells were harvested 18–21 days later for RT-ddPCR and Western blotting. The indicated sequences were inserted between the ITRs of AAV2.7m8. a dual hybrid molecule of an HTT exon editor and an HTT miRNA-1 (SEQ ID NO: 354), wherein the dual hybrid comprises SEQ ID NO: 341 and SEQ ID NO: 204; a dual hybrid molecule of an HTT exon editor and an MSH3 splice modulator (SEQ ID NO: 357), wherein the dual hybrid comprises SEQ ID NO: 331 and SEQ ID NO: 204 in a head-to-head orientation; and a triple hybrid molecule of an HTT exon editor, an HTT miRNA-1, and an MSH3 splice modulator (SEQ ID NO: 358), wherein the triple hybrid comprises SEQ ID NO: 331, SEQ ID NO: 341, and SEQ ID NO: 204. [Figure 56]Figure 1 shows the HTT miRNA knockdown profile in iCell GlutaNeurons measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced with either an HTT exon editor + HTT miRNA dual hybrid molecule or an HTT exon editor + MSH3 splice modulator dual hybrid molecule (both packaged in AAV2.7m8). Cells were harvested 18 days later for RT-ddPCR and Western blotting. The indicated sequences were inserted between the ITRs of AAV2.7m8. The HTT exon editor and HTT miRNA-1 dual hybrid molecule (SEQ ID NO: 354) contains SEQ ID NO: 341 and SEQ ID NO: 204; and the HTT exon editor + MSH3 splice modulator dual hybrid molecule (SEQ ID NO: 357) contains SEQ ID NO: 331 and SEQ ID NO: 204 in a head-to-head orientation. [Figure 57] Figure 1 shows the MSH3 knockdown profile of the MSH3 splice modulator in iCell GlutaNeurons, as measured by RT-ddPCR and Western blotting. iCell GlutaNeurons were transduced with either the HTT exon editor + HTT miRNA-1 dual hybrid molecule or the HTT exon editor + MSH3 splice modulator dual hybrid molecule, both packaged in AAV2.7m8. Cells were harvested 18 days later for RT-ddPCR and Western blotting. The indicated sequences were inserted between the ITRs of AAV2.7m8. The HTT exon editor + HTT miRNA-1 dual hybrid molecule (SEQ ID NO: 354) contains SEQ ID NO: 341 and SEQ ID NO: 204; and the HTT exon editor + MSH3 splice modulator dual hybrid molecule (SEQ ID NO: 357) contains SEQ ID NO: 331 and SEQ ID NO: 204 in a head-to-head orientation. [Figure 58]Figure 1 shows the HTT trans-splicing profile in the BAC-CAG mouse brain. Neonatal ICV injections (1E+11 or 3E+11 vg / animal) of BAC-CAG mice were performed with the indicated HD molecules packaged in AAV9. Mouse cortices and striatum were harvested 4 weeks post-injection, and the efficiency of HTT exon replacement by trans-splicing was profiled by RT-ddPCR and Western blotting. SEQ ID NO: 369: scAAV, mouse Msh3 splice modulator + CMVp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 370: ssAAV, mouse Msh3 splice modulator + CMVp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 371: ssAAV, mouse Msh3 splice modulator + CAGGSp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation). [Figure 59] Figure 1 shows the observed relationship between exon editor RNA copy number and trans-splicing efficiency (% HTT replacement) in BAC-CAG mouse brain. SEQ ID NO: 369: scAAV, mouse Msh3 splice modulator + CMVp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 370: ssAAV, mouse Msh3 splice modulator + CMVp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation); SEQ ID NO: 371: ssAAV, mouse Msh3 splice modulator + CAGG Sp::exon editor (dual hybrid of SEQ ID NO: 362 and SEQ ID NO: 204, head-to-head orientation). DETAILED DESCRIPTION OF THE INVENTION

[0053] The following examples are provided to illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure, and it will be understood that by their illustrative nature, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0054] The compositions and methods described herein involve trans-splicing molecules (e.g., pre-mRNA trans-splicing molecules) for treating diseases or disorders caused by mutations in the HTT gene. Such mutations include an expanded CAG trinucleotide repeat in exon 1 of the HTT gene. The compositions and methods described herein use pre-mRNA trans-splicing molecules for gene therapy (e.g., in vivo gene therapy, such as delivered by adeno-associated virus) to treat diseases, such as HD, caused by an expanded CAG trinucleotide repeat in HTT. The compositions and methods described herein also use pre-mRNA trans-splicing molecules for gene therapy (e.g., in vivo gene therapy) in combination with other therapeutic agents described herein to treat diseases, such as HD, caused by at least one mutation in HTT.

[0055] The compositions and methods described herein also involve therapeutic molecules that knock down expression of MSH3. MSH3 knockdown constructs can be used alone in therapeutic methods 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, for example, other therapeutic agents designed to correct trinucleotide repeat expansion disorders.

[0056] As described herein, HD is a hereditary, progressive neurodegenerative disease for which only palliative treatments are available. Such treatments include medications, physical therapy, and counseling, which provide some symptomatic relief. While HD symptoms vary, the disease is characterized by a progressive loss of the ability to control movement, regulate emotions, and maintain cognitive function. HD typically occurs in people in their 30s and 40s. The disease is associated with loss of cortical pyramidal neurons, loss of striatal medium spiny neurons, and loss of hypothalamic neurons. The genetic cause of HD is autosomal dominant inheritance of an expanded CAG trinucleotide repeat in exon 1 of the HTT gene; the presence of more than 40 CAG repeats in this region contributes to the disease. See Figure 1.

[0057] The HTT locus is large, spanning 180 kb and consisting of 67 exons. Expression of the HTT gene is required for normal development. Although the HTT protein is widely expressed, the brain is most severely affected by pathological expansion of CAG trinucleotide repeats, with early pathological effects observed in the striatum and motor cortex. The mechanism underlying the development of HD is somatic CAG repeat expansion in HTT, which occurs in affected brain regions (e.g., striatum) in HD patients (see Figure 2). Human genetic evidence suggests the involvement of genes in the DNA mismatch repair pathway (e.g., MSH2, MSH3, FAN1, MLH1) in regulating this process and modifying the clinical outcome of HD.

[0058] Despite considerable efforts by many biotechnology and pharmaceutical companies toward developing drugs to treat HD, there are currently no disease-modifying treatments for HD. The inventors have attempted to address this need using a variety of different modalities, each of which can be performed alone or in combination to provide a therapeutic intervention for HD.

[0059] Exon replacement by HTT pre-mRNA trans-splicing Exon replacement by pre-mRNA trans-splicing is highly suitable as a therapeutic approach for HD because of its ability to replace mutant HTT exon 1 while sparing the wild-type copy of HTT. Furthermore, the approach can address the entire spectrum of genetic variability in HD patients, theoretically treating 100% of the HD population. In other words, it is not limited to addressing specific HD patient-specific mutations (e.g., SNPs), but rather functions as a pan-specific therapeutic agent that can correct genetic defects across an entire exon(s).

[0060] As described herein, the inventors designed and tested RNA exon editors that target intron 1, intron 2, or intron 3 of HTT pre-mRNA. Thus, these RNA exon editors can replace exon 1, exons 1 and 2, or all of exons 1-3 of HTT mRNA, thereby correcting any pathogenic mutations present in exon 1, exons 1 and 2, or exons 1-3, respectively (e.g., correcting the number of expanded CAG trinucleotide repeats in exon 1 to the wild-type number). As shown in Figure 3, RNA exon editing via trans-splicing allows replacement of mutant HTT exon 1 with wild-type HTT exon 1 using either an intron 1-directed exon editor, an intron 2-directed exon editor, or an intron 3-directed exon editor.

[0061] In some embodiments, an RNA exon editor targeting intron 1 of HTT pre-mRNA includes a CMV promoter (SEQ ID NO: 137) (used here for in vitro experiments in HEK293 cells), a 5'UTR (e.g., the 5'UTR of HTT (SEQ ID NO: 136 or 192)), an epitope tag for detection of the on-target (ONT) HTT protein generated after successful trans-splicing (e.g., a 3X FLAG tag (SEQ ID NO: 4)), HTT exon 1 (SEQ ID NO: 3) (codon-modified and may be followed by the native sequence), a splice donor sequence (GTAAGT), a linker (e.g., a 40-mer linker (SEQ ID NO: 37)), a binding domain targeting any one of various regions within the HTT intron 1 pre-mRNA, and a triple helix terminator (e.g., the MALAT1 terminator (SEQ ID NO: 5) or a modified version thereof, e.g., SEQ ID NO: 6)). See, e.g., Figure 4. In some embodiments, an RNA exon editor targeting intron 1 of HTT pre-mRNA does not comprise one or more of the following: a CMV promoter (SEQ ID NO: 137), a 5' UTR (e.g., the 5' UTR of HTT (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 linker (SEQ ID NO: 37), a triple helix terminator (e.g., the MALAT1 terminator (SEQ ID NO: 5 or a modified version thereof, e.g., SEQ ID NO: 6)). In some embodiments, the CMV promoter may be replaced with a different promoter. A promoter may be selected to have properties suitable for in vivo studies and suitable for a therapeutic agent comprising an RNA exon editor. In some embodiments, an exemplary RNA exon editor does not comprise a FLAG tag or any epitope tag. In such embodiments, a therapeutic agent comprising an RNA exon editor may not comprise any epitope tag, which may reduce the potential for immunogenicity. FIG. 5 shows that the level of HTT exon substitution (trans-splicing efficiency) varies depending on where within intron 1 the binding domain is targeted.The results presented here show that targeting the 3' end of intron 1 near the branch site correlates with improved trans-splicing efficiency into HTT pre-mRNA. See Figure 6.

[0062] The inventors next attempted to further improve the trans-splicing efficiency of the HTT intron 1-directed exon editor by selecting an exemplary binding domain, HTT_intron1_11704_100, and determining the effect of different linkers operably linked to this binding domain in the context of an RNA exon editor. See, e.g., Figure 7, which presents a schematic diagram of the sequence of an exemplary RNA exon editor comprising the binding domain, HTT_intron1_11704_100. The results presented herein demonstrate that several linkers identified by an extensive trial-and-error procedure (23mer GU linker, 33mer GU linker, 34mer GU linker, 41mer GU linker, 40mer_2, 41mer_2, 68mer, 84mer, and 60mer) confer higher levels of trans-splicing efficiency compared to that conferred by the 40mer (SEQ ID NO: 37). See, e.g., Figure 8. The sequences of the 23mer GU linker (approximately 69-70% guanine content), 33mer GU linker (approximately 66-67% guanine content), 34mer GU linker (approximately 67-68% guanine content), 41mer GU linker (approximately 73-74% guanine content), 40mer_2, 41mer_2, 68mer, 84mer, and 60mer correspond to SEQ ID NOs: 38-46, respectively.

[0063] We next tested RNA exon editors targeting HTT intron 2. See Figure 9 for a general schematic of an HTT intron 2-directed RNA exon editor. Similar to the intron 1-directed exon editor, we first performed a binding domain scan in intron 2 to identify binding domains that bind to target regions within intron 2 that are associated with and promote high levels of trans-splicing efficiency. See, e.g., Figure 10. We determined that the 3' end of the intron, 10-20 nucleotides (nt) upstream of branch site A, is a particularly effective region for targeting trans-splicing to HTT intron 2. See, e.g., Figure 11. Additional analyses examining the relationship between binding domain length and functionality in the intronic region upstream of the branch site identified binding domain lengths in the range of 125-200 nt as having the highest relative trans-splicing efficiency. See, e.g., Figure 12.

[0064] We further explored the combinatorial functionality of binding domains operably linked to different linkers, as illustrated in Figure 13. We examined the effect of various exemplary linkers on the trans-splicing activity of a binding domain (HTT_intron2_12061_150) that functions at a high level for HTT intron 2. As shown in Figure 14, there was no significant difference in the trans-splicing activity of RNA exon editors containing HTT_intron2_12061_150 operably linked to the indicated linkers. Indeed, only minor effects were observed when HTT_intron2_12061_150 was operably linked to different linkers, suggesting that trans-splicing using HTT_intron2_12061_150 is highly optimized.

[0065] The results presented herein, which show that none of the linkers tested significantly improved trans-splicing efficiency compared to that conferred by a 40-mer linker, suggest that the HTT_intron2_12061_150 exon editor achieved high efficiency through binding affinity and recruitment of the associated spliceosome machinery. The results also demonstrate that different linker sequences can be included in an HTT RNA exon editor effective at inducing trans-splicing. We experimentally tested whether inhibition of cis-splicing could contribute to enhanced trans-splicing by designing antisense oligonucleotides (ASOs) that specifically block competing cis-splice sites (ASO8 (SEQ ID NO: 133), ASO9 (SEQ ID NO: 134), and ASO10 (SEQ ID NO: 135)) and antisense oligonucleotides (ASOs) that specifically block cis-splice sites involved in splicing of the upstream intron (ASOs 2-7 (SEQ ID NOs: 127-132, respectively)). Each of these ASOs was co-expressed with an RNA exon editor targeting intron 2. See Figure 16. Figure 17 shows that an HTT intron 2-directed exon editor (HTT_intron2_12061_150) combined with ASO6 (SEQ ID NO: 131), which blocks cis-splicing of the upstream intron, resulted in improved trans-splicing efficiency in vitro. Some embodiments described herein involve a strategy to block these cis-splicing events using antisense RNA expressed on the same plasmid as the HTT RNA exon editor (see also the combination embodiments section below).

[0066] To confirm the presence of authentic chimeric HTT proteins generated after trans-splicing, Western blot analysis was performed on cell lysates prepared from HEK293 cells transfected with selected HTT exon editors, and the blots were probed with an antibody that specifically binds to the N-terminal FLAG epitope. As shown in Figure 18, Western blotting confirmed on-target (ONT) trans-splicing and the resulting on-target protein generated after trans-splicing by detecting FLAG-tagged HTT proteins with the predicted molecular weight. Notably, the intensity of the anti-FLAG ONT protein band correlated with the trans-splicing efficiency (% RNA replacement). See Figure 18.

[0067] The present inventors also designed and tested an RNA exon editor that targets HTT intron 3. See Figure 23 for a general schematic of an HTT intron 3-directed RNA exon editor. As with the exon editors targeting intron 1 and intron 2, a binding domain scan was first performed to identify binding domains that bind to target regions within intron 3 that are associated with and promote high levels of trans-splicing efficiency. See, for example, Figure 24. The present inventors determined that the 3' end of the intron, 5 to 15 nt upstream of branch site A, is a particularly effective region for targeting trans-splicing into HTT intron 3.

[0068] To compare how intron 2-directed exon editors perform compared to intron 3-directed exon editors, we performed parallel transfections of exon editors containing the best-performing binding domains for each target intron and analyzed the RNA by RT-ddPCR. The results presented herein show that HTT intron 3-directed exon editors exhibit slightly higher trans-splicing efficiency compared to intron 2-directed exon editors. See, for example, Figure 25.

[0069] To investigate potential self-splicing (either due to cis-splicing of AAV concatemers or intermolecular trans-splicing) in HTT exon editors and constructs containing them, in silico predictions of self-splicing sites that mitigate such undesired events (see Table 1) were used to identify cryptic splice sites. An exemplary cryptic splice site-reducing HTT intron 2-directed exon editor (SEQ ID NO: 203) (with modifications to four of the identified self-splicing sites) was created and tested for trans-splicing efficiency, comparing it to the original (unmodified) exon editor (SEQ ID NO: 95). The results presented herein confirm that the minor sequence changes applied do not affect the trans-splicing activity of the exemplary cryptic splice site-reducing exon editor. See, e.g., Figure 26.

[0070] The first HTT exon editor molecule was expressed from a CMV promoter (SEQ ID NO: 137) and contained the native HTT 5'UTR (SEQ ID NO: 136). We next investigated how the exon editor would function when expressed from the CAGGS promoter (SEQ ID NO: 196). Because the CAGGS promoter contains the 5'UTR (CBA exon + rabbit beta globin exon), this study included testing exon editors with or without the native HTT 5'UTR in combination with the CAGGS promoter. Of note, although the results are presented in the context of an HTT intron 2-directed exon editor, the effects of these modifications are applicable to exon editors targeting other introns.

[0071] In HEK293 cell transfection experiments, we found that a CAGGS promoter-driven exon editor containing both the CAGGS 5'UTR and HTT 5'UTR had a similar HTT mRNA replacement percentage to that of HTT mRNA driven by a CMV promoter. For example, see Figure 27. When the HTT 5'UTR was removed from the CAGGS promoter-driven exon editor, a decrease in the HTT RNA replacement percentage was observed, and a significant increase in ONT protein expression was observed by Western blot, suggesting the presence of a protein translation activator in the CAGGS 5'UTR. At the same time, we observed an increase in unspliced ​​protein (NSP) and background (potential OFT) protein signals following removal of the HTT 5'UTR.

[0072] To explore NSP reduction strategies in the context of the 5'HTT exon editor, various elements were incorporated into the HTT intron 1-directed exon editor and the HTT intron 2-directed exon editor and tested, alone and in combination, to assess their impact on the performance of the exon editor. In the context of minimal NSP (i.e., a stop codon within or immediately following the exon editor's splice donor sequence to minimize neoantigens), these NSP reduction strategies include 1) three tandem repeats of the U1 snRNA binding site (3X UBS) (SEQ ID NO: 345) and 2) an AU-rich element (ARE) (SEQ ID NO: 346).

[0073] In an HTT intron 1-directed exon editor, the inventors demonstrated that including 3X UBS in the linker domain reduced NSP levels by approximately 75% compared to the baseline 40-mer-only linker control. Including an ARE within the linker reduced NSP levels by approximately 40% compared to the baseline 40-mer-only linker control. Combining 3X UBS and ARE in the exon editor reduced NSP levels by approximately 88% compared to the baseline 40-mer-only linker control. See, for example, Figure 28.

[0074] Similarly, in an HTT intron 2-directed exon editor, the inventors demonstrated that including 3X UBS in the linker domain reduced NSP levels by approximately 38% compared to the baseline 40-mer-only linker control. Including an ARE within the linker reduced NSP levels by approximately 33% compared to the baseline 40-mer-only linker control. The combination of 3X UBS and ARE in the exon editor worked combinatorially to reduce NSP levels by approximately 66% compared to the baseline 40-mer-only linker control. See, for example, Figure 29.

[0075] RNA exon editors that target HTT introns (e.g., intron 1, intron 2, or intron 3) can include a target-specific element, such as a binding domain specific for the HTT target intron, and a coding domain sequence encoding the HTT coding sequence (e.g., a sequence encoding all or a portion of exon 1, exon 2, or exon 3, or any combination thereof (e.g., exons 1 and 2 or exons 1-3)). RNA exon editors that target HTT introns can include one or more target-independent elements that can improve the functionality of the exon editor. Target-independent elements can include, for example, an epitope tag, a linker, a splice donor sequence, one or more repeats of the U1 snRNA binding site, an AU-rich element, or a terminator. Embodiments described herein can include one or more of such target-independent elements. Embodiments described herein can exclude one or more of such target-independent elements. RNA exon editors lacking one or more target-independent elements described herein can be capable of effecting exon editing. For example, in some embodiments, the RNA exon editors described herein do not include a triple helix terminator, a MALAT-1 terminator, or any terminator sequence. In some embodiments, the RNA exon editors described herein do not include a CMV promoter or a CAGGS promoter. In some embodiments, the RNA exon editors described herein do not include any 5' UTR sequence or do not include an HTT 5' UTR sequence. In some embodiments, the RNA exon editors described herein do not include a GTAAGT splice donor sequence. In some embodiments, the RNA exon editors described herein do not include any epitope tag or do not include a 3X FLAG tag. In some embodiments, the RNA exon editors described herein do not include any of the linkers described herein, such as any of SEQ ID NOs: 37-46.In some embodiments, the RNA exon editors described herein do not include three tandem repeats of the U1 snRNA binding site (3X UBS) (SEQ ID NO: 345). In some embodiments, the RNA exon editors described herein do not include an AU-rich element (ARE) (SEQ ID NO: 346).

[0076] Inhibition of somatic CAG repeat expansion Increasing evidence suggests that somatic instability of CAG repeat sequences is the underlying mechanism behind the development of HD. This supports exploring factors that modify somatic expansion as a viable therapeutic target. We designed and tested strategies to inhibit somatic CAG expansion. See, for example, Figure 19. Recent genome-wide association studies (GWAS) conducted in HD patients have identified many components of the DNA mismatch repair pathway as key genetic modifiers associated with the rate of disease progression. For example, in HD patients, single-nucleotide variants (SNVs) in the MSH3 gene that reduce its expression are associated with a delayed age at disease onset. MSH3 is speculated to be a good target for HD therapeutics, and evidence suggests that MSH3 knockdown leads to the inhibition of somatic CAG repeat expansion both in vitro and in vivo. Here, we explored several approaches to reduce MSH3 levels.

[0077] The present inventors explored the use of trans-splicing in a novel manner to reduce the expression level of a target gene by trans-splicing into a target pre-mRNA, producing an RNA that is rapidly degraded or unable to produce a functional protein. The inventors hypothesized that either a 5' exon editor or a 3' exon editor could achieve this result. A 5' exon editor can replace one or more of the target mRNA's 5' exons, removing the start codon and part or all of the coding sequence and replacing it with an alternative coding or non-coding sequence. A 3' exon editor can replace one or more of the target's 3' exons, replacing them with an alternative coding or non-coding sequence. The resulting chimeric mRNA may lack translation of any amino acids from the target mRNA. A 5' exon editor can also insert a 5' untranslated region (UTR) that prevents translation. A 3' exon editor can insert a 3' UTR that destabilizes the transcript or prevents it from being exported from the nucleus or translated. The portion of the target mRNA that remains after the trans-splicing reaction is presumably rapidly degraded because it lacks either a 5' cap or a 3' polyA tail.

[0078] The inventors have determined that an exon editor that edits a target such as HTT can simultaneously knock down a second gene, such as MSH3, by including two distinct binding domains (one targeting HTT and the other targeting MSH3). One embodiment of how this dual effect can be achieved is that a 5'HTT exon editor containing the 5' portion of HTT can trans-splice to HTT to reconstitute functional wild-type HTT, while also trans-splicing to MSH3 to generate a nonfunctional MSH3 mRNA. In one embodiment, the nonfunctional HTT-MSH3 chimeric mRNA encodes an HTT polypeptide that terminates at the first in-frame stop codon within the MSH3 portion of the mRNA. Thus, the normal wild-type MSH3 pre-mRNA is transformed into a nonfunctional HTT-MSH3 chimeric mRNA that encodes few or no amino acids from the MSH3 coding sequence and is likely susceptible to nonsense-mediated decay. As shown herein, this approach works with several different MSH3 intron-targeting binding domains, and indeed with any MSH3 intron-targeting binding domain. Furthermore, although the examples presented here relate to 5' exon editors, a similar approach could be implemented with 3' exon editors and would reasonably be expected to be successful.

[0079] Therefore, the inventors have designed and tested a strategy to treat HD early in its development by inhibiting somatic CAG expansion. The embodiments described herein, such as those describing inhibition of somatic CAG expansion, can be used alone or in conjunction with RNA exon editor-mediated trans-splicing to correct pathogenic mutations. While inhibition of somatic expansion alone is a viable therapeutic approach for HD, the inventors have tested hybrid therapeutic approaches and dual-action / hybrid molecules designed to inhibit somatic CAG expansion and correct HTT exon 1 RNA. See, e.g., Figures 19 and 20. Thus, the embodiments described herein, such as those designed to inhibit somatic CAG expansion, can be used alone or in conjunction with RNA exon editor-mediated trans-splicing to correct pathogenic mutations.

[0080] In addition to the above, to disrupt the mechanism underlying HD pathogenesis (i.e., somatic CAG expansion) while simultaneously replacing mutant HTT exon 1, the inventors designed a set of dual-acting / hybrid RNA exon editors containing targeting sequences for both HTT and MSH3 pre-mRNAs. In some embodiments, expression of the exon editor is driven by a CMV promoter. In some embodiments, an exemplary exon editor comprises an HTT 5'UTR, an N-terminal 3X FLAG tag, an HTT exon 1 coding sequence, a splice domain (splice donor site), a linker, an MSH3-binding domain, an HTT-binding domain, and a terminator sequence. The HTT-binding domain targets the exon editor to the HTT pre-mRNA, resulting in the production of a corrected HTT RNA after successful trans-splicing. The MSH3-binding domain targets the exon editor to the MSH3 pre-mRNA, resulting in the generation of an HTT exon 1-MSH3 chimeric RNA molecule with a premature stop codon (due to a frameshift resulting from replacement of the upstream MSH3 exon with HTT exon 1), which is subject to nonsense-mediated decay (NMD) and therefore results in a subsequent decrease in MSH3 expression. See, e.g., Figure 20.

