Methods for treating huntington's disease

Abstract

Aspects of the present disclosure relate to compositions and methods useful for treating Huntington's disease. In some embodiments, the present disclosure provides interfering nucleic acids (e.g., artificial miRNAs) targeting the Huntingtin gene (HTT) and methods of using the same to treat Huntington's disease. Accordingly, in some aspects, the present disclosure provides an isolated nucleic acid comprising or encoding the sequence set forth in any one of SEQ ID NO: 1-SEQ ID NO: 22.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 951,582, filed December 20, 2019, pursuant to 35 U.S. SC §119(e), the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 17, 2020, named 046192-096790WOPT_SL.txt, and has a size of 40,419 bytes. Technical Field

[0005] The techniques described in this article relate to methods for treating Huntington's disease. Background Technology

[0006] Huntington's disease (HD) is a devastating inherited neurodegenerative disorder caused by an amplification of the CAG repeat region in exon 1 of the huntingtin gene. Although huntingtin (HTT) is expressed systemically, the polyglutamine-amplified protein is particularly toxic to medium-sized polyspinous neurons in the striatum and their cortical junctions. Patients experience mood symptoms including depression and anxiety, as well as characteristic motor disturbances and chorea. Huntington's disease is currently incurable; treatment options are limited to improving disease symptoms. Summary of the Invention

[0007] This disclosure relates to compositions and methods for treating Huntington's disease (HD). In some embodiments, a repressive nucleic acid (e.g., miRNA, such as an artificial miRNA) is provided, which specifically hybridizes to human huntingtin protein (HTT) and inhibits its expression.

[0008] Therefore, in some aspects, this disclosure provides isolated nucleic acids that comprise or encode the sequence of any one of SEQ ID NO: 1-SEQ ID NO: 22.

[0009] One aspect described herein is an isolated nucleic acid comprising: (a) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and (b) a second region comprising a transgene encoding one or more miRNAs, wherein each miRNA comprises a seed sequence complementary to SEQ ID NO: 25.

[0010] One aspect described herein is an isolated nucleic acid comprising: (a) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and (b) a second region comprising a transgene encoding one or more miRNAs, wherein each miRNA is encoded by a sequence comprising a sequence comprising any one of SEQ ID NO: 1-SEQ ID NO: 22 with a miRNA backbone sequence side-attached.

[0011] In some aspects, this disclosure provides isolated nucleic acids comprising: a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and a second region comprising a transgene encoding one or more miRNAs.

[0012] In some embodiments, the sequence encoding each miRNA comprises the sequence described in any one of SEQ ID NO: 1-SEQ ID NO: 22. In some embodiments, the sequence encoding each miRNA comprises a sequence flanked by a sequence from a pre-miR. In some embodiments, the isolated nucleic acid comprises a pre-miR sequence corresponding to a mature miRNA sequence described in any one of SEQ ID NO: 1-SEQ ID NO: 22. In some embodiments, the sequence encoding each miRNA comprises a sequence flanked by a sequence encoding a miRNA backbone sequence described in any one of SEQ ID NO: 1-SEQ ID NO: 22. In some aspects, this disclosure provides isolated nucleic acids comprising transgenes encoding one or more miRNAs, wherein the sequence encoding each miRNA comprises a sequence flanked by a miRNA backbone sequence described in SEQ ID NO: 1-SEQ ID NO: 22.

[0013] In some embodiments, the transgene comprises two tandem miRNAs with introns attached to them. In some embodiments, the transgene comprises two tandem precursor miRNAs (pre-miRNAs) with introns attached to them (see, for example, SEQ ID NO: 35).

[0014] In some embodiments, the transgene comprises two miRNAs or two precursor miRNAs tandemly attached to introns.

[0015] In some implementations, the side-connected introns are identical.

[0016] In some implementations, the side-connected introns come from the same species.

[0017] In some implementations, the side-connected intron is the hCG intron.

[0018] In some embodiments, the transgene further comprises a nucleic acid sequence encoding a promoter.

[0019] In some embodiments, the promoter is the synaptic protein (Syn1) promoter.

[0020] In some embodiments, the transgene further comprises a nucleic acid sequence encoding a protein.

[0021] In some embodiments, the protein is CYP46A1.

[0022] In some embodiments, the protein is a therapeutic protein (e.g., non-mutated huntingtin) or a reporter protein (e.g., a fluorescent protein, such as GFP).

[0023] In some embodiments, the human huntingtin protein comprises the sequence described in SEQ ID NO: 25.

[0024] In some embodiments, this disclosure provides a nucleic acid (e.g., miRNA) that is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive bases of SEQ ID NO: 25.

[0025] In some embodiments, one or more miRNAs are located in the untranslated portion of the transgene.

[0026] In some implementations, the untranslated portion is an intron.

[0027] In some embodiments, the untranslated portion is located between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence.

[0028] In some embodiments, the untranslated portion is located between the last nucleic acid base of the promoter sequence and the first base of the poly-A tail sequence.

[0029] In some implementations, the polyA sequence is a small polyA sequence.

[0030] In some embodiments, the transgene is side-conjugated with an adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

[0031] In some embodiments, the isolated nucleic acid further comprises a third region containing a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

[0032] In some implementations, the first or second ITR variant lacks a functional end-resolver site (TRS), optionally wherein the ITR variant is an ATRS ITR.

[0033] In some embodiments, at least one of the miRNAs hybridizes with human huntingtin protein (e.g., SEQ ID NO: 25) and inhibits its expression.

[0034] In some aspects, this disclosure provides vectors comprising isolated nucleic acids as described in this disclosure.

[0035] In some aspects, this disclosure provides a vector comprising isolated nucleic acids containing transgenes encoding one or more miRNAs, wherein the sequence of the transgene encoding various miRNAs comprises the sequences described in SEQ ID NO: 1-SEQ ID NO: 22 with miRNA backbone sequences side-attached.

[0036] In some embodiments, the vector is a plasmid.

[0037] In some embodiments, each miRNA backbone sequence of the transgene is a mir-155 backbone sequence, a mir-30 backbone sequence, or a mir-64 backbone sequence.

[0038] In some aspects, this disclosure provides host cells comprising isolated nucleic acids or vectors as described in this disclosure.

[0039] In some aspects, this disclosure provides recombinant AAV (rAAV) comprising: (a) a capsid protein; and (b) isolated nucleic acid as described in this disclosure.

[0040] In some aspects, this disclosure provides a recombinant AAV (rAAV) comprising a capsid protein and isolated nucleic acid, the isolated nucleic acid comprising a transgene encoding one or more miRNAs, wherein the sequence of the transgene encoding various miRNAs comprises the sequences described in SEQ ID NO: 1-SEQ ID NO: 22 with miRNA backbone sequences side-attached.

[0041] In some embodiments, the capsid protein is AAV9 capsid protein.

[0042] In some embodiments, the capsid protein is AAVrh10 capsid protein.

[0043] In some embodiments, the capsid protein is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, or AAVrh10 capsid protein, or any chimera thereof.

[0044] In some implementations, the recombinant AAV (rAAV) is a haploid rAAV.

[0045] In some embodiments, the haploid rAAV comprises a chimeric capsid protein.

[0046] In some implementations, the rAAV is a self-complementary AAV (scAAV).

[0047] In some embodiments, the rAAV is formulated for delivery to the central nervous system (CNS).

[0048] This disclosure relates to compositions comprising any of the isolated nucleic acids described herein.

[0049] This disclosure relates to compositions comprising any of the carriers described herein.

[0050] This disclosure relates to compositions comprising any of the rAAVs described herein.

[0051] This disclosure relates to isolated nucleic acids that are capable of reducing (e.g., inhibiting) the expression of pathogenic huntingtin proteins and are therefore useful in the treatment of huntingtin disease.

[0052] Therefore, in some aspects, this disclosure provides a method for treating Huntington's disease in a subject in need, the method comprising administering a therapeutically effective amount of an isolated nucleic acid, rAAV, or composition as described in this disclosure to a subject who has Huntington's disease or is at risk of developing Huntington's disease.

[0053] In some aspects, this disclosure provides a method for treating Huntington's disease in a subject in need, the method comprising administering a therapeutically effective amount of rAAV as described herein (e.g., rAAV comprising a transgene encoding one or more miRNAs, wherein the sequence of the transgene encoding various miRNAs comprises the sequences described in SEQ ID NO: 1-SEQ ID NO: 22 with miRNA backbone sequences side-attached).

[0054] In some implementations, the subject contains the huntingtin gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

[0055] In some implementations, the subject is under 20 years of age or has been diagnosed with adolescent HD.

[0056] In some embodiments, administration is made to deliver the isolated nucleic acid or rAAV to the central nervous system (CNS) of the subject.

[0057] In some embodiments, the administration is performed by injection, optionally intravenous injection or intrastriatal injection.

[0058] In some embodiments, the administration is performed via a catheter or related device.

[0059] In some embodiments of any aspect, the method further includes the step of diagnosing the subject with Huntington's disease or at risk of developing Huntington's disease prior to administration.

[0060] In some embodiments of any aspect, the method further includes the step of receiving, prior to administration, the results of a assay that diagnoses a subject with Huntington's disease or at risk of developing Huntington's disease. Exemplary assays for diagnosing a subject with Huntington's disease or at risk of developing Huntington's disease are described herein, such as genetic screening for at least 36 CAG repeats, at least 40 CAG repeats, or at least 100 CAG repeats or more. Attached Figure Description

[0061] Figure 1This diagram illustrates the construct of the artificial miRNA. The pEMBL-D(+)-Syn1-hCG intron is a control vector containing a blank human chorionic gonadotropin (hCG) intron, driven by a synaptic protein promoter. Two copies of the control miRNA precursor (random sequence or non-functional mutation) are inserted into the hCGin in the vector pEMBL-D(+)-Syn1-hCGin-2x control pre-miR. Two copies of the artificial pre-miR (perfectly matched to the 3'-UTR targeting sequence, containing approximately 100-150 bp flanking upstream and downstream sequences) are cloned between the hCG introns. The vector pEMBL-D(+)-Syn1-CYP46A1-hCGin-2x artificial pre-miR is a combo construct that can simultaneously generate both CYP46A1 and the artificial miRNA. To determine whether pre-miRNA could be processed into mature miRNA and bind to an HTT targeting sequence containing CAG amplification (which is perfectly complementary to mature miRNA), the HTT targeting sequence was inserted after the luciferase gene. Due to packaging size limitations, a small poly A was used in the construct.

[0062] Figure 2 This is a schematic diagram illustrating the mechanism of Huntington's disease (HD).

[0063] Figure 3 This is a schematic diagram illustrating an exemplary method for treating HD.

[0064] Figure 4 This is a schematic diagram illustrating the process of screening artificial miRNAs for HD.

[0065] Figure 5 This is a schematic diagram showing the location of artificial miRNAs in the HTT gene (or mRNA). miHTT-H2 is located in region I; miHTT-H4 and miHTT-H5 are located in the 5' and 3' crossover regions of the CAG repeat; miHTT-H14 is located in region IV; and miHTT-H15, H17, H19, and H21 are located in region V.

[0066] Figure 6 This is a schematic diagram showing the regions of the HTT gene. The CAG repeat is located in region I.

[0067] Figure 7 This is a schematic diagram illustrating the first round of screening of artificial miRNAs via in vitro plasmid transfection (e.g., in the 293 cell line; phase I).

