Methods and materials for treating huntington's disease

Abstract

This document provides methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, Huntington's disease (HD). For example, this document provides agents that can reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mutant huntingtin (mHtt) polypeptide as well as methods for using such agents to treat a mammal (e.g., a human) having, or at risk of developing, HD to treat the mammal.

Description

[0001] METHODS AND MATERIALS FOR TREATING HUNTINGTON'S DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 653,046, filed on May 29, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “51229-001 lW01_SL.xml ” The XML file, created on May 21, 2025, is 60,144 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, Huntington's disease (HD). For example, this document provides agents that can reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mutant huntingtin (mHtt) polypeptide as well as methods for using such agents to treat a mammal (e.g., a human) having, or at risk of developing, HD to treat the mammal.

[0008] BACKGROUND

[0009] Huntington's disease is an autosomal dominant neurodegenerative disease that has a wide impact on a person's functional abilities and usually results in movement, cognitive, and psychiatric disorders. See, e.g., Tabrizi et al., Nat. Rev. Neurol. , 16:529-546 (2020).

[0010] The huntingtin (HTT) gene can encode a huntingtin (Htt) polypeptide. When one or both copies of a HTT gene present in a mammal (e.g., a human) includes a cytosine-adenine- guanine (CAG) trinucleotide repeat expansion, the gene encodes a mutant Htt (mHtt) polypeptide including an expanded glutamine (poly-Q) repeat, which can cause progressive degeneration of nerve cells in the brain resulting in the development of HD. Unaffected individuals have up to 35 CAG repeats in the HTT gene, while individuals at a high risk of developing HD carry 36 or more CAG repeats in the HTT ene. Individuals with 30-37 CAG repeats in the HTT gene have a high risk of passing on an HTT gene containing affected CAG repeat sizes to their offspring (Moncke-Buchner et al., NucL Acid. Res., 30(16): e83 (2002)). Analyzing the number of CAG repeats found in an individual’s HTT alleles can have predictive value for determining their risk for developing HD.

[0011] SUMMARY

[0012] A treatment for HD that can selectively reduce expression of a mutant copy of the HTT gene (e.g., while having little to no effect on expression of a normal copy of the HTT gene) is highly desirable. This document provides methods and materials for selectively reducing expression of a mutant HTT allele without reducing expression of the normal HTT allele.

[0013] This document provides methods and materials for treating a mammal (e g., a human) having, or at risk of developing, HD. In some cases, this document provides agents that can reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mHtt polypeptide. For example, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide can include (a) a nucleic acid molecule (e.g., a guide RNA (gRNA)) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mutant huntingtin (mHtt) polypeptide and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases. In some cases, one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered to a mammal (e.g., a human) having, or at risk of developing, HD to treat the mammal.

[0014] HD is an autosomal dominant neurodegenerative disease, with one mutant copy of the HTT gene being sufficient to cause disease. The vast majority of HD patients have one normal copy of the HTT gene (i.e., with 35 or fewer CAG repeats) and one mutant copy of the HTT gene (i.e., with 36 or more CAG repeats).

[0015] Single nucleotide polymorphisms (SNPs) present in the regulatory region of the HTT gene can be used to distinguish between an HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) and an HTT allele that lacks a pathogenic CAG trinucleotide repeat (i.e., an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat). For example, when a HD patient is heterozygous for a particular SNP within the regulatory region of an HTT gene, a first SNP allele (e.g., a sequence having a first nucleotide variation of that SNP) can be present in one copy of the 7 / 77'gene and a second SNP allele (e.g., a sequence having a second nucleotide variation of that SNP) can be present in the second copy of the HTT gene. As described herein, one or more SNPs present in the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be used to target (e.g., to selectively target) one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) to the regulatory region of the nucleic acid that can encode a mHtt polypeptide such that the presence of the agent can reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat). In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can reduce expression of that nucleic acid by covalent modification of the nucleic acid and / or by covalent modification of a chromatin environment of the nucleic acid.

[0016] Having the ability to reduce or eliminate a level of a mHtt polypeptide as described herein (e.g., by administering one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein) provides a unique and unrealized opportunity to treat mammal (e.g., humans) having, or at risk of developing, HD.

[0017] In general, one aspect of this document features agents that can reduce expression of a genomic nucleic acid that can encode a mHtt polypeptide, where the agent comprises (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of the genomic nucleic acid that can encode the mHtt polypeptide and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase. The nucleic acid sequence can include a SNP, where the SNP is not present in a regulatory region of genomic nucleic acid that can encode a Htt polypeptide that lacks an expanded glutamine repeat. The SNP can be rsl3102260, rsl3122415, rsl3132932, rs 112396951, or rs 149624523. The nucleic acid sequence can have a sequence set forth in SEQ ID NO:17 or SEQ ID NO: 18. The nucleic acid molecule can be from about 16 nucleotides to about 24 nucleotides in length. The nucleic acid molecule can comprise, consist essentially of, or consist of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32. The catalytically dead endonuclease can include a catalytically dead Cas9 (dCas9) polypeptide. The repressor polypeptide can be a Krtippel associated box (KRAB) polypeptide. The methyltransferase can be a DNMT3 A polypeptide, a DNMT3L polypeptide, a DNMT3 A polypeptide, or a DNMT3B polypeptide. The agent can include the repressor polypeptide and the methyltransferase.

[0018] In another aspect, this document features agents that can reduce expression of a genomic nucleic acid that can encode a mHtt polypeptide, where the agent comprises (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of the genomic nucleic acid that can encode the mHtt polypeptide, where the nucleic acid molecule comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase. The nucleic acid sequence can include a SNP, where the SNP is not present in a regulatory region of genomic nucleic acid that can encode a Htt polypeptide that lacks an expanded glutamine repeat. The SNP can be rsl3102260, rsl3122415, rsl3132932, rsll2396951, or rsl49624523. The nucleic acid sequence can have a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. The catalytically dead endonuclease can include a dCas9 polypeptide. The repressor polypeptide can be a KRAB polypeptide. The methyltransferase can be a DNMT3A polypeptide, a DNMT3L polypeptide, a DNMT3 A polypeptide, or a DNMT3B polypeptide. The agent can include the repressor polypeptide and the methyltransferase.

[0019] In another aspect, this document features methods for treating a mammal having HD.

[0020] The methods can include, or consist essentially of, administering to a mammal having HD an agent including (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of the genomic nucleic acid that can encode the mHtt polypeptide (e.g., a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32), and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase. The CAG trinucleotide repeat expansion can include at least 36 CAG trinucleotide repeats. The CAG trinucleotide repeat expansion can include from about 36 CAG trinucleotide repeats to about 350 CAG trinucleotide repeats. The mammal can be a human. In some cases, the level of a Htt polypeptide that lacks an expanded glutamine repeat is not reduced.

[0021] In another aspect, this document features methods for treating a mammal at risk of developing HD. The methods can include, or consist essentially of, administering to an agent including (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of the genomic nucleic acid that can encode the mHtt polypeptide (e.g., a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32), and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase to a mammal identified as having a pathogenic CAG trinucleotide repeat expansion in one or both copies of a HTT gene. The CAG trinucleotide repeat expansion can include at least 36 CAG trinucleotide repeats. The CAG trinucleotide repeat expansion can include from about 36 CAG trinucleotide repeats to about 350 CAG trinucleotide repeats. The mammal can be a human. In some cases, the level of a Htt polypeptide that lacks an expanded glutamine repeat is not reduced.

[0022] In another aspect, this document features methods for reducing a level of mHtt polypeptides within a mammal. The methods can include, or consist essentially of, determining that the mammal comprises a pathogenic CAG trinucleotide repeat expansion in one or both copies of a HTT gene; and administering to the mammal an agent including (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of the genomic nucleic acid that can encode the mHtt polypeptide (e.g., a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32), and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase. The CAG trinucleotide repeat expansion can include at least 36 CAG trinucleotide repeats. The CAG trinucleotide repeat expansion can include from about 36 CAG trinucleotide repeats to about 350 CAG trinucleotide repeats. The mammal can be a human. In some cases, the level of a Htt polypeptide that lacks an expanded glutamine repeat is not reduced.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0024] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1. gRNAs that can target SNPs near the transcription start site of a HTT gene. Exemplary target sequences shown include a regulatory region of a HTT gene including a rsl3102260 (S260) SNP (SEQ ID NO:1; top) and a regulatory region of a HTT gene including a rs 13122415 (S415) SNP (residues 20-58 of SEQ ID NO:2; bottom). The target sites of exemplary gRNAs shown include a target site for S260gl (G) (SEQ ID NO:3), a target site for S260g2 (G) (SEQ ID NO:4), a target site for S260g4 (A) (SEQ ID NO:5), a target site for S260g4G (G) (SEQ ID NO: 6), a target site for S415g 1 (C) (SEQ ID NO: 7), a target site for S415g2 (C) (SEQ ID NO:8), a target site for S415g4G (G) (SEQ ID NO:9), and a target site for S415g4C (C) (SEQ ID NOTO).

[0027] Figure 2. Hep3B cells were heterozygous at rsl3102260, rsl3122415, and rs362331. Sequence reads shown are SEQ ID NOs: 11-13, from top to bottom. Figure 3. Total HTT levels decreased in Hep3B cells transfected with CRISPR interference (CRISPRi) mRNA and SNP targeting guides (RT qPCR). NT refers to a nontargeting guide.