[0081] When exon editors containing tandem binding domains were used, our experiments demonstrated successful trans-splicing of both HTT and MSH3 into pre-mRNA, as measured by RT-qPCR designed to detect the "corrected" HTT chimeric RNA molecules and the HTT exon 1-MSH3 chimeric RNA molecules, respectively. See, e.g., Figures 21 and 22. The sequences of the MSH3 binding domains used in the tested constructs are set forth in SEQ ID NOs: 140 and 142 (Figure 21) and SEQ ID NOs: 144 and 146 (Figure 22). The sequences of the HTT binding domains used in the tested constructs are set forth in SEQ ID NOs: 20, 141, and 145 (Figures 21 and 22). The sequence of the HTT CDS in the tested construct is set forth in SEQ ID NO: 3. It is worth noting that HTT exon 1-MSH3 chimeric RNA levels may be underestimated due to NMD. To address this, we also measured MSH3 RNA transcript levels.

[0082] In another embodiment, the inventors also designed and tested a tandem binding domain exon editor that targets HTT intron 2 (using the HTT_intron2_12061_150 binding domain (SEQ ID NO: 72)) in conjunction with the MSH3 pre-mRNA. See, e.g., Figures 30-32. The sequences of the MSH3 binding domains used in the tested constructs are set forth in SEQ ID NOs: 140, 209, 144, and 210, and the sequence of the HTT CDS of the tested construct is set forth in SEQ ID NO: 59.

[0083] RT-qPCR analysis of tandem binding domain exon editors targeting HTT intron 2 and MSH3 intron 5 demonstrated successful trans-splicing to both HTT and MSH3, as measured by RT-qPCR designed to detect modified HTT chimeric RNA molecules and HTT exon 2-MSH3 exon 6 chimeric RNA molecules. See, e.g., Figure 31, A and B. MSH3_intron5_188_150 (SEQ ID NO: 209) exhibited higher efficiency of MSH3 trans-splicing compared to MSH3_intron5_213_100 (SEQ ID NO: 140). Similarly, tandem binding domain exon editors targeting HTT intron 2 and MSH3 intron 15 demonstrated successful trans-splicing to both HTT and MSH3. See, e.g., Figure 32, A and B. It is noteworthy that HTT exon 2-MSH3 chimeric RNA levels may be underestimated due to NMD.

[0084] The results presented herein demonstrate that tandem binding domain exon editors are effective and likely to have therapeutic benefits. Furthermore, these experiments identified binding domains that are efficient for trans-splicing targeting of MSH3 pre-mRNA. Beyond their use in conjunction with the tandem binding domains described herein, these MSH3-directed binding domains can also be used in conjunction with exon editors with NMD-induced coding sequences, and such exon editors can be used alone to target MSH3 knockdown or packaged in the same AAV vector with an HTT-directed exon editor for combination approaches and co-delivered to target tissues.

[0085] In some embodiments, an MSH3 exon editor can be used to knock down MSH3 expression, where the MSH3 exon editor has a binding domain that targets an intron of the MSH3 pre-mRNA, as described above, but does not contain a binding domain that targets the HTT pre-mRNA or any other pre-mRNA. The dual-binding domain RNA exon editor described above can be modified by removing the binding domain that targets the HTT intron. In some embodiments, an MSH3 exon editor can be administered without also administering a treatment that targets or corrects another gene sequence. In some embodiments, such an MSH3 exon editor can be used in a method for treating or preventing a trinucleotide repeat expansion disorder.

[0086] MSH3 knockdown by RNAi RNA interference (RNAi) is a natural mechanism by which double-stranded RNA (dsRNA) induces sequence-specific gene silencing by targeting mRNA for degradation. In the initial design to identify vectored RNAi constructs that can efficiently knock down MSH3, various constructs based on a single miRNA sequence (MSH3 Ex23-directed, TTAATCCATAACTCCTTGC; SEQ ID NO: 224) were designed. Pol III promoter-driven shRNAs were constructed as a potential positive control. These U6 promoter-driven shRNAs were tested together with constructs expressing Pol II promoter (CMV promoter)-driven primary miRNA (pri-miRNA)-like transcripts targeting MSH3. See, for example, Figure 33.

[0087] For example, as shown in Figure 34, Western blot analysis revealed that approximately 50% MSH3 protein knockdown was observed with this MSH3 Ex23-directed miRNA (TTAATCCATAACTCCTTGC; SEQ ID NO: 224) when expressed in the context of a miR-30a scaffold. Furthermore, placing the guide RNA on the 5' arm or adding a bulge to the stem structure did not improve the efficiency of knockdown (37% and 23% knockdown, respectively).

[0088] Additional designs focused on altering the miRNA active sequence showed that, in addition to the exon 23-directed sequence tested in Figure 34, miRNA sequences directed to exon 22 (SEQ ID NO: 246), exon 9 (SEQ ID NO: 248), exon 12 (SEQ ID NO: 256), and exon 15 (SEQ ID NO: 257) performed similarly well, with approximately 50% MSH3 protein knockdown observed. See, e.g., Figure 35. This MSH3 RNAi modality may be therapeutically relevant alone for many repeat expansion disorders and / or can be combined with exon editors for enhanced therapeutic potential.

[0089] In some embodiments, the MSH3-directed RNAi constructs disclosed herein can be used in conjunction with RNA exon editors that target HTT or other genes associated with trinucleotide repeat expansion diseases, as described above. In other embodiments, the MSH3-directed RNAi constructs disclosed herein can be used independently of HTT-targeted therapeutic approaches. In some embodiments, the MSH3-directed RNAi constructs disclosed herein can be administered to treat or prevent trinucleotide repeat expansion diseases.

[0090] MSH3 splice regulation MSH3 can be inactivated by incorporating sequences complementary to the MSH3 splice junction into snRNA sequences, such as U7 snRNA. We designed and tested a strategy to block cis-splicing events with antisense RNA expressed from the same plasmid as the HTT-directed RNA exon editor. Modified snRNAs, such as U7 Sm OPT, were designed by: 1) changing the targeting sequence (e.g., the histone-binding sequence in the 5' region of U7 snRNA) to the complementary sequence of the gene to be modified; and 2) changing the binding site (AAUUUGUCUAG; SEQ ID NO: 367; U7 Sm WT) for U7 snRNP-specific proteins (Lsm10 and Lsm11) to a consensus sequence (AAUUUUUGGAG; SEQ ID NO: 368; U7 Sm OPT) derived from the major spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP), resulting in the formation of a spliceosomal heptameric protein core surrounding U7 Sm OPT. In some embodiments, the asRNA molecules described herein consist of a U1 promoter, snRNA (with an asRNA sequence targeting the intron-exon boundary and consensus Sm binding site of MSH3), and a U1 terminator. See, for example, Figure 36.

[0091] We designed and tested MSH3 splice modulators that target skipping of MSH3 exons 2, 3, and 4. For each set, we tested asRNA sequences directed against upstream intron-exon boundaries (e.g., the intron 1 / exon 2 boundary of the exon 2 splice modulator (SEQ ID NO: 274)), downstream exon-intron boundaries (e.g., the exon 2 / intron 2 boundary of the exon 2 splice modulator (SEQ ID NO: 275)), and tandem sequences consisting of the two asRNAs (SEQ ID NOs: 276 and 277). Exemplary MSH3 splice modulator sequences that mediate exon 2 skipping are shown in SEQ ID NOs: 284-286, which contain the MSH3 splice modulator elements SEQ ID NOs: 274-276, respectively. For MSH3 exon 2 skipping molecules, asRNA molecules targeting both the intron 1 / exon 2 boundary and the exon 2 / intron 2 boundary without the U7SmOPT snRNA scaffold were also tested. RT-qPCR assays measuring the levels of spliced ​​MSH3 mRNA product demonstrated that the U7SmOPT splice modulator (but not the asRNA sequence without the U7SmOPT scaffold) exhibited the intended target exon skipping. Exemplary MSH3 splice modulator sequences mediating exon 3 skipping are shown in SEQ ID NOS: 288-290, which contain MSH3 splice modulator elements SEQ ID NOS: 278-280, respectively. See, for example, Figures 37-39. Notably, MSH3 exon 4 skipping does not result in NMD but generates a shorter MSH3 product due to exon 4 containing 213 nucleotides, resulting in an mRNA product in which exon 4 is skipped. The sequences of exemplary MSH3 splice modulators that mediate exon 4 skipping are set forth in SEQ ID NOs: 291-293, which comprise the MSH3 splice modulator elements SEQ ID NOs: 281-283, respectively.We observed a truncated protein product of the exon 4-targeted splice modulator in Western blots probed with an antibody to visualize MSH3, providing evidence that the splice modulator skipped the targeted exon as intended.

[0092] Subsequent rounds of design included MSH3 splice modulators targeting skipping in exon 3 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:324-326, which comprise MSH3 splice modulator elements SEQ ID NOS:299, 300+298+301, and 302+298+303, respectively), exon 6 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:327-329, which comprise MSH3 splice modulator elements SEQ ID NOS:304, 305+298+306, and 307+298+308, respectively), exon 7 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:330-332, which comprise MSH3 splice modulator elements SEQ ID NOS:331-333, respectively), exon 8 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:332-333, which comprise MSH3 splice modulator elements SEQ ID NOS:333-334, respectively), exon 9 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:334-335, which comprise MSH3 splice modulator elements SEQ ID NOS:335-336, respectively), exon 10 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:336-337, respectively), exon 11 (sequences of exemplary MSH3 splice modulators are set forth in SEQ ID NOS:337-338, which comprise MSH3 splice modulator elements SEQ ID NOS:338-339, respectively), exon 12 (sequences of exemplary MSH3 splice modulators are set forth in MSH3 splice modulators were tested that target skipping of exon 8 (exemplary MSH3 splice modulator sequences are set forth in SEQ ID NOS: 333-335, which comprise MSH3 splice modulator elements SEQ ID NOS: 314, 315-298-316, and 317-298-318, respectively), and exon 15 (exemplary MSH3 splice modulator sequences are set forth in SEQ ID NOS: 336-338, which comprise MSH3 splice modulator elements SEQ ID NOS: 319, 320-298-321, and 322-298-323, respectively). These exons were selected for their suitability for splice regulation, taking into account factors such as neoepitope length, splicing properties such as small versus large spliceosome-mediated splicing, and alternative splicing, if a protein is produced rather than an NMD-affected transcript.

[0093] We designed and tested MSH3 splice modulators targeting skipping of MSH3 exons 3, 6, 7, 8, and 15. For each set, we tested 1) U7SmOPT molecules containing antisense sequences directed against the entire length of the target exon and 12–13 nucleotides in both flanking introns, 2) U7SmOPT molecules containing antisense sequences directed against the downstream exon / intron boundary and the upstream intron / exon boundary separated by an unstructured linker, and 3) U2 snRNA molecules containing antisense sequences directed against the downstream exon / intron boundary and the upstream intron / exon boundary separated by an unstructured linker. The sequences of the MSH3 splice modulators tested are set forth in SEQ ID NOS: 290, and 324-326 (Figure 40); SEQ ID NOS: 327-329 (Figure 41); SEQ ID NOS: 330-332 (Figure 42); SEQ ID NOS: 333-335 (Figure 43); and SEQ ID NOS: 336-338 (Figure 44). The SEQ ID NOS and target regions, showing the corresponding asRNA region SEQ ID NOS, are shown in each of Figures 40-44, respectively. The inventors identified effective splice modulators for all target exons tested. In general, U7SmOPT molecules containing antisense sequences to downstream exon / intron boundaries and upstream intron / exon boundaries separated by an unstructured linker (e.g., SEQ ID NOS: 328, 331, 334, and 337) showed particularly high levels of MSH3 expression reduction, with some exceptions. See, for example, Figures 40-44. Western blots probed with antibodies to visualize MSH3 showed that the MSH3 protein knockdown levels were consistent with the RNA exon skipping efficiency profile. The sequences of the tested MSH3 splice modulators are set forth in SEQ ID NOs: 330-332. See, e.g., Figure 45.

[0094] In some embodiments, the MSH3 splice modulators disclosed herein may be used in conjunction with RNA exon editors that target HTT, as described above, or that target other genes associated with trinucleotide repeat expansion diseases. In some embodiments, the MSH3 splice modulators disclosed herein may be used independently of HTT-targeted therapeutic approaches. In some embodiments, the MSH3 splice modulators disclosed herein may be administered to treat or prevent trinucleotide repeat expansion diseases.

[0095] Knockdown of HTT virulent species To enhance the efficacy of the HTT exon editor described herein, the inventors envisioned a combinatorial approach that further addresses toxic species such as HTT1a. To this end, they designed and tested a vectorized hybrid molecule containing an HTT exon editor and a miRNA targeting exon 1 of unedited HTT, where the elements are intended to be packaged into a single AAV. See, e.g., Figure 46. The sequences of the miRNAs targeting unedited HTT (HTT miRNA-1 and HTT miRNA-2) are set forth in SEQ ID NOs: 339 and 342. Codon modifications were made to the CDS of the HTT exon editor to prevent interactions between the miRNA and the exon editor and to avoid degradation of the exon editor itself and the modified HTT product. See, e.g., Figure 47.

[0096] We first evaluated the efficacy of an exon editor alone, an HTT miRNA alone, and the combined exon editor and HTT miRNA molecule when the two modalities were combined. The HTT intron 2-directed exon editor (SEQ ID NO: 204) and HTT miRNA-1 (active sequence SEQ ID NO: 339) were tested in this experiment. SEQ ID NO: 341 corresponds to the HTT miRNA-1 (SEQ ID NO: 339) encoding the pri-miRNA sequence in the mir-33 scaffold (SEQ ID NOs: 259, 260, 261). For example, as shown in Figure 48, the proportion of trans-spliced ​​(edited) HTT transcripts among total HTT transcripts increased in the presence of HTT miRNA due to a decrease in total HTT transcript copy number. However, the copy number of trans-spliced ​​edited HTT transcripts remained unchanged regardless of the presence or absence of HTT miRNA-1, indicating minimal or no interaction between HTT miRNA-1 and the exon editor.

[0097] An additional comparison was performed between two independent miRNAs targeting HTT exon 1: HTT miRNA-1 (active sequence SEQ ID NO: 339; encoding the pri-miRNA sequence (SEQ ID NO: 341)) and HTT miRNA-2 (active sequence SEQ ID NO: 342). SEQ ID NO: 344 corresponds to the HTT miRNA-2 (SEQ ID NO: 342) encoding pri-miRNA sequence in the mir-33 scaffold (SEQ ID NOs: 259, 260, 261). To confer and ensure resistance to both miRNAs, further codon optimization was performed on the coding sequence of the HTT intron 2-directed exon editor (exemplary variant coding domain sequences are listed in SEQ ID NOs: 59 and 349-351). No adverse effect of HTT miRNA-1 or HTT miRNA-2 on trans-splicing was observed, as evidenced by an unchanged (or potentially increased) number of trans-spliced ​​HTT copies and a higher percentage of HTT mRNA trans-spliced ​​by the exon editor in the presence of the miRNA. See, for example, Figure 49. We demonstrated similar efficiency of HTT knockdown using HTT miRNA-1 and HTT miRNA-2, and neither miRNA adversely affected the performance of the exon editor, as evidenced by unchanged copy numbers of trans-spliced ​​HTT transcripts with or without each miRNA. See, e.g., Figure 49.

[0098] We next evaluated the HTT exon editor + miRNA dual molecule at the protein level. Testing of the HTT intron 2-directed exon editor + HTT miRNA dual hybrid molecule showed that in the presence of HTT miRNA (HTT miRNA-1; SEQ ID NO: 339), approximately 50% of unedited HTT transcript knockdown was achieved (Figure 50, left panel). Analysis of exon editor trans-splicing activity showed that in the presence of HTT miRNA, the percentage of edited HTT RNA increased due to the reduction in unedited, and therefore total, HTT copy number by miRNA (Figure 50, right panel).

[0099] Construction of hybrid molecules As described herein, the inventors have explored hybrid approaches that target multiple mechanisms and / or pathogenic species. As detailed herein, these approaches can include replacement of mutant HTT via exon editors, knockdown of mutant HTT and related transcripts (e.g., HTT1a) via RNAi, and reduction of MSH3 via trans-splicing or vectored splice modulation. When combined, these approaches exhibit multimodality mechanisms of action yet can be delivered with a single AAV. A subset of designs aimed at knocking down MSH3 relies on the design of hybrid molecules (e.g., tandem-binding domain exon editors). Similarly, the inventors designed HTT-directed miRNAs with the intention that they would be combined with HTT exon editors, thus incorporating additional considerations regarding the codon-modifying miRNA target site within the exon editor to minimize interactions between the HTT-directed miRNA and the exon editor. Such combination approaches may be used to achieve a high degree of therapeutic efficacy in some, if not all, HD patients. Such an approach could indeed confer dramatic disease-modifying effects.

[0100] HTT exon editor + MSH3 splice modulator Although inhibition of somatic expansion alone may have a positive therapeutic effect on HD, a hybrid molecule that inhibits somatic CAG expansion and corrects HTT exon 1 RNA may hold significant therapeutic promise, as this approach can combat disease in multiple areas simultaneously. Any of the HTT-directed RNA exon editors described herein may be used in combination with any one or more of the MSH3 splice modulators described herein. Any of the HTT-directed RNA exon editors described herein may be used without any of the MSH3 modulators described herein, and any of the MSH3 splice modulators described herein may also be used without any of the HTT-directed RNA exon editors.

[0101] To achieve this combinatorial effect, hybrid vectors encoding an HTT exon editor plus an MSH3 splice modulator were designed. See, for example, Figure 51. Initial testing of these dual hybrid molecules used an HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) and an exemplary MSH3 exon 7 skipping splice modulator (SEQ ID NO: 331). However, the combinatorial effect is equally applicable to any combination of an HTT exon editor and an MSH3 splice modulator (exemplary MSH3 splice modulator sequences are shown, for example, in SEQ ID NOs: 330-332 (comprising MSH3 splice modulator elements SEQ ID NOs: 309, 310+298+311, and 312+298+313, respectively), as well as in SEQ ID NOs: 284-293, 324-329, and 333-338). The exemplary HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) and an exemplary MSH3 exon 7 skipping splice modulator (SEQ ID NO: 331) are described herein. It will be further understood that the combinatorial effects observed with the HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) are similarly applicable to other HTT intron 2-directed (HTT_intron2_12061_150) exon editors, e.g., those comprising any one of SEQ ID NOs: 59, 350, or 351 corresponding to modified coding domain sequences. Thus, an HTT intron 2-directed exon editor may comprise any one of the variant coding domain sequences set forth in any one of SEQ ID NOs: 59 and 349-351). It will be further understood that the combinatorial effects observed with the exemplary HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein are equally applicable to either an HTT intron 3-directed (e.g., comprising HTT_intron3_4223_150 or HTT_intron3_4233_150) exon editor or an HTT intron 1-directed (e.g., comprising HTT_intron1_11704_100 or HTT_intron1_11724_100) exon editor.As shown herein, we examined the trans-splicing profiles and MSH3 knockdown efficiencies of exon editors alone, MSH3 splice modulators alone, and dual hybrid molecules of exon editors and MSH3 splice modulators. RT-qPCR analysis showed no change in the performance of the exon editor with or without the MSH3 splice modulator, as indicated by similar % HTT replacement. See, e.g., Figure 52A. Similarly, the performance of the MSH3 splice modulator was unchanged with or without the exon editor. See, e.g., Figure 52B. This experiment demonstrated that there was no interaction between the exon editor and the MSH3 splice modulator when co-delivered on the same DNA fragment.

[0102] Efficient trans-splicing requires sufficient levels of AAV delivery to target tissues 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, an exemplary HTT intron 2-directed exon editor + MSH3 exon 7 skipping splice modulator dual hybrid molecule was inserted into an ssAAV-compatible plasmid (containing wild-type ITRs) and an scAAV-compatible plasmid (ITR / ITR-Δtrs), packaged into AAV2, and used to transduce HEK293 cells. Characterization of MSH3 expression at both the RNA and protein levels suggested that the dual hybrid molecule had better performance for both modalities when packaged into scAAV (see, e.g., Figure 53, left panel). Characterization of trans-splicing also supported the enhanced performance of molecules packaged in scAAV compared to ssAAV, evidenced by a higher percent displacement of HTT transcripts and a corresponding increase in trans-spliced ​​HTT protein detected by Western blot (see, e.g., Figure 53, right panel). We also assayed the expression levels of the exon editor and observed higher transgene expression in cells transduced with scAAV. A consideration when using scAAV vectors is their limited cargo space (approximately half the size compared to ssAAV), which limits their use when driving expression of exon editors from large promoters (e.g., the CAGGS promoter).

[0103] HTT exon editor + HTT miRNA + MSH3 splice modulator We have also designed, constructed, and tested triple hybrid molecules containing multiple modalities targeting multiple mechanisms and / or pathogenic species. These approaches include replacement of mutant HTT via exon editing, knockdown of mutant HTT and related transcripts (e.g., HTT1a) via RNAi, and reduction of MSH3 via splice modulation. When combined, these approaches exhibit multimodality mechanisms of action yet can be delivered with a single AAV. Such a combined approach may be used to achieve high therapeutic efficacy in some, if not all, HD patients. Such an approach may indeed confer dramatic disease-modifying effects. As noted above, each of these modalities is designed to minimize interactions with the other (e.g., codon modification of an exon editor resistant to HTT miRNA).

[0104] To explore this approach, we analyzed triple hybrid molecules containing a CAGGS promoter (SEQ ID NO: 196)-driven HTT intron 2-directed exon editor (SEQ ID NO: 204), an exemplary MSH3 exon 7-skipping splice modulator (SEQ ID NO: 331), and either SEQ ID NO: 341 [HTT miRNA-1 (SEQ ID NO: 339) encoding a pri-miRNA sequence on a mir-33 scaffold (SEQ ID NOs: 259, 260, 261)] or SEQ ID NO: 344 [HTT miRNA-2 (SEQ ID NO: 342) encoding a pri-miRNA sequence on a mir-33 scaffold (SEQ ID NOs: 259, 260, 261)]. The activity of the triple hybrids was compared to control hybrid molecules containing the splice variant exon editor, a splice modulator containing a scrambled regulatory sequence, and a miRNA containing a scrambled regulatory sequence. The combinatorial effect is equally applicable to any combination of an HTT exon editor, an MSH3 splice modulator (exemplary MSH3 splice modulator sequences are set forth, for example, in SEQ ID NOs: 330-332 (comprising MSH3 splice modulator elements SEQ ID NOs: 309, 310+298+311, and 312+298+313, respectively), and SEQ ID NOs: 284-293, 324-329, and 333-338), and HTT miRNA-1 or HTT miRNA-2. It will be further understood that the combinatorial effects observed with the exemplary HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein are similarly applicable to other HTT intron 2-directed (HTT_intron2_12061_150) exon editors, e.g., those comprising any one of SEQ ID NOs: 59, 350, or 351, which correspond to modified coding domain sequences. Thus, an HTT intron 2-directed exon editor may comprise any one of the variant coding domain sequences set forth in any one of SEQ ID NOs: 59 and 349-351.It will be further understood that the combinatorial effects observed with the exemplary HTT intron 2-directed (HTT_intron2_12061_150) exon editor (SEQ ID NO: 204) described herein are equally applicable to either an HTT intron 3-directed (e.g., comprising HTT_intron3_4223_150 or HTT_intron3_4233_150) exon editor or an HTT intron 1-directed (e.g., comprising HTT_intron1_11704_100 or HTT_intron1_11724_100) exon editor.