[0068] Figures 8A-8B This is a series of schematic diagrams and graphs illustrating the first round of screening (e.g., phase I) of artificial miRNAs transfected in vitro via plasmids in 293 cells. Figure 8A This is a schematic diagram showing the selected artificial miRNA and its target region on the HTT gene. Figure 8B This is a bar chart showing the inhibition of luciferase gene expression with target sequences by artificial miRNAs through co-transfection. After 48 hours of co-transfection, pEMBL-CMV-hCGin-miHTT-H2 and miHTT-H5 effectively inhibited luciferase activity by approximately 46.4% and 54.8%, respectively, compared to pEMBL-CMV-hCGin (as a control). **p<0.01 vs Pembl-CMV-hCGin.

[0069] Figure 9 This is a schematic diagram illustrating the process (e.g., phase I) of screening artificial miRNAs for HD.

[0070] Figure 10 This is a schematic diagram illustrating the second round of screening of artificial miRNAs via in vitro AAV infection.

[0071] Figures 11A-11B This is a series of bar charts showing the testing of AAVRH10-mediated artificial miRNAs in the human neural cell line U87 (human primary glioblastoma cell line). Figure 11A The activity of luciferase was shown, and Figure 11B The percentage of luciferase activity compared to the control is shown. Figures 11A-11B This study demonstrates that AAVRH10-mediated artificial miRNAs inhibit luciferase gene expression in vitro via target sequences. Compared to AAVRH10-CMV-hCGin (as a control), AAVRH10-CMV-hCGin-miHTT-H2 and H5, in combination with the respective target sequences inserted into the luciferase gene, significantly inhibited luciferase activity by approximately 84.9% and 76.9%, respectively. *p<0.05; ***p<0.001 vs. AAVRH10-CMV-hCGin.

[0072] Figure 12 This is a schematic diagram illustrating the testing of the inhibition (e.g., phase I) of HTT protein in human U87 nerve cells by artificial miRNA.

[0073] Figure 13 Western blots showing HTT protein levels in the human neural cell line U87 were presented. Treatment with AAVRH10-CMV-hCGin-miHTT-H1-H5 in U87 cells reduced HTT protein expression by AAVRH10-CMV-hCGin-miHTT-H2, -miHTT-H4, and –miHTT-H5. β-actin was used as a loading control.

[0074] Figure 14 This is a bar chart showing quantitative data of HTT protein blots in the human neural cell line U87 (see example...). Figure 12 AAVRH10-CMV-hCGin-miHTT-H2 inhibited HTT protein expression by up to 73.2%, miHTT4 by up to 58.5%, and miHTT-H5 by up to 41.5%. (*p<0.05, **p<0.01, n=4).

[0075] Figure 15 This is a schematic diagram illustrating the process (e.g., phase II) of screening artificial miRNAs for HD.

[0076] Figure 16 This is a schematic diagram illustrating the first round of screening of artificial miRNAs via in vitro plasmid transfection (e.g., in the 293 cell line; phase II).

[0077] Figure 17 This is a bar chart showing the results of the second round of screening using sequences from the 3'-UTR. Artificial miRNAs inhibited luciferase gene expression by targeting sequences through co-transfection. In the second round of screening, after 48 hours of co-transfection, pEMBL-CMV-hCGin-miHTT-H14, H15, H17, H19 (miR-137), and miHTT-H21 (miR-216) effectively inhibited luciferase activity compared to pEMBL-CMV-hCGin (as a control). MiHTT-H2, H4, and H5 were used as positive controls. MiDMPK-M5, M7, and M9, targeting the myotonic dystrophy protein kinase (DMPK) gene, were also used as negative controls. ***p<0.001 vs. pEMBL-CMV-hCGin.

[0078] Figure 18This is a bar chart showing the results of a second round of screening using sequences from the 3'-UTR. Artificial miRNAs were used to inhibit luciferase gene expression via co-transfection with the target sequences. 48 hours after co-transfection, pEMBL-CMV-hCGin-miHTT-H14, H15, H17, H19 (miR-137), and miHTT-H21 (miR-216) effectively inhibited luciferase activity compared to pEMBL-CMV-hCGin (as a control). The luciferase activities relative to the control (100%) were 2.45% (H14), 8.75% (H15), 9.2% (H17), 12.89% (miR-137), and 4.17% (miR-216), respectively. MiHTT-H2, H4, and H5 were used as positive controls. MiDMPK-M5, M7, and M9, used to target the DMPK gene, were also used as negative controls. ***p<0.001 vs.pEMBL-CMV-hCGin.

[0079] Figure 19 This diagram illustrates a second round of screening using sequences from the 3'-UTR. Specifically, the diagram shows the locations of artificial miRNAs in the HTT gene. miHTT-H2 is located in region I; miHTT-H4 and miHTT-H5 are located in the 5' and 3' crossover regions of the CAG repeat, respectively; miHTT-H14 is located in region IV; and miHTT-H15, H17, H19, and H21 are located in region V.

[0080] Figure 20 This is a schematic diagram illustrating the testing of the inhibition (e.g., phase II) of HTT protein by artificial miRNA in human U87 nerve cells.

[0081] Figure 21 This diagram illustrates the testing of artificial miRNAs in human fibroblasts from HD patients. As a non-limiting example, transfected / infected samples from 2-3 top-performing miHTTs can be sent for off-target analysis.

[0082] Figure 22This is a schematic diagram showing the artificial miRNA construct. EMBL-D(+)-Syn1-hCGintron is a double-stranded vector used as a control and contains a blank human chorionic gonadotropin (hCG) intron (i.e., no miRNA) driven by a synaptic protein promoter. Two copies of artificial miHTT (perfectly matched to the target sequence, containing approximately 100-150 bp flanked upstream and downstream sequences) were cloned between the hCG introns. To determine whether miHTT could be processed into a mature miRNA, an HTT target sequence containing CAG amplification was inserted after the luciferase gene; this HTT target sequence is perfectly complementary to the mature miRNA. Small polyA was used in the construct due to packaging size limitations. Note that Syn1 stands for synaptic protein 1.

[0083] Figure 23 This is a schematic diagram showing the spectrum of pEMBL-D(+)-Syn1-hCGin-2x miHTT.

[0084] Figure 24 This is a schematic diagram showing the optimized CYP46A1 expression vector. pAAV2.1-Syn1-GFP-sPA is a single-stranded vector used as a control. pAAV2.1-Syn1-CYP46A1-sPA is used to overexpress CYP46A1 driven by the muscle-specific promoter Syn1. The vector pAAV2.1-Syn1-CYP46A1-hCGin-2x miHTT is a combo construct capable of simultaneously generating CYP46A1 and 2 copies of artificial miHTT.

[0085] Figure 25 This is a schematic diagram showing the spectrum of pAAV2.1-Syn1-CYP46A1-hCGin-2x miHTT.

[0086] Figure 26 This diagram illustrates the process of screening artificial miRNAs and identifying their target sequences in vitro. Two copies of the artificial miRNA precursor are cleaved and processed into mature miRNAs. The miRNAs are then precisely matched to the HTT target sequence containing CAG amplification and inhibit luciferase expression. Simultaneously, control miRNAs can also be processed but cannot bind to the HTT target sequence, thus having no effect on luciferase expression. This method is commonly used to identify the target sequences of miRNAs in vitro.

[0087] Figure 27 This is a series of schematic diagrams and images showing artificial miRNA constructs based on the miR-30 precursor backbone and related imprints. Detailed Implementation

[0088] Aspects of the present invention relate to certain interfering RNAs (e.g., miRNAs, such as artificial miRNAs) that, when delivered to a subject, effectively reduce the expression of pathogenic huntingtin protein (HTT) in the subject. Therefore, in some embodiments, the methods and compositions disclosed herein are useful for the treatment of Huntington's disease.

[0089] Repressive RNA

[0090] One aspect described herein is a repressive RNA that can be used to treat Huntington's disease. In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1-SEQ ID NO: 24, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1-SEQ ID NO: 24, said sequence retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 1-SEQ ID NO: 24.

[0091] Table 1: Locations and sequences of exemplary artificial miRNAs; reference HTT mRNA (see, for example, SEQ ID NO: 25; e.g., Hu 128TG mouse).

[0092]

[0093]

[0094] Any combination of the repressive RNAs (e.g., SEQ ID NO: 1-SEQ ID NO: 24) can be used in, for example, the vectors, rAAV compositions or treatments described herein. As a non-limiting example, specific consideration may be given to the following combinations: at least one of SEQ ID NO: 1-SEQ ID NO: 22; at least one of SEQ ID NO: 1-SEQ ID NO: 10; at least one of SEQ ID NO: 1-SEQ ID NO: 5; at least one of SEQ ID NO: 6-SEQ ID NO: 7; at least one of SEQ ID NO: 8-SEQ ID NO: 10; at least one of SEQ ID NO: 11-SEQ ID NO: 24; at least one of SEQ ID NO: 11-SEQ ID NO: 14; at least one of SEQ ID NO: 15-SEQ ID NO: 24; at least one of SEQ ID NO: 15-SEQ ID NO: 22; at least one of SEQ ID NO: 15-SEQ ID NO: 18; at least one of SEQ ID NO: 19-SEQ ID NO: 22; at least one of SEQ ID NO: 23-SEQ ID NO: 24; at least one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9; SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 9; At least one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21; at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 17; at least one of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5; at least one of SEQ ID NO: 2 or SEQ ID NO: 5; or at least one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21; at least one of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 17.

[0095] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1-SEQ ID NO: 22, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1-SEQ ID NO: 10 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 1-SEQ ID NO: 22.

[0096] In some embodiments of any aspect, the repressive RNA targets at least one of regions I-III of the HTT gene (e.g., CAG repeat, CAG 5' crossover, CAG 3' crossover; 5'-UTR; or exon 1). Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1-SEQ ID NO: 10, or has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1-SEQ ID NO: 10 and retains the same function (e.g., HTT repression) as at least one of SEQ ID NO: 1-SEQ ID NO: 10.

[0097] In some embodiments of any aspect, the repressive RNA targets region I of the HTT gene (e.g., the CAG repeat, the CAG 5' crossover, or the CAG 3' crossover). Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1-SEQ ID NO: 5, or has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1-SEQ ID NO: 5 and retains the same function (e.g., HTT repression) as at least one of SEQ ID NO: 1-SEQ ID NO: 5.

[0098] In some embodiments of any aspect, the repressive RNA targets region II (e.g., 5'-UTR) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 6-SEQ ID NO: 7, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 6-SEQ ID NO: 7 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 6-SEQ ID NO: 7.

[0099] In some embodiments of any aspect, the repressive RNA targets region III (exon 1) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 8-SEQ ID NO: 10, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 8-SEQ ID NO: 10 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 8-SEQ ID NO: 10.

[0100] In some embodiments of any aspect, the repressive RNA targets at least one of regions VI-V of the HTT gene (e.g., exons 2-67 or 3'UTR). Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 11-SEQ ID NO: 24, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 11-SEQ ID NO: 24 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 11-SEQ ID NO: 24.

[0101] In some embodiments of any aspect, the repressive RNA targets region VI of the HTT gene (e.g., exons 2-67). Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 11-SEQ ID NO: 14, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 11-SEQ ID NO: 14 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 11-SEQ ID NO: 14.

[0102] In some embodiments of any aspect, the repressive RNA targets regions III and VI (e.g., 5'-UTR and exon 2-exon 67) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 8 or SEQ ID NO: 13, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 8 or SEQ ID NO: 13 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 8 or SEQ ID NO: 13.