[0028] Figure 4. Allele selective RTqPCR can detect each allele separately using S331. Sequences shown include SEQ ID NO: 14 (top), sequencing read from a C allele cDNA template (SEQ ID NO: 15), a sequencing read from a T allele cDNA template (SEQ ID NO: 16), a primer pair to amplify the region (SEQ ID NO:37 and SEQ ID NO:38), a C probe (SEQ ID NO:39), and a T probe (SEQ ID NO:40).

[0029] Figure 5. Relative HTT allele expression changed in Hep3B cells transfected with CRISPRi mRNA and S260 guides.

[0030] Figure 6. Transfection of S260 gRNAs and CRISPRoff mRNA changed HTT allele ratios in Hep3B cells.

[0031] Figure 7. Transfection of S260 gRNAs and CRISPRoff mRNA changed the level of targeted HTT allele in Hep3B cells.

[0032] Figures 8A-8C. Allele selective repression in HD patient fibroblasts. Figure 8A) Sequencing reads show that HD patient fibroblasts (ND31551), carrying HTT mutant allele with 39 CAG repeats, are heterozygous for both S260 (targeting SNP; SEQ ID NO:50) and S331 (SNP used for allele selective RTqPCR; SEQ ID NO:51)). Figure 8B) Relative levels of HTT expression (C allele versus T allele) in cells transfected with each guide compared to non-targeting guide (NT). Figure 8C) Levels of each allele to levels of that same allele in non-targeting samples (normalized to geometric mean of GAPDH and B2M).

[0033] Figures 9A-9B. Different repressor constructs can lower HTT expression in human neural progenitor cells (NPCs). Figure 9A) Schematic showing design of constructs with different repressor domains on N-terminus fused to dCas9 with or without Zim3-KRAB on C-terminus. Figure 9B) Relative levels of HTT expression in NPCs 3 days post transfection.

[0034] Figure 10. MBZ3 construct can lower HTT expression in human iPSC-derived neurons.

[0035] Figure 11. MBZ3 construct can lower HTT expression in human NGN2 iNeurons using LNP delivery. Figure 12. Prime editing can be used to create S260 heterozygous iPSCs with NGN2 insertion for making iNeurons. Sequences shown include NGN2-iPSCs (SEQ ID NO:52) and prime edited NGN2-iPSCs (SEQ ID NO: 53).

[0036] DETAILED DESCRIPTION

[0037] This document provides methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, HD. In some cases, this document provides agents that can reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mHtt. For example, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide can include (a) a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases. In some cases, one or more agents that can reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mHtt polypeptide provided herein can be administered to a mammal (e.g., a human) having, or at risk of developing, HD to treat the mammal.

[0038] As described herein, SNPs present in the regulatory region of the HTT gene can be used to distinguish between an HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) and an HTT allele that lacks a pathogenic CAG trinucleotide repeat (i.e., an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat). For example, when a HD patient is heterozygous at a particular SNP, a first SNP allele (e.g., a sequence having a first nucleotide variation of that SNP) can be present in one copy of the HTT gene and a second SNP allele (e.g., a sequence having a second nucleotide variation of that SNP) can be present in the second copy of the HTT gene. In some cases, when a HD patient is heterozygous at a particular SNP, a first SNP allele (e.g., a sequence having a first nucleotide variation of that SNP) can be present in an HTT gene allele that encodes a mHtt polypeptide and a second SNP allele (e g., a sequence having a second nucleotide variation of that SNP) can be present in an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat. Examples of SNPs that can be present in the regulatory region of the HTT gene include, without limitation, rsl 3102260 (S260), rsl3122415 (S415), rsl3132932, rsl 12396951, and rsl49624523. For example, a SNP that can be present in the regulatory region of the HTT gene can include the SNP shown in SEQ ID NO: 17 below.

[0039] CGCAAGCGTCTGGGACGCAAGGCGCCGTG (G / A) GGGCTGCCGGGACGGGTCCAAGATGGAC (SEQ ID NO: 17)

[0040] In another example, a SNP that can be present in the regulatory region of the HTT gene can include the SNP shown in SEQ ID NO: 18 below.

[0041] CCCCATTACAGTCTCACCACGCCCCGTCC (C / G) CTCTCCGTTGAGCCCCGCGCCTTCGCCC (SEQ ID NO: 18)

[0042] When a mammal (e.g., a human) having, or at risk of developing, HD is heterozygous for a particular SNP within the regulatory region of an HTT gene, a first SNP allele (e.g., a sequence having a first nucleotide variation of that SNP) can be present in one copy of the HTT gene and a second SNP allele (e.g., a sequence having a second nucleotide variation of that SNP) can be present in the second copy of the HTT gene. In some cases, a mammal that is heterozygous for a particular SNP present in the regulatory region of the HTT gene can have one SNP sequence the regulatory region of in one copy of the HTT gene (e.g., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) and can have a second SNP sequence in the regulatory region of the second copy of the HTT gene (e.g., an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat). In such cases, the particular SNP allele present in the HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) can be targeted to reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat). In some cases, a mammal (e.g., a human) having, or at risk of developing, HD can be heterozygous for the SNP shown in SEQ ID NO: 17. For example, a mammal that is heterozygous for the SNP shown in SEQ ID NO: 17 can have SEQ ID NO: 17 with the “G” at residue 30 in the regulatory region of an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat and can have SEQ ID NO: 17 with the “A” at residue 30 in the regulatory region of an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat. In such examples, the SNP allele shown in SEQ ID NO: 17 with the “G” at residue 30 present in the regulatory region of the HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) can be targeted with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) to reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0043] In another example, a mammal that is heterozygous for the SNP shown in SEQ ID NO: 17 can have SEQ ID NO: 17 with the “A” at residue 30 in the regulatory region of an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat and can have SEQ ID NO: 17 with the “G” at residue 30 in the regulatory region of an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat. In such examples, the SNP allele shown in SEQ ID NO: 17 with the “A” at residue 30 present in the regulatory region of the HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) can be targeted with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) to reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0044] In some cases, a mammal (e g., a human) having, or at risk of developing, HD can be heterozygous for the SNP shown in SEQ ID NO: 18. For example, a mammal that is heterozygous for the SNP shown in SEQ ID NO: 18 can have SEQ ID NO: 18 with the “C” at residue 30 in the regulatory region of an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat and can have SEQ ID NO: 18 with the “G” at residue 30 in the regulatory region of an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat. In such examples, the SNP allele shown in SEQ ID NO: 18 with the “C” at residue 30 present in the regulatory region of the HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) can be targeted with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e g., one or more agents each including (a) a nucleic acid molecule (e g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) to reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0045] In another example, a mammal that is heterozygous for the SNP shown in SEQ ID NO: 18 can have SEQ ID NO: 18 with the “G” at residue 30 in the regulatory region of an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat and can have SEQ ID NO: 18 with the “C” at residue 30 in the regulatory region of an HTT gene allele that encodes a Htt polypeptide that lacks an expanded glutamine repeat. In such examples, the SNP allele shown in SEQ ID NO: 18 with the “G” at residue 30 present in the regulatory region of the HTT gene allele containing a pathogenic CAG trinucleotide repeat expansion (i.e., an HTT gene allele that encodes a mHtt polypeptide including an expanded glutamine repeat) can be targeted with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) to reduce or eliminate transcription of that nucleic acid thereby resulting in a reduced or eliminated level of the mHtt polypeptide (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0046] In some cases, the methods provided herein can include determining that a mammal (e.g., a human) having, or at risk of developing, HD is heterozygous for a SNP present in the regulatory region of an HTT gene. Any appropriate method can be used to assess a mammal for heterozygosity of a SNP present in the regulatory region of an HTT gene. For example, the regulatory region of a HTT gene can be sequenced. In some cases, the regulatory region of a HTT gene can be assessed for heterozygosity as described in Example 1.

[0047] In some cases, the methods and materials described herein can be used in a CRISPRi system. A CRISPRi system uses a gRNA to target a nuclease deficient endonuclease (e.g., a catalytically dead Cas9 (dCas9) polypeptide) fused to a repressor polypeptide to a specific locus in the genome to repress gene expression (e.g., to transiently repress gene expression) at that locus by blocking transcriptional initiation or elongation. For example, a CRISPRi system designed to reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mHtt as described herein can include one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including a catalytically dead endonuclease and one or more repressor polypeptides (e.g., a Kriippel associated box (KRAB) polypeptide).

[0048] In some cases, the methods and materials described herein can be used in a CRISPRoff system. A CRISPRoff system uses a gRNA to target a nuclease deficient endonuclease (e.g., a dCas9 polypeptide) fused to a methyltransferase (e.g., a DNA methyltransferase) to a specific locus in the genome to repress gene expression (e.g., to heritably repress gene expression) at that locus by methylation-induced gene silencing. For example, a CRISPRoff system designed to reduce expression of a nucleic acid (e.g., a genomic nucleic acid such as a gene) that can encode a mHtt as described herein can include one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including a catalytically dead endonuclease and one or more methyltransferases (e.g., one or more DNA methyltransferases).