[0105] The trans-splicing efficiency of the HTT exon editor was comparable with or without the MSH3 splice modulator, as previously seen in Figure 52 (see, e.g., Figure 54; top panel). In the presence of HTT-miRNA1 or HTT-miRNA2, the percentage of edited HTT transcripts increased due to the reduction of unedited HTT transcripts (and thus total HTT transcripts) by the HTT miRNA (see, e.g., Figure 54; top panel). The middle panel of Figure 54 evaluates the performance of HTT miRNAs, and comparable efficiencies were observed for HTT miRNA-1 and HTT miRNA-2, regardless of whether the situation was a dual hybrid (exon editor + HTT miRNA) or triple hybrid (exon editor + HTT miRNA + MSH3 splice modulator). Evaluation of MSH3 knockdown efficiency confirmed that the knockdown efficiency of splice modulators was unchanged in the presence of both the exon editor and the HTT miRNA. See, e.g., the bottom panel of Figure 54. These results demonstrate the lack of interaction between the exon editor, HTT miRNA, and MSH3 splice modulator when co-delivered on the same DNA fragment. Notably, the combinatorial effect is equally applicable to any combination of HTT exon editor, HTT miRNA, and MSH3 splice modulator.

[0106] Evidence for mechanism in iCell GlutaNeurons To confirm and validate the effectiveness of the aforementioned molecules in additional in vitro cell systems, we utilized iCell GlutaNeurons (FujiFilm), human induced pluripotent stem (iPS) cell-derived glutamate-enriched cortical neurons. Exemplary molecules were packaged into AAV2.7m8 and used to transduce iCell GlutaNeurons to express the molecules of interest.

[0107] We tested two different AAV2.7m8 vectors for mechanistic proof-of-concept experiments using three separate modalities: 1) HTT exon replacement by trans-splicing, 2) HTT knockdown by miRNA targeting HTT exon 1, and 3) reduction of MSH3 by splice modulation of MSH3 exon 7. Notably, one of the tested AAVs (SEQ ID NO: 354) packaged a dual hybrid molecule expressing both an HTT intron 2-directed exon editor (SEQ ID NO: 204) and HTT miRNA-1 (encoding the active sequence SEQ ID NO: 339; pri-miRNA sequence (SEQ ID NO: 341)), while the other tested AAV (SEQ ID NO: 357) packaged a dual hybrid molecule expressing an HTT intron 2-directed exon editor (SEQ ID NO: 204) and an MSH3 exon 7-skipping splice modulator (SEQ ID NO: 331), each packaged within AAV2.7m8.

[0108] RT-ddPCR analysis measuring the percent level of edited (trans-spliced) HTT RNA showed that, without HTT miRNA, the dual hybrid molecule HTT exon editor + MSH3 splice modulator resulted in approximately 37% trans-splicing replacement in iCell GlutaNeurons. With the dual hybrid molecule exon editor + HTT miRNA, we observed an approximately 50% percent edited RNA, due in part to a reduction in native HTT copy number as a result of HTT miRNA. Western blot analysis of the N-terminal FLAG tag confirmed the presence of trans-spliced ​​full-length HTT protein. See, for example, Figure 55.

[0109] Further analysis of unedited HTT transcript levels suggested that, without HTT miRNA, the dual hybrid molecule containing the HTT exon editor and MSH3 splice modulator exhibited a 47% reduction in unedited HTT RNA in iCell GlutaNeurons as a result of trans-splicing (replacement of native RNA with modified RNA). In the presence of the HTT miRNA modality, the dual hybrid molecule containing the exon editor and HTT miRNA knocked down 50% of the miRNA-mediated unedited HTT transcript, resulting in a more favorable ratio of edited to unedited HTT transcripts. Western blot analysis to visualize total HTT (trans-spliced ​​and unedited HTT protein) confirmed the knockdown of unedited HTT with the additional HTT miRNA modality. See, for example, Figure 56.

[0110] We also tested MSH3 splice modulation modalities in iCell GlutaNeurons. Western blot analysis of MSH3 protein showed that the dual hybrid molecule exon editor + MSH3 exon 7 splice modulator (SEQ ID NO: 331) resulted in greater than 40% MSH3 protein knockdown in these cells. See, e.g., Figure 57. Overall, we observed comparable activity for each of the three HD modalities (HTT exon editor, HTT miRNA, and MSH3 splice modulator) in HEK293 cells and iCell GlutaNeurons.

[0111] Additional combination embodiments and related details: Improving exon substitution by HTT pre-mRNA trans-splicing Results presented herein (e.g., Figure 17) demonstrate that blocking HTT cis-splicing events can result in enhanced trans-splicing efficiency and HTT exon 1 replacement. Accordingly, embodiments of the present disclosure include strategies to block these cis-splicing events with antisense RNA (asRNA). Modified snRNAs, such as modified U7 snRNA (U7 Sm OPT), previously shown to act as splice modulators, can be used to express cis-splicing blockers from the same plasmid as the HTT-directed RNA exon editor. As described in detail herein, snRNAs such as U7 Sm OPT are engineered by 1) changing the targeting sequence (e.g., the histone-binding sequence in the 5' region of the U7 snRNA) to the complementary sequence of the gene to be modified, and 2) changing the binding site (AAUUUGUCUAG; SEQ ID NO: 367; U7 Sm WT) for U7 snRNP-specific proteins (Lsm10 and Lsm11) to the consensus binding sequence from the major spliceosomal U snRNP (AAUUUUUGGAG; SEQ ID NO: 368; U7 Sm OPT), resulting in binding of Sm proteins found in spliceosomal snRNPs to U7 Sm OPT. In some embodiments, the asRNA molecule can include a U1 promoter, a snRNA (with an asRNA sequence targeting the HTT intron-exon boundary and the consensus Sm binding site), and a U1 terminator. Exemplary asRNA sequences include (SEQ ID NOS: 127-135). Among these, the combination of the HTT intron 2-directed exon editor (HTT_intron2_12061_150) with ASO6 (SEQ ID NO: 131), which blocks cis-splicing of the upstream intron, resulted in particularly significant improvements in trans-splicing efficiency in vitro. See, for example, Figures 16 and 17.

[0112] Embodiments Directed to Inhibition of Somatic CAG Repeat Expansion Without wishing to be bound by theory, it is believed that the underlying mechanism of pathogenesis in HD and many other repeat expansion disorders is the somatic expansion of repeat sequences. As detailed herein, human genetic evidence strongly supports the idea that somatic instability resulting from somatic expansion of CAG repeat sequences can at least partially explain the clinical outcome of HD. Therefore, inhibiting the underlying pathogenic mechanisms resulting from somatic instability should alter the disease course and may be a potential treatment for HD and many other repeat disorders.

[0113] Therefore, the inventors designed and tested a strategy to treat HD early in its development by inhibiting somatic CAG expansion. The embodiments described herein, such as those describing inhibition of somatic CAG expansion, can be used alone or in conjunction with RNA exon editor-mediated trans-splicing to correct pathogenic mutations. While inhibition of somatic expansion alone can have a positive therapeutic effect on HD (see the combination embodiments section above), the inventors tested a hybrid therapeutic approach and a dual-action / hybrid molecule designed to inhibit somatic CAG expansion and correct HTT exon 1 RNA. See, e.g., Figures 19 and 20. The inventors also demonstrated the efficacy of this hybrid therapeutic approach using a dual-action / hybrid molecule designed to inhibit somatic CAG expansion and correct HTT exon 2 RNA. See, e.g., Figures 30-32, 52, and 53. Thus, embodiments described herein, such as those designed to inhibit somatic CAG expansions, can be used alone or in conjunction with RNA exon editor-mediated trans-splicing to correct pathogenic mutations.

[0114] The mechanisms that control this process involve cellular mechanisms, such as DNA mismatch repair enzymes such as MSH2 and MSH3. MSH3 is presumed to be a good target for inhibiting somatic CAG repeat expansion. Here, we outline several ongoing approaches designed to reduce MSH3 levels and inhibit somatic CAG repeat expansion.

[0115] Knockdown of MSH3 and other genes by trans-splicing: As described herein, trans-splicing can be used either alone or in combination with other applications of trans-splicing to reduce expression of MSH3 or other genes.

[0116] Embodiment A1.1. Trans-splicing for knockdown of MSH3 and other genes: As described herein, trans-splicing can be used to edit mRNA, but it is also practiced herein to reduce the expression level of a target gene. As described herein, reducing the expression level of a target gene can be achieved by trans-splicing a target pre-mRNA or mature mRNA to produce an RNA that is rapidly degraded or cannot produce a functional protein. See, for example, Figures 20-22 and 30-32. Both 5' exon editors and 3' exon editors can achieve this result. A 5' exon editor can, for example, replace one or more of the 5' exons of a target mRNA to remove the start codon and some or all of the coding sequence, which can be replaced with an alternative coding or non-coding sequence. A 3' exon editor can, for example, replace one or more of the 3' exons of a target mRNA, which can be replaced with an alternative coding or non-coding sequence. The resulting chimeric mRNA may be free of translation of any amino acids from the target mRNA. 5' exon editors can also insert 5' untranslated regions (UTRs), for example, that block translation. 3' exon editors can also insert 3' UTRs, for example, that destabilize the transcript or prevent it from being exported from the nucleus or translated. The portion of the target mRNA that remains after the trans-splicing reaction is presumed to be rapidly degraded because it lacks either a 5' cap (5' exon editor) or a 3' polyA tail (3' exon editor).

[0117] Embodiment A1.2. Tandem Binding Domain Exon Editor: Exon editors that edit a target such as HTT can simultaneously knock down a second gene, such as MSH3, by including two distinct binding domains (one targeting HTT and the other targeting MSH3). Examples of how this dual effect can be achieved are presented in Figures 19-22 and 30-32. Briefly, a 5'HTT exon editor containing the 5' portion of HTT can trans-splice to HTT to reconstitute a functional wild-type HTT mRNA, while also trans-splicing to MSH3 to generate a nonfunctional MSH3 mRNA. In this embodiment, the nonfunctional HTT-MSH3 chimeric mRNA encodes an HTT polypeptide that terminates at the first in-frame stop codon within the MSH3 portion of the mRNA. As a result, MSH3 pre-mRNAs affected by such a trans-splicing process are altered to encode little or no amino acids from the MSH3 coding sequence, making them likely susceptible to nonsense-mediated decay. This approach is applicable to binding domains targeting any MSH3 intron. Although the examples presented herein (Figures 19-22 and 30-32) use a 5' exon editor, a similar approach can also be carried out using a 3' exon editor.

[0118] Embodiment A.1.3. Tandem RNA Exon Editors: Tandem exon editors are two distinct exon editors inserted into the same plasmid, AAV, or other delivery vehicle. They can be organized in a head-to-head or head-to-tail configuration. Each exon editor may have its own regulatory sequence, including a promoter, or may be generated via cleavage events from a single bicistronic transcript. One exon editor can target one gene or intron, and the second exon editor can target a different gene or intron. In some embodiments, one exon editor can replace mutant HTT with wild-type HTT, and the second exon editor can reduce MSH3 expression. In some embodiments, one exon editor can target one intron of HTT, and the second editor can target a second intron of HTT, thereby improving overall HTT exon editing efficiency.

[0119] Embodiment A.2. Reduction of MSH3 or other genes by RNAi in combination with HTT trans-splicing: Short hairpin RNAs (shRNAs) or microRNAs (miRNAs) designed to reduce gene expression can be added to exon editors either within the same cistron (e.g., within an intron of the exon editor) or as separate cistrons with their own regulatory sequences. RNAi can reduce expression of the unedited target (e.g., mutant HTT) or another gene (e.g., MSH3). To selectively reduce mutant HTT using RNAi, the exon editor replaces a portion of the mutant HTT transcript with a sequence resistant to shRNA or miRNA. See, for example, Figures 33-35 and 46-50.

[0120] Embodiment A.3. Reduction of MSH3 by Vectorized Splice Modulation or Translational Blockade: In some embodiments, MSH3 is inactivated by blocking the inclusion of one or more MSH3 exons, the absence of which results in premature translation termination and thereby prevents translation of a functional protein. In some embodiments, MSH3 is inactivated by incorporating a sequence complementary to an MSH3 splice junction into a small nuclear RNA (snRNA) sequence that blocks exon inclusion, such as U7 snRNA. Here, splice modulation can be vectorized, i.e., delivered, by an AAV, which may also include an exon editor. See, e.g., Figures 36-45 and 51-54.

[0121] Embodiment A.3.1. asRNA against MSH3 using small nuclear RNA (e.g., U7SmOPT): In some embodiments, MSH3 is inactivated by incorporating a sequence complementary to an MSH3 splice junction into a snRNA sequence, such as U7 snRNA (see, e.g., Figures 36-45).

[0122] Embodiment A.3.2. MSH3 Reduction in Combination with HTT Trans-Splicing: In some embodiments, strategy A.3.1 and the other strategies described herein are integrated into the same DNA fragment, or the same fragment as the exon editor, or are co-packaged within a single AAV for co-delivery to patient tissue. See, e.g., Figures 33-54.

[0123] Potential reduction of HTT toxic species: Short hairpin RNAs (shRNAs) or microRNAs (miRNAs) designed to reduce gene expression can be added to exon editors as separate cistrons with their own regulatory sequences. RNAi can reduce the expression of unedited targets (e.g., mutant HTT). To selectively reduce mutant HTT using RNAi, the exon editor replaces a portion of the mutant HTT transcript with a sequence resistant to shRNA or miRNA. See Figures 46-50 and 54-56.

[0124] To address the possibility that treatment of HD may be enhanced by using combinatorial therapeutic approaches, the inventors designed hybrid, multifaceted approaches that target multiple mechanisms and / or pathogenic species. In some embodiments, these approaches included replacement of mutant HTT via an exon editor, knockdown of mutant HTT and related transcripts (e.g., HTT1a) via RNAi, and reduction of MSH3 via, for example, trans-splicing, antisense RNA, RNAi, vectored splice modulation, or translation blockade, or any combination thereof. These approaches, when integrated into the same DNA fragment or the same fragment as an exon editor, exhibit multimodality mechanisms of action yet can be delivered by a single AAV for co-delivery to patient tissue. Embodiments utilizing such combinatorial approaches may be used to achieve improved therapeutic efficacy in some, if not all, HD patients. Such approaches may confer additional disease-modifying effects.

[0125] Animal models BACHD Mouse Model: This animal model is a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) that expresses full-length human mutant huntingtin (fl-mhtt). It was developed by expressing fl-mhtt, which contains 97 glutamine repeats, on a BAC under the control of endogenous htt regulatory mechanisms. The glutamine repeats are encoded by 97 mixed CAG-CAA repeats. BACHD mice recapitulate human HD disease and disease progression, exhibiting progressive motor dysfunction, neuronal synaptic dysfunction, and delayed selective neuropathology, including significant cortical and striatal atrophy and striatal neuron degeneration. Based on robust behavioral and neuropathological phenotypes, which are well documented in the literature, BACHD mice are recognized as a suitable fl-mhtt mouse model for preclinical studies. BACHD mice are described in detail in Gray et al. (2008, J Neuroscience 28:6182, the contents of which are incorporated herein in their entirety) and are commercially available.

[0126] In some embodiments, the therapeutic efficacy of the nucleic acid trans-splicing molecules / RNA exon editors described herein in the context of a BACHD mouse model may be measured by at least one of an increase in percent replacement of pathogenic HTT RNA, a reduction in pathogenic HTT, a reduction in pathogenic HTT aggregates, improved motor coordination and balance (e.g., as measured by the rotarod test), a reduction in forebrain atrophy, or any combination thereof.

[0127] BAC-CAG mouse model: This animal model is a human genome-wide BAC transgenic mouse model of HD. It expresses human mutant huntingtin (mHTT) containing a long, uninterrupted, somatically unstable CAG repeat (120–130 pure CAG repeats) and exhibits a progressive disease-associated phenotype. Unlike other mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice exhibit robust striatal-selective nuclear inclusions and also exhibit the transcriptional dysregulation observed in HD patients and huntingtin knock-in models. BAC-CAG mice are described in detail in Gu et al. (2022, Neuron 110:1173, the contents of which are incorporated herein in their entirety) and are commercially available. As described there, striatal transcriptionopathy in HD models correlates with the length of their uninterrupted CAG repeats, rather than the length of the polyglutamine repeats. Also, as described therein, somatic CAG repeat instability and nuclear mHTT aggregates best correlate with early-onset striatal-selective molecular mechanisms of pathogenesis and locomotor and sleep disorders, whereas repeat RNA-associated pathology and repeat-associated non-AUG-dependent (RAN) translation may have less selective or delayed effects on pathogenic roles, respectively.

[0128] In some embodiments, in the context of a BAC-CAG mouse model, the therapeutic efficacy of a nucleic acid trans-splicing molecule / RNA exon editor described herein can be measured by at least one of an increase in percent replacement of pathogenic HTT RNA, a decrease in pathogenic HTT, a decrease in pathogenic HTT aggregates, an improvement in motor coordination and balance (e.g., as measured by the rotarod test), a decrease in forebrain atrophy, or a reduction in striatal-specific transcriptional dysfunction, or any combination thereof.

[0129] To demonstrate in vivo mechanistic evidence in the brain, we utilized the BAC-CAG mouse model (Gu et al. 2022). As described above, this transgenic mouse line contains the human mutant HTT genomic locus (more than 120 uninterrupted CAG repeats within HTT exon 1) and was engineered to exhibit abnormal features resembling disease phenotypes, including nuclear HTT aggregates and transcriptional dysregulation, from 12 months of age onward. We conducted mechanistic evidence-based studies of HTT trans-splicing in the BAC-CAG mouse brain using exemplary molecules packaged in AAV9. Three different AAV test articles were tested in this study: a CMV promoter-driven HTT intron 2-directed exon editor (SEQ ID NO:204) packaged into a self-complementary AAV (scAAV) (SEQ ID NO:363, which comprises SEQ ID NO:204), a CMV promoter-driven HTT intron 2-directed exon editor (SEQ ID NO:204) packaged into a single-stranded AAV (ssAAV) (SEQ ID NO:363, which comprises SEQ ID NO:204), and a CAGGS promoter-driven HTT intron 2-directed exon editor (SEQ ID NO:204) packaged into a ssAAV (SEQ ID NO:364, which comprises SEQ ID NO:204). All three test articles also contained a mouse-directed alternative MSH3 exon 7 splice modulator (SEQ ID NO:362) co-packaged as a hybrid molecule. The full sequences of the test articles are as follows: a CMV promoter-driven HTT intron 2-directed exon editor packaged into a scAAV (SEQ ID NO: 369), a CMV promoter-driven HTT intron 2-directed exon editor packaged into a ssAAV (SEQ ID NO: 370), and a CAGGS promoter-driven HTT intron 2-directed exon editor packaged into a ssAAV (SEQ ID NO: 371). To test the dose response of the exon editors, two different doses were selected for injection: 1E+11 vector genomes (vg) and 3E+11 vg (bilateral injections per animal).Two independent routes of administration were selected: intracerebroventricular (ICV) injection at neonatal P0 (Table 2) and intrastriatal injection at 8 weeks of age. Mouse cortices and striatum were harvested 4 weeks after injection, and the efficiency of HTT exon replacement by trans-splicing and MSH3 knockdown by splice regulation were profiled by RT-ddPCR and Western blotting.

[0130] [Table 1]

[0131] As shown in Figure 58, analysis of HTT trans-splicing profiles demonstrated that neonatal ICV injection achieved greater than 30% HTT replacement in the mouse brain. The CAGGS promoter-driven exon editor performed better than its CMV promoter-driven counterpart, as indicated by the higher % HTT replacement in both the cortex and striatum. A clear dose-response was observed among the CMV promoter-driven exon editors, such that animals treated with 3E+11vg had a higher % HTT replacement than animals treated with 1E+11vg. Importantly, trans-spliced ​​full-length HTT protein was detected by Western blotting against 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), suggesting that identifying a stronger promoter or administering a higher dose may result in even greater exon editor activity.

[0132] I. Definition As used herein, "trans-splicing" refers to the ligation of a first RNA molecule containing one or more exons (e.g., exogenous exons or exons that are part of the coding domain of the trans-splicing molecule) to a second RNA molecule (e.g., a pre-mRNA molecule, e.g., an endogenous pre-mRNA molecule) and replacing a portion of the second RNA molecule with a portion of the first RNA molecule via a spliceosome-mediated mechanism. The mechanism of a general RNA trans-splicing reaction is illustrated, for example, in Figure 3.

[0133] A "nucleic acid trans-splicing molecule" or "trans-splicing molecule" has three major elements: (a) a binding domain that confers specificity by anchoring the trans-splicing molecule to its target gene (e.g., pre-mRNA), (b) a splicing domain (e.g., a splicing domain with a 3' or 5' splice site), and (c) a coding domain configured to be trans-spliced ​​to a target nucleic acid that can replace one or more exons (e.g., one or more mutant exons) in the target nucleic acid. A "pre-mRNA trans-splicing molecule" or "RTM" refers to a nucleic acid trans-splicing molecule that targets a pre-mRNA. The terms "nucleic acid trans-splicing molecule" and "trans-splicing molecule" refer to both (1) DNA that encodes an RNA, where the RNA transcript is the effector molecule that physically associates with the target pre-mRNA, and (2) the RNA transcript itself. For clarity, the term "coding sequence" (e.g., trans-splicing molecule coding sequence) is used herein to clarify that the subject encodes an effector (e.g., the coding sequence is DNA and the effector is RNA). In some embodiments, the trans-splicing molecule coding sequence can include a cDNA, e.g., as part of a functional exon (e.g., a functional HTT exon) for replacement of a mutant HTT exon.

[0134] As used herein, the term "exon editor" can be used to refer to a trans-splicing molecule or a vector containing it (e.g., an AAV vector containing DNA encoding an RNA transcript that is a trans-splicing molecule).

[0135] As used herein, "trans-splicing efficiency" refers to the ratio between the detected expression level of the desired trans-spliced ​​RNA product (i.e., a chimeric RNA molecule that includes a functional exon of the trans-splicing molecule operably linked to the endogenous target pre-mRNA generated by the RNA trans-splicing reaction) and the amount of DNA or RNA introduced into the trans-splicing molecule (or reference molecule). In some cases, the expression level of the trans-spliced ​​RNA product is detected from RNA isolated from cells or tissues using RNA-seq.

[0136] As used herein, "% RNA replacement" refers to the fraction of the total target mRNA population that has successfully undergone trans-splicing (TS) and is calculated by the following equation: On-target (ONT) TS% = 100 * (ONT copy number / (ONT copy number + native copy number)).

[0137] As used herein, "relative trans-splicing efficiency" refers to the ratio of the test trans-splicing efficiency to the reference trans-splicing efficiency, where the test 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 the reference molecule (e.g., a reference molecule having the same elements as the nucleic acid trans-splicing molecule, except that the binding domain is replaced with a scrambled binding domain or a non-targeting binding domain (e.g., a binding domain comprising or consisting of SEQ ID NO: 7)). The relative trans-splicing efficiency of a trans-splicing molecule can be given as the ratio (also known as the fold increase) of the test RNA trans-splicing efficiency to the reference trans-splicing efficiency tested under similar conditions.

[0138] As used herein, the terms "operably linked" or "operatively linked" refer to the arrangement of elements so described, wherein the components are configured to perform their normal function. A nucleic acid is "operably linked" to another nucleic acid when it is placed into a functional relationship with the other nucleic acid sequence. Elements need not be contiguous to be operably linked. Thus, for example, intervening sequences can be present between operably linked sequences (e.g., a binding domain and coding sequence can be separated by an intervening sequence and the binding domain would still be considered "operably linked" to the coding sequence).

[0139] 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 protein (e.g., a target protein in which a mutation has been corrected). Thus, a coding domain can include one or more functional exons (e.g., a sequence of functional exons). In some cases, the one or more functional exons of a coding domain are not separated by introns (e.g., as in endogenous pre-mRNA) but are adjacent to each other (e.g., as in cDNA). In some cases, a coding domain can include one or more introns (e.g., naturally occurring introns) or untranslated regions (UTRs, e.g., naturally occurring UTRs) between or otherwise adjacent to (e.g., upstream or downstream of) the exons.

[0140] As used herein, "native 5'HTT untranslated region" or "native 5'HTT UTR" refers to a sequence greater than 20 nucleotides in length that has at least 90% sequence identity to a region of a native HTT gene (e.g., a human HTT gene) that is 5' to the ATG start codon. An example of a native 5'HTT untranslated region is represented by the DNA sequence of SEQ ID NO: 136. An example of a modified version of the 5'HTT untranslated region is represented by the DNA sequence of SEQ ID NO: 192.