[0103] In some embodiments of any aspect, the repressive RNA targets region V (e.g., 3'UTR) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 15-SEQ ID NO: 24, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 15-SEQ ID NO: 24 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 15-SEQ ID NO: 24.

[0104] In some embodiments of any aspect, the repressive RNA targets region V (e.g., 3'UTR) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 15-SEQ ID NO: 22, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 15-SEQ ID NO: 22 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 15-SEQ ID NO: 22.

[0105] In some embodiments of any aspect, the repressive RNA targets region V (e.g., 3'UTR) of the HTT gene and is an artificial miRNA. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 15-SEQ ID NO: 18, or has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 15-SEQ ID NO: 18 and retains the same function (e.g., HTT repression) as at least one of SEQ ID NO: 15-SEQ ID NO: 18.

[0106] In some embodiments of any aspect, the repressive RNA targets region V (e.g., 3'UTR) of the HTT gene and is a human-expressed miRNA. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 19-SEQ ID NO: 22, or has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 19-SEQ ID NO: 22 and retains the same function (e.g., HTT repression) as at least one of SEQ ID NO: 19-SEQ ID NO: 22.

[0107] In some embodiments of any aspect, the repressive RNA targets region V (e.g., 3'UTR) of the HTT gene. Therefore, in some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 23-SEQ ID NO: 24, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 23-SEQ ID NO: 24 and retaining the same function (e.g., HTT repression) as at least one of SEQ ID NO: 23-SEQ ID NO: 24. In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA does not contain one of SEQ ID NO: 23-SEQ ID NO: 24, or has at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 23-SEQ ID NO: 24 and retains the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 23-SEQ ID NO: 24.

[0108] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9.

[0109] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or the following sequence: having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21.

[0110] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or the following sequence: having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17.

[0111] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, or the following sequence: having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5.

[0112] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 2, SEQ ID NO: 5, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 2 or SEQ ID NO: 5 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 2 or SEQ ID NO: 5.

[0113] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or the following sequence: having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21.

[0114] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or the following sequence: having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 and retaining the same function (e.g., HTT inhibition) as at least one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17.

[0115] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H2 (SEQ ID NO: 2). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H4 (SEQ ID NO: 4). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H5 (SEQ ID NO: 5). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H14 (SEQ ID NO: 14). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H15 (SEQ ID NO: 15). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H17 (SEQ ID NO: 17). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H19 (SEQ ID NO: 19; miR-137). In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises miHTT-H21 (SEQ ID NO: 21; miR-216).

[0116] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets at least a portion of an HTT nucleic acid (see, for example, SEQ ID NO: 25). In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5' untranslated region of an HTT nucleic acid (e.g., mRNA).

[0117] In some implementations of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets exon 1 (i.e., the first nucleic acid segment encoding a polypeptide) of a target (e.g., HTT).

[0118] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the CAG repeat of the HTT nucleic acid (e.g., mRNA). The term "CAG repeat" refers to a region of exon 1 of the HTT gene containing repeats of CAG trinucleotides (i.e., cytosine, adenine, and guanine). Typically, CAG trinucleotides can be repeated 10 to 35 times within the HTT gene. In individuals with Huntington's disease, the CAG region can be repeated 36 to over 120 times.

[0119] In some implementations of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the CAG 5'-transfer region of the HTT nucleic acid (e.g., mRNA). The term "CAG 5'-transfer region" refers to the region of the HTT gene that contains the 3' end of exon 1 and the 5' end of the CAG repeat.

[0120] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the CAG 3'-transfer region of an HTT nucleic acid (e.g., mRNA). The term "CAG 3'-transfer region" refers to the region of the HTT gene containing the 3' end of the CAG repeat and the 5' end of exon 2-exon 67.

[0121] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets exon 2-exon 67 of the HTT nucleic acid (e.g., mRNA). The term "exon 2-exon 67" in the HTT gene refers to the region comprised of exons 2-exons 67 of the HTT gene. In some embodiments of any aspect, the repressive RNA (e.g., miRNA) binds to and / or targets at least one of the following exons of the HTT nucleic acid (e.g., mRNA): exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 2 ... Exon 30, Exon 31, Exon 32, Exon 33, Exon 34, Exon 35, Exon 36, Exon 37, Exon 38, Exon 39, Exon 40, Exon 41, Exon 42, Exon 43, Exon 44, Exon 45, Exon 46, Exon 47, Exon 48, Exon 49, Exon 50, Exon 51, Exon 52, Exon 53, Exon 54, Exon 55, Exon 56, Exon 57, Exon 58, Exon 59, Exon 60, Exon 61, Exon 62, Exon 63, Exon 64, Exon 65, Exon 66, or Exon 67.

[0122] In some implementations of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 3' untranslated region (UTR) of an HTT nucleic acid (e.g., mRNA).

[0123] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5' UTR, exon 1, CAG repeat, CAG 5'-transfer region, or CAG 3'-transfer region of a target (e.g., HTT). In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5' UTR, exon 1, CAG repeat, CAG 5'-transfer region, CAG 3'-transfer region, exon 2-exon 67, or 3' UTR of a target (e.g., HTT). In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5' UTR, exon 1, CAG repeat, CAG 5'-transfer region, CAG 3'-transfer region, exon 2-exon 67, or 3' UTR of a target (e.g., HTT).

[0124] In some embodiments of any aspect, the repressive RNA (e.g., miRNA) binds to at least one binding site in the HTT nucleic acid (e.g., mRNA). In some embodiments of any aspect, the repressive RNA (e.g., miRNA) binds to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 binding sites in the HTT nucleic acid (e.g., mRNA).

[0125] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in humans, non-human primates, or mice. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in humans, non-human primates, and mice. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in humans. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in non-human primates. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in mice. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets HTT nucleic acids (e.g., mRNA) in humans and non-human primates.

[0126] In some embodiments of any aspect, the agent for treating Huntington's disease is a repressive nucleic acid. In some embodiments of any aspect, the inhibitor of expression of a given gene may be a repressive nucleic acid. As used herein, "repressive nucleic acid" refers to a nucleic acid molecule (e.g., double-stranded RNA (dsRNA), repressive RNA (iRNA), etc.) that can inhibit the expression of a target.

[0127] Double-stranded RNA molecules (dsRNAs) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The repressive nucleic acids described herein may comprise an RNA strand (antisense strand) having a region of less than 30 nucleotides in length (i.e., 15-30 nucleotides in length, typically 19-24 nucleotides in length) that is at least partially substantially complementary to the target mRNA transcript. Using these iRNAs enables targeted degradation of the mRNA transcript, thereby reducing the expression and / or activity of the target.

[0128] As used herein, the term "iRNA" refers to an agent that contains RNA (or a nucleic acid modified as described below) and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of any aspect, iRNA, as described herein, affects the inhibition of target expression and / or activity. In some embodiments of any aspect, exposing cells to an inhibitor (e.g., iRNA) results in a reduction of target mRNA levels in cells by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA levels present in the absence of said iRNA. In some embodiments of any aspect, administering an inhibitor (e.g., iRNA) to a subject reduces the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level present in the absence of said iRNA.

[0129] In some embodiments of any aspect, the iRNA may be a dsRNA. The dsRNA comprises two RNA strands sufficiently complementary to hybridize under the conditions under which the dsRNA will be used to form a duplex structure. One strand of the dsRNA (the antisense strand) contains a region complementary to (substantially complementary and usually perfectly complementary to) the target sequence. The target sequence may be derived from the sequence of mRNA formed during target expression, for example, it may cross one or more intron boundaries. The other strand (the sense strand) contains a region complementary to the antisense strand such that the two strands hybridize and form a duplex structure when bound under suitable conditions. Typically, the length of the duplex structure is between 15 and 30 base pairs (inclusive), more typically between 18 and 25 base pairs (inclusive), even more typically between 19 and 24 base pairs (inclusive), and most typically between 19 and 21 base pairs (inclusive). Similarly, the length of regions complementary to the target sequence is between 15 and 30 base pairs (inclusive), more typically between 18 and 25 base pairs (inclusive), even more typically between 19 and 24 base pairs (inclusive), and most typically between 19 and 21 base pairs (inclusive). In some embodiments of any aspect, the dsRNA is between 15 and 20 nucleotides (inclusive), and in other embodiments, the dsRNA is between 25 and 30 nucleotides (inclusive). As those skilled in the art will recognize, the target region targeting RNA for cleavage will most often be a portion of a larger RNA molecule, frequently an mRNA molecule. In relevant cases, a “portion” of the mRNA target is a contiguous sequence of the mRNA target whose length is sufficient to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNAs with double strands as short as 9 base pairs can mediate RNAi-directed RNA cleavage in certain circumstances. In most cases, the target length will be at least 15 nucleotides, preferably 15-30 nucleotides.

[0130] Exemplary implementations of the type of inhibitory nucleic acid may include, for example, siRNA, shRNA, miRNA, and / or amiRNA, all of which are well known in the art.

[0131] In some embodiments of any aspect, the repressive RNA used to treat Huntington's disease is a miRNA. MicroRNAs (miRNAs) are small RNAs of 17-25 nucleotides that function as regulators of gene expression in eukaryotes. miRNAs are initially expressed in the nucleus as part of a long primary transcript called primary miRNA (pri-miRNA). Within the nucleus, pri-miRNA is partially digested by the Drosha enzyme to form a hairpin precursor miRNA (pre-miRNA) of 65-120 nucleotides in length. This pre-miRNA is exported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs, which are the active molecules. In animals, these short RNAs contain a 5' proximal "seed" region (nucleotides 2 to 8), which appears to be the primary determinant of the miRNA's pairing specificity with the 3' untranslated region of the target mRNA.

[0132] In the context of this invention, a miRNA molecule or its equivalent or mimetic, or isomiR, can be synthetic, natural, recombinant, mature, or a portion of a mature miRNA or human miRNA, or a human-derived miRNA, as further defined in the section dedicated to general definitions. Human miRNA molecules are miRNA molecules found in human cells, tissues, organs, or body fluids (i.e., endogenous human miRNA molecules). Human miRNA molecules can also be human miRNA molecules derived from endogenous human miRNA molecules through nucleotide substitution, deletion, and / or addition. A miRNA molecule or its equivalent or mimetic can be a single-stranded or double-stranded RNA molecule. Preferably, the miRNA molecule or its equivalent or mimic has a length of 6 to 30 nucleotides, more preferably 12 to 30 nucleotides, more preferably 15 to 28 nucleotides, and more preferably the molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

[0133] In a preferred embodiment, the miRNA molecule or its equivalent or mimetic or isomiR comprises at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule or its equivalent or mimetic or isomiR. Preferably, in this embodiment, the miRNA molecule or its equivalent or mimetic or isomiR is 6 to 30 nucleotides in length, and more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule or its equivalent. Even more preferably, the miRNA molecule or its equivalent or analogue or isomiR has a length of 15 to 28 nucleotides, and more preferably contains at least 6 of the 7 nucleotides present in the seed sequence, and even more preferably, the miRNA molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

[0134] Therefore, the preferred miRNA molecule or its equivalent or analogue or isomiR contains at least 6 of the 7 nucleotides present in the seed sequence identified as at least one of SEQ ID NO: 1-SEQ ID NO: 24, and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides.

[0135] Delivery media for miRNAs include, but are not limited to, liposomes, polymer nanoparticles, viral systems, conjugates of lipid or receptor-binding molecules, exosomes, and phages; see, for example, Baumann and Winkler, miRNA-based therapies: Strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents, Future Med Chem. 2014, 6(17): 1967–1984; U.S. Patents 8,900,627, 9,421,173, and 9,555,060; WO2019 / 177550; the contents of each of these patents are incorporated herein by reference in their entirety.