[0049] An agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide can be any appropriate type of agent. In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide can be an epieditor. An epieditor can be any compound that is designed to modify and / or influence genomic DNA function without changing the genomic DNA sequence itself. In some cases, an epieditor can heritably reduce expression of a nucleic acid that can encode a mHtt polypeptide. In some cases, an epieditor can methylate one or more nucleotides within a genomic DNA sequence provided that the DNA is not altered. For example, an epieditor can methylate a cytosine (C) to create a 5-methylcytosine. In some cases, an epieditor can be a ribonucleoprotein complex that includes (a) a DNA binding domain that can target (e.g., target and bind) a specific sequence and (b) a polypeptide including a catalytically dead endonuclease and a polypeptide that provides a desired epigenome modification (e.g., a methyltransferase). For example, an epieditor typically includes (a) a nucleic acid molecule (e.g., a gRNA) and (b) a polypeptide including a catalytically dead endonuclease and a methyltransferase. As described herein, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide can include (a) a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransf erases.

[0050] In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can reduce or eliminate expression of a nucleic acid (e.g., an RNA such as a messenger RNA (mRNA)) that can encode a mHtt polypeptide. A nucleic acid (e.g., an RNA such as a mRNA) that can encode a mHtt polypeptide can be any nucleic acid that can encode a mHtt polypeptide and that includes at least 36 CAG trinucleotide repeats. A nucleic acid (e.g., an RNA such as a mRNA) that can encode a mHtt polypeptide can include any number of CAG trinucleotide repeat expansion that causes, or increases risk for developing, HD. In some cases, a nucleic acid (e.g., an RNA such as a mRNA) that can encode a mHtt polypeptide can include 36 or more CAG trinucleotide repeats. For example, a nucleic acid (e.g., an RNA such as a mRNA) that can encode a mHtt polypeptide can include from about 36 CAG trinucleotide repeats to about 350 CAG trinucleotide repeats (e.g., from about 36 to about 300, from about 36 to about 250, from about 36 to about 200, from about 36 to about 150, from about 36 to about 100, from about 36 to about 75, from about 36 to about 50, from about 50 to about 350, from about 75 to about 350, from about 100 to about 350, from about 150 to about 350, from about 200 to about 350, from about 250 to about 350, from about 50 to about 300, from about 75 to about 250, from about 100 to about 200, from about 50 to about 150, from about 150 to about 250, or from about 200 to about 300 CAG trinucleotide repeats). Examples of nucleic acids (e.g., RNA s such as a mRNAs) that can encode mHtt polypeptides include, without limitation, those sequences set forth in the National Center for Biotechnology Information (NCBI) database at Accession Nos. VCV000000409.5, VCV000031916.4, and VCVOO 1687507.1. In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be used to reduce a level of RNA (e.g. mRNA) mHtt polypeptides in a mammal (e.g., a human) having, or at risk of developing, HD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0051] In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can reduce or eliminate expression of a mHtt polypeptide. A mHtt polypeptide can be any Htt polypeptide that includes an expanded glutamine repeat (e.g., as compared to a Htt polypeptide that does not cause HD). A mHtt polypeptide can include any number of glutamine residues in a glutamine repeat that can cause HD. In some cases, a mHtt polypeptide can include greater than 36 glutamine residues in a glutamine repeat. For example, a mHtt polypeptide can include from about 36 glutamine residues in a glutamine repeat to about 350 glutamine residues in a glutamine repeat (e.g., from about 36 to about 300, from about 36 to about 250, from about 36 to about 200, from about 36 to about 150, from about 36 to about 100, from about 36 to about 75, from about 36 to about 50, from about 50 to about 350, from about 75 to about 350, from about 100 to about 350, from about 150 to about 350, from about 200 to about 350, from about 250 to about 350, from about 50 to about 300, from about 75 to about 250, from about 100 to about 200, from about 50 to about 150, from about 150 to about 250, or from about 200 to about 300 CAG glutamine residues in a glutamine repeat). Examples of mHtt polypeptides include, without limitation, those polypeptides set forth in the NCBI databases at Accession Nos.

[0052] NP_001375421.1:p.Glnl8_Gln38dup and NP_002102.4:p.Gln22_Gln40dup. In some cases, a mHtt polypeptide can be as described elsewhere (see, e.g., Neueder et al., Sci. Rep., 7(1): 1307 (2017)). In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be used to reduce expression of one or more mHtt polypeptides in a mammal (e.g., a human) having, or at risk of developing, HD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0053] In some cases, an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) does not reduce expression of a Htt polypeptide that lacks an expanded glutamine repeat.

[0054] A nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can target any appropriate site. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind) a nucleic acid sequence downstream of a transcription start site present in the regulatory region of a nucleic acid that can encode a mHtt polypeptide. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind) genomic DNA. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind) the regulatory region of HTT gene (e.g., a human HTT gene). In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind) one or more SNPs present in the regulatory region of the nucleic acid that can encode a mHtt polypeptide. Examples of SNPs that can be present in the regulatory region of a nucleic acid that can encode a mHtt polypeptide include, without limitation, rsl3102260 (S260), rsl3122415 (S415), rsl3132932, and rsll2396951, rsl49624523.

[0055] For example, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind to) any portion of SEQ ID NO: 17, provided that it targets the SNP present at residue 30 as numbered in SEQ ID NO: 17.

[0056] In another example, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can target (e.g., target and bind to) any portion of SEQ ID NO: 18, provided that it targets the SNP at residue 30 as numbered in SEQ ID NO: 18.

[0057] A nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be any appropriate type of nucleic acid molecule. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be an RNA molecule. For example, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide that is an RNA molecule can be a gRNA. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be deoxyribonucleic acid molecule.

[0058] A nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be any appropriate length (e.g., can include any appropriate number of nucleotides). In some cases, nucleic acid molecule that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be from about 16 nucleotides to about 24 nucleotides in length (e.g., from about 16 to about 22, from about 16 to about 20, from about 16 to about 18, from about 18 to about 24, from about 20 to about 24, from about 22 to about 24, or from about 18 to about 22 nucleotides in length).

[0059] A nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide present in an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can have any appropriate nucleic acid sequence. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can have a nucleic acid sequence that is complementary to at least a portion of the regulatory region of a nucleic acid that can encode a mHtt polypeptide. For example, a nucleic acid molecule (e.g., a gRNA) that can target (e g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can have a nucleic acid sequence that is complementary to at least a portion of SEQ ID NO: 17, provided that it targets residue 30 as numbered in SEQ ID NO: 17. For example, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can have a nucleic acid sequence that is complementary to at least a portion of SEQ ID NO: 18, provided that it targets residue 30 as numbered in SEQ ID NO: 18. In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can comprise, consist essentially of, or consist of the nucleic acid sequences set forth in Table 1.

[0060] Table 1. Exemplary nucleic acid molecules that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide.

[0061] In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be 100% complementary to at least a portion of SEQ ID NO: 17 or SEQ ID NO: 18.

[0062] In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can be at least 85% (e.g., at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) complementary to at least a portion of SEQ ID NO: 17 or SEQ ID NO: 18, provided that the nucleic acid molecule maintains its basic ability to target (e.g., target and bind) the regulatory region of the nucleic acid that can encode a mHtt polypeptide.

[0063] In some cases, a nucleic acid molecule (e.g., a gRNA) that can target (e.g., target and bind) the regulatory region of a nucleic acid that can encode a mHtt polypeptide can have one or more (e.g., one, two, three, four, or more) nucleotide substitutions, one or more (e.g., one, two, three, four, or more) additional 5' nucleotides, and / or one or more (e.g., one, two, three, four, or more) additional 3' nucleotides relative to any one of SEQ ID NOs: 19-32, provided that the nucleic acid molecule maintains its basic ability to target (e.g., target and bind) the regulatory region of the nucleic acid that can encode a mHtt polypeptide.

[0064] A polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases can include any appropriate catalytically dead endonuclease. In some cases, a catalytically dead endonuclease can be a catalytically dead Cas polypeptide such as a dCas9 polypeptide. In some cases, a catalytically dead endonuclease can be as set forth in the NCBI databases at Accession Nos: AKA60242 and UXX62623.

[0065] When a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases includes one or more repressor polypeptides, the one or more repressor polypeptides can be any appropriate one or more repressor polypeptides. Examples of repressor polypeptides includes, without limitation, KRAB polypeptides (e.g., Zim3KRAB polypeptides).

[0066] When a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases includes one or more methyltransferases, the one or more methyltransferases can be any appropriate one or more methyltransferases. For example, a methyltransferase can be a DNA methyltransferase. In some cases, a methyltransferase can methylate a nucleic acid (e.g., a DNA sequence) without altering the sequence of the nucleic acid. In some cases, a methyltransf erase that can modulate one or more methylation sensitive transcription factors and / or CCCTC-binding factors (CTCFs). In some cases, a methyltransferase can methylate a target sequence that lacks any CpG islands. Examples of methyltransferases include, without limitation, DNMT3A polypeptides, DNMT3L polypeptides, DNMT3A polypeptides, and DNMT3B polypeptides.

[0067] In some cases, an agent that can be used to reduce expression of a nucleic acid that can encode a mHtt polypeptide as described herein can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2021 / 247570; and Nunez et al., Cell, 184:2503-2519 (2021)).

[0068] Also provided herein are vectors (e.g., expression vectors) containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases). Vectors can carry nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases), where it can be replicated and / or expressed.

[0069] A vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be any appropriate type of expression vector. In some cases, a vector can be a non-viral vector. In some cases, a vector can be a viral vector.

[0070] When a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) is a non- viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

[0071] When a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) is a viral vector, any appropriate viral vector can be used. A viral vector can be derived from a positive-strand virus or a negative-strand virus. A viral vector can be derived from a virus with a DNA genome or an RNA genome. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells. Examples virus-based vectors that can be used to deliver nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein include, without limitation, vectors based on adeno-associated viruses (e.g., AAV vectors).