[0141] As used herein, a "functional sequence of a 5' HTT exon" refers to a nucleic acid sequence comprising one or more of HTT exons 1-3 (e.g., exon 1; or exon 1 and exon 2; or exon, exon 2, and exon 3) that encodes a functional (biologically active) portion of the HTT protein. In some embodiments, a "functional sequence of a 5' HTT exon" refers to a nucleic acid sequence comprising HTT exon 1, HTT exons 1-2, or HTT exons 1-3 that encodes a functional (biologically active) portion of the HTT protein. When trans-spliced ​​to an endogenous HTT exon 3' of the binding site, the functional sequence of a 5' HTT exon results in expression of a functional HTT protein (e.g., a non-mutated HTT protein). In some cases, the functional sequence of a 5' HTT exon includes the sequence of an exon adjacent to the exon to which the trans-splicing molecule is trans-spliced ​​(e.g., a trans-splicing molecule that binds to HTT intron 2 and trans-splices with endogenous HTT exon 3 may include the functional sequence of a 5' HTT exon that includes exons 1 and 2, or a trans-splicing molecule that binds to HTT intron 3 and trans-splices with endogenous HTT exon 4 may include the functional sequence of a 5' HTT exon that includes exons 1-3).

[0142] As used herein, the term "functional," when used in reference to a protein, refers to a biologically active protein. The term "functional" may also refer to the amount of protein activity necessary to support normal cellular function. With respect to HTT, the term "functional" may refer to the amount of HTT protein activity necessary to restore HTT activity levels and support normal cellular function in the context of, for example, cortical pyramidal neurons, striatal medium spiny neurons, and / or hypothalamic neurons. Furthermore, reducing the amount of mutant HTT and / or shifting the mutant:wild-type ratio significantly contributes to reducing neurodegeneration in cortical pyramidal neurons, striatal medium spiny neurons, and / or hypothalamic neurons. In the context of treating a condition associated with pathogenic HTT activity (e.g., HD), or using a therapeutic agent comprising a nucleic acid trans-splicing molecule described herein, "functional" refers to reducing the amount of defective (non-functional) HTT protein, e.g., containing a polyglutamine sequence greater than 35 or 40 glutamine repeats, to eliminate one or more symptoms of the condition 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 reduces the level of pathogenic HTT activity 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 compared to that of control (untreated) cells expressing mutant pathogenic HTT.In some embodiments, such an increase in HTT protein activity restores HTT activity levels 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 compared to that of cells in which HTT is present at normal wild-type levels as present in cells expressing non-mutated HTT.

[0143] As used herein, "cryptic splice site corrected," "cryptic splice site mitigated," or "cryptic splice site resistance" 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 to reduce the frequency of splicing occurring at a cryptic splice site in the context of the nucleic acid trans-splicing molecule. In some embodiments, the modification does not result in any change to the amino acid sequence encoded thereby. In some embodiments, the cryptic splice site resistance nucleic acid sequence within the nucleic acid trans-splicing molecule is within a coding domain sequence (CDS). In some embodiments, the cryptic splice site-resistant HTT CDS comprises, consists essentially of, or consists of exon 1, or exons 1 and 2, or exons 1, 2, and 3 of the HTT gene, wherein the cryptic splice sites have been identified in association with a nucleic acid trans-splicing molecule, and at least one of the cryptic splice sites has been modified to reduce the frequency of splicing at the at least one site but not alter the amino acid encoded thereby.

[0144] As used herein, "splice domain" refers to a nucleic acid sequence having a motif that is recognized by the spliceosome and mediates trans-splicing. A splice domain includes a splice site (e.g., a single splice site, i.e., only one splice site), which may be a 3' splice site or a 5' splice site. A splice domain may include other regulatory elements. In some embodiments, the splice domain includes GUAAGT or GTAAGT. In some embodiments, the splice site consists essentially of GUAAGT or GTAAGT. In some embodiments, the splice domain consists of GUAAGT or GTAAGT.

[0145] As used herein, a "binding domain" of a trans-splicing molecule is a polynucleotide sequence that binds to a target gene at a binding site through hybridization (i.e., complete or partial complementarity to the binding site).

[0146] As used herein, the term "binding site" refers to an endogenous polynucleotide sequence within a target pre-mRNA (e.g., an endogenous gene, e.g., the pre-mRNA of HTT) to which a binding domain of a nucleic acid trans-splicing molecule binds. The binding site extends from the 5'-most nucleotide bound by the binding domain to the 3'-most nucleotide bound by the binding domain. In some embodiments, the binding site is the same length as the binding domain. In other embodiments, the binding site is longer or shorter than the binding domain (i.e., some of the nucleotides of either the binding site or the binding domain are not hybridized), ranging from 1-10 nucleotides. In embodiments involving binding domains with at least two non-overlapping sequences having at least 80% complementarity to the binding site, the binding site may be substantially shorter than the binding domain.

[0147] As used herein, "complementarity" and grammatical variations thereof refer to the percentage of nucleotide bases of a given sequence that pair with a reference sequence through hydrogen bonding.

[0148] As used herein, a given sequence (e.g., a binding domain sequence) is "100% complementary" to, or has "100% complementarity" with, a reference sequence (e.g., an endogenous pre-mRNA binding site) if each of the nucleotide bases of the given sequence pairs with the reference sequence through hydrogen bonds, thereby hybridizing to form a double-stranded sequence (e.g., through Watson-Crick base pairing, e.g., each A pairs with a T or a U, and each C pairs with a G). For example, a binding domain in antisense orientation to a binding site is complementary to the binding site. RNA pairing includes G pairing with U; thus, an RNA binding domain having GU pairing with the binding site may be 100% complementary to the binding site. Thus, a binding domain that is exactly the reverse complement of its binding site (i.e., an A in the binding domain pairs with a U in the binding site) can be modified to replace any one or more of the As with G, or replace a C with a T, without substantially affecting binding.

[0149] As used herein, a given sequence (e.g., a binding domain sequence) is "at least X% complementary" to, or "X% complementary to," a reference sequence (e.g., an endogenous pre-mRNA binding site) if X% of the nucleotide bases in the given sequence pair with, e.g., hybridize to form a double-stranded sequence (e.g., by Watson-Crick base pairing, e.g., A pairs with T or U, C pairs with G) through hydrogen bonding with the reference sequence. For example, a binding domain sequence 150 bases in length is at least 90% complementary to a 150-base long binding site if at least 135 of its 150 residues hydrogen bond with the binding site through Watson-Crick base pairing, leaving 15 or fewer mismatched nucleotides.

[0150] With respect to the sequences presented herein and in the accompanying sequence listing, it is understood that the RNA transcript encoded by the DNA sequence will include a uridine (U) at the position corresponding to the thymidine (T) set forth in the corresponding DNA sequence. In some cases, the sequence of an RNA exon editor component is disclosed herein as a DNA sequence. For any sequence disclosed herein as a DNA sequence, an RNA sequence having a U in place of each T in the sequence is also contemplated. Thus, when a given SEQ ID NO is identified as having a sequence that may be included in an RNA exon editor, a version of the SEQ ID NO having a U in place of each T is also contemplated.

[0151] As used herein, "binding" between a binding domain and an intron refers to hydrogen bonding (e.g., double helix formation, or Watson-Crick pairing) between the binding domain and the target intron sufficient to mediate trans-splicing by associating the trans-splicing molecule with a target (e.g., pre-mRNA). In some embodiments, the hydrogen bonding between the binding domain and the target intron is between nucleotide bases that are complementary to each other and in antisense orientation (e.g., hybridize to each other).

[0152] As used herein, an "artificial intron" refers to a non-coding nucleic acid sequence that links (directly or indirectly) a binding domain to a coding domain. The artificial intron includes a splicing domain and may further include one or more spacer sequences and / or other regulatory elements.

[0153] As used herein, the term "mutation" can refer to any abnormal nucleic acid sequence that encodes a defective RNA or protein product (e.g., a non-functional protein product, a protein that is not biologically active, a protein product with reduced function, a protein product with pathogenic or abnormal function, and / or a protein product that is produced less than or more than normal). Mutations include base pair mutations (e.g., single nucleotide polymorphisms), duplications, missense mutations, frameshift mutations, deletions, insertions, and splice mutations. In some embodiments, a mutation refers to a nucleic acid sequence in which one or more portions of its sequence differ from the corresponding wild-type nucleic acid sequence or a functional variant thereof. In some embodiments, a mutation refers to a nucleic acid sequence that encodes a protein whose amino acid sequence differs from the corresponding wild-type protein or a functional variant thereof. A "mutated exon" (e.g., a mutant HTT exon) refers to an exon containing a mutation or an exon sequence that reflects a mutation in a different region, e.g., a cryptic exon resulting from a mutation in an intron.

[0154] The term "HTT" (huntingtin), unless otherwise indicated, refers to any native HTT from any vertebrate source, e.g., 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, muteins, analogs, subunits, receptor complexes, isotypes, splice variants, and fragments thereof. Functionally equivalent improved variants can be determined based on known HTT signaling. HTT encompasses full-length, unprocessed HTT and any form of HTT resulting from natural processes in cells. An exemplary human HTT sequence is provided as National Center for Biotechnology Information (NCBI) Reference Sequence: NG_009378. In some cases, the HTT fragment is encoded by a therapeutic agent that includes a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 3, 59, 157, 349, 350, 351, 352, or 353 (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 59, 157, 349, 350, 351, 352, or 353), a functional portion thereof, and / or a codon-modified variant thereof.

[0155] As used herein, "variant" refers to a polynucleotide that differs from a reference polynucleotide sequence, such as a naturally occurring polynucleotide sequence, by at least one nucleic acid residue, or a polypeptide that differs from a reference polypeptide sequence, such as a naturally occurring polypeptide sequence, by at least one amino acid residue (e.g., an AAV capsid sequence), or, for example, any of the rAAV sequences described herein. In this context, a difference in at least one residue can include, for example, a substitution, deletion, or insertion of a nucleic acid residue with another nucleic acid, or a substitution of an amino acid residue with another amino acid. A variant can be a homolog, isoform, or transcript variant of a polynucleotide defined herein, where the homolog, isoform, or transcript variant is characterized by a degree of identity or homology, respectively, as defined herein.

[0156] In some cases, a variant of a polynucleotide or polypeptide comprises at least one nucleic acid substitution (e.g., 1 to 100 nucleic acid or amino acid substitutions, 1 to 50 nucleic acid or amino acid substitutions, 1 to 20 nucleic acid or amino acid substitutions, 1 to 10 nucleic acid or amino acid substitutions, e.g., 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 result in an expressed polypeptide with replaced amino acids from the same class are referred to herein as conservative substitutions. In particular, these are side chains with aliphatic side chains, positively or negatively charged side chains, or aromatic groups that are capable of forming hydrogen bridges, e.g., side chains with hydroxyl functions. A conservative substitution may, for example, replace an amino acid having a polar side chain with another amino acid having a corresponding polar side chain, or may, for example, replace an amino acid characterized by a hydrophobic side chain with another amino acid having a corresponding hydrophobic side chain, such as serine (threonine) with threonine (serine) or leucine (isoleucine) with isoleucine (leucine).

[0157] In some cases, for example, insertions, deletions, and / or non-conservative substitutions at positions that do not result in a substantial change in the three-dimensional structure of the protein are also encompassed by the term variant. Alterations in the three-dimensional structure due to insertion(s) or deletion(s) can be readily identified by those skilled in the art, for example, using CD spectroscopy (circular dichroism spectroscopy).

[0158] The term "homology" refers to the degree of identity between two nucleic acid sequences. Sequence homology is determined by comparing two sequences aligned under standard conditions across the length of the sequences being compared. The sequences compared herein may have additions or deletions (e.g., gaps) in the optimal alignment of the two sequences. In some embodiments, sequence homology is calculated by, for example, creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 1994, 22(22):46734680). Commonly available sequence analysis software such as Vector NTI, GENETYX, BLAST, or analysis tools provided by public databases can also be used.

[0159] As used herein, the term "AAV" or "AAV serotype" refers to dozens of naturally occurring, available adeno-associated viruses, and engineered AAVs. Among the well-characterized AAVs isolated or engineered from humans or non-human primates (NHPs), human AAV2 was the first AAV developed as a gene transfer vector and has been widely used for efficient gene transfer experiments in different target tissues and animal models. Among AAV serotypes, for example, AAV9, AAV-retro, AAV1, AAV4, AAV8, AAV5, and AAV-PHP.eB are neurotropic in nature.

[0160] As used herein, in the context of AAV, the term variant refers to any AAV sequence derived from a known AAV sequence, e.g., sharing 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 more sequence identity to the amino acid or nucleic acid sequence. In another embodiment, the AAV capsid includes variants that may include up to about 10% variation from any described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity to an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV capsid. When determining the percent identity of an AAV capsid, the comparison can be made to any of the proteins (e.g., vp1, vp2, or vp3), which may differ.

[0161] ITRs or other AAV components can be readily isolated or engineered from AAV using techniques available to those skilled in the art. Such AAV can be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences can be engineered synthetically or via other suitable means by reference to published sequences available in the literature or in databases such as GenBank, PubMed, etc. AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimized immunogenicity, tailored stability and particle lifespan, efficient degradation, precise delivery to the nucleus, etc.

[0162] As used herein, the terms "subject," "individual," or "patient" include any mammal in need of these therapeutic or prophylactic methods, including primates such as humans. Other mammals in need of such treatment or prevention include non-human primates (NHPs; e.g., cynomolgus monkeys and African green monkeys), dogs, cats, or other domestic animals, horses, livestock, laboratory animals, etc. The individual may be male or female. In one embodiment, the individual has a disease or disorder caused by a mutation in the HTT gene (e.g., HD). In another embodiment, the individual is at risk of developing a disease or disorder caused by a mutation in the HTT gene. In another embodiment, the individual exhibits clinical signs of a disease or disorder caused by a mutation in the HTT gene, such as HD. The individual may be of any age that may benefit from therapeutic or prophylactic treatment. For example, in some embodiments, the individual is between 0 and 5 years old, between 5 and 10 years old, between 10 and 20 years old, between 20 and 30 years old, between 30 and 40 years old, between 30 and 50 years old, between 40 and 50 years old, between 50 and 60 years old, between 60 and 70 years old, or over 70 years old.

[0163] As used herein, the term "mutation-associated disorder" or "mutation associated with disorder" refers to a correlation between a disorder and a mutation. In some embodiments, a mutation-associated disorder is known or suspected to be caused, in whole or in part, or directly or indirectly, by the mutation. For example, an individual with a mutation may be at risk of developing the disorder, and the risk may further depend on other factors, such as other (e.g., independent) mutations (e.g., in the same gene or different genes), or environmental factors.

[0164] As used herein, the term "treatment," or grammatical derivatives thereof, is defined as alleviating the progression of a disease, reducing the severity of disease symptoms, delaying the progression of disease symptoms, eliminating disease symptoms, or delaying the onset of disease. In some embodiments, the term "treatment" is used to refer to a durable, lasting effect of a therapeutic agent, such as an RNA exon editor described herein.

[0165] As used herein, the term "prevention" of a disorder, or its grammatical derivatives, is defined as reducing the risk of developing a disease, for example, as prophylactic treatment for an individual at risk of developing a disorder associated with a mutation. An individual can be characterized as being "at risk" of developing a disorder by identifying a mutation associated with the disorder according to any suitable method known in the art or described herein. In some embodiments, an individual at risk of developing a disorder has one or more HTT mutations associated with the disorder. Additionally or alternatively, an individual can be characterized as being "at risk" of developing a disorder if they have a family history of the disorder.

[0166] Treatment or prevention of a disorder in an individual can be carried out by directly administering a trans-splicing molecule or RNA exon editor (e.g., in a vector, e.g., an AAV vector or AAV particle) to the individual. Alternatively, host cells containing the trans-splicing molecule can be administered to the individual.

[0167] As used in the methods described herein, the term "administering" or grammatical derivatives thereof refers to administering a trans-splicing molecule or RNA exon editor (e.g., in a vector, e.g., in an AAV vector or AAV particle) or composition thereof, or ex vivo treated cells, to an individual in need thereof, e.g., an individual with a mutation or defect in HTT. For example, in one embodiment in which striatal cells (e.g., medium spiny neurons) or cortical cells (e.g., pyramidal neurons) are targeted, the method involves delivering a trans-splicing molecule or RNA exon editor (e.g., in a vector, e.g., in an AAV vector or AAV particle) or composition thereof to an individual via intracerebral (IC) delivery (e.g., slow delivery injection or convection-enhanced diffusion injection), intracerebroventricular (ICV) delivery, or intrathecal delivery. In some embodiments, IC injection involves stereotactic implantation of a microinjection guide sleeve to improve delivery to specific loci in the brain. In another embodiment, the composition is administered systemically (e.g., intravenously). Still other administration methods may be selected by those skilled in the art in light of the present disclosure.

[0168] As used herein, "modulating the expression of HTT" refers to reducing the expression of endogenous mutant HTT and / or increasing the expression of trans-spliced ​​HTT. Modulating the expression of HTT can refer to, for example, reducing the expression of endogenous (e.g., mutated) HTT and / or increasing the expression of trans-spliced ​​HTT (e.g., an HTT transcript or protein product having a corrected mutation site mediated by a trans-splicing molecule) compared to its endogenous mutant transcript or protein product. When the endogenous HTT exon containing the mutation site is replaced via trans-splicing, functional HTT protein is expressed.

[0169] As used herein, "codon optimization" refers to modifying a nucleic acid sequence to alter individual nucleic acids without resulting in any changes to the encoded amino acids. Such modified sequences are referred to herein as "codon optimization." This process can be performed on any of the sequences described herein to enhance performance or stability. Codon optimization can be performed, for example, in the manner described in U.S. Pat. Nos. 7,561,972, 7,561,973, or 7,888,112, each of which is incorporated herein by reference in its entirety. The sequence surrounding the translation start site can be converted to a consensus Kozak sequence according to known methods. See, e.g., Kozak et al., 1987. Nucleic Acids Res. 15(20):8125-8148, incorporated herein by reference in its entirety. The term "pharmaceutically acceptable" means safe for administration to mammals, such as humans. In some embodiments, a pharmaceutically acceptable composition is approved by a federal or state regulatory agency or is listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic molecule (e.g., a trans-splicing molecule, or a trans-splicing molecule comprising a vector or cell of the present invention) is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition.

[0170] The terms "a" and "an" mean "one or more of." For example, "a gene" is understood to refer to one or more such genes. Thus, the terms "a" and "an," "one or more of," and "at least one of" are used interchangeably herein.

[0171] As used herein, the term "about" refers to a value within ±10% variation from the reference value, unless otherwise specified.

[0172] II. Trans-splicing molecules Provided herein is a nucleic acid trans-splicing molecule useful for correcting HTT mutations by replacing at least one mutant HTT exon with a functional HTT exon (e.g., the HTT exon 5' of the binding site, for example, exon 1 (SEQ ID NO: 348; HTT 5'UTR + exon 1, which contains 21 CAG repeats (within the normal range of repeat number)), exon 2 (SEQ ID NO: 365), and exon 3 (SEQ ID NO: 366 of HTT)). Note that although the sequence of HTT exon 1 described in SEQ ID NO: 348 contains 21 CAG repeats, the number of CAG repeats in individual genomic sequences may vary (i.e., be more or less). The corresponding nucleotide numbering positions in sequences with different numbers of CAG repeats can be easily determined, taking into account any variations in the number of CAG repeats. In some embodiments, the nucleic acid trans-splicing molecule is an RNA trans-splicing molecule (RTM). In designing a trans-splicing molecule, the defective or mutated portion of a pre-mRNA exon(s) can be replaced with a nucleic acid sequence, e.g., an exon(s) having a functional (e.g., normal) sequence without the mutation. The functional sequence can be a wild-type, naturally occurring sequence or a corrected sequence with some other modification, e.g., codon optimization.

[0173] 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 with a native 5' HTT untranslated region (e.g., a sequence having at least 80% sequence identity to either SEQ ID NO: 136 or 192). In some embodiments, the nucleic acid trans-splicing molecule has a GTAAGT splice site. In some embodiments, the nucleic acid trans-splicing molecule has a linker domain greater than 25 nucleotides in length. In some embodiments, the nucleic acid trans-splicing molecule has a linker domain that comprises, consists essentially of, or consists of any one of SEQ ID NOs: 37-46 and 106-112, or a sequence having at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 37-46 and 106-112.

[0174] In some embodiments, the trans-splicing molecules described herein include, operably linked in a 5' to 3' direction, a 5' untranslated region, a coding domain sequence (e.g., a CDS, e.g., a sequence encoding a functional sequence of an HTT exon, e.g., a functional sequence of an HTT exon 5' to a binding site), a splicing domain (e.g., a splice site), a linker domain, a binding domain, a 3' downstream region, and a terminator domain.

[0175] In some embodiments, the nucleic acid trans-splicing molecules described herein are configured to correct at least one mutation in an allele of a subject's HTT gene located in a 5' region of the HTT gene (e.g., a region 5' to intron 1, 2, or 3) by binding to target intron 1, 2, or 3 and mediating trans-splicing of a coding domain having a functional sequence of the 5' HTT exon to an endogenous HTT exon 3' to the target intron. Such trans-splicing thereby repairs the defective HTT gene in a target cell of an individual by replacing the defective exon(s) and removing the defective portion of the target pre-mRNA, resulting in a wild-type HTT mRNA capable of transcribing functional HTT protein in the cell.

[0176] HTT The HTT gene targeted by the trans-splicing molecules described herein can contain one or more mutations associated with HD. An exemplary human HTT sequence is provided as the National Center for Biotechnology Information (NCBI) reference sequence: NG_009378. In addition to the published sequence, any subsequent modifications or naturally occurring conservative and non-disease-causing variant sequences occurring in human or other mammalian populations are also included. Additional conservative nucleotide substitutions or codon optimizations are also included. Sequences provided by database accession numbers can be used to search for homologous sequences in the same or other mammalian organisms.

[0177] HTT nucleic acid sequences and the resulting expressed proteins can tolerate certain minor modifications at the nucleic acid level, including, for example, modifications to nucleotide bases that are silent with respect to the encoded amino acid. In other embodiments, nucleic acid base modifications that change amino acids are contemplated, for example, to improve expression of the resulting peptide / protein. In some embodiments, modifications of allelic variations caused by the natural degeneracy of the genetic code are contemplated.

[0178] Modifications of the HTT gene also include analogs or variants of the encoded amino acid sequence. Typically, such analogs differ from the specifically identified protein by only one to four codon changes. Conservative substitutions are those that occur within a family of amino acids that are related in side chains and chemical properties.

[0179] The nucleic acid sequence of functional HTT gene can be derived from any mammal that naturally expresses functional HTT or its homologue.In other embodiments, in order to enhance expression in target cells, certain modifications are made to the HTT gene sequence.Such modifications can include codon optimization.

[0180] As described herein above, HD is caused by an expansion of CAG trinucleotide repeats in exon 1 of the HTT gene, greater than 35 CAG repeats (incomplete penetrance) or greater than 40 CAG repeats (juvenile or adult onset), and is inherited in an autosomal dominant manner. See Figure 1. Compositions comprising the trans-splicing molecules described herein can correct expanded CAG repeats in exon 1, regardless of the number of repeats present, because the trans-splicing molecule replaces the entire pathogenic exon 1 of the HTT gene.

[0181] Code Domain In some embodiments, the coding domain of the 5' trans-splicing molecule includes all HTT exons (e.g., functional HTT exons) 5' to the targeted HTT intron (e.g., HTT intron 1 [(SEQ ID NO: 348; HTT 5'UTR + exon 1, which may include 21 CAG repeats (within the normal range of repeat number), or 35-39 CAG repeats (incomplete penetrance) or 40+ CAG repeats (adult-onset or juvenile-onset HD)], intron 2 (SEQ ID NO: 365), and / or intron 3 (SEQ ID NO: 366)). For example, in embodiments in which the 5' trans-splicing molecule targets HTT intron 2, the coding domain includes all HTT exons (e.g., functional HTT exons) 5' to the targeted HTT intron (e.g., HTT intron 1 [(SEQ ID NO: 348; HTT 5'UTR + exon 1, which may include 21 CAG repeats (within the normal range of repeat number), or 35-39 CAG repeats (incomplete penetrance) or 40+ CAG repeats (adult-onset or juvenile-onset HD)], intron 2 (SEQ ID NO: 365), and / or intron 3 (SEQ ID NO: 366)). In some embodiments, functional HTT exons 1-2 may be encoded by a sequence comprising any one of SEQ ID NOs: 59, 349, 350, or 351. In some embodiments, functional HTT exons 1-2 are encoded by a sequence comprising any one of SEQ ID NOs: 59, 349, 350, or 351, which further comprises an ATG start codon at the 5' end. In some embodiments, the binding domain binds to intron 2, and the coding domain comprises functional HTT exons 1-2.