[0136] Nucleic acid

[0137] In some aspects, this disclosure provides isolated nucleic acids useful for reducing (e.g., inhibiting) the expression of human huntingtin protein (HTT). A “nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of this disclosure are isolated. As used herein, the term “isolated” means artificially produced. As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) produced by clonal recombination; (iii) purified, such as by shearing and gel separation; or (iv) synthesized by, for example, chemical synthesis. Isolated nucleic acids are nucleic acids readily manipulated using recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which the 5' and 3' restriction sites are known or the polymerase chain reaction (PCR) primer sequences are disclosed is considered isolated, but a nucleic acid sequence present in its natural host in its native state is not. Isolated nucleic acids may be substantially purified, but are not required to be. For example, nucleic acids isolated in a cloning or expression vector are not pure because they may only constitute a small percentage of the material in the cell in which they reside. However, this nucleic acid is isolated, as the term is used herein, because it can be easily manipulated using standard techniques known to those skilled in the art. As used herein with respect to proteins or peptides, the term "isolated" means a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., through chemical synthesis, through recombinant DNA technology, etc.).

[0138] Those skilled in the art will also recognize that conserved amino acid substitutions can be performed to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, this disclosure includes sequence alterations that result in conserved amino acid substitutions. As used herein, a conserved amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is performed. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, such as those found in references compiling such methods, e.g., *Molecular Cloning: A Laboratory Manual*, eds. J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; or *Current Protocols in Molecular Biology*, eds. F.A. Mosurubel et al., John Wiley & Sons, Inc., New York. Conserved amino acid substitutions include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, conserved amino acid substitutions can be performed on the amino acid sequences of the proteins and peptides disclosed herein.

[0139] The isolated nucleic acid of the present invention can be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described in this disclosure comprises a region containing a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof (e.g., a first region). The isolated nucleic acid (e.g., a recombinant AAV vector) can be packaged into a capsid protein and administered to a subject and / or delivered to selected target cells. A “recombinant AAV (rAAV) vector” typically consists at least of a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). As disclosed elsewhere herein, the transgene may comprise one or more regions encoding one or more repressive RNAs (e.g., miRNAs) containing nucleic acids targeting endogenous mRNAs of a subject. The transgene may also comprise regions encoding, for example, proteins and / or expression control sequences (e.g., poly-A tails) as described elsewhere in this disclosure.

[0140] Typically, the ITR sequence is about 145 bp in length. Preferably, a substantially complete sequence encoding the ITR is used in the molecule, although some degree of minor modification of these sequences is permitted. The ability to modify these ITR sequences is within the scope of the art. (See, for example, the texts of Sambrook et al., “Molecular Cloning. A Laboratory Manual,” 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996).) An example of such a molecule used in this invention is a “cis-acting” plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory element are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequence can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., an rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region encoding an AAV2 ITR (e.g., a first region).

[0141] In some embodiments, the isolated nucleic acid further comprises a region containing a second AAV ITR (e.g., a second region, a third region, a fourth region, etc.). In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, and variants thereof. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal resolution site (TRS). The term "lacking a terminal resolution site" can refer to an AAV ITR containing a mutation that disables the function of the terminal resolution site (TRS) of the ITR (e.g., a sense mutation (e.g., a nonsynonymous mutation) or a missense mutation), or a truncated AAVTR (e.g., an ATRS ITR) lacking a nucleic acid sequence encoding a functional TRS. Without being bound by any particular theory, rAAV vectors containing ITRs lacking functional TRS generate self-complementary rAAV vectors, such as those described by McCarthy (2008) Molecular Therapy, 16(10):1648-1656.

[0142] In addition to the principal elements identified above for the recombinant AAV vector, the vector also includes conventional control elements operatively linked to the transgenic elements to allow the transgenic gene to be transcribed, translated, and / or expressed in cells transfected with the vector or infected with a virus produced in this invention. As used herein, "operatively linked" sequences include both expression control sequences adjacent to the gene of interest and expression control sequences that act in a trans-acting or distant-acting manner to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals (e.g., splicing and polyadenylation (polyA) signals); sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product when needed. Many expression control sequences (including native, constitutive, inducible, and / or tissue-specific promoters) are known and available in the art.

[0143] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are considered operatively linked when they are covalently linked in such a manner that the expression or transcription of the nucleic acid sequence is placed under the influence or control of the regulatory sequence. Two DNA sequences are considered operatively linked if the translation of the nucleic acid sequence into a functional protein is desired, if the induction of a promoter in the 5' regulatory sequence causes transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, if a promoter region is capable of influencing the transcription of the DNA sequence, resulting in a transcript that may be translated into the desired protein or polypeptide, then the promoter region is operatively linked to the nucleic acid sequence. Similarly, when two or more coding regions are linked in such a manner that transcription from a common promoter results in the expression of two or more proteins already translated within the frame. In some embodiments, operatively linked coding sequences produce fusion proteins. In some implementations, the operatively linked coding sequences produce functional RNA (e.g., miRNA).

[0144] In some aspects, this disclosure provides isolated nucleic acids comprising transgenes, wherein the transgenes comprise nucleic acid sequences encoding one or more microRNAs (e.g., miRNAs). “MicroRNA” or “miRNA” is a small non-coding RNA molecule capable of mediating transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed into hairpin or stem-loop (e.g., having a self-complementary, single-stranded backbone) double-stranded structure, called primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into pre-miRNA. The length of pri-miRNA can vary. In some embodiments, the length of pri-miRNA ranges from about 100 to about 5000 base pairs (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base pairs). In some implementations, the pri-miRNA is longer than 200 base pairs (e.g., 2,500, 5,000, 7,000, 9,000 or more base pairs).

[0145] Pre-miRNAs are characterized by a hairpin or stem-loop double-stranded structure, and their lengths can vary. In some embodiments, the pre-miRNA ranges in size from about 40 base pairs to about 500 base pairs in length. In some embodiments, the pre-miRNA ranges in length from about 50 to about 100 base pairs. In some embodiments, the pre-miRNA ranges in length from about 50 to about 90 base pairs (e.g., lengths of about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 66, about 68, about 70, about 72, about 74, about 76, about 78, about 80, about 82, about 84, about 86, about 88, or about 90 base pairs).

[0146] Typically, pre-miRNA is exported to the cytoplasm and processed by Dicer enzymatic processing to first produce an incomplete miRNA / miRNA* duplex, then a single-stranded mature miRNA molecule, which is subsequently loaded into an RNA-induced silencing complex (RISC). Typically, the size of the mature miRNA molecule ranges from about 19 to about 30 base pairs in length. In some embodiments, the length of the mature miRNA molecule is about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or 30 base pairs. In some embodiments, the isolated nucleic acid of this disclosure comprises a sequence encoding a mature miRNA, pri-miRNA, or pre-miRNA comprising any one of the sequences described in SEQ ID NO: 1-SEQ ID NO: 24.

[0147] It should be understood that, in some embodiments, the isolated nucleic acid or vector (e.g., an rAAV vector) contains nucleic acid sequences encoding more than one (e.g., multiple, such as 2, 3, 4, 5, 10, or more) miRNAs. In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds) the same target gene (e.g., an isolated nucleic acid encoding three unique miRNAs, each miRNA targeting the HTT gene). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds) a different region of the same target gene (e.g., HTT). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds) a different target gene.

[0148] In some aspects, this disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) encoding one or more artificial miRNAs. As used herein, “artificial miRNA” or “amiRNA” refers to endogenous pri-miRNA or pre-miRNA (e.g., miRNA backbone, which is a precursor miRNA capable of producing a functional mature miRNA), wherein the miRNA and miRNA* (e.g., the passenger strand of the miRNA duplex) sequences have been replaced by corresponding amiRNA / amiRNA* sequences that guide efficient RNA silencing of target genes, as described, for example, by Eamens et al., (2014), Methods Mol. Biol. 1062:211-224. For example, in some embodiments, the artificial miRNA comprises a miR-155 pri-miRNA backbone into which the sequence encoding a mature HTT-specific miRNA (e.g., any one of SEQ ID NO: 1-SEQ ID NO: 24) has been inserted to replace the endogenous miR-155 mature miRNA coding sequence. In some embodiments, the miRNA described in this disclosure (e.g., an artificial miRNA; for example, one of SEQ ID NO: 1-SEQ ID NO: 24) comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a miR-64 backbone sequence, or a miR-122 backbone sequence. In some embodiments, the miRNA described in this disclosure (e.g., an artificial miRNA; for example, one of SEQ ID NO: 1-SEQ ID NO: 24) comprises a backbone as disclosed in SEQ ID NO: 35.

[0149] The transgenic region (e.g., second region, third region, fourth region, etc.) may be located at any suitable location on the isolated nucleic acid. The region may be located in any untranslated portion of the nucleic acid (including, for example, introns, 5' or 3' untranslated regions, etc.).

[0150] In some cases, it may be desirable to locate the region (e.g., a second region, a third region, a fourth region, etc.) upstream of the first codon of the nucleic acid sequence encoding a protein (e.g., a protein-coding sequence). For example, the region may be located between the first codon of the protein-coding sequence and 2000 nucleotides upstream of the first codon. The region may be located between the first codon of the protein-coding sequence and 1000 nucleotides upstream of the first codon. The region may be located between the first codon of the protein-coding sequence and 500 nucleotides upstream of the first codon. The region may be located between the first codon of the protein-coding sequence and 250 nucleotides upstream of the first codon. The region may be located between the first codon of the protein-coding sequence and 150 nucleotides upstream of the first codon. In some cases (e.g., when the transgene lacks a protein-coding sequence), it may be desirable to locate the region (e.g., a second region, a third region, a fourth region, etc.) upstream of the poly-A tail of the transgene. For example, the region may be located between the first base of the poly-A tail and 2000 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 1000 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 500 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 250 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 150 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 100 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 50 nucleotides upstream of the first base. The region may be located between the first base of the poly-A tail and 20 nucleotides upstream of the first base. In some embodiments, the region is located between the last nucleotide base of the promoter sequence and the first nucleotide base of the poly-A tail sequence.

[0151] In some cases, the region may be located downstream of the last base of the poly-A tail of the transgene. The region may be located between the last base of the poly-A tail and 2000 nucleotides downstream of that last base. The region may be located between the last base of the poly-A tail and 1000 nucleotides downstream of that last base. The region may be located between the last base of the poly-A tail and 500 nucleotides downstream of that last base. The region may be located between the last base of the poly-A tail and 250 nucleotides downstream of that last base. The region may be located between the last base of the poly-A tail and 150 nucleotides downstream of that last base.

[0152] It should be understood that when a transgene encodes more than one miRNA, each miRNA can be located at any suitable position within the transgene. For example, the nucleic acid encoding the first miRNA can be located in an intron of the transgene, while the nucleic acid sequence encoding the second miRNA can be located in another untranslated region (e.g., between the last codon of the protein-coding sequence and the first base of the poly-A tail of the transgene).

[0153] In some embodiments, the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., promoters, etc.). Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals (e.g., splicing and polyadenylation (polyA) signals); sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance the secretion of encoded products when desired. Many expression control sequences (including native, constitutive, inducible, and / or tissue-specific promoters) are known in the art and can be utilized.

[0154] A promoter is a DNA sequence recognized by the cell's synthetic mechanisms or introduced synthetic mechanisms and required to initiate the specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and gene expression.