[0072] In some cases, a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) also can include one or more regulatory elements (e.g., an enhancer or a promoter sequence such as a constitutive, inducible, and / or tissue-specific promoter sequence) operably linked to the nucleic acid that can encode the agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) that can be included in a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein to facilitate transcription of a gRNA. For example, a promoter can be included in a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein to facilitate transcription of nucleic acid encoding a repressor domain. In some cases, a promoter can be included in a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein to facilitate expression of a gRNA fused to a repressor domain. A promoter can be a naturally occurring promoter or a recombinant promoter (e.g., a chimeric promoter). A promoter can be ubiquitous or inducible, and can affect the expression of a nucleic acid encoding a polypeptide in a general or tissue-specific manner. Examples of promoters that can be used to drive expression of a nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein in cells include, without limitation, cytomegalovirus (CMV) promoters, Synapsin 1 promoters, pol3 (polIII) promoters (e.g., U6), and pol2 promoters. Expression of a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide (e.g., a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32), and expression of a repressor domain can be under the control of the same regulatory elements or can be under the control of separate regulatory elements. For example, a vector containing nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can include nucleic acid that can encode (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide (e.g., a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32) that is operably linked to a first promoter and can include nucleic acid that can encode a repressor domain that is operably linked to a second promoter. As used herein, “operably linked” refers to positioning of a regulatory element in a vector relative to a nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein in such a way as to permit or facilitate expression of the encoded agent. For example, a vector can contain a promoter and nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein. In this case, the promoter is operably linked to nucleic acid encoding an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein such that it drives expression of the agent in cells.

[0073] One or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having, or at risk of developing, HD. For example, a therapeutically effective amount of one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0074] This document also provides methods for using one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases). In some cases, one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered to a mammal having, or at risk of developing, HD (e.g., to treat the mammal).

[0075] In some cases, a mammal (e.g., a human) can be identified as having, or as being at risk of developing, HD. For example, neurological examinations (e.g., to evaluate motor symptoms, sensory symptoms, and / or psychiatric symptoms), neuropsychological testing (e.g., to evaluate, memory, reasoning, mental agility, language skills, and / or spatial reasoning), brain-imaging (e.g., to evaluate the structure and / or function of the brain), genetic testing (e g., to determine the presence or absence of a pathogenic CAG trinucleotide repeat expansion in one or both copies of &HTT gene) can be used to identify a mammal as having, or as being at risk of developing, HD.

[0076] Once identified as having, or as being at risk of developing, HD, the mammal (e.g., the human) can be administered, or instructed to self-administer, one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) as described herein. In some cases, one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered to a mammal (e.g., a human) identified as having, or at risk of developing, HD based, at least in part, on the presence of a pathogenic CAG trinucleotide repeat expansion (e.g., the presence of at least 36 trinucleotide repeat expansions) in one or both copies of a HTT gene present in a mammal (e.g., a human) to treat the mammal.

[0077] One or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to any appropriate mammal. Examples of mammals that can have, or can be at risk of developing, HD include, without limitation, humans, non-human primates (e.g., monkeys), and mice (e.g., transgenic mice).

[0078] One or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to a mammal (e.g., a human) by any appropriate methods. In some cases, a composition (e.g., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered locally or systemically. Examples of administration routes that can be used to deliver one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein include, without limitation, intraparenchymal administration, intracerebral administration, intra cisterna magna administration, intracerebroventricular administration, intrathecal administration, and intravenous administration. For example, a composition containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered locally by direct injection (e.g., an intraparenchymal injection) to the brain of a mammal (e.g., a human).

[0079] An effective amount of a composition (e.g., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be any amount that can treat a mammal (e.g., a human) having, or at risk of developing, HD without producing significant toxicity to the mammal. For example, an effective amount of one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be from about 0.1 milligrams of polypeptide(s) per kilogram body weight of the mammal (mg / kg) per dose to about 1 mg / kg. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.

[0080] In some cases, a mammal (e.g., a human) having, or at risk of developing, HD can be administered a single dose of a composition (e.g., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases).

[0081] In some cases, a mammal (e g., a human) having, or at risk of developing, HD can be administered two or more doses of a composition (e.g., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases). When a mammal having, or at risk of developing, HD is administered two or more doses of a composition containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein, the frequency of administration of the composition containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be any frequency that can treat a mammal (e.g., a human) having, or at risk of developing, HD without producing significant toxicity to the mammal. For example, the frequency of administration can be once every three months. The frequency of administration can remain constant or can be variable during the duration of treatment. In some cases, a course of treatment with a composition containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency. An effective duration for administering a composition (e. ., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be any duration that treat a mammal (e.g., a human) having, or at risk of developing, HD without producing significant toxicity to the mammal. For example, the effective duration can vary from several months to several years. For example, the effective duration can be the life of the mammal. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.

[0082] In some cases, a composition (e.g., a pharmaceutical composition) containing one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to a mammal (e.g., a human) having, or at risk of developing, HD as a single administration (e.g., as a once-administered therapy).

[0083] In some cases, the one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be used as the sole active agent used to treat a mammal (e.g., a human) having, or at risk of developing, HD.

[0084] In some cases, the methods and materials described herein can include one or more (e.g, one, two, three, four, five or more) additional agents / therapies used to treat a mammal (e.g., a human) having, or at risk of developing, HD (e.g., to treat one or more symptoms of HD). In some cases, an agent used to treat HD (or one or more symptoms of HD) can be an antipsychotic agent. In some cases, an agent used to treat HD (or one or more symptoms of HD) can be an antidepressant. In some cases, an agent used to treat HD (or one or more symptoms of HD) can be an antipsychotic agent. In some cases, an agent used to treat HD (or one or more symptoms of HD) can be a mood-stabilizing agent. Examples of agents used to treat HD (or one or more symptoms of HD) that can be administered to a mammal (e.g., a human) having, or at risk of developing, HD together with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) include, without limitation, tetrabenazine (e g., XENAZINE®), deutetrabenazine (e.g., AUSTEDO®), haloperidol, fluphenazine, olanzapine (e.g., ZYPREXA®), aripiprazole (e.g., ABILIFY®), amantadine (e.g., GOCOVRI® and OSMOLEX® ER), levetiracetam (e.g, KEPPRA™, ELEPSIA™ XR, and SPRITAM®), clonazepam (e.g, KLONOPIN®), citalopram (e.g, CELEXA®), escitalopram (e.g, LEXAPRO®), fluoxetine (e.g, PROZAC®), sertraline (e.g, ZOLOFT®), quetiapine (e.g, SEROQUEL®), olanzapine (e.g, ZYPREXA®), divalproex (e g, DEPAKOTE®), carbamazepine (e.g, TEGRETOL®, CARBATROL®, and EPITOL®), and lamotrigine (e.g, LAMICTAL™). In some cases, the one or more additional agents can be administered together with one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g, in the same composition). In some cases, the one or more additional agents can be administered independent of the one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein. When the one or more additional agents are administered independent of the one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein, the one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered first, and the one or more additional agents administered second, or vice versa.

[0085] Examples of therapies that can be used as described herein to treat HD (or one or more symptoms of HD) include, without limitation, psychotherapy (e.g., to help with behavioral problems, develop coping strategies, and / or managing expectations during progression of the disease), speech therapy (e.g., to improve the ability to speak clearly and / or address difficulties with muscles used in eating and swallowing), physical therapy (e.g., to enhance strength, flexibility, balance, and / or coordination), therapies that reduce involuntary physical movements (chorea) associated with HD, and / or occupational therapy (e.g., to use assistive devices that improve functional abilities). In cases where one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) are used in combination with one or more additional therapies used to treat HD (or one or more symptoms of HD), the one or more additional therapies can be performed at the same time or independently of the administration of one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein. For example, the one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein can be administered before, during, and / or after the one or more additional therapies are performed.

[0086] In some cases, the methods and materials described herein can be used to reduce or eliminate one or more symptoms of HD. For example, one or more (e.g., one, two, three, or more) agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, HD) to reduce or eliminate one or more symptoms of HD in the mammal. Examples of symptoms of HD include, without limitation, involuntary j erking or writhing movements (chorea), muscle problems, such as rigidity or muscle contracture (dystonia), slow or unusual eye movements, impaired gait, impaired posture, impaired balance, difficulty with speech, difficulty with swallowing, difficulty organizing, difficulty prioritizing or focusing on tasks, slowness in processing thoughts, difficulty in learning new information, depression, irritability, sadness, apathy, social withdrawal, insomnia, fatigue, loss of energy, obsessive-compulsive disorder, mania, and bipolar disorder. In some cases, the materials and methods described herein can be used to reduce the severity of one or more symptoms of HD in a mammal (e.g., a human) having HD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0087] In some cases, the methods and materials described herein can be used to delay or prevent the development of HD. For example, one or more (e.g., one, two, three, or more) agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, HD) to delay or prevent the development of HD in the mammal. In some cases, the materials and methods described herein can be used to delay the development of HD in a mammal (e.g., a human) at risk of developing HD by, for example, at least 6 months (e.g., at least about 6 months, at least about 8 months, at least about 10 months, at least about 1 year, at least about 1.5 years, at least about 2 years, at least about 2.5 years, or at least about 3 years).