[0182] In some embodiments, in which the 5' trans-splicing molecule targets HTT intron 3, the coding domain can include functional HTT exons 1-3. In some embodiments, the functional HTT exons 1-3 are encoded by a sequence comprising any one of SEQ ID NOs: 157, 352, or 353. In some embodiments, the functional HTT exons 1-3 are encoded by a sequence comprising SEQ ID NOs: 157, 352, or 353, which further comprises an ATG start codon at the 5' end. In some embodiments, the binding domain binds to intron 3, and the coding domain includes functional HTT exons 1-3.

[0183] In some embodiments, in which the 5' trans-splicing molecule targets HTT intron 1, the coding domain can include functional HTT exon 1. In some embodiments, the functional HTT exon 1 is encoded by a sequence comprising SEQ ID NO: 3. In some embodiments, the functional 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, the binding domain binds to intron 1, and the coding domain includes functional HTT exon 1.

[0184] In some embodiments, the sequence encoding the coding domain (e.g., of a transgene encoding an RTM) includes a cDNA of an HTT exon (e.g., an HTT exon) for replacement of a mutant HTT exon(s). For example, one or more functional HTT exons within the coding domain can be a cDNA sequence. In some embodiments, the entire coding domain is a cDNA sequence. Additionally or alternatively, all or a portion of the coding domain or one or more functional HTT exons thereof can be a naturally occurring sequence (e.g., a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an endogenous HTT exon).

[0185] In some embodiments, the coding domain or all or a portion of the sequence encoding the coding domain, or one or more functional HTT exons thereof, is a codon-optimized sequence, in which the nucleic acid sequence has been modified, for example, to enhance expression or stability, without causing changes in the encoded amino acids. Codon optimization can be performed, for example, as described in 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. As described herein, for delivery via recombinant AAV, in one embodiment, the coding domain can be a nucleic acid sequence up to 4,000 nucleotide bases in length.

[0186] In some embodiments, a nucleic acid trans-splicing molecule is described herein, comprising, in a 5' to 3' direction, (a) a cDNA coding domain sequence, (b) a splice donor sequence, and (c) a binding domain sequence configured to bind to an intron of an endogenous RNA molecule, wherein the coding domain sequence comprises at least one nucleotide mutation relative to the endogenous RNA molecule sequence, wherein the at least one nucleotide mutation disrupts a cryptic splice site within the coding domain sequence. 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 in silico analysis.

[0187] Also encompassed herein are methods for modifying an RNA molecule in a cell, the methods comprising providing an exogenous RNA molecule to a cell, the molecule comprising, in a 5' to 3' direction: (a) a cDNA coding domain sequence comprising a nucleotide mutation that differs 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 in 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 in silico analysis.

[0188] Also encompassed herein are methods for increasing the trans-splicing efficiency or therapeutic effect of an RNA exon editor, comprising introducing a mutation into the coding domain sequence of the RNA exon editor, wherein the mutation disrupts a cryptic splice site within 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 splice site is experimentally identified. In some embodiments, the cryptic splice site is predicted based on in silico analysis.

[0189] Nucleotide changes that alleviate cryptic splice sites can include changes that eliminate or reduce the ability of the cryptic splice site to be used in a splicing reaction. For example, cryptic splice sites identified in association with an RNA exon editor can include a splice site, a polypyrimidine sequence, and a branch site. In some embodiments, one or more nucleotide changes can be introduced into at least one of the splice site, the polypyrimidine sequence, or the branch site, or any combination thereof, of a cryptic splice site identified in association with an RNA exon editor. In some embodiments, the nucleotide changes are determined to minimize the potential impact on the protein encoded thereby. Those skilled in the art will understand that if a nucleotide change made to reduce the frequency of cryptic splice site usage also changes the amino acids encoded by the trans-splicing RNA, a conservative amino acid change is preferable to a non-conservative amino acid change. Furthermore, such skilled artisans can easily analyze the sequence and structure of a protein with an eye toward functional domains and important sequences therein to assess whether such changes can reasonably be expected to alter the function of the protein encoded by the trans-splicing protein. One skilled in the art can also use assays known in the art to test proteins containing such amino acid changes to determine whether biological activity is altered. In some embodiments, multiple nucleotides are altered within the identified cryptic splice site. Under some circumstances, the determination of the number of nucleotides to be altered is performed empirically based on in silico predictions and / or experimental results. In some embodiments, one or more (also referred to herein as at least one) synonymous mutations can be introduced into at least one of a splice site, a polypyrimidine tract, or a branch point, or any combination thereof, of a cryptic splice site identified in the context of an RNA exon editor. Synonymous mutations do not change the amino acid sequence of the protein encoded by the trans-splicing RNA.In some embodiments, two or more synonymous mutations may be introduced into at least one of a splice site, a polypyrimidine tract, or a branch site, or any combination thereof, of a cryptic splice site identified in association with an RNA exon editor.

[0190] In addition to the above, experimental results and sequence information are analyzed broadly as follows. Modifications to eliminate experimentally identified cryptic splice sites are performed by searching for splice acceptor sites and replacing specific elements within those sites. Nucleotide changes are prioritized for AG sites (more likely CAG sites) at the ends of the splice acceptor sites. If an AG site is not found or cannot be modified without introducing nonsynonymous mutations, the sequence 42–4 base pairs upstream of the splice site is scanned for branch sites (sequences matching YNAH). Any such branch site sequences identified are then analyzed and considered for the introduction of one or more nucleotide mutations to reduce cryptic splice site utilization at the experimentally identified cryptic splice site. Furthermore, sequences are also scanned for the presence of polypyrimidine sequences (multiple Ys adjacent to the terminal AG). Typically, such polypyrimidine sequences contain at least five pyrimidines within 10 base pairs upstream of the splice site. Once identified, such polypyrimidine sequences are then analyzed and considered for the introduction of nucleotide mutation(s) to reduce cryptic splice site utilization at the experimentally identified cryptic splice sites.

[0191] In some embodiments, the cryptic splice site or off-target splice site that is modified to reduce off-target splicing is the site that has been empirically identified as the site of off-target splicing.Such site can be identified, for example, by using the technique described in Example 6 of WO2023 / 220742, the entirety of which is incorporated herein by reference.In some embodiments, all cryptic splice sites that have a usage frequency above a predetermined threshold are modified by the nucleotide change that reduces the cryptic splice site.

[0192] In some embodiments, the cryptic splice sites, or off-target splice sites, that are altered to mitigate off-target splicing are predicted sites of off-target splicing. Such predictions can be made based on sequence analysis to identify canonical splice sites, polypyrimidine sequences, and / or branch sites of putative cryptic splice sites therein.

[0193] In some embodiments, a nucleotide change that reduces a cryptic splice site causes a nucleotide sequence that matches a canonical splice site consensus sequence to no longer match the canonical sequence. In some embodiments, a nucleotide change that reduces a cryptic splice site eliminates a cryptic splice site nucleotide. In some embodiments, a nucleotide change that reduces a cryptic splice site eliminates a potential polypyrimidine tract nucleotide. In some embodiments, a nucleotide change that reduces a cryptic splice site eliminates a potential branch point nucleotide. In some embodiments, a nucleotide change that reduces a cryptic splice site is a synonymous nucleotide change. In some embodiments, a nucleotide change that reduces a cryptic splice site causes a change in an amino acid encoded by the exon editor. In some embodiments, the amino acid change is a conservative amino acid substitution.

[0194] To address the possibility of potential self-splicing (either due to cis-splicing or intermolecular trans-splicing of AAV concatemers), we used in silico prediction of self-splicing sites to mitigate such undesired events (see, e.g., Table 1). [Table 2]

[0195] Binding domain The HTT trans-splicing molecules described herein are characterized by a binding domain (BD) configured to bind / anneal with a targeted HTT intron and / or exon. In some cases, the targeted HTT intron is HTT intron 2. In one embodiment, the binding domain is a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., a target HTT intron) (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto), which can suppress endogenous target cis-splicing while enhancing trans-splicing between a trans-splicing molecule and a target HTT pre-mRNA (e.g., endogenous HTT by creating a chimeric molecule having a portion of the mRNA and a coding domain having one or more functional HTT exons encoding the wild-type HTT amino acid sequence.In one embodiment involving a sequence encoding a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto).

[0196] In some cases, the invention provides trans-splicing molecules (or vectors thereof) that bind to HTT in intron 2, e.g., a nucleic acid trans-splicing molecule configured to trans-splice a coding domain into endogenous HTT exon 3. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to a binding site having any one or more (e.g., 6 or more, 8 or more, 10 or more, or 12 or more) of nucleotides 1-200, 1,500-2,500, or 10,500-12,251 of SEQ ID NO:57.

[0197] In some cases, the binding site includes any six or more contiguous nucleotides within nucleotides 1-200, 1,500-2,500, or 10,500-12,251 of HTT intron 2 (e.g., any eight or more contiguous nucleic acids within nucleotides 1-200, 1,500-2,500, or 10,500-12,251 of HTT intron 2 ... Any 10 or more consecutive nucleic acids, any 12 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2, any 20 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2, any 30 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2 any 40 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2; any 50 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2; any 100 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2; any 150 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of intron 2; any 200 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2; or any 250 or more consecutive nucleic acids within nucleotides 1 to 200, 1,500 to 2,500, or 10,500 to 12,251 of HTT intron 2).

[0198] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 10,000-12,251 of HTT intron 2 (e.g., any 8 or more contiguous nucleic acids within nucleotides 10,000-12,251 of HTT intron 2, any 10 or more contiguous nucleic acids within nucleotides 10,000-12,251 of HTT intron 2, any 12 or more contiguous nucleic acids within nucleotides 10,000-12,251 of HTT intron 2, any 20 or more contiguous nucleic acids within nucleotides 10,000-12,251 of HTT intron 2, any 30 or more contiguous nucleic acids within nucleotides 10,000-12,251 of HTT intron 2). any 40 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2; any 50 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2; any 100 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2; any 150 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2; any 200 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2; or any 250 or more consecutive nucleic acids within nucleotides 10,000 to 12,251 of HTT intron 2.

[0199] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 11,000-12,251 of HTT intron 2 (e.g., any 8 or more contiguous nucleic acids within nucleotides 11,000-12,251 of HTT intron 2, any 10 or more contiguous nucleic acids within nucleotides 11,000-12,251 of HTT intron 2, any 12 or more contiguous nucleic acids within nucleotides 11,000-12,251 of HTT intron 2, any 20 or more contiguous nucleic acids within nucleotides 11,000-12,251 of HTT intron 2, any 30 or more contiguous nucleic acids within nucleotides 11,000-12,251 of HTT intron 2). any 40 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2; any 50 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2; any 100 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2; any 150 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2; any 200 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2; or any 250 or more consecutive nucleic acids within nucleotides 11,000 to 12,251 of HTT intron 2.

[0200] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 11,950-12,251 of HTT intron 2 (e.g., any 8 or more contiguous nucleic acids within nucleotides 11,950-12,251 of HTT intron 2, any 10 or more contiguous nucleic acids within nucleotides 11,950-12,251 of HTT intron 2, any 12 or more contiguous nucleic acids within nucleotides 11,950-12,251 of HTT intron 2, any 20 or more contiguous nucleic acids within nucleotides 11,950-12,251 of HTT intron 2, any 30 or more contiguous nucleic acids within nucleotides 11,950-12,251 of HTT intron 2). any 40 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2; any 50 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2; any 100 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2; any 150 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2; any 200 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2; or any 250 or more consecutive nucleic acids within nucleotides 11,950 to 12,251 of HTT intron 2.

[0201] In some cases, the binding domain has at least two non-overlapping sequences that have at least 80% complementarity to the binding site.

[0202] In some cases, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to any one of SEQ ID NOs:60-81.

[0203] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 60, 62, or 67-81.

[0204] In some cases, the binding domain includes 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, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 60 or 67-81.

[0205] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 67 or 70-81.

[0206] In some cases, the binding domain includes 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, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 71-74 or 77-79.

[0207] In some cases, the target HTT intron is HTT intron 3. In one embodiment, the binding domain is a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., a target HTT intron) (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto), which can suppress endogenous target cis-splicing while enhancing trans-splicing between a trans-splicing molecule and a target HTT pre-mRNA (e.g., endogenous HTT by creating a chimeric molecule having a portion of the mRNA and a coding domain having one or more functional HTT exons encoding the wild-type HTT amino acid sequence. In one embodiment involving a sequence encoding a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto).

[0208] In some cases, the invention provides trans-splicing molecules (or vectors thereof) that bind to HTT in intron 3, e.g., a nucleic acid trans-splicing molecule configured to trans-splice a coding domain into endogenous HTT exon 4. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to a binding site having any one or more (e.g., 6 or more, 8 or more, 10 or more, or 12 or more) of nucleotides 3,100-4,429 of SEQ ID NO:155.

[0209] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 3,100-4,429 of HTT intron 3 (e.g., any 8 or more contiguous nucleic acids within nucleotides 3,100-4,429 of HTT intron 3, any 10 or more contiguous nucleic acids within nucleotides 3,100-4,429 of HTT intron 3, any 12 or more contiguous nucleic acids within nucleotides 3,100-4,429 of HTT intron 3, any 20 or more contiguous nucleic acids within nucleotides 3,100-4,429 of HTT intron 3, any 30 or more contiguous nucleic acids within nucleotides 3,100-4,429 of HTT intron 3). any 40 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3; any 50 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3; any 100 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3; any 150 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3; any 200 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3; or any 250 or more consecutive nucleic acids within nucleotides 3,100 to 4,429 of HTT intron 3.

[0210] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 4,100-4,429 of HTT intron 3 (e.g., any 8 or more contiguous nucleic acids within nucleotides 4,100-4,429 of HTT intron 3, any 10 or more contiguous nucleic acids within nucleotides 4,100-4,429 of HTT intron 3, any 12 or more contiguous nucleic acids within nucleotides 4,100-4,429 of HTT intron 3, any 20 or more contiguous nucleic acids within nucleotides 4,100-4,429 of HTT intron 3, any 30 or more contiguous nucleic acids within nucleotides 4,100-4,429 of HTT intron 3). any 40 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3; any 50 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3; any 100 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3; any 150 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3; any 200 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3; or any 250 or more consecutive nucleic acids within nucleotides 4,100 to 4,429 of HTT intron 3.

[0211] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 4,100-4,388 of HTT intron 3 (e.g., any 8 or more contiguous nucleic acids within nucleotides 4,100-4,388 of HTT intron 3, any 10 or more contiguous nucleic acids within nucleotides 4,100-4,388 of HTT intron 3, any 12 or more contiguous nucleic acids within nucleotides 4,100-4,388 of HTT intron 3, any 20 or more contiguous nucleic acids within nucleotides 4,100-4,388 of HTT intron 3, any 30 or more contiguous nucleic acids within nucleotides 4,100-4,388 of HTT intron 3). any 40 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3; any 50 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3; any 100 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3; any 150 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3; any 200 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3; or any 250 or more consecutive nucleic acids within nucleotides 4,100 to 4,388 of HTT intron 3.

[0212] In some cases, the binding domain has at least two non-overlapping sequences that have at least 80% complementarity to the binding site.

[0213] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 158-174.

[0214] In some cases, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to any one of SEQ ID NOs: 164-174.

[0215] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 170-174.

[0216] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 171-172.

[0217] The binding domain can be operably linked 3' to the splicing domain (e.g., directly connected to the splicing domain or with an intervening sequence connecting the 3' end of the splicing domain and the 5' end of the binding domain).

[0218] In some cases, the target HTT intron is HTT intron 1. In one embodiment, the binding domain is a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., a target HTT intron) (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto), which can suppress endogenous target cis-splicing while enhancing trans-splicing between a trans-splicing molecule and a target HTT pre-mRNA (e.g., endogenous HTT by creating a chimeric molecule having a portion of the mRNA and a coding domain having one or more functional HTT exons encoding the wild-type HTT amino acid sequence. In one embodiment involving a sequence encoding a trans-splicing molecule (e.g., a vector encoding a trans-splicing molecule), the binding domain-encoding sequence encodes a nucleic acid sequence that is at least 80% complementary to a sequence of a target HTT intron pre-mRNA (e.g., at least 85% complementary thereto, at least 86% complementary thereto, at least 87% complementary thereto, at least 88% complementary thereto, at least 89% complementary thereto, at least 90% complementary thereto, at least 91% complementary thereto, at least 92% complementary thereto, at least 93% complementary thereto, at least 94% complementary thereto, at least 95% complementary thereto, at least 96% complementary thereto, at least 97% complementary thereto, at least 98% complementary thereto, at least 99% complementary thereto, or 100% complementary thereto).

[0219] In some cases, the invention provides trans-splicing molecules (or vectors thereof) that bind to HTT in intron 1, e.g., a nucleic acid trans-splicing molecule configured to trans-splice a coding domain into endogenous HTT exon 2. In particular, the trans-splicing molecules described herein include those in which the binding domain binds to a binding site having any one or more (e.g., 6 or more, 8 or more, 10 or more, or 12 or more) of nucleotides 1-1000 or 11,500-11,850 of SEQ ID NO:1.

[0220] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1 (e.g., any 8 or more contiguous nucleic acids within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1, any 10 or more contiguous nucleic acids within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1, any 12 or more contiguous nucleic acids within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1, any 20 or more contiguous nucleic acids within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1, any 30 or more contiguous nucleic acids within nucleotides 1-1000 or 11,500-11,850 of HTT intron 1). any 40 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1; any 50 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1; any 100 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1; any 150 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1; any 200 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1; or any 250 or more consecutive nucleic acids within nucleotides 1 to 1000 or 11,500 to 11,850 of HTT intron 1.

[0221] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 11,500-11,850 of HTT intron 1 (e.g., any 8 or more contiguous nucleic acids within nucleotides 11,500-11,850 of HTT intron 1, any 10 or more contiguous nucleic acids within nucleotides 11,500-11,850 of HTT intron 1, any 12 or more contiguous nucleic acids within nucleotides 11,500-11,850 of HTT intron 1, any 20 or more contiguous nucleic acids within nucleotides 11,500-11,850 of HTT intron 1, any 30 or more contiguous nucleic acids within nucleotides 11,500-11,850 of HTT intron 1). any 40 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1; any 50 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1; any 100 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1; any 150 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1; any 200 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1; or any 250 or more consecutive nucleic acids within nucleotides 11,500 to 11,850 of HTT intron 1.

[0222] In some cases, the binding site may include any 6 or more contiguous nucleotides within nucleotides 11,650-11,850 of HTT intron 1 (e.g., any 8 or more contiguous nucleic acids within nucleotides 11,650-11,850 of HTT intron 1, any 10 or more contiguous nucleic acids within nucleotides 11,650-11,850 of HTT intron 1, any 12 or more contiguous nucleic acids within nucleotides 11,650-11,850 of HTT intron 1, any 20 or more contiguous nucleic acids within nucleotides 11,650-11,850 of HTT intron 1, any 30 or more contiguous nucleic acids within nucleotides 11,650-11,850 of HTT intron 1). any 40 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1; any 50 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1; any 100 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1; any 150 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1; any 200 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1; or any 250 or more consecutive nucleic acids within nucleotides 11,650 to 11,850 of HTT intron 1.

[0223] In some cases, the binding domain has at least two non-overlapping sequences that have at least 80% complementarity to the binding site.

[0224] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 8 or 14-21.

[0225] In some cases, the binding domain includes a nucleic acid sequence having at least 80% identity (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) to any one of SEQ ID NOs: 16-21.

[0226] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 17, 18, 20, or 21.

[0227] In some embodiments, the binding domain is a DNA sequence having at least 80% identity (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) to any one of SEQ ID NOs: 17 or 18.

[0228] The binding domain can be operably linked 3' to the splicing domain (e.g., directly connected to the splicing domain or with an intervening sequence connecting the 3' end of the splicing domain and the 5' end of the binding domain).

[0229] As detailed herein, the first phase of HTT exon editor design began with screening and selection of highly efficient BD sequences complementary to target pre-mRNA introns. As shown herein, BDs targeting HTT intron 2 and HTT intron 3 exhibited the highest levels of trans-splicing and have therefore been identified as effective elements of exemplary HTT-directed exon editors for the treatment of HD patient populations. See Figures 5, 10, 12, 24, and 25.

[0230] Splicing domain In the case of a 5' exon editor, the splicing domain may include a splice donor site (5' splice site) to mediate trans-splicing.

[0231] In the case of a 3' exon editor, the splicing domain may include a splice site, a branch site, and / or a polypyrimidine tract (PPT) to mediate trans-splicing. In some embodiments, the splicing domain has a single splice site, indicating that the splice site is designed for alternative trans-splicing rather than cis-splicing due to the lack of a corresponding splice site.

[0232] Alternative splicing domains can be selected by those skilled in the art according to well-known methods and principles. In one embodiment, the 5' splice site consensus sequence is the nucleic acid sequence AG / GURAGU (where / indicates the splice site). In another embodiment, endogenous splice sites corresponding to the exons and introns proximal to the splice site can be used to maintain any splicing regulatory signals.

[0233] In one embodiment, the preferred 5' splice site comprises GTAAGT or GUAAGT.

[0234] The splicing domain can be operably linked 5' to the terminator domain (e.g., directly connected to the terminator domain or with an intervening sequence, e.g., a linker domain and / or a binding domain and / or a 3' downstream sequence, connecting the 3' end of the splicing domain to the 5' end of the terminator domain).

[0235] 5' untranslated region In some cases, the nucleic acid trans-splicing molecule includes a 5' untranslated region. In some embodiments, the 5' untranslated region comprises, consists essentially of, or consists of a native 5' HTT untranslated region. In some embodiments, the 5' untranslated region includes a sequence having at least 80% sequence identity to 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).

[0236] In some embodiments, the 5' untranslated region can be operably linked 5' to the coding domain (e.g., directly connected to the coding domain or with an intervening sequence connecting the 3' end of the 5' untranslated region to the 5' end of the coding domain).

[0237] In some embodiments, the nucleic acid trans-splicing molecule does not include a 5' untranslated region or does not include the native 5' HTT untranslated region.

[0238] 5' regulatory domain In some cases, the nucleic acid trans-splicing molecule is operably linked to a 5' regulatory domain operably linked to the 5' side of the coding domain (e.g., directly linked to the coding domain or linked via an intermediate domain, e.g., an untranslated region). The 5' regulatory domain can include a promoter (e.g., a constitutive promoter, e.g., a CMV promoter or an EF1-alpha promoter). In some cases, the 5' regulatory domain includes a native 5' HTT untranslated region operably linked to a promoter (e.g., a constitutive promoter, e.g., a CMV / CMV promoter). In some embodiments, the 5' regulatory domain operably linked to the native 5'HTT untranslated region comprises a sequence having at least 80% sequence identity to SEQ ID NO: 137 (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).

[0239] In some embodiments, the 5' regulatory domain operably linked to the native 5'HTT untranslated region comprises a sequence having at least 80% sequence identity to SEQ ID NO: 196 (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).

[0240] In some embodiments, the CMV promoter is replaced with a CAGGS promoter, which is used to drive expression of the RNA exon editor described herein. See, e.g., Figure 27. As shown in the figure, comparing the CAGGS 5'UTR with and without the HTT 5'UTR, protein translation appears to be regulated via the HTT 5'UTR when the CAGGS 5'UTR and HTT 5'UTR are operably linked. We observed stronger protein expression when the HTT 5'UTR was removed, leaving only the CAGGS 5'UTR; this activity may be due to some factor upregulating translation in the CAGGS 5'UTR. Transduction experiments presented herein, in which RNA exon editors were introduced into cells via AAV, also demonstrated that the CAGGs promoter drove significant expression of the RNA exon editor, MSH3 splice modulator, and miRNA-encoding construct. See, e.g., Figures 55, 56, 58, and 59.

[0241] In some embodiments, the 5' regulatory domain can be operably linked 5' to the coding domain (e.g., directly connected to the coding domain or with an intervening sequence connecting the 3' end of the 5' regulatory domain to the 5' end of the coding domain).