[0155] For nucleic acids encoding proteins, polyadenylated sequences are typically inserted after the transgene sequence and before the 3'AAVITR sequence. The rAAV constructs useful in this disclosure may also contain introns, ideally located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence. Another vector element that can be used is the internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences will be used to produce proteins containing more than one polypeptide chain. The selection of these and other common vector elements is routine and many such sequences are available (see, for example, Sambrook et al., and references cited therein, e.g., pp. 3.18, 3.26 and 16.17, 16.27; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). In some embodiments, the foot-and-mouth disease virus 2A sequence is contained in a polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; pp.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6:198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11:1921-1931; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

[0156] Examples of constitutive promoters include, but are not limited to, the retroviral Rouss sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1α promoter (Invitrogen). In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is the U6 promoter.

[0157] Inducible promoters allow for the regulation of gene expression and can be modulated by the presence of exogenously supplied compounds, environmental factors (e.g., temperature), or specific physiological states (e.g., acute phase, specific differentiation state of cells, cells replicating only). Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; and the T7 polymerase promoter system (WO... 98 / 10088); Ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)); Tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); Tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)); RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)); and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). However, other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological states (e.g., temperature, acute phase, specific differentiation state of the cell, or cells in replication only).

[0158] In another embodiment, a natural promoter of the transgene will be used. A natural promoter may be preferred when it is desired that the expression of the transgene should mimic natural expression. A natural promoter may be used when the expression of the transgene must be regulated temporally, developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. In another embodiment, other natural expression control elements (such as enhancer elements, polyadenylation sites, or Kozak concordant sequences) may also be used to mimic natural expression.

[0159] In some embodiments, regulatory sequences confer the ability to express tissue-specific genes. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synaptic protein-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters include the β-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther., 3:1002-9 (1996)), the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)), and the bone sialic acid protein promoter (Chen et al., J. Bone The promoters include: Miner.Res., 11:654-64 (1996); CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998)); immunoglobulin heavy chain promoter; T cell receptor α chain promoter; neuron (e.g. neuron-specific) enolase (NSE) promoter (Andersen et al., Cell.Mol.Neurobiol., 13:503-15 (1993)); neurofilament light chain gene promoter (Piccioli et al., Proc.Natl.Acad.Sci.USA, 88:5611-5 (1991)); and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), which are obvious to a skilled technician.

[0160] This disclosure relates to isolated nucleic acids containing more than one promoter (e.g., two, three, four, five, or more promoters). For example, in the context of a construct having a transgene containing a first region encoding a protein and a second region encoding a repressive RNA (e.g., miRNA), it is desirable to use a first promoter sequence (e.g., a first promoter sequence operatively linked to the protein-coding region) to drive the expression of the protein-coding region and a second promoter sequence (e.g., a second promoter sequence operatively linked to the repressive RNA-coding region) to drive the expression of the repressive RNA-coding region. Typically, the first and second promoter sequences can be the same or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter driving the expression of the protein-coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include U6 and HI promoter sequences. In some embodiments, the second promoter sequence (e.g., the promoter sequence driving the expression of the repressive RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of the polII promoter sequence include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the polIII promoter sequence drives the expression of repressive RNA (e.g., miRNA) coding regions. In some embodiments, the polII promoter sequence drives the expression of protein-coding regions.

[0161] In some embodiments, the nucleic acid comprises a transgene encoding a protein. The protein may be a therapeutic protein (e.g., a peptide, protein, or polypeptide useful for treating or preventing a disease state in a mammalian subject) or a reporter protein. In some embodiments, the therapeutic protein is useful for treating or preventing Huntington's disease, such as CYP46A1, polyglutamine-binding peptide 1 (QBP1), PTD-QBP1, ED11, C4 intracellular antibody, VL12.3 intracellular antibody, MW7 intracellular antibody, Happ1 antibody, Happ3 antibody, mEM48 intracellular antibody, certain monoclonal antibodies (e.g., 1C2), and peptide P42, and variants thereof, as described in Marelli et al., (2016) Orphanet Journal of Rare Disease 11:24. In some embodiments, the therapeutic protein is a wild-type Huntington's protein (e.g., a Huntington's protein having a PolyQ repeat region containing fewer than 36 repeats).

[0162] Without wishing to be bound by any particular theory, allele-specific silencing of the mutant huntingtin protein (HTT) can provide an improved safety profile in subjects compared to non-allelic-specific silencing (e.g., silencing of both wild-type and mutant HTT alleles), since the expression and function of wild-type HTT are preserved in the cells. Aspects of the invention relate to the inventors' knowledge and understanding that isolated nucleic acids and vectors containing one or more repressive RNA (e.g., miRNA) sequences that target the HTT gene in a non-allelic-specific manner, while driving the expression of a hardened wild-type HTT gene (a wild-type HTT gene not targeted by miRNAs) can achieve concomitant mutant HTT knockdown (e.g., increased expression of wild-type HTT in CNS tissues). Typically, the sequences of the nucleic acids encoding endogenous wild-type and mutant HTT mRNAs, and the nucleic acids encoding the "hardened" wild-type HTT transgenic mRNA, are quite different, such that the "hardened" wild-type HTT transgenic mRNA is not targeted by said one or more repressive RNAs (e.g., miRNAs). For example, this can be achieved by introducing one or more silent mutations into the HTT transgenic sequence to encode the same protein as the endogenous wild-type HTT gene but with a different nucleic acid sequence. In this case, the exogenous mRNA can be referred to as "robust". Alternatively, repressive RNAs (such as miRNAs) can target the 5' and / or 3' untranslated regions of the endogenous wild-type HTT mRNA. These 5' and / or 3' regions in the transgenic mRNA can then be removed or replaced so that the transgenic mRNA is not targeted by the one or more repressive RNAs.

[0163] Reporter sequences that can be provided in transgenes (e.g., nucleic acid sequences encoding reporter proteins) include, but are not limited to, DNA sequences encoding β-lactamases, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other substances well known in the art. When bound to regulatory elements driving their expression, reporter sequences provide signals detectable by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorescence assays or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunoassays (including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry). For example, in the case where the marker sequence is the LacZ gene, the presence of a signal-carrying vector is detected by measuring β-galactosidase activity. In the case where the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visually measured by the generation of color or light in a photometer. For example, such reporters are useful for verifying the tissue-specific targeting ability of nucleic acids and the tissue-specific promoter regulatory activity. Recombinant adeno-associated virus (rAAV). In some aspects, this disclosure provides isolated AAVs. As used herein with respect to AAV, the term "isolated" refers to an artificially produced or obtained AAV. Isolated AAVs can be produced using recombinant methods. Such AAVs are referred to herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities, such that the transgene and / or nuclease of the rAAV will be specifically delivered to one or more predetermined tissues. The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Therefore, rAAVs with capsids suitable for the targeted tissues can be selected.

[0164] Methods for obtaining recombinant AAV with the desired capsid protein are well known in the art (see, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing host cells containing a nucleic acid sequence encoding an AAV capsid protein, a functional rep gene, an AAV inverted terminal repeat (ITR), and a transgene, as well as sufficient helper function to allow the recombinant AAV vector to be packaged into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the AAV cap gene. AAV contains three capsid proteins, viral particle proteins 1 through 3 (named VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, VP1, VP2, and VP3 have molecular weights of approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, after translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some implementations, the capsid protein functions to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0165] In some embodiments, the AAV capsid protein is selected from the AAV serotypes of the group consisting of: AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAV9, AAV10, and AAVrh10. In some embodiments, the AAV capsid protein is derived from a non-human primate serotype, such as AAVrh8 or AAVrh10. In some embodiments, the AAV capsid protein is the AAV9 serotype. In some embodiments, the AAV capsid protein is the AAVrh10 serotype. In some embodiments, the capsid protein is the AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10 capsid protein, or any chimera thereof. In some embodiments, the recombinant AAV (rAAV) is a haploid rAAV. In some implementations, haploid rAAV contains chimeric capsid proteins.

[0166] In one embodiment, the viral capsid is modified. In one embodiment, the modified viral capsid is a chimeric capsid. As used herein, a "chimeric" capsid protein means an AAV capsid protein modified by substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to the wild type, and by insertion or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to the wild type (e.g., any one or more of VP1, VP2, or VP3). In some embodiments, complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace corresponding wild-type domains, functional regions, epitopes, etc., from different AAV serotypes in any combination to produce the chimeric capsid protein of the present invention. The generation of chimeric capsid proteins can be carried out according to methods well known in the art, and a significant number of chimeric capsid proteins are described in the literature and herein, which can be included in the capsid of the present invention.

[0167] In one embodiment, the modified viral capsid is a haploid capsid. As used herein, the term "haploid AAV" should mean the AAV described in International Application WO2018 / 170310 or U.S. Application US2018 / 037149, which are incorporated herein by reference in their entirety. In some embodiments, the viral particle population is a haploid AAV population in which viral particles can be constructed, wherein at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins as required to form a viral particle capable of encapsulating the AAV genome. For each viral protein present (VP1, VP2, and / or VP3), the protein is of the same type (e.g., all AAV2 VP1). In one case, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric, while only VP3 is non-chimeric. For example, a viral particle composed solely of VP1 / VP2 derived from chimeric AAV2 / 8 (the N-terminus of AAV2 and the C-terminus of AAV8) pairs with VP3 derived solely from AAV2; or a chimeric VP1 / VP2 28m-2P3 (derived from the N-terminus of AAV8 and the C-terminus of AAV2, without a VP3 start codon mutation) pairs with VP3 derived solely from AAV2. In another embodiment, only VP3 is chimeric, while VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins originates from a completely different serotype. For example, a chimeric VP1 / VP2 28m-2P3 pairs with VP3 derived solely from AAV3. In another instance, no chimera exists. See, for example, U.S. Patent Application 2019 / 0002841, or U.S. Patent 8,906,675; the contents of which are incorporated herein by reference in their entirety.

[0168] The components cultured in host cells to package the rAAV vector in an AAV capsid can be provided to the host cells in trans form. Alternatively, any one or more of the desired components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper function) can be provided by stable host cells engineered to contain one or more desired components, using methods known to those skilled in the art. Most preferably, such stable host cells will contain the desired components under the control of an inducible promoter. However, the desired components may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided in the discussion of regulatory elements applicable to transgenes. In yet another alternative, the selected stable host cells may contain the selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells derived from 293 cells (which contain E1 helper function under the control of a constitutive promoter) but containing rep and / or cap proteins under the control of an inducible promoter can be generated. Other stable host cells may also be generated by those skilled in the art. In some embodiments, this disclosure relates to a host cell comprising nucleic acids containing a coding sequence for a protein (e.g., wild-type huntingtin, optionally "robust" wild-type huntingtin). In some embodiments, this disclosure relates to a composition comprising the aforementioned host cell. In some embodiments, the composition comprising the aforementioned host cell further comprises a cryopreservative.