[0088] In some cases, the methods and materials described herein can be used to slow expansion of a CAG trinucleotide repeat expansion present in one or both copies of a HTT gene present in a mammal (e.g., a human). For example, one or more (e.g., one, two, three, or more) agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, HD) to slow expansion of a CAG trinucleotide repeat expansion present in one or both copies of a HTT gene present in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0089] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0090] EXAMPLES

[0091] Example 1: Allele selective epiediting as a treatment for Huntington ’s disease

[0092] This Example describes the design of epieditors that can target SNPs present in the regulatory region of a HTT gene alleles that can encode a mHtt polypeptide. In heterozygous patients, the SNP allele that is in phase with the pathogenic CAG trinucleotide repeat expansion can be targeted to selectively reduce levels of mHTT mRNA.

[0093] Methods:

[0094] Cell culture

[0095] Hep3B cells were cultured in DMEM with 10% FBS. DNA was extracted from Hep3B cells using QuickExtract (Lucigen) and the region of interest was PCR amplified using OneTaq2X Master Mix with corresponding primers and purified with QIAquick PCR purification kit (Qiagen). PCR product was sanger sequenced at UC Berkeley DNA Sequencing facility to check for heterozygosity at different SNPs.

[0096] Hep3B Transfection

[0097] Hep3B cells were transfected using 1.5ul Lipofectamine MessengerMax (Invitrogen) with in vitro transcribed CRISPRi mRNA (2ug per reaction) or CRISPRoff mRNA (2.5ug per reaction) and corresponding single guide RNA (lOOpmol per reaction) (Synthego). After 3 or 14 days, RNA was extracted using Direct-zol (Zymo) and converted to cDNA using Maxima cDNA first strand synthesis kit (Thermo Scientific).

[0098] In vitro transcription

[0099] HiScribe T7 mRNA kit with CleanCap Reagent AG (New England Biolabs) was used for in vitro transcription from a PCR product from plasmid with CRISPRoff construct (CRISPRoff-V2.1) or PCR product from plasmid with CRISPRi construct, according to manufacturer’s instructions and with complete substitution of UTP with Nl-methyl- Pseudouridine-5’ -Triphosphate (Trilink). After transcription, RNA was cleaned using a Monarch RNA cleanup kit (New England Biolabs) and RNA concentration was measured with Nanodrop.

[0100] Allele-selective RTqPCR

[0101] Allele selective RTqPCR was performed using Taqman Fast Advanced Master Mix with 500nM primers and 200nM custom Taqman probes (S331 C allele probe was labeled with FAM and S33 IT allele labeled with VIC). Primer and probe sequences were obtained from Miller et al (Sci Rep 2017). Thermocycling conditions were the following: 50°C for 2min, 95°C for 2min and 40 cycles of 95°C for 15sec, 65°C for Imin. Ct values were analyzed using the 2-DDCt method.

[0102] Total HTT RTqPCR

[0103] Taqman Fast Advanced Master Mix and Taqman Assays were used to detect total HTT levels which were normalized to GAPDH levels (HTT assay ID: Hs00918174_ml and GAPDH assay ID: Hs99999905_ml). Thermocycling conditions were the following: 50°C for 2min, 95°C for 20sec and 40 cycles of 95°C for 3sec, 60°C for 30sec. Ct values were analyzed using the 2-DDCt method.

[0104] Sanger Sequencing Primers

[0105] To check for heterozygous SNPs near regulatory region:

[0106] HTT reg FWD: CGACCACGCGCATTCTCTG (SEQ ID NO:33)

[0107] HTT reg REV: CCTTCATCAGCTTTTCCAGGGTC (SEQ ID NO:34)

[0108] To check SNP rs362331 at Exon 50:

[0109] HTT int49 FWD : GGGCATTCTGTGACTCGGTA (SEQ ID NO: 35) HTT int50 REV: GATAGGAACCCACCGTTCAT (SEQ ID NO:36)

[0110] Allele selective RTqPCR primer / probes

[0111] S331 qPCR FWD CTGGAGCGTGGTCTCCTCCACA (SEQ ID NO:37) S331 qPCR REV GTGTGTTTGGATCTACTTCCTCC (SEQ ID NO 38) rs362331 C probe (FAM) TCCCTCATCCACTGTGTGC (SEQ ID NO:39) rs362331 T probe (VIC) TCCCTCATCTACTGTGTGC (SEQ ID NO:40)

[0112] Spacer Guide Sequences

[0113] Non-targeting (NT) AACGACTAGTTAGGCGTGTA (SEQ ID NO:41) S260gl (G) TCTGGGACGCAAGGCGCCGT (SEQ ID NO: 19)

[0114] S260g2 (G) GTCTGGGACGCAAGGCGCCG (SEQ ID NO:20)

[0115] S260g4 (G) ACCCGTCCCGGCAGCCCCCA (SEQ ID NO:21)

[0116] S260g4 (A) ACCCGTCCCGGCAGCCCTCA (SEQ ID NO: 22)

[0117] S415gl (C) AGGCGCGGGGCTCAACGGAG (SEQ ID NO:25)

[0118] S415g2 (C) GGCGCGGGGCTCAACGGAGA (SEQ ID NO:26)

[0119] S415g4 (C) GGGCTCAACGGAGAGGGGAC (SEQ ID NO: 27)

[0120] S415g4 (G) GGGCTCAACGGAGAGGGGAC (SEQ ID NO:28) Results: gRNAs that can target rsl3102260 (S260) or rsl 3122415 (S415) SNPs near the transcription start site of the HTT gene were designed (Figure 1). Some targeted SNPs were in the protospacer adjacent motif (PAM). gRNAs S260gl and S260g2 target alleles with a G at S260. gRNAs S415g 1 and S415g2 targeted alleles with a C at S415. Other targeted SNPS were in the seed region of the guide. gRNA S260g4 (G) targeted the G at S260, gRNA S260g4 (A) targeted the A at S260, gRNA S415g4C (C) targeted the C at S415, and S415g4G (G) targeted the G at S415.

[0121] The presence of SNPs in HTT alleles in Hep3B cells was confirmed by PCR amplification and sequencing of the region of interest. Hep3B cells were heterozygous at rs!3102260, rsl3122415, and rs362331 (Figure 2).

[0122] Hep3B cells were transfected with CRISPR interference (CRISPRi) mRNA and corresponding sgRNA. RNA was extracted 3 days later and RTqPCR was performed to measure relative HTT levels normalized to GAPDH. Each guide targeted only one of the alleles in Hep3B cells. Total HTT levels were decreased in Hep3B cells transfected with CRISPRi mRNA and SNP targeting guides (Figure 3).

[0123] To validate allele selectivity, PCR amplified cDNA from cell lines homozygous at S331 was used to make mixes with defined amounts of each template. These templates were used for allele selective qPCR to confirm that each allele can be detected separately. Allele selective RTqPCR detected each allele separately using S331 (Figure 4).

[0124] Hep3B cells were transfected with CRISPRi mRNA and corresponding sgRNA. RNA was extracted 3 days later and allele selective RTqPCR was performed. The 2-DDCt method was used to compare relative levels C allele compared to T allele in cells transfected with each guide compared to non-targeting guide (NT). S260 guides targeting S260 G allele (S260gl, S260g2 and S260g4G) showed decreased levels of C allele relative to T allele, while S260 guide targeting S260 A allele (S260g4A) shows higher levels of C allele compared to T allele (Figure 5).

[0125] Hep3B cells were transfected with CRISPRoff mRNA and corresponding sgRNA. RNA was extracted on day 3 and day 14 post transfection (pt). Allele selective RTqPCR performed and the 2-DDCt method were used to compare relative levels C allele compared to T allele. S260 guides targeting S260 G allele (S260gl, S260g2 and S260g4G) showed decreased levels of C allele relative to T allele, while S260 guide targeting S260 A allele (S260g4A) showed higher levels of C allele compared to T allele. While the changes in allele ratios were more pronounced at day 3 pt, allele ratio differences were still present at day 14 pt (Figure 6).

[0126] Hep3B cells were transfected with CRISPRoff mRNA and corresponding sgRNA.

[0127] Allele selective RTqPCR from day 3 post transfection compared relative levels of each allele to levels of that same allele in non-targeting samples (normalized to geometric mean of GAPDH and B2M). In Hep3B cells, the S260 G allele was in phase with S331 C allele (Allele 1) and S260 A allele was in phase with S331 T allele (Allele 2). When using CRISPRoff mRNA with S260 guides targeting Allele 1 (S260gl, S260g2 and S260g4G) there was a decrease in levels of the targeted allele (Allele 1) but not the other allele (Allele 2). When targeting Allele 2 (S260g4A) there was a decrease in levels of targeted allele (Allele 2) but not the other allele (Allele 1). See, e.g., Figure 7.

[0128] Together, these results demonstrate that epieditors that can target SNPs present in the regulatory region of HTT gene alleles that can encode a mHtt polypeptide can be used to reduce or eliminate expression of mHtt polypeptides, and can thus be used to treat a mammal (e.g., a human) having, or at risk of developing, HD.

[0129] Example 2: Exemplary Sequences

[0130] The results in this Example re-present and expand on at least some of the results provided in other Examples.