[0242] In some embodiments, other types of promoters may be used. In some embodiments, the nucleic acid trans-splicing molecule is not operably linked to a CMV promoter or a CAGGS promoter.

[0243] Linker Domain As discussed herein, improving trans-splicing efficiency remains an important goal for the implementation of nucleic acid trans-splicing molecules as therapeutic agents.In order to engineer nucleic acid trans-splicing molecules with trans-splicing efficiency that can meet the requirements for use in therapeutic intervention, the present inventors have tested multiple linker sequences between splice domain (SD) and binding domain (BD), and identified a series of exemplary linkers that have statistically significant improvement in performance compared with 40-mer linker.For example, see Figure 8.

[0244] In addition to the above, a nucleic acid trans-splicing molecule can include a linker domain at one or more positions along with the molecule. In some embodiments, the linker domain is operably linked 3' to the splicing domain or splice site (e.g., directly connected to the splicing domain or splice site). The linker domain can be of any suitable size. In some embodiments, the linker domain is greater than 20 nucleotides in length (e.g., 20-100 nucleotides in length or 20-85 nucleotides in length). In some cases, the linker domain comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 80% identical to any one of SEQ ID NOs: 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).

[0245] Linker sequences are often included in trans-splicing molecules, located between the splice donor and the binding domain, providing flexibility and accessibility to each element. As described herein, the functional contributions of different elements were evaluated in different combinations for the activity they confer in the context of the trans-splicing molecule. In some embodiments, the nucleic acid trans-splicing molecule may not include any of SEQ ID NOS: 37-46 or 106-112, or the linker sequence may be absent.

[0246] The linker domains described herein can also be included in embodiments of RNA exon editors that do not target HTT or MSH3. Embodiments disclosed herein include an RNA exon editor that includes a binding domain that targets an endogenous pre-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 includes one or more of SEQ ID NOs: 37-46 or 106-112.

[0247] 3' transcription terminator domain In some embodiments, the trans-splicing molecule includes a 3' transcription terminator domain. In some embodiments, the 3' transcription terminator domain forms a triple helix structure that effectively caps the 3' end of the trans-splicing molecule. In some embodiments, the 3' transcription terminator domain is derived from human long non-coding RNA MALAT1 (e.g., wild-type MALAT1). In some embodiments, the 3' transcription terminator domain includes a tRNA-like domain. 3' transcription terminator domains useful as part of the HTT trans-splicing molecule are described in International Patent Publication No. WO2020 / 214973, which is incorporated herein by reference in its entirety. For example, in some embodiments, the region of the RTM operably linked to the 3' end of the binding domain includes a terminator domain that includes, consists essentially of, or consists of a wild-type MALAT1 + mascRNA domain, such as SEQ ID NO:5. In some embodiments, the region of the RNA exon editor operably linked to the 3' end of the binding domain includes a terminator domain that comprises, consists essentially of, or consists of a mutated MALAT1+masc RNA (anti-Mut1 masc RNA) domain, such as SEQ ID NO:6.

[0248] 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.

[0249] Splicing domain In some embodiments, exemplary RNA exon editors described herein include those that comprise a binding domain that binds to intron 1 of HTT; such exemplary intron 1-binding RNA exon editors can comprise any one of SEQ ID NOS: 23-36 and 47-56, which sequences include a coding sequence (e.g., SEQ ID NO: 3), a splice domain, a linker 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.

[0250] In some embodiments, exemplary RNA exon editors described herein include those that comprise a binding domain that binds to intron 2 of HTT; such exemplary intron 2-binding RNA exon editors can comprise any one of SEQ ID NOs: 83-105 and 113-125, which sequence includes a coding sequence (e.g., SEQ ID NOs: 59, 349, 350, or 351), a splice domain, a linker 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.

[0251] In some embodiments, exemplary RNA exon editors described herein include those that comprise a binding domain that binds to intron 3 of HTT; such exemplary intron 3-binding RNA exon editors may comprise any one of SEQ ID NOs: 175-191, which sequence includes a coding sequence (e.g., any one of SEQ ID NOs: 157, 352, or 353), a splice domain, a linker 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.

[0252] In some embodiments, exemplary RNA exon editors described herein include those that contain a binding domain that binds to HTT intron 1 and a binding domain that binds to the MSH3 intron; such exemplary hybrid / dual HTT / MSH3 RNA exon editors may comprise any one of SEQ ID NOS: 149-154, which sequence includes a coding sequence (e.g., SEQ ID NO: 3), a splice domain, a linker domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence further includes an ATG start codon at the 5' end. In some embodiments, exemplary hybrid / dual HTT / MSH3 RNA exon editors include binding domains that can bind to both the HTT intron and the MSH3 intron.

[0253] In some embodiments, exemplary RNA exon editors described herein include those that contain a binding domain that binds to HTT intron 2 and a binding domain that binds to the MSH3 intron; such exemplary hybrid / dual HTT / MSH3 RNA exon editors may comprise any one of SEQ ID NOs: 212-223, which sequence includes a coding sequence (e.g., SEQ ID NO: 59), a splice domain, a linker domain, a binding domain, and a 3' transcription terminator. In some embodiments, the coding sequence further includes an ATG start codon at the 5' end. In some embodiments, exemplary hybrid / dual HTT / MSH3 RNA exon editors include binding domains that can bind to both the HTT intron and the MSH3 intron.

[0254] In some embodiments, exemplary RNA exon editors include a binding domain that binds an HTT intron (e.g., intron 2) and a binding domain that binds an MSH3 intron (e.g., intron 5 or intron 15). In some embodiments, the MSH3 binding 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 HTT binding domain includes HTT_intron2_12061_150 (SEQ ID NO: 95). In some embodiments, the MSH3 binding domain is 5' to the HTT binding domain. In some embodiments, the MSH3 binding domain is 3' to the HTT binding domain. In some embodiments, a MALAT1 terminator is between the MSH3 binding domain and the HTT binding domain. In some embodiments, the MALAT1 terminator is not between the MSH3-binding domain and the HTT-binding domain. In some embodiments, the MALAT1 terminator is 3' to both the MSH3-binding domain and the HTT-binding domain.

[0255] In some embodiments, exemplary RNA exon editors include a binding domain that binds to an MSH3 intron (e.g., intron 5 or intron 15). In some embodiments, the MSH3 binding domain includes intron 5_213_100 (SEQ ID NO: 140), intron 5_188_150 (SEQ ID NO: 209), intron 15_6523_120 (SEQ ID NO: 144), or intron 15_6498_150 (SEQ ID NO: 210). In some embodiments, the MSH3-directed RNA exon editor further includes any splice domain disclosed herein, any 3X UBS sequence described herein, any AU-rich element described herein, any linker domain described herein, and / or any terminator sequence disclosed herein. In some embodiments, an MSH3-directed RNA exon editor is administered in conjunction with an HTT-directed exon editor. In some embodiments, an MSH3-directed exon editor is not used in conjunction with an HTT-directed exon editor.

[0256] In some embodiments, MSH3 expression is reduced by miRNAs targeting MSH3 mRNA. In some embodiments, the pri-miRNA comprises one or more of 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 pri-miRNA comprises one or more of SEQ ID NOs: 234, 235, 238-241, or 262-269. In some embodiments, the miRNA active sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257, or a sequence 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 NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257. MiRNAs targeting MSH3 mRNA can be used in combination with any of the RNA exon editors disclosed herein, such as, for example, RNA exon editors that target HTT, MSH3, or both. miRNAs targeting MSH3 mRNA can be used without any of the RNA exon editors disclosed herein, but instead can be used independently to reduce MSH3 expression. miRNAs targeting MSH3 mRNA can be used in methods of treating or preventing trinucleotide repeat expansion disorders.

[0257] In some embodiments, MSH3 expression is reduced by snRNA-based antisense RNAs that can induce exon skipping during pre-mRNA processing. In some embodiments, the snRNA construct comprises one or more of the following: SEQ ID NO: 274 (targets the junction between MSH3 intron 1 and exon 2); SEQ ID NO: 275 (targets the junction between MSH3 exon 2 and intron 2); SEQ ID NO: 278, 301, or 303 (targets the junction between MSH3 intron 2 and exon 3); SEQ ID NO: 279, 300, or 302 (targets the junction between MSH3 exon 3 and intron 3); SEQ ID NO: 281 (targets the junction between MSH3 intron 3 and exon 4); SEQ ID NO: 282 (targets the junction between MSH3 exon 4 and intron 4); SEQ ID NO: 306 or 308 (targets the junction between MSH3 intron 5 and exon 6) SEQ ID NO: 305 or 307 (targets the junction between MSH3 exon 6 and intron 6); SEQ ID NO: 311 or 313 (targets the junction between MSH3 intron 6 and exon 7); SEQ ID NO: 310 or 312 (targets the junction between MSH3 exon 7 and intron 7); SEQ ID NO: 316 or 318 (targets the junction between MSH3 intron 7 and exon 8); SEQ ID NO: 315 or 317 (targets the junction between MSH3 exon 8 and intron 8); SEQ ID NO: 321 or 323 (targets the junction between MSH3 intron 14 and exon 15); or SEQ ID NO: 320 or 322 (targets the junction between MSH3 exon 15 and intron 15).

[0258] In some embodiments, 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 embodiments, a construct targeting the intron 1-exon 2 junction is used in combination with a construct targeting the exon 2-intron 2 junction; a construct targeting the intron 2-exon 3 junction is used in combination with a construct targeting the exon 3-intron 3 junction; a construct targeting the intron 3-exon 4 junction is used in combination with a construct targeting the exon 4-intron 4 junction; a construct targeting the intron 5-exon 6 junction is used in combination with a construct targeting the exon 6-intron 6 junction. A construct targeting the intron 6-exon 6 junction is used in conjunction with a construct targeting the intron 6-exon 6 junction; a construct targeting the intron 6-exon 7 junction is used in conjunction with a construct targeting the exon 7-intron 7 junction; a construct targeting the intron 7-exon 8 junction is used in conjunction with a construct targeting the exon 8-intron 8 junction; or a construct targeting the intron 14-exon 15 junction and / or a construct targeting the exon 15-intron 15 junction is used in conjunction with a construct targeting the intron 14-exon 15 junction.

[0259] In some embodiments, a single asRNA construct is used to target two intron-exon junctions. In some embodiments, the asRNA construct includes a sequence at least partially complementary to the complete exon sequence and a portion of the intron sequence on either side of the exon, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of intron sequence on either side of the exon sequence. Such a construct may target, for example, the intron-exon junctions on either side of any one of MSH3 exons 2, 3, 4, 5, 6, 7, 8, or 15. In some embodiments, the asRNA construct is a U7SmOPT construct containing SEQ ID NO: 324 (In3 / Ex3 / In2 sequence; SEQ ID NO: 299), targeting the entire length of exon 3 and its adjacent junctions on both sides. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 327 (In6 / Ex6 / In5 sequence; comprising SEQ ID NO: 304) and targets the entire length and both flanking junctions of exon 6. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 330 (In7 / Ex7 / In6 sequence; comprising SEQ ID NO: 309) and targets the entire length and both flanking junctions of exon 7. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 333 (In8 / Ex8 / In7 sequence; comprising SEQ ID NO: 314) and targets the entire length and both flanking junctions of exon 8. In some embodiments, the asRNA construct is a U7SmOPT construct comprising SEQ ID NO: 336 (In15 / Ex15 / In14 sequence; comprising SEQ ID NO: 319) and targets the entire length and both flanking junctions of exon 15.

[0260] In some embodiments, a single asRNA construct is used that targets two intron-exon junctions. In some embodiments, 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 linker connecting these two sequences. Such a construct may target, for example, the intron-exon junctions on both sides of MSH3 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) + linker + Ex3 / In2 (SEQ ID NO: 301)], targeting both junctions 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) + linker + Ex3 / In2 (SEQ ID NO: 303)] and targets 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) + linker + Ex6 / In5 (SEQ ID NO: 306)] and targets the junction 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) + linker + Ex6 / In5 (SEQ ID NO: 308)] and targets the junction 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) + linker + Ex7 / In6 (SEQ ID NO: 311)], which targets the junction on either side of exon 7. In some embodiments, the asRNA construct is a U2 snRNA construct comprising SEQ ID NO: 332 [U2 In7 / Ex7 (SEQ ID NO: 312) + linker + Ex7 / In6 (SEQ ID NO: 313)], which targets the junction 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) + linker + Ex8 / In7 (SEQ ID NO: 316)] and targets the junction 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) + linker + Ex8 / In7 (SEQ ID NO: 318)] and targets the junction 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) + linker + Ex15 / In14 (SEQ ID NO: 321)] and targets the junction 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) + linker + Ex15 / In14 (SEQ ID NO: 323)], targeting both junctions of exon 15.

[0261] Some embodiments of MSH3 splice modulator constructs include an operably linked 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 that promotes exon skipping of a target exon in the MSH3 pre-mRNA. Exon skipping may introduce a frameshift and / or a premature stop codon, which may induce nonsense-mediated decay or otherwise impair the production of functional MSH3. The antisense RNA that promotes exon skipping of a target exon may target one or both of the 5' exon-intron junction and the 3' exon-intron junction of the target exon in the MSH3 pre-mRNA. As used herein, an antisense RNA is said to "target" a particular exon-intron junction if it has sufficient complementarity to the sequence surrounding the exon-intron junction such that it can promote skipping of the target exon during pre-mRNA processing. In some embodiments, the antisense RNA sequence includes a flanking region 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 flanking region of the same length of nucleotides on a pre-mRNA that includes an exon-intron junction. In some embodiments, the antisense RNA sequence includes a 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 nucleotide contiguous region that 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 contiguous region of the same nucleotide length on a pre-mRNA that includes an exon-intron junction.In some embodiments, the antisense RNA sequence includes a 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 nucleotide contiguous region that 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 contiguous region of the same nucleotide length on a pre-mRNA that includes an exon-intron junction or is within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of an exon-intron junction. Those skilled in the art will understand that when less than perfect complementarity exists, the functionality of exon skipping can be enhanced by increasing the length of the flanking region of partially complementary nucleotides.Those skilled in the art will also understand that the GC content of the sequence around the exon-intron junction can affect the ability of antisense RNA sequences to effectively target the exon-intron junction and induce skipping.Higher GC content increases the strength of annealing, which can reduce the required complementary region.

[0262] In some embodiments, the MSH3 splice modulator comprises an antisense RNA sequence that targets two exon-intron junctions. In some embodiments, the antisense RNA sequence that targets two exon-intron junctions comprises a sequence that anneals to most or all of the complete exon sequence of the pre-mRNA. In some embodiments, the antisense RNA is 100% complementary to the complete pre-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 pre-mRNA exon sequence. In some embodiments, the antisense RNA further comprises a nucleotide sequence that is at least partially complementary to intron sequence upstream and / or downstream of the target pre-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 in length or more. In some embodiments, the antisense RNA sequence comprises a sequence that 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 such intron sequences.

[0263] In some embodiments, an antisense RNA sequence targeting two exon-intron junctions of a target exon comprises (a) two non-adjacent sequences, each of which anneals to a sequence surrounding the exon-intron junction of the target exon, and (b) a linker sequence between the two non-adjacent sequences, which has a low degree of complementarity (e.g., less than 50%) to 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 to all adjacent regions of the target exon sequence that are the same length as the linker.

[0264] In some embodiments, any of the MSH3 splice modulators disclosed herein may be used in combination with any of the HTT RNA exon editors disclosed herein. In some embodiments, an MSH3 splice modulator disclosed herein is not used in combination with any of the RNA exon editors disclosed herein. Any of the MSH3 splice modulators described herein may be used independently in methods of treating a trinucleotide repeat expansion disorder.

[0265] In some embodiments, multiple constructs are encoded on a single vector, such as an AAV vector. Any of the 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 target the same gene, if applicable. For example, a single vector may encode an HTT exon editor to generate corrected HTT mRNA and an HTT-directed miRNA and / or HTT-directed asRNA to reduce the amount of defective HTT. In some embodiments, a single vector encodes an HTT exon editor (e.g., an exon editor comprising any one of SEQ ID NOs: 83-104, 113-125, 175-191, 199-206, or 23-36) and an HTT-directed miRNA (e.g., SEQ ID NO: 339 and / or 342). In some embodiments, the vector comprises a sequence set forth in any one of SEQ ID NOs: 354 or 355, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence. In some embodiments, the exon editor, miRNA, and / or asRNA, if applicable, target different genes. In some embodiments, for example, a single vector encodes an HTT exon editor to generate a corrected HTT mRNA and an MSH3 exon editor, MSH3 miRNA, and / or MSH3 asRNA (e.g., an snRNA construct) to knock down MSH3 expression. In some embodiments, a single vector encodes an HTT exon editor (e.g., an exon editor comprising any one of SEQ ID NOs: 83-104, 113-125, 175-191, 199-206, or 23-36) and an MSH3-directed asRNA (e.g., a construct comprising any one or more of SEQ ID NOs: 284-293 or 324-338).In some embodiments, the vector comprises the sequence set forth in SEQ ID NO: 356 or 357, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to such a sequence. In some embodiments, the vector encodes an HTT exon editor (e.g., an exon editor comprising any one of SEQ ID NOs: 83-104, 113-125, 175-191, 199-206, or 23-36), an MSH3-directed asRNA (e.g., a construct comprising any one or more of SEQ ID NOs: 284-293 or 324-338), and an HTT-directed miRNA (e.g., SEQ ID NO: 339 and / or 342). In some embodiments, the vector comprises the sequence set forth in SEQ ID NO: 358 or 359, or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to such a sequence.

[0266] The different therapeutic approaches disclosed herein can be combined in various combinations, whether they are encoded on the same vector or on different vectors. For example, any of the HTT exon editors disclosed herein having a binding domain targeting intron 1 can be combined with any of the MSH3-directed asRNA constructs disclosed herein and / or any of the HTT-directed miRNA constructs disclosed herein. In some embodiments, an HTT exon editor having a binding domain targeting intron 1, an MSH3-directed asRNA, and an HTT-directed miRNA are contained on the same vector. As another example, any of the HTT exon editors disclosed herein having a binding domain targeting intron 2 can be combined with any of the MSH3-directed asRNA constructs disclosed herein and / or any of the HTT-directed miRNA constructs disclosed herein. In some embodiments, an HTT exon editor having a binding domain targeting intron 2, an MSH3-directed asRNA, and an HTT-directed miRNA are contained on the same vector. As another example, any of the HTT exon editors disclosed herein having a binding domain that targets intron 3 can be combined with any of the MSH3-directed asRNA constructs disclosed herein and / or any of the HTT-directed miRNA constructs disclosed herein. In some embodiments, the HTT exon editor having a binding domain that targets intron 3, the MSH3-directed asRNA, and the HTT-directed miRNA are contained on the same vector.

[0267] In some embodiments, binding of the trans-splicing molecule to the target pre-mRNA is mediated by percent complementarity (i.e., based on the base-pairing properties of the nucleic acid), triple helix formation, or protein-nucleic acid interactions (as described in the documents cited herein), or any combination thereof. In one embodiment, the nucleic acid trans-splicing molecule includes a DNA, RNA, or DNA / RNA hybrid molecule, where the DNA or RNA is either single-stranded or double-stranded. Also included herein are RNAs or DNAs that can hybridize to one of the aforementioned RNAs or DNAs, preferably under stringent conditions, e.g., in 2.5x SSC buffer at 60°C and several washes in lower buffer concentrations, e.g., 0.5x SSC buffer at 37°C. These nucleic acids can encode proteins that exhibit lipid phosphate phosphatase activity and / or plasma membrane association. When trans-splicing molecules are synthesized in vitro, they can be modified in the base moiety, sugar moiety, or phosphate backbone to improve, for example, the molecule's stability, hybridization to target mRNA, or transport into cells. For example, modifying a trans-splicing molecule to reduce its total charge can enhance the molecule's cellular uptake. Furthermore, modifications can be made to reduce its susceptibility to nucleases or chemical degradation. Nucleic acid molecules can be synthesized in such a way that they are conjugated to another molecule, such as a peptide, a cross-linking agent that induces hybridization, a transport agent, a cleavage agent that induces hybridization, etc.

[0268] Various other well-known modifications to nucleic acid molecules can be introduced as a means of increasing intracellular stability and half-life (see also above for oligonucleotides). Possible modifications are well known in the art. Modifications that can be made to the structure of synthetic trans-splicing molecules include backbone modifications.

[0269] Cell line assays In some cases, the trans-splicing molecules described herein are tested in cultured cell lines. Cultured cell lines may be obtained or engineered to express the target HTT pre-mRNA at sufficient levels to screen, select, and improve the functionality of the RNA exon editor.

[0270] RNA exon editor screening platform As described herein, 5' RNA exon editors contain several functional sequence elements, such as a binding domain (BD) for pre-mRNA targeting and a linker that allows access to a splice donor (SD) site. When engineering an RNA exon editor for a given gene target, various sequence options for each of these elements are tested for their ability to contribute to high trans-splicing (TS) efficiency. Such testing can be accomplished by (A) cloning and transfecting individual RNA exon editor variants and analyzing their efficiency by RT-qPCR / ddPCR and Western blot, and / or (B) cloning and pooling RNA exon editors in a high-throughput (HT) library-based approach that relies on next-generation sequencing (NGS) and computational analysis to evaluate efficiency. Both approaches are described below.

[0271] Screening for RNA exon editors in individual formats: This approach can be applied to test the number of small variable elements in RNA exon editor sequences before 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 the RNA and protein levels.

[0272] At the RNA level, TS activity is assessed by isolating total RNA from cells, followed by reverse transcription, and real-time quantitative PCR (RT-qPCR) to measure RNA copy numbers of, for example, the following targets: RNF20 (a housekeeping gene for normalization); native (HTT) mRNA; exon editor RNA; on-target exon-edited RNA (ONT), which is the product of a positive TS; and ONT + exon editor + OFT (off-target)—a single assay that captures all three of these targets. OFT represents the incorrect RNA molecule that the RNA exon editor can trans-splice.

[0273] ONT TS efficiency, also referred to as percent replacement or percent editing, represents the fraction of the total HTT mRNA population that successfully underwent TS and is calculated by the following equation: ONT TS%=100 * (ONT copy number / (ONT copy number + native copy number)).

[0274] The TS efficiency of an RNA exon editor is the fraction of the RNA exon editor transcript population that is correctly trans-spliced ​​into HTT RNA and is calculated by the following equation: exon editor TS% = 100 * (ONT copy number / (ONT copy number + exon editor copy number + OFT copy number)).

[0275] At the protein level, TS activity is measured by Western blot analysis applied to proteins extracted from cell or tissue samples. Cytoskeletal proteins, beta-actin or tubulin, can be used as loading controls. For constructs containing a tag, such as a FLAG tag at their N-terminus, an antibody (Ab) specific for the tag (e.g., a FLAG-specific Ab) can be used to probe Western blots to assess ONT protein levels.

[0276] III. Vector Trans-splicing molecules can be delivered to target cells of an individual using various techniques, for example, using recombinant adeno-associated virus (AAV) vectors or other vector modalities, such as non-viral vectors. Accordingly, provided herein are vectors containing / encoding trans-splicing molecules (e.g., viral or non-viral vectors containing / encoding trans-splicing molecules, e.g., DNA vectors containing / encoding trans-splicing molecules). Any suitable nucleic acid vector can be used in conjunction with the compositions and methods of the present invention to design and assemble trans-splicing molecules and recombinant AAV components. In one embodiment, the vector is a recombinant AAV carrying a trans-splicing molecule driven by a promoter that expresses the trans-splicing molecule in selected cells of the 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(1):33-40; and Walther W. and Stein U., Drugs 2000, 60(2):249-71.

[0277] In certain embodiments described herein, trans-splicing molecules are delivered to selected cells, e.g., neuronal cells, in need of treatment with an AAV vector. A variety of naturally occurring AAV serotypes are available. Many natural variants of AAV capsids exist, allowing for the identification and use of AAVs with properties specifically suited to neuronal cells. Artificial AAV vectors can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of trans-splicing molecule nucleic acid sequences, minimized immunogenicity, tuned stability and particle lifespan, efficient degradation, precise delivery to the nucleus, and the like. For example, such artificial capsids can be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that can be obtained from a different selected AAV, a non-adjacent portion of the same AAV, a non-AAV viral source, or a non-viral source. Artificial AAVs can be, but are not limited to, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. Pseudotyped vectors in which one AAV capsid is replaced with a heterologous capsid protein are useful for delivering the trans-splicing molecules described herein.