[0169] The recombinant AAV vector, rep sequence, cap sequence, and helper function required to generate the rAAV of this disclosure can be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element can be delivered by any suitable method, including those described herein. Methods for constructing any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV viral particles are well known, and the choice of suitable method is not limited to this disclosure. See, for example, K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0170] In some embodiments, a triple transfection method (described in detail in U.S. Patent No. 6,001,650) can be used to generate recombinant AAV. Typically, recombinant AAV is generated by transfecting host cells with a recombinant AAV vector (containing a transgene), an AAV helper functional vector, and an accessory functional vector to be packaged into AAV particles. The AAV helper functional vector encodes “AAV helper function” sequences (i.e., rep and cap), which function in trans form for productive AAV replication and encapsulation. Preferably, the AAV helper functional vector supports efficient AAV vector production without generating any detectable wild-type AAV virus particles (i.e., AAV virus particles containing functional rep and cap genes). Non-limiting examples of vectors suitable for use in this disclosure include the pHLP19 vector described in U.S. Patent No. 6,001,650 and the pRep6cap6 vector described in U.S. Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety. Helper vectors encode nucleotide sequences for viral and / or cellular functions derived from non-AAV sources, functions that AAV depends on for replication (i.e., "helper functions"). Helper functions include those required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Virus-based helper functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0171] In some respects, this disclosure provides transfected host cells. The term “transfection” is used to refer to the presence of foreign DNA within a cellular community, and when foreign DNA is introduced into the cell membrane, the cell has been “transfected.” Many transfection techniques are generally known in the art. See, for example, Graham et al., (1973) Virology, 52:456; Sambrook et al., (1989) Molecular Cloning, alaboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al., (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al., (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign nucleic acids (e.g., nucleotide integrative vectors and other nucleic acid molecules) into a suitable host cell.

[0172] "Host cell" refers to any cell that carries or is capable of carrying the substance of interest. Typically, host cells are mammalian cells. Host cells can serve as recipients of AAV helper constructs, AAV minigene plasmids, auxiliary functional vectors, or other transferred DNA in connection with recombinant AAV production. The term includes the offspring of the transfected original cell. Therefore, as used herein, "host cell" can refer to a cell transfected with a foreign DNA sequence. It should be understood that, due to natural, accidental, or intentional mutations, the offspring of a single parent cell need not necessarily be identical to the original parent in morphology, genome, or total DNA set.

[0173] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in the karyotype of such a clonal population during storage or transfer. Therefore, cells derived from the cell lines mentioned may not be identical to the ancestral cells or cultures, and the cell lines mentioned include such variants.

[0174] As used herein, the term “recombinant cell” refers to a cell in which a foreign DNA segment has been introduced (e.g., a DNA segment that induces transcription of bioactive polypeptides or bioactive nucleic acids (e.g., RNA)).

[0175] As used herein, the term "vector" includes any genetic element, such as plasmids, bacteriophages, transposons, granules, chromosomes, artificial chromosomes, viruses, viral particles, etc., which, when associated with appropriate control elements, are capable of replicating and can transfer gene sequences between cells. Therefore, the term includes cloning media and expression media, as well as viral vectors. In some embodiments, vectors intended to be useful are those in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter. "Promoter" refers to a DNA sequence recognized by or introduced into the cell's synthetic mechanisms and required to initiate specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term "expression vector or construct" refers to any type of genetic construct containing nucleic acids in which some or all of the nucleic acid coding sequences can be transcribed. In some embodiments, expression includes the transcription of nucleic acids, for example, to generate a biologically active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.

[0176] The foregoing methods for packaging the recombinant vector into a desired AAV capsid to produce the rAAV of this disclosure are not intended to be limiting, and other suitable methods will be apparent to those skilled in the art.

[0177] In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequences provided herein (including those provided in the sequence listing) may be replaced by any other nucleotide suitable for base pairing with adenosine nucleotides (e.g., via Watson-Crick base pairs). For example, in some embodiments, any one or more thymidine (T) nucleotides in the sequences provided herein (including those provided in the sequence listing) may be suitably replaced with uridine (U) nucleotides, and vice versa.

[0178] In some embodiments of any aspect, nucleic acids (e.g., miRNAs) are chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and / or modified by methods well-established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5' modifications (phosphorylation, conjugation, reverse linkage, etc.), 3' modifications (conjugation, DNA nucleotides, reverse linkage, etc.); (b) base modifications, such as substitution with a stable base, destabilization of a base, or base pairing with an extended chaperone library, base removal (base-free nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids comprising a modified backbone or lacking natural internucleotide links. Nucleic acids having a modified backbone particularly include those without phosphorus atoms in their backbone. For the purposes of this specification, and as sometimes mentioned in the art, modified nucleic acids that do not have phosphorus atoms in their internucleotide backbone may also be considered oligonucleotides. In some embodiments in any aspect, the modified RNA will have phosphorus atoms in its internucleotide backbone.

[0179] For example, modified nucleic acid backbones may include thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphates and aminoalkylaminophosphates), borophosphates, thioaminophosphates, thioalkylphosphonates, and thioalkylphosphotriesters with normal 3'-5' linkages, their 2'-5' linked analogs, and those with opposite polarities, wherein adjacent nucleoside unit pairs are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' manner. Various salts, mixed salts, and free acid forms are also included. Nucleic acid backbones not containing phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocycles. These backbones include those with the following: morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformyl and thioformacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; other backbones with mixed N, O, S, and CH2 components; and those with... Oligonucleotides with heteroatomic skeletons, especially --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [called methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [where the native phosphodiester skeleton is represented as --O--P--O--CH2--].

[0180] In other nucleic acid mimics, both the sugar and nucleoside links (i.e., the backbone) of the nucleotide units are replaced with new groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomer (showing an RNA mimic with excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the RNA sugar backbone is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bound to the nitrogen atom of the amide portion of the backbone.

[0181] Nucleic acids can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety contains an additional bridging link between the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-inner structural conformation. It has been shown that adding locked nucleic acids to siRNA increases the stability of siRNA in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al., (2007) Mol. Canc. Ther. 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0182] The modified nucleic acid may also contain one or more substituted sugar moieties. The nucleic acid described herein may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, wherein n and m are 1 to about 10. 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl groups, or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of nucleic acids, or groups that improve the pharmacodynamic properties of nucleic acids, and other substituents with similar properties. In some embodiments in any aspect, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e. 2'-O--CH2--O--CH2--N(CH2)2, which is also described in the examples below.

[0183] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorine (2'-F). Similar modifications can also be made at other positions on nucleic acids, particularly at the 3' end nucleotide or the 5' end of the sugar in 2'-5' linked dsRNAs. Nucleic acids can also have sugar mimics, such as a cyclobutyl moiety, to replace the furanopentose.

[0184] Nucleic acids may also include modifications or substitutions of nucleobases (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, including but not limited to 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo Uracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thionuracil; 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenine and guanine; 5-halogenated (especially 5-bromo), 5-trifluoromethyl, and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazoguanine and 7-deazoadenine; and 3-deazoguanine and 3-deazoadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids characteristic of this invention. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine). The 5-methylcytosine substitution has been shown to improve the stability of nucleic acid duplexes by 0.6–1.2 °C (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution, even more so when combined with 2'-O-methoxyethyl sugar modifications. In some embodiments of any aspect, the modified nucleobases may include d5SICS and dNAM, which are non-limiting examples of non-natural nucleobases that may be used alone or together as base pairs (see, for example, Leconte et al., J. Am. Chem. Soc. 2008, 130, 7, 2336-2343; Malyshev et al., PNAS. 2012, 109(30) 12005-12010). In some embodiments of any aspect, the oligonucleotide tag (e.g., Oligopaint) contains any modified nucleobases known in the art, i.e., any modified nucleobase derived from unmodified and / or natural nucleobases.

[0185] The preparation of the modified nucleic acids, backbones, and nucleobases described above is well known in the art.

[0186] Another modification of the nucleic acid characterized in this invention involves chemically linking the nucleic acid to one or more ligands, portions or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties or cellular uptake of the nucleic acid. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556); bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060); thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3: 2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538); and aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO). J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, such as di-hexadecyl-racemic glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphonate / salt (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 1996, 1996, 1996); Lett., 1995, 36: 3651-3654); palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229-237); or octadecylamine or hexano-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923-937).

[0187] Treatment

[0188] This disclosure provides a method for delivering a transgene (e.g., a repressive RNA, such as miRNA) to a subject. The method typically involves administering to the subject an effective amount of isolated nucleic acid or rAAV, said isolated nucleic acid encoding interfering RNA capable of reducing the expression of the huntingtin protein (htt) protein, said rAAV containing nucleic acid for expressing the repressive RNA capable of reducing huntingtin protein expression.

[0189] In some aspects, this disclosure provides repressive miRNAs that specifically bind (e.g., hybridize) to (e.g., human huntingtin protein) (e.g., SEQ ID NO: 25) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive bases. As used herein, “consecutive bases” means two or more nucleotide bases that are covalently bound (e.g., via one or more phosphodiester bonds, etc.) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, the at least one miRNA has about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% identity with two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleotide bases of SEQ ID NO: 25. In some embodiments, the repressive RNA is a miRNA encoding a sequence comprising any one of SEQ ID NO: 1-SEQ ID NO: 24 or a sequence comprising any one of SEQ ID NO: 1-SEQ ID NO: 24.

[0190] As used herein, "huntington disease" or "HD" refers to a neurodegenerative disease characterized by progressively worsening motor, cognitive, and behavioral changes caused by pathogenic mutant huntingtin protein (HTT or mHTT) resulting from trinucleotide repeat amplification (e.g., CAG, which is translated as polyglutamine or PolyQ bundle) in the HTT gene. In some embodiments, the mutant huntingtin protein accelerates the rate of neuronal cell death in certain regions of the brain. Typically, the severity of HD is related to the size of the trinucleotide repeat amplification in the subject. For example, subjects with a CAG repeat region containing between 36 and 39 repeats are characterized as having "reduced penetrance" HD, while subjects with more than 40 repeats are characterized as having "full penetrance" HD. Thus, in some embodiments, subjects with HD or at risk of developing HD have an HTT gene containing between approximately 36 and approximately 39 CAG repeats (e.g., 36, 37, 38, or 39 repeats). In some embodiments, subjects with or at risk of developing HD have an HTT gene containing 40 or more (e.g., 40, 45, 50, 60, 70, 80, 90, 100, 200 or more) CAG repeats. In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats develop HD earlier than subjects with fewer than 100 CAG repeats. In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats may develop HD symptoms before approximately age 20 and are referred to as having juvenile HD (also known as akinetic-rigid HD, or Westphal variant HD). The number of CAG repeats in the subject's HTT gene alleles can be determined by any suitable method known in the art. For example, nucleic acids (e.g., DNA) can be isolated from a subject's biological sample (e.g., blood), and the number of CAG repeats in the HTT allele can be determined by hybridization-based methods (e.g., PCR or nucleic acid sequencing, such as Illumina sequencing, Sanger sequencing, SMRT sequencing, etc.).

[0191] In some embodiments of any aspect, the method further includes the step of diagnosing the subject with Huntington's disease or at risk of developing Huntington's disease prior to administration.

[0192] In some embodiments of any aspect, the method further includes the step of receiving, prior to administration, the results of a assay that diagnoses a subject with Huntington's disease or at risk of developing Huntington's disease. Exemplary assays for diagnosing a subject with Huntington's disease or at risk of developing Huntington's disease are described herein, such as genetic screening for at least 36 CAG repeats, at least 40 CAG repeats, or at least 100 CAG repeats or more.