[0131] Methods

[0132] HD Fibroblast cell culture

[0133] HD fibroblast cells (ND31551 fibroblats with HD mutation from NINDS) were cultured in DMEM with 15% FBS, 1% Pen / Strep and 1% NonEssential Amino Acids. HD Fibroblasts were transfected using Lipofectamine MessengerMax with CRISPRoff mRNA and corresponding synthetic sgRNAs. RNA was extracted 6 days post transfection and allele selective RTqPCR was performed as described above. Repressor constructs

[0134] Potential repressor domains (MBD1, EHMT2, RNF2 and YAF2) were used (DelRosso et al., Nature, 616:365-372 (2023); and Policarpi et al., Nature Genetics, 56: 1168- 1180 (2024)). Constructs were designed to fuse each repressor domain (at N-terminus) to dCas9 with or without Zim3KRAB at C-terminus. For example, the MBZ3 contract consists of the transcriptional repressor domain of human Methyl-CpG Binding Domain Protein 1 (MBD1) fused to dCas9 and Zim3KRAB. Sequences were ordered from Azenta with restriction sites compatible with Fragmid system. After cloning, plasmid sequence was verified by Nanopore Sequencing and plasmids were linearized and used as templates for in vitro transcription to generate mRNA used in experiments. iPSC-derived neurons iPSCs (MSC-iPSl) were differentiated into NPCs using dual SMAD inhibition with small molecules (LDN193189, SB431542 and XAV939). NPCs were expanded in NPC media (DMEM / F12, N2 supplement, B27 supplement and FGF2). For neuronal differentiation was achieved using the B-27 Plus Neuronal Culture System (Gibco). Briefly, NPC were plated at density of 152,000 cells per well of 24-well plate coated in Poly-D- Lysine (PDL) and laminin. For the first 6 days, cells were plated with Differentiation Media (Neurobasal media, B27 supplement, GlutaMAX, CultureOne Supplement and Ascorbic Acid). On day 7, half of the media was changed to B27 plus maturation media (Neurobasal Plus media, B27 Plus supplement, GlutaMAX, CultureOne Supplement, and Ascorbic Acid). Day 25 neurons were transfected with CRISPRi (Ci) or MBZ3 mRNA and corresponding sgRNA. RNA was extracted 3 days post transfection and RTqPCR was performed to measure relative HTT levels normalized to GAPDH. iNeuron differentiation

[0135] WTC1 1 NGN2 iPSCs were cultured in mTeSR Plus media on matrigel coated plates. iNeurons were induced as described in Tian et al., Neuron, 104:239-255. el2 (2019). In brief, iPSCs were dettached with Accutase and plated for pre-differentiation in matrigel coated plates at a density of 750,000 cells per well of a 6-well plate in N2 pre-differentiation media (Knockout DMEM / F12, NonEssential Amino Acids, N2 supplement, NT-3, BDNF and mouse laminin) with 2 pg / mL doxycycline (DOX) and 10 nM ROCK inhibitor. After 3 days of pre-differentiation, cells were dettached with Accutase and replated onto a 24-well plate coated with PDL, at a density of 100,000 cells per well in neuronal differentiation media (DMEM / F12, Neurobasal -A, NonEssential amino acids, Glutamax, N2 supplement, B27 without vitamin A, NT-3, BDNF and mouse laminin) with 2 pg / mL DOX. Half media changes were performed with neuronal differentiation media (without DOX) once a week until cells were used for an experiment. For transfection, LNPs (L42) were prepared so that 2 pg of MBZ3 mRNA and 2ug of guide (chemically modified for increased stability as described in Finn et al., Cell Rep., 22:2227-2235 (2018)) were added per well of iNeurons. RNA was extracted 3 days post transfection and RTqPCR was performed to measure relative HTT levels normalized to GAPDH.

[0136] Prime Editing

[0137] WTC1 1 NGN2 iPSCs were cultured in mTeSR Plus in matrigel coated plates. iPSCs were dettached with Accutase to create a single cell suspension. iPSCs were nucleofected using Lonza 4D Nucleofector and P3 buffer with PE7 mRNA (encoding prime editor) and synthetic pegRNA (designed to edit S260 from G to A) and a nicking guide as described in Yan et al., Nature, 628:639-647 (2024). Genomic DNA was extracted 3 days post nucleofection to assay editing. Bulk edited cells were plated at very low density for single cell clones picking. HTT reg FWD and HTT reg REV primers were used to amplify region of interest and amplicon was sanger sequenced to determine editing. pegRNA (S260peg 1 GtoA, designed to edit S260 from G to A)

[0138] Sequence as DNA for reference: tCTGGGACGCAAGGCGCCGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCCCGGCAGCCCt CACGGCGCCTTGCGTCCTTTTTT (SEQ ID NO:54) Sequence as RNA with modifications, as ordered from IDT, where a m indicates that the base is a 2'-0Me base, an * indicates the presence of a phosphorothioate bond, and a r indicates that the base is a ribonucleotide base: mU*mC*mU*rGrGrGrArCrGrCrArArGrGrCrGrCrCrGrUrGrUrUrUrUrArGrArGrCrUrArG rArArArUrArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAr ArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCrUrCrCrCrGrGrCrAr GrCrCrCrUrCrArCrGrGrCrGrCrCrUrUrGrCrGrUrCrCrUrU*mU*mU*mUrU (SEQ ID NO 55)

[0139] Nicking guide (s260_nick) spacer sequence: CCCGTCCCGGCAGCCCtCA (SEQ ID NO:56)

[0140] Results

[0141] Allele selective repression in HD patient fibroblasts

[0142] The genome of HD patient fibroblasts (ND31551) carrying HTT mutant allele with 39 CAG repeats was sequenced to confirm that the cells were heterozygous for both S260 (targeting SNP) and S331 (SNP used for allele selective RTqPCR (Figure 8A). HD fibroblasts were transfected with CRISPRoff mRNA and corresponding sgRNA (nontargeting guide or guide targeting either HTT S260 G allele or HTT S260 A allele) using Lipofectamine MessengerMax. RNA was extracted 6 days post transfection and used for allele selective RTqPCR and 2- ACtmethod was used to compare relative levels of each allele. The 2-AACtmethod was used to compare relative levels of C allele to T allele in cells transfected with each guide compared to non-targeting guide (Figure 8B). S260 guides targeting S260 G allele (S260g2 and S260g4G) show decreased levels of C allele relative to T allele. When the other allele is targeted (S260g4A) there are higher levels of C allele compared to T allele. The 2"AACtmethod was also used to compare levels of each allele to levels of that same allele in non-targeting samples normalized to geometric mean of GAPDH and B2M (Figure 8C). When using CRISPRoff mRNA with S260 guides targeting Allele 1 (S260g2 and S260g4G) there was a decrease in levels of the targeted allele (Allele 1) but not the other (Allele 2). When targeting Allele 2 (S260g4A) there was a decrease in levels of targeted allele (Allele 2) but not the other (Allele 1) demostrating that allele selective repression was achieved in the context of HD repeat expansion.

[0143] Different repressor constructs can lower HTT expression in human NPCs.

[0144] A schematic showing the design of constructs with different repressor domains on N- terminus fused to dCas9 with or without Zim3-KRAB on C-terminus is shown in Figure 9 A. Repressor constructs were screened in NPCs to determine which constructs led to higher HTT repression (Figure 9B). NPCs were transfected with non-targeting guide or S260gl targeting HTT and mRNA coding for repressor construct. RNA was extracted 3 days post transfection and RTqPCR was performed to measure relative 7 / TZlevels normalized to GAPDH. MBZ3 (MBDl-dCas9-Zim3) construct was selected for neuronal experiments since it led to the highest level of HTT repression.

[0145] Repressor construct (MBZ3) can lower HTT expression in human iPSC-derived neurons

[0146] Neurons were transfected with Lipofectamine MessengerMax (LMM) on day 25 with CRISPRi (Ci) or MBZ3 mRNA and guide targeting a different locus (AAVS1) or HTT targeting guide S260gl. RNA was extracted 3 days post transfection and RTqPCR was performed to measure relative HTT levels normalized to GAPDH (Figure 10). Also in Figure 10 is a schematic showing differentiation of iPSC-derived neurons. iPSCs were first differentiated into NPCs (neural progenitor cells) using dual SMAD inhibition. NPCs were expanded and then differentiated into neurons using B-27 Plus Neuronal Culture System (Gib co).

[0147] Repressor construct (MBZ3) can lower HTT expression in human NGN2 iNeurons using LNP delivery iNeurons (day 20) were transfected LNPs containing MBZ3 mRNA and guide targeting a AAVS1 or HTT targeting guide S260gl. RNA was extracted 3 days post transfection and RTqPCR was performed to measure relative HTT levels normalized to GAPDH (Figure 11). Also in Figure 11 is a schematic showing differentiation of NGN2 induced neurons (iNeurons). In brief, iPSCs with a stably integrated NGN2 under a doxy cycling (DOX) inducible promoter (WTC11-NGN2) were first pre-differentiated in the presence of DOX for 3 days. Pre-differentiated cells were then replated onto PDL-coated plates in presence of DOX to produce iNeurons. Representative images of iNeurons at day 2, day 7 and day 22 post-PDL plating.

[0148] Prime editing can be used to create S260 heterozygous iPSCs with NGN2 insertion for making iNeurons

[0149] WTC1 1 NGN2 iPSCs were prime edited using PE7 mRNA, pegRNA (designed to edit S260 from G to A) and a nicking guide (Figure 12). Sanger sequencing of bulk edited iPSCs showed the presence of the desired edit (S260 G to A). Edited iPSCs were plated as single cell suspension at very low density and allowed to expand. Single cell clones were picked and screened for establishing a line that is heterozygous at S260 and has NGN2 integrated for iNeuron differentiation.