[0278] Expression of the trans-splicing molecules described herein can be achieved in selected cells via delivery of recombinantly engineered or engineered AAVs containing sequences encoding / including the desired trans-splicing molecules. The use of AAVs is a common mode of exogenous DNA delivery because they are relatively non-toxic, provide efficient gene transfer, and can be easily optimized for specific purposes. Among the well-characterized serotypes of AAV 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.

[0279] In some embodiments, 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 7 of US20030138772, 1), AAV3a or a variant thereof, AAV3b or a variant thereof (e.g., SEQ ID NOs: 1 and 10 in U.S. Pat. No. 6,156,303), AAV3-3 or a variant thereof (e.g., SEQ ID NOs: 200 and 217 in WO2005033321), AAV4 or a variant thereof (e.g., SEQ ID NO: 63 in US20030138772), AAV4-4 or a variant thereof (e.g., SEQ ID NO: 201 or 218 in WO2005033321), AAV5 or a variant thereof (e.g., SEQ ID NO: 114 in US20030138772), AAV6 or a variant thereof (e.g., SEQ ID NO: 65 of US20030138772), AAV6.1 or a variant thereof (e.g., SEQ ID NO: 29 of US20150159173), AAV6.2 or a variant thereof, AAV6.1.2 or a variant thereof, AAV7 or a variant thereof (e.g., SEQ ID NOs: 1-3 of US20030138772), AAV7.2 or a variant thereof, AAV8 or a variant thereof (e.g., SEQ ID NOs: 4 and 95 of US20030138772 or AAV8(b) (incorporated herein by reference in their entirety). No. 9,567,376, incorporated herein by reference), AAV9 or a variant thereof (e.g., SEQ ID NOs: 5 and 100 of US20030138772), AAV9.9 or a variant thereof, AAV9.11 or a variant thereof, AAV9.13 or a variant thereof, AAV9.16 or a variant thereof, AAV9.24 or a variant thereof, AAV9.AAV16.3 or a variant thereof, AAV24.1 or a variant thereof, AAV27.3 or a variant thereof, AAV28.1 or a variant thereof, AAV29.1 or a variant thereof, AAV30.1 or a variant thereof, AAV31.1 or a variant thereof, AAV32.1 or a variant thereof, AAV33.1 or a variant thereof, AAV34.1 or a variant thereof, AAV35.1 or a variant thereof, AAV36.1 or a variant thereof, AAV37.1 or a variant thereof, AAV38.1 or a variant thereof, AAV39 ... 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, AAV4 2-11 or variant thereof, AAV42-12 or variant thereof, AAV42-13 or variant thereof, AAV42-15 or variant thereof, AAV42-aa or variant thereof, AAV43-1 or variant thereof, AAV43-12 or variant thereof, AAV43-20 or variant thereof, AAV43-21 or variant thereof, AAV43-23 or variant thereof, AAV43-25 or variant thereof, AAV43-5 or variants thereof, AAV44.1 or variants thereof, AAV44.2 or variants thereof, AAV44.5 or variants thereof, AAV223.1 or variants thereof, AAV223.2 or variants thereof, AAV223.4 or variants thereof, AAV223.5 or variants thereof, AAV223.6 or variants thereof, AAV223.7 or variants thereof, AAV1-7 / rh.48 or variants thereof, AAV1-8 / rh.49 or variant thereof, AAV2-15 / rh.62 or variant thereof, AAV2-3 / rh.61 or variant thereof, AAV2-4 / rh.50 or variant thereof, AAV2-5 / rh.51 or variant thereof, AAV3.1 / hu.6 or variant thereof, AAV3.1 / hu.9 or variant thereof, AAV3-9 / rh.52 or variant thereof, AAV3-11 / rh.53 or variant thereof, AAV4-8 / rh.64 or variant thereof, AAV4-9 / rh.54 or variant thereof AAV4-19 / rh.55 or a variant thereof (e.g., SEQ ID NO: 116 in WO2005033321), AAV4-19 / rh.55 or a variant thereof (e.g., SEQ ID NO: 117 in WO2005033321), AAV5-3 / rh.57 or a variant thereof, AAV5-22 / rh.58 or a variant thereof, AAV7.3 / hu.7 or a variant thereof, AAV16.8 / hu.10 or a variant thereof, AAV16.12 / hu.11 or a variant thereof, AAV29.3 / bb.1 or a variant thereof, AAV29.5 / bb.2 or a variant thereof t, AAV106.1 / hu.37 or a variant thereof, AAV114.3 / hu.40 or a variant thereof, AAV127.2 / hu.41 or a variant thereof, AAV127.5 / hu.42 or a variant thereof, AAV128.3 / hu.44 or a variant thereof, AAV130.4 / hu.48 or a variant thereof, AAV145.1 / hu.53 or a variant thereof, AAV145.5 / hu.54 or a variant thereof, AAV145.6 / hu.55 or a variant thereof, AAV161.10 / hu. 60 or a variant thereof, AAV161.6 / hu.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 variants thereof, AAVC1 or variants thereof, AAVC2 or variants thereof, AAVC5 or variants thereof, AAV-DJ or variants thereof (e.g., SEQ ID NO: 2 or 3 of US20140359799), AAV-DJ8 or variants thereof, AAVF3 or variants thereof, AAVF5 or variants thereof, AAVH2 or variants thereof, AAVH6 or variants thereof, AAVLK03 or variants thereof, AAVH-1 / hu.1 or variants thereof, AAVH-5 / hu.3 or variants 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: 47 of US20150159173), 50159173), AAVCy.5R1 or a variant thereof, AAVCy.5R2 or a variant thereof, AAVCy.5R3 or a variant thereof, AAVCy.5R4 or a variant thereof, AAVcy.6 or a variant thereof, AAVhu.1 or a variant thereof (e.g., SEQ ID NO: 144 of WO2005033321), AAVhu.2 or a variant thereof (e.g., SEQ ID NO: 143 of WO2005033321), AAVhu.3 or a variant thereof (e.g., SEQ ID NO: 14 5), AAVhu.4 or a variant thereof (e.g., SEQ ID NO: 141 in WO2005033321), AAVhu.5 or a variant thereof, AAVhu.6 or a variant thereof (e.g., SEQ ID NO: 84 in WO2005033321), AAVhu.7 or a variant thereof (e.g., SEQ ID NO: 150 in WO2005033321), AAVhu.9 or a variant thereof (e.g., SEQ ID NO: 155 in WO2005033321), AAVhu.10 or a variant thereof (e.g., SEQ ID NO: 156 in WO2005033321), AAVhu.AAVhu.11 or a variant thereof (e.g., SEQ ID NO: 153 in WO2005033321), AAVhu.13 or a variant thereof (SEQ ID NOs: 16 and 32 in US20150159173), AAVhu.15 or a variant thereof (e.g., SEQ ID NO: 147 in WO2005033321), AAVhu.16 or a variant thereof (e.g., SEQ ID NO: 148 in WO2005033321), AAVhu.17 or a variant thereof (e.g., SEQ ID NO: 83 in WO2005033321), AAVhu.18 or a variant thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.19 or a variant thereof (e.g., SEQ ID NO: 133 of WO2005033321), AAVhu.20 or a variant thereof (e.g., SEQ ID NO: 134 of WO2005033321), AAVhu.21 or a variant thereof (e.g., SEQ ID NO: 135 of WO2005033321), AAVhu.22 or a variant thereof (e.g., SEQ ID NO: 138 of WO2005033321), AAVhu.23.2 or a variant thereof (e.g., SEQ ID NO: 139 of WO2005033321), AAVhu.24.2 or a variant thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.19 or a variant thereof (e.g., SEQ ID NO: 133 of WO2005033321), AAVhu.20 or a variant thereof (e.g., SEQ ID NO: 134 of WO2005033321), AAVhu.21 or a variant thereof (e.g., SEQ ID NO: 135 of WO2005033321), AAVhu.22 or a variant thereof (e.g., SEQ ID NO: 138 of WO2005033321), AAVhu.24.2 or a variant thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.24.3 or a variant thereof (e.g., SEQ ID NO: 149 of WO2005033321), AAVhu.24.4 or a variant thereof SEQ ID NO: 137), AAVhu.24 or a variant thereof (e.g., SEQ ID NO: 136 in WO2005033321), AAVhu.25 or a variant thereof (e.g., SEQ ID NO: 146 in WO2005033321), AAVhu.26 or a variant thereof (e.g., SEQ ID NOs: 17 and 33 in US20150159173), AAVhu.27 or a variant thereof (e.g., SEQ ID NO: 140 in WO2005033321), AAVhu.28 or a variant thereof (e.g., SEQ ID NO: 42 in US20150159173), AAVhu.29 or a variant thereof (e.g., SEQ ID NO: 144 in WO2005033321), AAVhu.30 or a variant thereof (e.g., SEQ ID NO: 146 in WO2005033321), AAVhu.31 or a variant thereof (e.g., SEQ ID NO: 148 in US20150159173), AAVhu.32 or a variant thereof (e.g., SEQ ID NO: 149 in WO2005033321), AAVhu.33 or a variant thereof (e.g., SEQ ID NO: 150 in WO2005033321), AAVhu.34 or a variant thereof (e.g., SEQ ID NO: 151 in US20150159173), AAVhu.35 or a variant thereof (e.g., SEQ ID NO: 152 in US20150159173), AAVhu.36 or a variant thereof (e.g., SEQ ID NO: 153 in US201 .29 or a variant thereof (e.g., SEQ ID NO: 132 in WO2005033321), AAVhu.29R or a variant thereof, AAVhu.31 or a variant thereof (e.g., SEQ ID NO: 121 in WO2005033321), AAVhu.32 or a variant thereof (e.g., SEQ ID NO: 122 in WO2005033321), AAVhu.34 or a variant thereof (e.g., SEQ ID NO: 125 in WO2005033321), AAVhu.35 or a variant thereof (e.g., SEQ ID NO: 164 in WO2005033321), AAVhu.37 or a variant thereof (e.g., SEQ ID NOs: 18 and 34 in US20150159173), AAVhu.39 or a variant thereof (e.g., SEQ ID NO: 102 in WO2005033321), AAVhu.40 or a variant thereof (e.g., SEQ ID NO: 87 in WO2005033321), AAVhu.41 or a variant thereof (e.g., SEQ ID NO: 91 in WO2005033321), AAVhu.42 or a variant thereof (e.g., SEQ ID NO: 85 in WO2005033321), AAVhu. AAVhu.43 or a variant thereof (e.g., SEQ ID NO: 160 in WO2005033321), AAVhu.44 or a variant thereof (e.g., SEQ ID NO: 45 in US20150159173), AAVhu.44R1 or a variant thereof, AAVhu.44R2 or a variant thereof, AAVhu.44R3 or a variant thereof, AAVhu.45 or a variant thereof (e.g., SEQ ID NO: 127 in WO2005033321), AAVhu.46 or a variant thereof (e.g., SEQ ID NO: 159 in WO2005033321), AAVhu.47 or or a variant thereof (e.g., SEQ ID NO: 128 in WO2005033321), AAVhu.48 or a variant thereof (e.g., SEQ ID NO: 38 in US20150159173), AAVhu.48R1 or a variant thereof, AAVhu.48R2 or a variant thereof, AAVhu.48R3 or a variant thereof, AAVhu.49 or a variant thereof (e.g., SEQ ID NO: 189 in WO2005033321), AAVhu.51 or a variant thereof (e.g., SEQ ID NO: 190 in WO2005033321), AAVhu.52 or a variant thereof or a variant thereof (e.g., SEQ ID NO: 191 in WO2005033321), AAVhu.53 or a variant thereof (e.g., SEQ ID NOs: 19 and 35 in US20150159173), AAVhu.54 or a variant thereof (e.g., SEQ ID NO: 188 in WO2005033321), AAVhu.55 or a variant thereof (e.g., SEQ ID NO: 187 in WO2005033321), AAVhu.56 or a variant thereof (e.g., SEQ ID NO: 192 in WO2005 033321), AAVhu.58 or a variant thereof (e.g., SEQ ID NO: 194 of WO2005033321), AAVhu.60 or a variant thereof (e.g., SEQ ID NO: 184 of WO2005033321), AAVhu.61 or a variant thereof (e.g., SEQ ID NO: 185 of WO2005033321), AAVhu.63 or a variant thereof (e.g., SEQ ID NO: 195 of WO2005033321), AAVhu.64 or a variant thereof (e.g., SEQ ID NO: 196 of WO2005033321), AAVhu.AAVhu.66 or a variant thereof (e.g., SEQ ID NO: 197 in WO2005033321), AAVhu.67 or a variant thereof (e.g., SEQ ID NO: 198 in WO2005033321), AAVhu.14 / 9 or a variant thereof, AAVhu.t19 or a variant thereof, AAVrh.2 or a variant thereof (e.g., SEQ ID NO: 39 in US20150159173), AAVrh.2R or a variant thereof, AAVrh.8 or a variant thereof (e.g., SEQ ID NO: 41 in US20150159173), AAVrh.8R or a variant thereof or variants thereof, AAVrh.10 or variants thereof (e.g., SEQ ID NOs: 9 and 25 of US20150159173), AAVrh.12 or variants thereof, AAVrh.13 or variants thereof (e.g., SEQ ID NOs: 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 NOs: 11 and 12 of US20150159173), 0150159173), AAVrh.21 or a variant thereof, AAVrh.22 or a variant thereof, AAVrh.23 or a variant thereof, AAVrh.24 or a variant thereof, AAVrh.25 or a variant thereof, AAVrh.31 or a variant thereof, AAVrh.32 or a variant thereof, AAVrh.33 or a variant thereof, AAVrh.34 or a variant thereof, AAVrh.35 or a variant thereof, AAVrh.36 or a variant thereof, AAVrh.37 or a variant thereof (e.g., SEQ ID NO: 40 in US20150159173), AAVrh.37R2 or a variant thereof, AAVrh.38 or a variant thereof (e.g., SEQ ID NO: 86 in WO2005033321), AAVrh.39 or a variant thereof (e.g., SEQ ID NO: 3, 20, or 36 in US20150159173), AAVrh.40 or a variant thereof (e.g., SEQ ID NO: 92 in WO2005033321), AAVrh.43 or a variant thereof (e.g., SEQ ID NO: 21 and 37 in US20150159173), AAVrh.AAVrh.46 or a variant thereof (e.g., SEQ ID NOs: 4 and 22 of US20150159173), AAVrh.48 or a variant thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1 or a variant thereof (e.g., SEQ ID NO: 44 of US20150159173), AAVrh.48.1.2 or a variant thereof, AAVrh.48.2 or a variant thereof, AAVrh.49 or a variant thereof (e.g., SEQ ID NO: 5 of WO2005033321), SEQ ID NO: 103), AAVrh.50 or a variant thereof (e.g., SEQ ID NO: 108 of WO2005033321), AAVrh.51 or a variant thereof (e.g., SEQ ID NO: 104 of WO2005033321), AAVrh.52 or a variant thereof (e.g., SEQ ID NO: 96 of WO2005033321), AAVrh.53 or a variant thereof (e.g., SEQ ID NO: 97 of WO2005033321), AAVrh.54 or a variant thereof (e.g., US20 SEQ ID NO: 49 in US20150159173), AAVrh.56 or a variant thereof (e.g., SEQ ID NO: 152 in WO2005033321), AAVrh.57 or a variant thereof (e.g., SEQ ID NO: 105 in WO2005033321), AAVrh.58 or a variant thereof (e.g., SEQ ID NO: 48 in US20150159173), AAVrh.61 or a variant thereof (e.g., SEQ ID NO: 107 in WO2005033321), AAVrh.62 or a variant thereof AAVrh.64 or a variant thereof (e.g., SEQ ID NO: 114 in WO2005033321), AAVrh.64 or a variant thereof (e.g., SEQ ID NO: 43 in US20150159173), AAVrh.64R1 or a variant thereof, AAVrh.64R2 or a variant thereof, AAVrh.67 or a variant thereof (e.g., SEQ ID NO: 47 in US20150159173), AAVrh.73 or a variant thereof (e.g., SEQ ID NO: 5 in US20150159173), or AAVrh.74 or a variant thereof (e.g., SEQ ID NO: 6 of US2015015917). Non-limiting examples of variants 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 US2021 / 0189430, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence of the AAV can include one or more amino acid substitutions in the AAV capsid protein at one or more positions that interact with heparan sulfate proteoglycans or at one or more positions corresponding to amino acids 484, 487, 527, 532, 585, or 588 (numbering based on the numbering of VP1 of AAV2).

[0280] Unless otherwise specified, AAV ITRs and other selected AAV components described herein can be readily selected from any of the AAV serotypes, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or known and unknown AAV serotypes. In one embodiment, the ITRs are derived from AAV2. ITRs or other AAV components can be readily isolated from AAV using techniques available to those of skill in the art. Such AAVs can be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences can be obtained via synthesis or other suitable means by reference to published sequences available in the literature or in databases such as GenBank, PubMed, etc.

[0281] Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3, and hypervariable regions; rep proteins, including rep78, rep68, rep52, and rep40; and sequences encoding these proteins. These fragments can be readily utilized 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 containing non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences that can be obtained from a different selected AAV, non-contiguous portions of the same AAV, from a non-AAV viral source, or from a non-viral source. The artificial AAV can be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotype vectors in which the capsid of one AAV is utilized with ITRs from an AAV with a different capsid protein are useful as described herein. In one embodiment, the AAV is AAV2 / 5 (i.e., an AAV with AAV2 ITRs and an AAV5 capsid). In another embodiment, the AAV is AAV2 / 8 (i.e., an AAV with AAV2 ITRs and an AAV8 capsid). In one embodiment, the AAV comprises an AAV8 capsid. Such an AAV8 capsid comprises the amino acid sequence found in NCBI Reference Sequence: YP_077180.1. In another embodiment, the AAV8 capsid comprises the capsid encoded by nt 2121 to 4337 of GenBank Accession: AF513852.1.

[0282] In one embodiment, vectors useful in the compositions and methods described herein minimally include sequences encoding a selected AAV serotype capsid, e.g., an AAV2 capsid, or fragment thereof. In another embodiment, useful vectors minimally include sequences encoding a selected AAV serotype rep protein, e.g., an AAV2 rep protein, or fragment thereof. Optionally, such vectors may include both AAV cap and AAV rep proteins. In vectors in which both AAV rep and AAV cap are provided, the AAV rep and AAV cap sequences may both be of one serotype origin, e.g., AAV2 origin.

[0283] Alternatively, a vector can be used in which the rep sequence is derived from an AAV serotype different from that providing the cap sequence. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, the rep sequences are fused in frame to the cap sequence of a different AAV serotype to form a chimeric AAV vector, such as those described in U.S. Patent No. 7,282,199 (incorporated herein by reference).

[0284] Suitable recombinant AAV (rAAV) is produced by culturing host cells containing nucleic acid sequences encoding an AAV serotype capsid protein, or fragments thereof, as defined herein; a functional rep gene; a minigene composed of, for example, AAV ITRs and trans-splicing molecule nucleic acid sequences; and sufficient helper functions to allow packaging of the minigene into an AAV capsid protein. The components required for culturing the host cell to package the AAV minigene into an AAV capsid can be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper functions) can be provided by a stable host cell engineered to contain one or more of the required components using methods well known to those of skill in the art.

[0285] In one embodiment, the AAV comprises a promoter (or a functional fragment of a promoter). The promoter used in the rAAV can be selected from among a number of constitutive or inducible promoters that can drive expression of a selected transgene in a desired target cell. 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 particular promoter selected for the recombinant vector can drive expression of a selected transgene in a specific cell type. In some embodiments, the promoter is specific for expression of a transgene in neural cells. In some embodiments, the promoter is specific for expression in cortical neurons (e.g., cortical pyramidal neurons). In some embodiments, the promoter is specific for expression of a transgene in striatal neurons (medium spiny neurons of the striatum). In some embodiments, the promoter is specific for expression of a transgene in hypothalamic neurons. In some embodiments, the transgene is expressed in at least one of a cell type or cells.

[0286] In another embodiment, the promoter is the native promoter of the target gene to be expressed. Useful promoters include, but are not limited to, the promoter CAGGS and neuron-specific promoters, including, but not limited to, the human synapsin 1 gene promoter, the neuron-specific enolase (NSE) promoter, the human synapsin 1 promoter, the CaMK kinase promoter, or the MeCP2 promoter. Other suitable promoters include inducible promoters, which initiate transcription only when the host cell is exposed to a stimulus that acts as a trigger to activate the promoter.

[0287] Other conventional regulatory sequences that may be contained in minigenes or rAAVs are also disclosed in documents such as WO2014 / 124282 and other documents cited and incorporated by reference herein. One skilled in the art can select from among these and other expression control sequences without departing from the scope described herein.

[0288] The selected genetic elements may be delivered by any suitable method, including those described herein. The methods used to construct any of the embodiments described herein are well known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not limited to the methods and constructs described herein. See, e.g., K. Fisher et al., J. Virol., 1993 70:520-532 and U.S. Pat. No. 5,478,745, each of which is incorporated herein by reference.

[0289] In some embodiments, the trans-splicing molecule is included in a proviral plasmid, such as that disclosed in International Patent Publication No. WO 2012 / 158757, which is incorporated herein by reference. Such a proviral plasmid contains a modular recombinant AAV genome, which includes a operably associated wild-type 5' AAV2 ITR sequence flanked by unique restriction sites that allow for rapid removal or replacement; a cytomegalovirus (CMV)-promoter containing 49 nucleic acids of a cytomegalovirus sequence upstream of a chicken beta-actin sequence, or a neuron-specific promoter / enhancer, wherein the promoter is flanked by unique restriction sites that allow for rapid removal or replacement of the entire promoter sequence, and the upstream sequence is flanked by unique restriction sites that allow for rapid removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. The trans-splicing molecule described herein can be inserted into the multicloning polylinker site, where the trans-splicing molecule is operably linked to the promoter and under its regulatory control. Also part of such plasmids are a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that allow for the rapid removal or replacement of the polyA sequence, and the wild-type 3' AAV2 ITR sequence, also flanked by unique restriction sites that allow for the rapid removal or replacement of the ITR. The plasmid backbone contains elements necessary for replication in bacterial cells, such as a kanamycin resistance gene, which is itself flanked by transcription terminator / insulator sequences.

[0290] In some embodiments, the proviral plasmid comprises (a) a modular recombinant AAV genome comprising, operably associated: (i) sequences of the wild-type 5' AAV2 ITRs flanked by unique restriction sites that allow for rapid removal or replacement of said ITRs; and (ii) a promoter comprising (A) a CMV sequence of 49 nucleic acids upstream of a CMV-chicken beta-actin sequence or (B) a neuron-specific promoter / enhancer. The promoter is flanked by unique restriction sites that allow for rapid removal or replacement of the entire promoter sequence, and the upstream sequence is flanked by unique restriction sites that allow for rapid removal or replacement of only the upstream CMV or enhancer sequence from the promoter sequence. Also included in this proviral plasmid is a multicloning polylinker sequence that allows for the insertion of a trans-splicing molecule sequence, including any of those described herein, operably linked to and under the control of a promoter; a bovine growth hormone polyadenylation sequence flanked by unique restriction sites that allow for easy removal or replacement of the polyA sequence; and a wild-type 3' AAV2 ITR sequence flanked by unique restriction sites that allow for easy removal or replacement of the 3' ITR. The proviral plasmid also contains elements necessary for replication in bacterial cells and includes a plasmid backbone that further includes a kanamycin resistance gene, the plasmid backbone flanked by transcription terminator / insulator sequences. The proviral plasmids described herein may also include a non-coding lambda phage 5.1 kb stuffer sequence in the plasmid backbone to increase the backbone length and prevent reverse packaging of non-functional AAV genomes.

[0291] In yet another aspect, the promoter of the proviral plasmid is modified to reduce the size of the promoter to allow for larger trans-splicing molecule sequences to be inserted into the rAAV. In one embodiment, the CMV / CBA hybrid promo...

Claims

1. 1. 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 an HTT pre-mRNA, wherein the target intron comprises intron 2.

2. 2. The HTT nucleic acid trans-splicing molecule of claim 1, wherein the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 60-81, or a sequence having at least 90% identity to any one of SEQ ID NOs: 60-81.