[0193] The “effective amount” of a substance is an amount sufficient to produce the desired effect. In some embodiments, the effective amount of isolated nucleic acid is an amount sufficient to transfect (or infect) a sufficient number of target cells in a subject’s target tissue in the context of rAAV-mediated delivery. In some embodiments, the target tissue is central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, the effective amount of isolated nucleic acid (e.g., which can be delivered via rAAV) may be an amount sufficient to have a therapeutic benefit in the subject, such as reducing the expression of a pathogenic gene or protein (e.g., HTT), prolonging the lifespan of the subject, improving symptoms of one or more diseases in the subject (e.g., symptoms of Huntington's disease), etc. The effective amount will depend on a variety of factors, such as the subject’s species, age, weight, health status, and the tissue to be targeted, and therefore, as described elsewhere in this disclosure, the effective amount may vary between subjects and tissues.

[0194] give

[0195] The rAAV disclosed herein can be delivered to a subject in a composition according to any suitable method known in the art. For example, rAAV preferably suspended in a physiologically compatible carrier (i.e., in the composition) can be administered to a subject (i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque)). In some embodiments, the host animal does not include humans.

[0196] Delivery of rAAV to mammalian subjects can be performed, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream can be achieved by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream via isolated limb perfusion, a technique well-known in the surgical field, which essentially allows a technician to isolate the limb from the systemic circulation before administering the rAAV viral particles. A technician can also use variations of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 to administer viral particles into the vascular system of the isolated limb to potentially enhance transduction into muscle cells or tissues. Furthermore, in some cases, delivery of viral particles to the subject's CNS may be desired. "CNS" refers to all cells and tissues of the brain and spinal cord in vertebrates. Therefore, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAV can be delivered directly to the CNS or brain using neurosurgical techniques known in the art (e.g., stereotactic injection) via injection into, for example, ventricular regions and the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobules using needles, catheters, or related devices (see, for example, Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, rAAV as described in this disclosure is administered via intravenous injection. In some embodiments, rAAV is administered via intracerebral injection. In some embodiments, the rAAV is administered via intrathecal injection. In some embodiments, the rAAV is administered via intrathecal injection. In some embodiments, the rAAV is delivered via intracranial injection. In some embodiments, the rAAV is delivered via injection into the cerebellomedullary cistern. In some embodiments, the rAAV is delivered via injection into the lateral ventricle.

[0197] This disclosure relates to compositions comprising recombinant AAV, said recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein said transgene comprises a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises the sequence described in any one of SEQ ID NO: 1-SEQ ID NO: 24. In some embodiments, said nucleic acid further comprises an AAV ITR. In some embodiments, said ITR is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10 ITR. In some embodiments, the composition also comprises a pharmaceutically acceptable vector. The compositions of this disclosure may comprise rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition comprises one, two, three, four, five, six, seven, eight, nine, ten, or more different rAAVs, each having one or more different transgenes.

[0198] Given the indications targeted by rAAV, those skilled in the art can readily select a suitable carrier. For example, a suitable carrier includes saline solution, which can be prepared with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of this disclosure.

[0199] Optionally, in addition to rAAV and the carrier, the compositions disclosed herein may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0200] Administer rAAV in sufficient quantities to transfect cells of the desired tissue and provide adequate levels of gene transfer and expression without excessive adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intravenous delivery to the liver), oral administration, inhalation (including intranasal and intratracheal delivery), intraocular administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, and other parenteral administration routes. Routes of administration may be combined if desired.

[0201] The dosage of rAAV viral particles required to achieve a specific "therapeutic effect" (e.g., dosage units in genome copies per kilogram of body weight (GC / kg)) will vary based on several factors, including but not limited to: the route of administration of the rAAV viral particles, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. Based on the above factors and other factors well known in the art, those skilled in the art can readily determine the dosage range of rAAV viral particles for treating patients with specific diseases or disorders.

[0202] An effective amount of rAAV is an amount sufficient to target and infect an animal and the desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health status of the subject, and the tissue to be targeted, and therefore can vary from animal to tissue. For example, the effective amount of rAAV is typically found in an amount containing about 10 9 One to 10 16 The range is approximately 1 mL to 100 mL of solution containing one genome copy. In some cases, approximately 10 11 One to 10 13 The dosage between rAAV genome copies is appropriate. In some implementations, 10 12 One or 10 13 One rAAV genome copy is effective in targeting CNS tissues. In some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0203] In some embodiments, the subject is given a dose of rAAV no more than once per calendar day (e.g., during a 24-hour period). In some embodiments, the subject is given a dose of rAAV no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the subject is given a dose of rAAV no more than once per calendar week (e.g., during 7 calendar days). In some embodiments, the subject is given a dose of rAAV no more than once every two weeks (e.g., once between two calendar weeks). In some embodiments, the subject is given a dose of rAAV no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, the subject is given a dose of rAAV no more than once every six calendar months. In some embodiments, the subject is given a dose of rAAV no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0204] In some embodiments, the rAAV composition is formulated to reduce the aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., -10).13 In cases of GC / mL or higher. Methods for reducing rAAV aggregation are well known in the art, including, for example, the addition of surfactants, pH adjustment, salt concentration adjustment, etc. (see, for example, Wright FR et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0205] Pharmaceutically acceptable excipient and carrier solutions are well known to those skilled in the art, as are suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens.

[0206] Typically, these dosage forms may contain at least about 0.1% or more of the active compound, although the percentage of the active ingredient can, of course, vary and can conveniently range from about 1% or 2% to about 70% or 80% or more of the total dosage form by weight or volume. Naturally, the amount of the active compound in each therapeutically useful composition can be prepared in such a manner that a suitable dose will be obtained at any given unit dose of the compound. Those skilled in the art in preparing such pharmaceutical dosage forms will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations; therefore, a variety of dosages and treatment regimens may be desirable.

[0207] In some cases, rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein may be delivered subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, administration methods as described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety) may be used to deliver rAAV. In some embodiments, a preferred mode of administration is via portal vein injection.

[0208] Suitable injectable drug forms include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In many cases, the form is sterile and fluid, to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms (e.g., bacteria and fungi). The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable fluidity can be maintained, for example, by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved by a variety of antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents (e.g., sugars or sodium chloride) will be preferred. The absorption of injectable compositions can be prolonged by using agents that delay absorption (such as aluminum monostearate and gelatin) in the composition.

[0209] For example, for the administration of injectable aqueous solutions, the solution may be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media that can be used in this regard will be known to those skilled in the art. For example, a dose may be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous perfusion fluid or injected at the recommended site of infusion (see, for example, Remington's Pharmaceutical Sciences, 15th edition, pp. 1035-1038 and 1570-1580). Some dosage variations will inevitably occur depending on the host's condition. In any case, the person responsible for administration will determine the appropriate dose for the individual host.

[0210] As needed, sterile injectable solutions are prepared by incorporating the desired amount of active rAAV with various other ingredients listed herein into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile medium containing those basic dispersion media listed above and other desired ingredients. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from their previously sterile filtered solutions.

[0211] The rAAV compositions disclosed herein can also be formulated into neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (forming with the free amino groups of proteins) and those formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., hydroxides of sodium, potassium, ammonium, calcium, or iron) and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. In formulation, the solution will be administered in a dosage form compatible with the dosage form and at a therapeutically effective amount. The dosage form is readily available for administration in various formulations (e.g., injectable solutions, drug-release capsules, etc.).

[0212] As used herein, "carrier" includes any and all solvents, dispersion media, media, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption-retarding agents, buffers, carrier solutions, suspending agents, colloids, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be incorporated into the compositions. The phrase "pharmaceutically acceptable" means a molecular entity and composition that does not produce an allergic reaction or similar adverse reaction when administered to a host.

[0213] Delivery media (e.g., liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc.) can be used to introduce the compositions of this disclosure into suitable host cells. In particular, rAAV vectors for delivering transgenes can be formulated for delivery encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0214] Such dosage forms are preferably used for pharmaceutically acceptable formulations of the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulating half-life have been developed (US Patent No. 5,741,516). Furthermore, various methods for using liposomes and liposome-like dosage forms as potential drug delivery carriers have been described (US Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0215] Liposomes have been successfully used with many cell types that are typically resistant to transfection by other procedures. Furthermore, liposomes are not limited by DNA length (as is typical in virus-based delivery systems). Liposomes have been effectively used to introduce genes, drugs, radiotherapy agents, viruses, transcription factors, and allosteric effectors into a wide variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to test the effectiveness of liposome-mediated drug delivery.

[0216] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayered concentric bilayered vesicles (also known as multilayered vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayered vesicles (SUVs) with diameters ranging from 200 Å to 500 Å, whose cores contain an aqueous solution.

[0217] Alternatively, rAAV nanocapsule formulations can be used. Nanocapsules typically capture substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, these ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that are biodegradable in vivo. Consider using biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements.

[0218] In addition to the delivery methods described above, the following technologies are considered as alternatives for delivering rAAV compositions to the host. Ultrasound delivery (i.e., ultrasound) has been used and is described in U.S. Patent No. 5,656,016 as a device to improve the rate and efficacy of drug penetration into and through the circulatory system. Other drug delivery alternatives considered include intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic dosage forms (Bourlais et al., 1998), transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0219] In some embodiments, the methods described herein involve treating a subject with or diagnosed with Huntington's disease using the nucleic acids described herein. Subjects with Huntington's disease can be identified by a physician using current methods for diagnosing Huntington's disease. Symptoms and / or complications of Huntington's disease that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, depression and anxiety, as well as characteristic movement disorders and chorea. Tests that may aid in the diagnosis of Huntington's disease include, but are not limited to, genetic testing. A family history of Huntington's disease also helps determine whether a subject is likely to have Huntington's disease or aids in the diagnosis of Huntington's disease.

[0220] The compositions and methods described herein can be administered to subjects who have or have been diagnosed with Huntington's disease. In some embodiments, the methods described herein include administering to a subject an effective amount of the composition described herein (e.g., a nucleic acid described herein) to alleviate symptoms of Huntington's disease. As used herein, "alleviate symptoms of Huntington's disease" means improvement of any symptom or condition associated with Huntington's disease. Such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique, compared to an equivalent untreated control.

[0221] Effective doses, toxicity, and therapeutic efficacy can be determined through standard pharmaceutical procedures in cell cultures or laboratory animals, for example, to determine the minimum effective dose and / or maximum tolerated dose. Dosage can vary depending on the dosage form used and the route of administration. The therapeutically effective dose can initially be estimated through cell culture assays. Furthermore, doses can be formulated in animal models to achieve a dose range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dose can be monitored through appropriate bioassays, such as assays for tumor growth and / or size. Dosage can be determined and adjusted (if necessary) by a physician to accommodate observed therapeutic effects.