[0150] Example 3: Exemplary Sequences

[0151] This Example provides sequences of exemplary components of an agent that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein and nucleic acids that can encode such components.

[0152] Exemplary dCAS9 polypeptide sequence

[0153] MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHE RHPIFGNIVDEVAYHEI<YPTIYHLRI<I<LVDSTDI<ADLRLIYLALAHMII<FRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGF ANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLT RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQ FYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ

[0154] KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO:42)

[0155] Nucleic acid sequence that can encode an exemplary dCAS9 polypeptide

[0156] ATGGACAAGAAGTATTCTATCGGACTGGCCATCGGGACTAATAGCGTCGGGTGG GCCGTGATCACTGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTGCTCGGG AACACCGACCGGCATTCCATCAAGAAAAATCTGATCGGAGCTCTCCTCTTTGATT CAGGGGAGACCGCTGAAGCAACCCGCCTCAAGCGGACTGCTAGACGGCGGTAC ACCAGGAGGAAGAACCGGATTTGTTACCTTCAAGAGATATTCTCCAACGAAATG GCAAAGGTCGACGACAGCTTCTTCCATAGGCTGGAAGAATCATTCCTCGTGGAA GAGGATAAGAAGCATGAACGGCATCCCATCTTCGGTAATATCGTCGACGAGGTG GCCTATCACGAGAAATACCCAACCATCTACCATCTTCGCAAAAAGCTGGTGGAC TCAACCGACAAGGCAGACCTCCGGCTTATCTACCTGGCCCTGGCCCACATGATCA AGTTCAGAGGCCACTTCCTGATCGAGGGCGACCTCAATCCTGACAATAGCGATG TGGATAAACTGTTCATCCAGCTGGTGCAGACTTACAACCAGCTCTTTGAAGAGAA CCCCATCAATGCAAGCGGAGTCGATGCCAAGGCCATTCTGTCAGCCCGGCTGTC AAAGAGCCGCAGACTTGAGAATCTTATCGCTCAGCTGCCGGGTGAAAAGAAAAA

[0157] TGGACTGTTCGGGAACCTGATTGCTCTTTCACTTGGGCTGACTCCCAATTTCAAG

[0158] TCTAATTTCGACCTGGCAGAGGATGCCAAGCTGCAACTGTCCAAGGACACCTAT

[0159] GATGACGATCTCGACAACCTCCTGGCCCAGATCGGTGACCAATACGCCGACCTTT

[0160] TCCTTGCTGCTAAGAATCTTTCTGACGCCATCCTGCTGTCTGACATTCTCCGCGTG

[0161] AACACTGAAATCACCAAGGCCCCTCTTTCAGCTTCAATGATTAAGCGGTATGATG

[0162] AGCACCACCAGGACCTGACCCTGCTTAAGGCACTCGTCCGGCAGCAGCTTCCGG

[0163] AGAAGTACAAGGAAATCTTCTTTGACCAGTCAAAGAATGGATACGCCGGCTACA

[0164] TCGACGGAGGTGCCTCCCAAGAGGAATTTTATAAGTTTATCAAACCTATCCTTGA

[0165] GAAGATGGACGGCACCGAAGAGCTCCTCGTGAAACTGAATCGGGAGGATCTGCT

[0166] GCGGAAGCAGCGCACTTTCGACAATGGGAGCATTCCCCACCAGATCCATCTTGG

[0167] GGAGCTTCACGCCATCCTTCGGCGCCAAGAGGACTTCTACCCCTTTCTTAAGGAC

[0168] AACAGGGAGAAGATTGAGAAAATTCTCACTTTCCGCATCCCCTACTACGTGGGA

[0169] CCCCTCGCCAGAGGAAATAGCCGGTTTGCTTGGATGACCAGAAAGTCAGAAGAA

[0170] ACTATCACTCCCTGGAACTTCGAAGAGGTGGTGGACAAGGGAGCCAGCGCTCAG

[0171] TCATTCATCGAACGGATGACTAACTTCGATAAGAACCTCCCCAATGAGAAGGTC

[0172] CTGCCGAAACATTCCCTGCTCTACGAGTACTTTACCGTGTACAACGAGCTGACCA

[0173] AGGTGAAATATGTCACCGAAGGGATGAGGAAGCCCGCATTCCTGTCAGGCGAAC

[0174] AAAAGAAGGCAATTGTGGACCTTCTGTTCAAGACCAATAGAAAGGTGACCGTGA

[0175] AGCAGCTGAAGGAGGACTATTTCAAGAAAATTGAATGCTTCGACTCTGTGGAGA

[0176] TTAGCGGGGTCGAAGATCGGTTCAACGCAAGCCTGGGTACCTACCATGATCTGCT

[0177] TAAGATCATCAAGGACAAGGATTTTCTGGACAATGAGGAGAACGAGGACATCCT

[0178] TGAGGACATTGTCCTGACTCTCACTCTGTTCGAGGACCGGGAAATGATCGAGGA

[0179] GAGGCTTAAGACCTACGCCCATCTGTTCGACGATAAAGTGATGAAGCAACTTAA

[0180] ACGGAGAAGATATACCGGATGGGGACGCCTTAGCCGCAAACTCATCAACGGAAT

[0181] CCGGGACAAACAGAGCGGAAAGACCATTCTTGATTTCCTTAAGAGCGACGGATT

[0182] CGCTAATCGCAACTTCATGCAACTTATCCATGATGATTCCCTGACCTTTAAGGAG

[0183] GACATCCAGAAGGCCCAAGTGTCTGGACAAGGTGACTCACTGCACGAGCATATC

[0184] GCAAATCTGGCTGGTTCACCCGCTATTAAGAAGGGTATTCTCCAGACCGTGAAA

[0185] GTCGTGGACGAGCTGGTCAAGGTGATGGGTCGCCATAAACCAGAGAACATTGTC ATCGAGATGGCCAGGGAAAACCAGACTACCCAGAAGGGACAGAAGAACAGCAG

[0186] GGAGCGGATGAAAAGAATTGAGGAAGGGATTAAGGAGCTCGGGTCACAGATCC

[0187] TTAAAGAGCACCCGGTGGAAAACACCCAGCTTCAGAATGAGAAGCTCTATCTGT

[0188] ACTACCTTCAAAATGGACGCGATATGTATGTGGACCAAGAGCTTGATATCAACA

[0189] GGCTCTCAGACTACGACGTGGACGCCATCGTCCCTCAGAGCTTCCTCAAAGACG

[0190] ACTCAATTGACAATAAGGTGCTGACTCGCTCAGACAAGAACCGGGGAAAGTCAG

[0191] ATAACGTGCCCTCAGAGGAAGTCGTGAAAAAGATGAAGAACTATTGGCGCCAGC

[0192] TTCTGAACGCAAAGCTGATCACTCAGCGGAAGTTCGACAATCTCACTAAGGCTG

[0193] AGAGGGGCGGACTGAGCGAACTGGACAAAGCAGGATTCATTAAACGGCAACTT

[0194] GTGGAGACTCGGCAGATTACTAAACATGTCGCCCAAATCCTTGACTCACGCATG

[0195] AATACCAAGTACGACGAAAACGACAAACTTATCCGCGAGGTGAAGGTGATTACC

[0196] CTGAAGTCCAAGCTGGTCAGCGATTTCAGAAAGGACTTTCAATTCTACAAAGTGC

[0197] GGGAGATCAATAACTATCATCATGCTCATGACGCATATCTGAATGCCGTGGTGG

[0198] GAACCGCCCTGATCAAGAAGTACCCAAAGCTGGAAAGCGAGTTCGTGTACGGAG

[0199] ACTACAAGGTCTACGACGTGCGCAAGATGATTGCCAAATCTGAGCAGGAGATCG

[0200] GAAAGGCCACCGCAAAGTACTTCTTCTACAGCAACATCATGAATTTCTTCAAGAC

[0201] CGAAATCACCCTTGCAAACGGTGAGATCCGGAAGAGGCCGCTCATCGAGACTAA

[0202] TGGGGAGACTGGCGAAATCGTGTGGGACAAGGGCAGAGATTTCGCTACCGTGCG

[0203] CAAAGTGCTTTCTATGCCTCAAGTGAACATCGTGAAGAAAACCGAGGTGCAAAC

[0204] CGGAGGCTTTTCTAAGGAATCAATCCTCCCCAAGCGCAACTCCGACAAGCTCATT

[0205] GCAAGGAAGAAGGATTGGGACCCTAAGAAGTACGGCGGATTCGATTCACCAACT

[0206] GTGGCTTATTCTGTCCTGGTCGTGGCTAAGGTGGAAAAAGGAAAGTCTAAGAAG

[0207] CTCAAGAGCGTGAAGGAACTGCTGGGTATCACCATTATGGAGCGCAGCTCCTTC

[0208] GAGAAGAACCCAATTGACTTTCTCGAAGCCAAAGGTTACAAGGAAGTCAAGAAG

[0209] GACCTTATCATCAAGCTCCCAAAGTATAGCCTGTTCGAACTGGAGAATGGGCGG

[0210] AAGCGGATGCTCGCCTCCGCTGGCGAACTTCAGAAGGGTAATGAGCTGGCTCTC

[0211] CCCTCCAAGTACGTGAATTTCCTCTACCTTGCAAGCCATTACGAGAAGCTGAAGG

[0212] GGAGCCCCGAGGACAACGAGCAAAAGCAACTGTTTGTGGAGCAGCATAAGCATT

[0213] ATCTGGACGAGATCATTGAGCAGATTTCCGAGTTTTCTAAACGCGTCATTCTCGC

[0214] TGATGCCAACCTCGATAAAGTCCTTAGCGCATACAATAAGCACAGAGACAAACC AATTCGGGAGCAGGCTGAGAATATCATCCACCTGTTCACCCTCACCAATCTTGGT

[0215] GCCCCTGCCGCATTCAAGTACTTCGACACCACCATCGACCGGAAACGCTATACCT

[0216] CCACCAAAGAAGTGCTGGACGCCACCCTCATCCACCAGAGCATCACCGGACTTT

[0217] ACGAAACTCGGATTGACCTCTCACAGCTCGGAGGGGAT

[0218] (SEQ ID NO:43)