3. 1. An HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exons 1-3; (b) a splicing domain; and (c) a binding domain that binds to a target intron of an HTT pre-mRNA, wherein the target intron comprises intron 3.

4. 4. The HTT nucleic acid trans-splicing molecule of claim 3, wherein the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 158-174, or a sequence having at least 90% identity to any one of SEQ ID NOs: 158-174.

5. 1. An HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exon 1; (b) a splicing domain; and (c) a binding domain that binds to a target intron of an HTT pre-mRNA, wherein the target intron comprises intron 1, and the binding domain comprises any one of SEQ ID NOs: 8-21.

6. 10. The HTT nucleic acid trans-splicing molecule according to any one of the preceding claims, wherein the coding domain comprises, consists essentially of, or consists of HTT exon 1; HTT exon 1 and HTT exon 2; or HTT exons 1-3.

7. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, wherein the coding domain comprises, consists essentially of or consists of any one of SEQ ID NOs: 3, 59, 157, or 349-353, or a sequence having at least 90% identity to SEQ ID NOs: 3, 59, 157, or 349-353.

8. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, wherein the coding domain, the splicing domain, and the binding domain are operably linked in a 5' to 3' direction.

9. 10. The HTT nucleic acid trans-splicing molecule of claim 1, further comprising a linker, wherein the coding domain, the splicing domain, the linker, and the binding domain are operably linked in a 5' to 3' direction.

10. The linker is a sequence ranging from 20 to 50 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine; a sequence ranging from 20 to 45 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine, or 10. The HTT nucleic acid trans-splicing molecule of claim 9, comprising, consisting essentially of, or consisting of a sequence ranging from 22 to 42 nucleotides in length, wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine.

11. The linker is SEQ ID NO: 38 or a sequence having at least 90% identity to SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity to SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity to SEQ ID NO: 40, or 11. The HTT nucleic acid trans-splicing molecule of claim 9, comprising, consisting essentially of, or consisting of any one of SEQ ID NO: 41 or a sequence having at least 90% identity to SEQ ID NO:

41.

12. The linker is SEQ ID NO: 37 or a sequence having at least 90% identity to SEQ ID NO: 37; SEQ ID NO: 42 or a sequence having at least 90% identity to SEQ ID NO: 42; SEQ ID NO: 43 or a sequence having at least 90% identity to SEQ ID NO: 43; SEQ ID NO: 44 or a sequence having at least 90% identity to SEQ ID NO: 44; SEQ ID NO: 45 or a sequence having at least 90% identity to SEQ ID NO: 45; SEQ ID NO: 46 or a sequence having at least 90% identity to SEQ ID NO: 46; SEQ ID NO: 106 or a sequence having at least 90% identity to SEQ ID NO: 106; SEQ ID NO: 107 or a sequence having at least 90% identity to SEQ ID NO: 107; SEQ ID NO: 108 or a sequence having at least 90% identity to SEQ ID NO: 108; SEQ ID NO: 109 or a sequence having at least 90% identity to SEQ ID NO: 109; SEQ ID NO: 110 or a sequence having at least 90% identity to SEQ ID NO: 110; SEQ ID NO: 111 or a sequence having at least 90% identity to SEQ ID NO: 111; SEQ ID NO: 112 or a sequence having at least 90% identity to SEQ ID NO: 112; SEQ ID NO: 197 or a sequence having at least 90% identity to SEQ ID NO: 197, or 10. The HTT nucleic acid trans-splicing molecule of claim 9, comprising, consisting essentially of, or consisting of any one of SEQ ID NO: 198 or a sequence having at least 90% identity to SEQ ID NO:

198.

13. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, 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 operably linked in a 5' to 3' direction.

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

5.

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

6.

16. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, 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 operably linked in a 5' to 3' direction.

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

18. 18. The HTT nucleic acid trans-splicing molecule of claim 17, wherein the HTT 5'UTR comprises, consists essentially of, or consists of any one of SEQ ID NOs: 136 or 192, or a sequence having at least 90% identity to any one of SEQ ID NOs: 136 or 192.

19. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, further comprising a sequence encoding an epitope tag, wherein the 5'UTR (if present), the epitope tag, the coding domain, the splicing domain, the linker (if present), the binding domain, and the triple helix terminator (if present) are operably linked in a 5' to 3' direction.

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

4.

21. 1. 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 linker; and (d) a binding domain that binds to a target intron of an HTT pre-mRNA, wherein the target intron comprises intron 2.

22. 22. The HTT nucleic acid trans-splicing molecule of claim 21, wherein the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 60-81, or a sequence having at least 90% identity to any one of SEQ ID NOs: 60-81.

23. 1. An HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exons 1-3; (b) a splicing domain; and (c) a linker; and (d) a binding domain that binds to a target intron of an HTT pre-mRNA, wherein the target intron comprises intron 3.

24. 24. The HTT nucleic acid trans-splicing molecule of claim 23, wherein the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 158-174, or a sequence having at least 90% identity to any one of SEQ ID NOs: 158-174.

25. 1. An HTT nucleic acid trans-splicing molecule comprising: (a) a coding domain comprising HTT exon 1; (b) a splicing domain; and (c) a linker; and (d) a binding domain that binds to a target intron of an HTT pre-mRNA, wherein the target intron comprises intron 1.

26. 26. The HTT nucleic acid trans-splicing molecule of claim 25, wherein the binding domain comprises, consists essentially of, or consists of any one of SEQ ID NOs: 8-21, or a sequence having at least 90% identity to any one of SEQ ID NOs: 8-21.

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

198.

28. 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 linked in a 5' to 3' direction, and optionally further comprising a 5' UTR, wherein the 5' UTR (if present), the coding domain, the splicing domain, the linker, the binding domain, and the triple helix terminator (if present) are operably linked in a 5' to 3' direction.

29. 1. A nucleic acid trans-splicing molecule comprising a linker, wherein said linker comprises, consists essentially of, or consists of a sequence in the range of 20-50 nucleotides in length, said linker comprising 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine.

30. 30. The nucleic acid trans-splicing molecule of claim 29, wherein the linker comprises, consists essentially of, or consists of a sequence ranging from 20 to 45 nucleotides in length, and wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine.

31. 31. The nucleic acid trans-splicing molecule of claim 29 or claim 30, wherein the linker comprises, consists essentially of, or consists of a sequence in the range of 22-42 nucleotides in length, and wherein the linker comprises 60-80% guanines interspersed with thymidine / uridine, 65-75% guanines interspersed with thymidine / uridine, or 66-74% guanines interspersed with thymidine / uridine.

32. The linker is SEQ ID NO: 38 or a sequence having at least 90% identity to SEQ ID NO: 38; SEQ ID NO: 39 or a sequence having at least 90% identity to SEQ ID NO: 39; SEQ ID NO: 40 or a sequence having at least 90% identity to SEQ ID NO: 40, or 32. The nucleic acid trans-splicing molecule of any one of claims 29 to 31, comprising, consisting essentially of, or consisting of SEQ ID NO: 41 or a sequence having at least 90% identity to SEQ ID NO:

41.

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

198.

34. 29. The HTT nucleic acid trans-splicing molecule of any one of claims 1 to 28, further comprising a binding domain that binds to a target intron of the MSH3 pre-mRNA.

35. 35. The HTT nucleic acid trans-splicing molecule of claim 34, wherein the MSH3 target intron comprises any one of intron 5 or intron 15 of MSH3.

36. 36. The HTT nucleic acid trans-splicing molecule of any one of claims 34 or 35, wherein the binding domain that binds to a target intron of an MSH3 pre-mRNA comprises, consists essentially of, or consists of any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210, or a sequence having at least 90% identity to any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210.

37. 37. The HTT nucleic acid trans-splicing molecule of any one of claims 34 to 36, wherein the nucleic acid trans-splicing molecule comprises any one of SEQ ID NOs: 149-154 or 212-223, or a sequence having at least 90% identity to any one of SEQ ID NOs: 149-154 or 212-223.

38. 10. The HTT nucleic acid trans-splicing molecule of any one of the preceding claims, further comprising a nucleic acid sequence encoding a pri-miRNA comprising a microRNA (miRNA) sequence specific for exon 1 of an endogenous HTT mRNA, wherein exon 1 of said nucleic acid trans-splicing molecule comprises a nucleotide sequence change that impairs binding of said miRNA to an mRNA at least partially encoded by said nucleic acid trans-splicing molecule.

39. 39. The HTT nucleic acid trans-splicing molecule of claim 38, wherein the miRNA sequence comprises any one of SEQ ID NOs: 339 or 342 or a nucleic acid sequence having at least 90% identity to any one of SEQ ID NOs: 339 or 342.

40. 40. The HTT nucleic acid trans-splicing molecule of claim 39, wherein the nucleic acid sequence encoding the pri-miRNA comprises any one of SEQ ID NOs: 341 or 344.

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

42. The HTT nucleic acid trans-splicing molecule of any one of claims 38 to 40, wherein the pri-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. 43. The HTT nucleic acid trans-splicing molecule of claim 42, wherein the mir-30a scaffold sequence comprises the 5' scaffold sequence set forth in SEQ ID NO:227 or the 3' scaffold sequence set forth in SEQ ID NO:228, the mir-30a loop sequence comprises SEQ ID NO:229, the mir-155 scaffold sequence comprises the 5' scaffold sequence set forth in SEQ ID NO:230 or the 3' scaffold sequence set forth in SEQ ID NO:231, the mir-155 loop sequence comprises SEQ ID NO:232, the mir-33 scaffold sequence comprises the 5' scaffold sequence set forth in SEQ ID NO:259 or the 3' scaffold sequence set forth in SEQ ID NO:260, or the mir-33 loop sequence comprises SEQ ID NO:

261.

44. Functionally linked, (a) a sequence encoding an antisense RNA that promotes exon skipping of a target exon in MSH3 pre-mRNA, wherein the target exon is any one of MSH3 exons 2 to 4, 6 to 8, or 15, and the target exon comprises a 5' exon-intron junction sequence and a 3' exon-intron junction sequence; and (b) an MSH3 exon-skipping nucleic acid construct comprising a sequence encoding a small nuclear RNA (snRNA) sequence.

45. 45. The MSH3 exon skipping nucleic acid construct of claim 44, further comprising a U1 promoter and a U1 terminator operably linked to (a) and (b).

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

47. 47. The MSH3 exon skipping nucleic acid construct of claim 46, wherein the modified snRNA comprises a U7 Sm OPT sequence or a U2 snRNA sequence.

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

49. The antisense RNA is selected from the group consisting of any one of SEQ ID NOs: 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, and 322, or any one of SEQ ID NOs: 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, and 322.

45. The MSH3 exon skipping nucleic acid construct of claim 44, comprising, consisting essentially of, or consisting of a sequence having at least 90% identity to any one of: 00, 302, 301, 303, 281, 282, 306, 308, 305, 307, 311, 313, 310, 312, 316, 318, 315, 317, 321, 323, 320, or 322.

50. 45. The MSH3 exon skipping nucleic acid construct of claim 44, comprising any one of SEQ ID NOs: 284, 285, 286, 287, 288, 289, 290, 325, 326, 291, 292, 328, 329, 331, 332, 334, 335, 337, and 338.

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

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

53. The antisense RNA is (a) a sequence that is at least 80% complementary to a 5-nucleotide sequence upstream of the 5' exon-intron junction; and 52. The MSH3 exon skipping nucleic acid construct of claim 51, further comprising (b) a sequence that is at least 80% complementary to a 5-nucleotide sequence downstream of the 3' exon-intron junction.

54. 54. The MSH3 exon skipping nucleic acid construct of Claim 52 or 53, wherein the antisense RNA comprises any one of SEQ ID NOs: 299, 304, 309, 314, or 319, or a sequence having at least 90% identity to any one of SEQ ID NOs: 299, 304, 309, 314, or 319.

55. 54. The MSH3 exon skipping nucleic acid construct of any one of claims 51 to 53, comprising any one of SEQ ID NOs: 324, 327, 330, 333, or 336.

56. The antisense RNA is operably linked in the 5' to 3' direction. (a) a sequence that targets the 3' exon-intron junction; (b) a linker sequence of at least 15 nucleotides that does not anneal to the target exon; and 52. The MSH3 exon skipping nucleic acid construct of claim 51, comprising (c) a sequence that targets the 5' exon-intron junction.

57. 57. The MSH3 exon skipping nucleic acid construct of Claim 56, wherein said linker sequence is less than 50% complementary to any sequence of said target exon that is the same length as said linker.

58. 58. The MSH3 exon skipping nucleic acid construct of claim 56 or 57, wherein the antisense RNA comprises any one of SEQ ID NOs: 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. 59. The MSH3 exon skipping nucleic acid construct of any one of claims 56 to 58, comprising any one of SEQ ID NOs: 325, 326, 328, 329, 331, 332, 334, 335, 337, or 338.

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

61. 61. The MSH3 exon skipping nucleic acid construct of claim 60, comprising SEQ ID NO:

309.

62. 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) the MSH3 exon skipping nucleic acid construct of claim 60, comprising SEQ ID NO: 312 (In7 / Ex7 asRNA), SEQ ID NO: 298 (linker), and SEQ ID NO: 313 (In7 / Ex7 asRNA).

63. 61. The MSH3 exon skipping nucleic acid construct of claim 60, wherein the antisense RNA comprises any one of SEQ ID NOs: 309, 310, 311, 312, or 313, or any combination thereof, or a sequence having at least 90% identity to any one of SEQ ID NOs: 309, 310, 311, 312, or 313.

64. 61. The MSH3 exon skipping nucleic acid construct of claim 60, comprising at least one of SEQ ID NOs: 330-332, or any combination thereof.

65. An MSH3 miRNA nucleic acid construct, comprising a sequence encoding a pri-miRNA comprising a scaffold sequence, a loop sequence, and a miRNA sequence that targets endogenous MSH3 mRNA; (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 MSH3 miRNA nucleic acid construct, wherein the miRNA sequence comprises any one of SEQ ID NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257, or a sequence having at least 90% identity to any one of SEQ ID NOs: 224, 244, 246, 248, 250, 252, 254, 256, or 257.

66. 66. The MSH3 miRNA nucleic acid construct of Claim 65, wherein the scaffold sequence comprises any one of SEQ ID NOs: 227, 228, 230, 231, 259 or 260.

67. 67. The MSH3 miRNA nucleic acid construct of claim 65 or 66, wherein the loop sequence comprises any one of SEQ ID NOs: 229, 232, or 261.

68. 68. The MSH3 miRNA nucleic acid construct of any one of claims 65-67, wherein the pri-miRNA sequence comprises any one of SEQ ID NOs: 234, 235, 238-241, or 262-269.

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

70. 1. 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 the MSH3 pre-mRNA; The MSH3 nucleic acid trans-splicing molecule, wherein the coding domain sequence is not an MSH3 coding domain sequence.

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

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

74. 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 NOs: 140, 142, 144, 146, 209, or 210, or a sequence having at least 90% identity to any one of SEQ ID NOs: 140, 142, 144, 146, 209, or 210.

75. 1. A nucleic acid construct for HTT trans-splicing and MSH3 exon skipping, comprising: (a) an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28; (b) the MSH3 exon skipping nucleic acid construct of any one of claims 44 to 64.

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

78. 78. The HTT trans-splicing and MSH3 exon skipping nucleic acid construct of claim 77, comprising any one of SEQ ID NOs: 356, 357, 363, or 364.

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

80. 80. The nucleic acid construct of claim 79 for HTT trans-splicing and MSH3 exon skipping, comprising any one of SEQ ID NOs: 369, 370, and 371.

81. A nucleic acid construct for HTT trans-splicing, HTT miRNA, and MSH3 exon skipping, comprising: (a) an HTT nucleic acid trans-splicing molecule according to any one of claims 38 to 43; (b) the MSH3 exon skipping nucleic acid construct of any one of claims 44 to 64.

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

83. 83. The HTT trans-splicing, HTT miRNA and MSH3 exon skipping nucleic acid construct of claim 82, comprising any one of SEQ ID NOs: 358 or 359.

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

85. A nucleic acid construct of HTT trans-splicing and MSH3 miRNA, comprising: (a) an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28; (b) the MSH3 miRNA nucleic acid construct of any one of claims 65 to 69.

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

87. 87. The nucleic acid construct of HTT trans-splicing and MSH3 miRNA of claim 86, comprising any one of SEQ ID NOs: 354 or 355.

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

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

90. 90. The AAV vector of claim 89, comprising any one of SEQ ID NOs: 356, 357, 363, or 364.

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

92. A ribonucleic acid trans-splicing molecule comprising any one of SEQ ID NOs: 23-36, 47-56, 83-105, 113-125, 175-191, or 199-206.

93. A ribonucleic acid trans-splicing molecule transcribed from an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 39 to 43, or a nucleic acid trans-splicing molecule according to any one of claims 29 to 33.

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

95. 95. The HTT nucleic acid trans-splicing molecule of claim 94, wherein the at least one mutation associated with HD comprises an expansion of a CAG repeat in an HTT gene allele.

96. 96. The HTT nucleic acid trans-splicing molecule of claim 95, wherein the CAG repeat expansion in the HTT gene allele comprises more than 35 CAG repeats.

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

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

99. a nucleic acid trans-splicing molecule of any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct of any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct of any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule of any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping of any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping of any one of claims 81 to 84; or a nucleic acid construct of HTT trans-splicing and MSH3 miRNA of any one of claims 85 to 88.

100. A vector comprising the HTT nucleic acid trans-splicing molecule of any one of claims 1 to 28 or 34 to 43.

101. 101. The vector of claim 100, wherein the vector comprises a 5' regulatory domain operably linked 5' to the coding domain.

102. 102. The vector of claim 101, wherein the 5' regulatory domain is operably linked to a 5' untranslated region.

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

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

105. a nucleic acid trans-splicing molecule of any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct of any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct of any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule of any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping of any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping of any one of claims 81 to 84, or a nucleic acid construct of HTT trans-splicing and MSH3 miRNA of any one of claims 85 to 88.

106. an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping according to any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping according to any one of claims 81 to 84; or a nucleic acid construct of HTT trans-splicing and MSH3 miRNA according to any one of claims 85 to 88.

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

108. The AAV of claim 107, wherein the AAV comprises a 5' regulatory domain operably linked to the 5' side of the coding domain.

109. 109. The AAV of any one of claims 107 or 108, wherein the 5' regulatory domain is operably linked to a 5' untranslated region.

110. The AAV of any one of claims 107 to 109, wherein the 5' regulatory 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 according to any one of claims 107 to 111, wherein the AAV exhibits neuronal tropism.

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

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

115. 115. The composition of claim 114, comprising a pharmaceutically acceptable excipient.

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

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

131.

119. 1. A method for expressing biologically active HTT in a target cell and restoring functional levels of HTT protein in said target cell, comprising: an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 34 to 43; a nucleic acid trans-splicing molecule according to any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping according to any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping according to any one of claims 81 to 84; The method comprising transducing the target cell with a nucleic acid construct with a miRNA; a vector of any one of claims 99-104, a proviral plasmid of claim 105; an AAV of any one of claims 89-91 or 106-113; or a composition of any one of claims 114-118.

120. 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 pre-mRNA containing at least one mutation associated with HD in the target cell is replaced.

121. 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 pre-mRNA containing at least one mutation associated with HD in the target cell is replaced.

122. 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 pre-mRNA containing at least one mutation associated with HD in the target cell is replaced.

123. 123. The method of any one of claims 119 to 122, wherein functional levels of HTT are restored in the target cell by expressing biologically functional HTT protein and / or mutant HTT RNA, and reducing related transcripts (e.g., HTT1a).

124. 1. A method for reducing expression of HTT containing a polyglutamine repeat of more than 35 consecutive glutamine residues in a subject, comprising administering to a target cell, more particularly a neuron, in said subject: an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 34 to 43; a nucleic acid trans-splicing molecule according to any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping according to any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping according to any one of claims 81 to 84; or a nucleic acid construct of HTT trans-splicing and MSH3 according to any one of claims 85 to 88. The method comprising transfecting or transducing a nucleic acid construct with a miRNA; a vector of any one of claims 99-104; a proviral plasmid of claim 105; an AAV of any one of claims 89-91 or 106-113; or a composition of any one of claims 114-118.

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

126. A method for treating Huntington's disease in a subject in need thereof, comprising administering to said subject an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 34 to 43; a nucleic acid trans-splicing molecule according to any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping according to any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping according to any one of claims 81 to 84; or a nucleic acid construct of HTT trans-splicing and MSH3 according to any one of claims 85 to 88. The method comprises administering to the subject a therapeutically effective amount of a nucleic acid construct with miRNA; a vector of any one of claims 99-104; a proviral plasmid of claim 105; an AAV of any one of claims 89-91 or 106-113; or a composition of any one of claims 114-118.

127. a nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 34 to 43; a nucleic acid trans-splicing molecule according to any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; a nucleic acid construct of HTT trans-splicing and MSH3 exon skipping according to any one of claims 75 to 80; a nucleic acid construct of HTT trans-splicing, HTT miRNA and MSH3 exon skipping according to any one of claims 81 to 84; or a nucleic acid construct of HTT trans-splicing and MSH3 according to any one of claims 85 to 88, to the brain of the subject.

127. The method of any one of claims 119 to 126, comprising administration of a nucleic acid construct with miRNA; a vector of any one of claims 99 to 104; a proviral plasmid of claim 105; an AAV of any one of claims 89 to 91 or 106 to 113; or a composition of any one of claims 114 to 118.

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

129. 129. The method of any one of claims 124 to 128, wherein the subject has a genetic predisposition to or has been diagnosed with HD.

130. an HTT nucleic acid trans-splicing molecule according to any one of claims 1 to 28 or 34 to 43; an nucleic acid trans-splicing molecule according to any one of claims 29 to 33; an MSH3 exon skipping nucleic acid construct according to any one of claims 44 to 64; an MSH3 miRNA nucleic acid construct according to any one of claims 65 to 69; an MSH3 nucleic acid trans-splicing molecule according to any one of claims 70 to 74; an HTT trans-splicing and MSH3 exon skipping nucleic acid construct according to any one of claims 75 to 80; an HTT trans-splicing, HTT miRNA and MSH3 exon skipping nucleic acid construct according to any one of claims 81 to 84; an HTT trans-splicing and MSH3 nucleic acid trans-splicing molecule according to any one of claims 85 to 88 for use in preventing or treating HD in a subject in need thereof. A nucleic acid construct with miRNA; a vector according to any one of claims 99 to 104; a proviral plasmid according to claim 105; an AAV according to any one of claims 89 to 91 or 106 to 113; or a composition according to any one of claims 114 to 118.

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

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

133. 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 pre-mRNA and a second binding domain configured to target an intron of the second target pre-mRNA.

134. 134. The method of Claim 132 or 133, wherein 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 pre-mRNA that corrects the defect in the first target pre-mRNA.

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

136. 136. The method of Claim 135, wherein said frameshift creates a premature stop codon in said second target pre-mRNA.

137. 137. The method of any one of claims 132 to 136, wherein the defect comprises an endogenous start codon of the second target pre-mRNA that is removed.

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

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

140. 140. The method of any one of claims 132 to 139, wherein said defect comprises removal of a 5' cap or a 3' polyA tail from said second target pre-mRNA.

141. 141. The method of any one of claims 132-140, wherein said defect results in nonsense-mediated decay of said second target pre-mRNA.

142. 142. The method of any one of claims 132-141, wherein said introducing reduces the abundance of a gene product of said second target pre-mRNA in said cell compared to the abundance of the gene product before said introducing.

143. 1. A method for reducing the abundance of a protein in a cell, comprising introducing into said cell a nucleic acid trans-splicing molecule that introduces a defect in a pre-mRNA that encodes said protein.

144. The defect is (a) a frameshift introduced within the coding sequence of the pre-mRNA; (b) removal of the endogenous start codon of said pre-mRNA; (c) introducing a premature stop codon into the coding sequence of the pre-mRNA; (d) replacing the endogenous coding sequence of the pre-mRNA with an alternative coding sequence; (e) insertion of a 5′ UTR that prevents translation of the endogenous coding sequence of the pre-mRNA; (f) inserting a 3′UTR that destabilizes the pre-mRNA; (g) insertion of a 3′UTR that prevents the pre-mRNA from being exported from the nucleus; (h) removing the 5' cap from the pre-mRNA; or (i) removal of the 3' polyA tail from the pre-mRNA.

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

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

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

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