[0219] Exemplary KRAB polypeptide sequence

[0220] RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRL

[0221] EKGEEP

[0222] (SEQ ID NO:44)

[0223] Nucleic acid sequence that can encode an exemplary KRAB polypeptide

[0224] CGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGG

[0225] AAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAAC

[0226] TATAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCC

[0227] GGTTGGAGAAGGGAGAAGAGCCC

[0228] (SEQ ID NO:45)

[0229] Exemplary DNMT3A polypeptide sequence

[0230] MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCED

[0231] SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYE

[0232] GTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA

[0233] KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNS

[0234] IKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWS

[0235] VPVIRHLFAPLKEYFACV

[0236] (SEQ ID NO:46)

[0237] Nucleic acid sequence that can encode an exemplary DNMT3A polypeptide

[0238] ATGAACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGCT

[0239] GAGAAGAGGAAGCCCATCCGCGTGCTGTCTCTCTTTGATGGGATTGCTACAGGG

[0240] CTCCTGGTGCTGAAGGACCTGGGCATCCAAGTGGACCGCTACATTGCCTCCGAG GTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGGAAAGATCATG

[0241] TACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCCCA

[0242] TTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCTCCATTGTCAACCCTG

[0243] CCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCT

[0244] CCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTT

[0245] GAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTT

[0246] GAGTCTAACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCC

[0247] CGTTACTTCTGGGGTAACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGA

[0248] ATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCAGAATAGCCAAGTTCA

[0249] GCAAAGTGAGGACCATTACCACCAGGTCAAACTCTATAAAGCAGGGCAAAGACC

[0250] AGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATCCTGTGGTGCACTGAAA

[0251] TGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAACATGAGCCG

[0252] CTTGGCGAGGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCCA

[0253] CCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTG

[0254] (SEQ ID NO:47)

[0255] Exemplary DNMT3L polypeptide sequence

[0256] MGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTN

[0257] VVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFW

[0258] IFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLT

[0259] PKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL

[0260] (SEQ ID NO:48)

[0261] Nucleic acid sequence that can encode an exemplary DNMT3L polypeptide

[0262] ATGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTG

[0263] CGGGTACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTGGGCTTCT

[0264] TGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGTCA

[0265] CAAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACG

[0266] GCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATGTT

[0267] CCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCGGCC CTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACA ACTACCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGA GACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAGAGCAAG CATGCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGC AGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTC CCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCTCTT (SEQ ID NO:49)

[0268] Example 4: Treating HD

[0269] A human identified as having HD is administered one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases). The administered agents(s) can reduce or eliminate expression of a nucleic acid that can encode a mHtt polypeptide (e.g., a nucleic acid that can encode a mHtt polypeptide and that includes from about 36 to about 350 CAG trinucleotide repeat expansions) such that the a level of a mHtt polypeptide (e.g., a mHtt polypeptide including from about 36 to about 350 glutamine residues in a glutamine repeat) within the human is reduced or eliminated (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0270] Example 5: Delaying or Preventing Development of HD

[0271] A human identified as being at risk of developing HD (e.g., identified as having the presence of a pathogenic CAG trinucleotide repeat expansion (e.g., the presence of at least 36 trinucleotide repeat expansions) in one or both copies of HTT gene) is administered one or more agents that can reduce expression of a nucleic acid that can encode a mHtt polypeptide provided herein (e.g., one or more agents each including (a) a nucleic acid molecule (e.g., a gRNA) that can target the regulatory region of a nucleic acid that can encode a mHtt polypeptide such as a nucleic acid molecule that comprises, consists essentially of, or consists of a sequence set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) one or more repressor polypeptides and / or one or more methyltransferases). The administered agents(s) can reduce or eliminate expression of a nucleic acid that can encode a mHtt polypeptide (e.g., a nucleic acid that can encode a mHtt polypeptide and that includes from about 36 to about 350 CAG trinucleotide repeat expansions) such that the a level of a mHtt polypeptide (e.g., a mHtt polypeptide including from about 36 to about 350 glutamine residues in a glutamine repeat) within the human is reduced or eliminated (e.g., without reducing a level of a Htt polypeptide that lacks an expanded glutamine repeat).

[0272] OTHER EMBODIMENTS

[0273] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT Is CLAIMED IS:

1. An agent that can reduce expression of a genomic nucleic acid that can encode a mutant huntingtin (mHtt) polypeptide, wherein said agent comprises (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of said genomic nucleic acid that can encode said mHtt polypeptide and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase.

2. The agent of claim 1, wherein said nucleic acid sequence comprises a single nucleotide polymorphism (SNP), wherein said SNP is not present in a regulatory region of genomic nucleic acid that can encode a Htt polypeptide that lacks an expanded glutamine repeat.

3. The agent of claim 2, wherein said SNP is selected from the group consisting of rsl3102260, rsl3122415, rsl3132932, rsll2396951, and rsl49624523.

4. The agent of any one of claims 1-2, wherein said nucleic acid sequence comprises a sequence set forth in SEQ ID NO : 17 or SEQ ID NO : 18.

5. The agent of any one of claims 1-4, wherein said nucleic acid molecule is from about 16 nucleotides to about 24 nucleotides in length.

6. The agent of any one of claims 1-5, wherein said nucleic acid molecule comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32.

7. The agent of any one of claims 1-6, wherein said catalytically dead endonuclease comprises a catalytically dead Cas9 (dCas9) polypeptide.

8. The agent of any one of claims 1-7, wherein said repressor polypeptide is a Kriippel associated box (KRAB) polypeptide.

9. The agent of any one of claims 1-7, wherein said methyltransferase is selected from the group consisting of a DNMT3A polypeptide, a DNMT3L polypeptide, a DNMT3A polypeptide, and a DNMT3B polypeptide.

10. The agent of any one of claims 1 -9, wherein said agent comprises said repressor polypeptide and comprises said methyltransferase.

11. An agent that can reduce expression of a genomic nucleic acid that can encode a mHtt polypeptide, wherein said agent comprises (a) a nucleic acid molecule that can bind a nucleic acid sequence present in a regulatory region of said genomic nucleic acid that can encode said mHtt polypeptide, where said nucleic acid molecule comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 19-32, and (b) a polypeptide including (i) a catalytically dead endonuclease and (ii) a repressor polypeptide and / or a methyltransferase.

12. The agent of claim 11, wherein said nucleic acid sequence comprises a SNP, wherein said SNP is not present in a regulatory region of genomic nucleic acid that can encode a Htt polypeptide that lacks an expanded glutamine repeat.

13. The agent of claim 12, wherein said SNP is selected from the group consisting of rsl3102260, rsl3122415, rsl3132932, rsl 12396951, and rsl49624523.

14. The agent of any one of claims 11-12, wherein said nucleic acid sequence comprises a sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

15. The agent of any one of claims 11-14, wherein said catalytically dead endonuclease comprises a dCas9 polypeptide.

16. The agent of any one of claims 11-15, wherein said repressor polypeptide is a KRAB polypeptide.

17. The agent of any one of claims 11-15, wherein said methyltransferase is selected from the group consisting of a DNMT3A polypeptide, a DNMT3L polypeptide, a DNMT3A polypeptide, and a DNMT3B polypeptide.

18. The agent of any one of claims 11-15, wherein said agent comprises said repressor polypeptide and comprises said methyltransferase.

19. A method for treating a mammal having Huntington's disease (HD), the method comprising: administering to said mammal an agent of any one of claims 1-18.

20. A method for treating a mammal at risk of developing HD, the method comprising: administering an agent of any one of claims 1-18 to a mammal identified as having a pathogenic CAG trinucleotide repeat expansion in one or both copies of a HTT gene.

21. A method for reducing a level of mHtt polypeptides within a mammal, the method comprising: determining that said mammal comprises a pathogenic CAG trinucleotide repeat expansion in one or both copies of a HTT gene; administering to said mammal an agent of any one of claims 1-18.

22. The method of any one of claims 20-21, wherein said CAG trinucleotide repeat expansion comprises at least 36 CAG trinucleotide repeats.

23. The method of any one of claims 20-21, wherein said CAG trinucleotide repeat expansion comprises from about 36 CAG trinucleotide repeats to about 350 CAG trinucleotide repeats.

24. The method of any one of claims 19-23, wherein said mammal is a human.

25. The method of any one of claims 19-23, wherein a level of a Htt polypeptide that lacks an expanded glutamine repeat is not reduced.

Images