[0222] This document provides in vitro and animal model assays that allow for the evaluation of a given dose of isolated nucleic acid (e.g., at least one of SEQ ID NO: 1-SEQ ID NO: 24) or a given dose of recombinant AAV (rAAV) containing isolated nucleic acid as described herein (see, for example, ...). Figure 4 , Figure 9 , Figure 15 As a non-limiting example, the efficacy of the dose of the isolated nucleic acid or rAAV described herein can be evaluated in vitro by any of the following methods: (1) co-transfecting the isolated nucleic acid (e.g., a plasmid containing at least one of SEQ ID NO: 1-SEQ ID NO: 24) and the target sequence (e.g., an HTT targeting sequence associated with luciferase expression) into cells (e.g., 293 cells) and measuring the target concentration and / or activity (see, for example, Figure 7 , Figure 8B , Figures 16-18 (2) Infecting cells (e.g., human nerve cells U87) with rAAV expressing isolated nucleic acids (e.g., at least one of SEQ ID NO: 1-SEQ ID NO: 24; e.g., operatively linked to a constitutive promoter, such as a CMV promoter) (e.g., AAVRH10) and rAAV expressing a target sequence (e.g., an HTT target sequence associated with luciferase expression), and measuring target concentration and / or activity (see, e.g., Figure 10 , Figures 11A-11B (3) Infect cells expressing HTT (e.g., human neural cells U87 or human lung fibroblasts from HD patients) with rAAV (e.g., AAVRH10) expressing isolated nucleic acids (e.g., at least one of SEQ ID NO: 1-SEQ ID NO: 24; e.g., operatively linked to a constitutive promoter, such as the CMV promoter), and measure the concentration and / or activity of HTT protein and / or mRNA (see e.g. Figures 12-14 , Figures 20-21 (4) Infecting cells expressing HTT (e.g., human neural cells U87 or human lung fibroblasts from HD patients) with rAAV (e.g., AAVRH10) expressing isolated nucleic acids (e.g., at least one of SEQ ID NO: 1-SEQ ID NO: 24; e.g., operably linked to a neuron-specific promoter (e.g., hSyn1), wherein the rAAV optionally further expresses a transgene encoding a protein, e.g., CYP46A1), and measuring the concentration and / or activity of HTT protein and / or mRNA (see, e.g., Figure 4 , Figure 9 , Figure 15 ).

[0223] The efficacy of the isolated nucleic acids or rAAVs described herein can also be evaluated in animal models, such as mouse models expressing HTT proteins. For example, the following HTT mouse models (see, for example, Table 3) can be used according to the desired HTT target: Hu128; B6CBA-R6 / 2 (CAG 120+ / -5); B6CBA-Tg(HDexon1) 62Gpb / 3J; B6CBA-R6 / 2 (CAG 160+ / -5); or B6CBA-Tg(HDexon1) 62Gpb / 1J. As a non-limiting example, mouse models as described herein were infected with an expression isolated nucleic acid (e.g., at least one of SEQ ID NO: 1-SEQ ID NO: 24; e.g., operably linked to a neuron-specific promoter (e.g., hSyn1), wherein the rAAV optionally further expresses a transgene encoding a protein, e.g., CYP46A1) (e.g., AAVRH10), and the concentration and / or activity of HTT protein and / or mRNA in the mice, and / or the pathogenesis of the disease, were measured. See, for example... Figure 4 , Figure 9 , Figure 15 .

[0224] carrier

[0225] In some embodiments, one or more miRNAs described herein are expressed in recombinant expression vectors or plasmids. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring transgenes into host cells. The term "vector" includes plasmids, mini-chromosomes, bacteriophages, naked DNA, etc. See, for example, U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and 5,919,670; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment is linked. Another type of vector is a viral vector, in which an additional DNA segment is linked into a viral genome. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attached mammalian vectors). Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "expression vectors". Typically, expression vectors used in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, this invention aims to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which provide equivalent functionality.

[0226] A cloning vector is a vector capable of autonomously replicating or integrating into the genome within a host cell, and is further characterized by one or more endonuclease restriction sites at which the vector can be cleaved in a deterministic manner, and whereby a desired DNA sequence can be ligated into the site, so that the new recombinant vector retains its ability to replicate within the host cell. In the case of plasmids, replication of the desired sequence can occur multiple times as the copy number of the plasmid increases within the host cell (e.g., host bacteria), or only once per host before the host reproduces through mitosis. In the case of bacteriophages, replication can occur actively during the lysis phase or passively during the lysogenic phase.

[0227] An expression vector is a vector into which a desired DNA sequence can be inserted by restriction and ligation, thereby operatively binding it to a regulatory sequence and expressing it as an RNA transcript. The vector may further comprise one or more marker sequences suitable for identifying cells transformed with or without the vector, or cells transfected with or without the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity can be detected by standards known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that significantly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In some embodiments, the vectors used herein are capable of autonomously replicating and expressing structural gene products present in the DNA segments to which they are operatively bound.

[0228] As used herein, when a coding sequence and a regulatory sequence are covalently linked in a manner that places the expression or transcription of the coding sequence under the influence or control of the regulatory sequence, they are referred to as “operably” joined. Two DNA sequences are referred to as operably joined if the desired translation of the coding sequence into a functional protein is desired, if the promoter induction in the 5' regulatory sequence causes transcription of the coding sequence, and if the nature of the connection between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, a promoter region is operably joined to a coding sequence if it can influence the transcription of a DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide.

[0229] When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, a variety of transcriptional control sequences (e.g., promoter / enhancer sequences) can be used to guide its expression. The promoter can be a natural promoter (i.e., the promoter of a gene in its endogenous environment), which provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive (i.e., the promoter is unregulated, allowing its associated gene to be continuously transcribed). A variety of conditional promoters (e.g., promoters controlled by the presence or absence of a molecule) can also be used.

[0230] The precise nature of the regulatory sequences required for gene expression can vary between species or cell types, but generally may include 5' non-transcriptional sequences and 5' non-translational sequences related to transcription initiation and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' non-transcriptional regulatory sequences will include promoter regions containing promoter sequences for transcriptional control of operatively conjugated genes. Depending on preference, the regulatory sequences may also include enhancer sequences or upstream activator sequences. The vectors of the present invention may optionally contain 5' leader or signal sequences. The selection and design of appropriate vectors are within the competence and judgment of those skilled in the art.

[0231] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into them. The heterologous DNA (RNA) is placed under the operative control of transcriptional elements to allow expression of the heterologous DNA in the host cell.

[0232] In some embodiments, the vector is adeno-associated virus (AAV) or recombinant AAV.

[0233] In some embodiments of any aspect, the vector is pEMBL. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-only intron. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x control pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-CYP46A1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-luc-HTT-3'UTR / mutant.

[0234] In some embodiments in any aspect, the vector or isolated nucleic acid as described herein comprises at least one of the following: at least one (e.g., two) ITR; a Syn1 promoter (see, for example, SEQ ID NO: 31-SEQ ID NO: 32); at least one (e.g., two) hCG introns (see, for example, SEQ ID NO: 34); at least one (e.g., two) copy of premiR (see, for example, SEQ ID NO: 35; for example, control pre-miR, artificial pre-miR; at least one of SEQ ID NO: 1-SEQ ID NO: 24); small polyA (see, for example, SEQ ID NO: 36); CYP46A1 (see, for example, SEQ ID NO: 26-SEQ ID NO: 27); luciferase (see, for example, SEQ ID NO: 28-SEQ ID NO: 29); and / or an HTT targeting sequence (see, for example, SEQ ID NO: 30), such as HTT-3'UTR / mutant). See, for example Figure 1 .

[0235] In some embodiments of any aspect, the isolated nucleic acid or vector described herein comprises CYP46A1. CYP46A1 is a member of the cytochrome P450 enzyme superfamily. Cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and the synthesis of cholesterol, steroids, and other lipids. This endoplasmic reticulum protein is expressed in the brain, where it converts cholesterol to 24S-hydroxycholesterol. Although cholesterol cannot cross the blood-brain barrier, 24S-hydroxycholesterol can be secreted in the brain into circulation and returned to the liver for catabolism. In some embodiments of any aspect, CYP46A1 may comprise human CYP46A1 (see, for example, NCBI ref number NG_007963.1RefSeqGene Range4881-47884; NM_006668.2; NP_006659.1; see, for example, SEQ ID NO: 26-SEQ ID NO: 27). CYP46A1 is the rate-limiting enzyme for cholesterol degradation and has neuroprotective effects in Huntington's disease (see, for example, Boussicault et al., CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease, Brain, March 2016, 139(Pt3):953-70; Kacher et al., CYP46A1 genetherapy deciphers the role of brain cholesterol metabolism in Huntington's disease, Brain, August 1, 2019; 142(8):2432-2450; the contents of each of these are incorporated herein by reference in their entirety).

[0236] In some embodiments in any aspect, the transgene described herein (e.g., CYP46A1) comprises SEQ ID NO: 27, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 27 and retains the same amino acid sequence as SEQ ID NO: 27 (e.g., a therapeutic protein for HD).

[0237] In some embodiments in any aspect, the transgene described herein (e.g., CYP46A1) is encoded by a nucleic acid sequence comprising: SEQ ID NO: 26, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 26 and retaining the same function or a codon-optimized form of SEQ ID NO: 26.

[0238] SEQ ID NO: 26, CYP46A1, 1503 nucleotides (nt) (see, for example, Homo sapiens cytochrome P450 family 46 subfamily A member 1 (CYP46A1), mRNA, NCBI reference sequence: NM_006668.2)

[0239]

[0240] SEQ ID NO: 27, CYP46A1, 500 amino acids (aa) (See, for example, cholesterol 24-hydroxylase precursor (Homo sapiens), NCBI reference sequence: NP_006659.1)

[0241]

[0242] In some implementations, one or more of the recombinantly expressed genes can be integrated into the cell's genome.

[0243] In some embodiments of any aspect, the isolated nucleic acid or vector as described herein contains at least one promoter. In some embodiments of any aspect, the promoter is the human Syn1 promoter. In some embodiments of any aspect, the promoter as described herein (e.g., Syn1) contains one of SEQ ID NO: 31-SEQ ID NO: 32, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of SEQ ID NO: 31-SEQ ID NO: 32 and retains a sequence with the same function (e.g., tissue-specific promoter) as one of SEQ ID NO: 31-SEQ ID NO: 32.

[0244] SEQ ID NO: 31, Syn1 promoter, 477nt

[0245]

[0246] SEQ ID NO: 32, Syn1 promoter, 448nt

[0247]

[0248] In some embodiments of any aspect, the promoter is a constitutive CMV promoter. In some embodiments of any aspect, the promoter (e.g., CMV) described herein comprises SEQ ID NO: 33, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 33 and retaining the same function as SEQ ID NO: 33 (e.g., constitutive promoter).

[0249] SEQ ID NO: 33, CMV promoter

[0250]

[0251] In some embodiments of any aspect, the isolated nucleic acid or vector as described herein comprises at least one intron. In some embodiments of any aspect, the intron is an hCG intron. In some embodiments of any aspect, the intron described herein (e.g., an hCG intron) comprises SEQ ID NO: 34, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 34 and retaining the same function as SEQ ID NO: 34. In some embodiments of any aspect, the isolated nucleic acid or vector as described herein comprises at least one (e.g., two) portions of SEQ ID NO: 34, such as approximately nucleotides 1-100, nucleotides 16-100, nucleotides 114-635, and / or nucleotides 122-635 of SEQ ID NO: 34.

[0252] SEQ ID NO: 34, hCG intron, 635nt (see, for example, the 6β subunit of the human chorionic gonadotropin (HCG) gene, GenBank: X00266.1)

[0253]

[0254] In some embodiments of any aspect, the isolated nucleic acid or vector as described herein comprises a premiR, which comprises two copies of an artificial miRNA as described herein (e.g., one of SEQ ID NO: 1-SEQ ID NO: 24). In some embodiments of any aspect, the premiR as described herein comprises SEQ ID NO: 35, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 35 and maintaining the same function as SEQ ID NO: 35 (e.g., expression of at least one miRNA). In some embodiments of any aspect, the isolated nucleic acid or vector as described herein contains a portion of SEQ ID NO: 35, for example, such that a copy of miRNA is expressed, such as approximately nt 1-191, nt 1-199, nt 191-386, or nt 199-386 of SEQ ID NO: 35.

[0255] SEQ ID NO: 35, 2x miHTT-H1; bold text indicates 5' flanking sequences (e.g., nt 1-65 or nt 199-260 of SEQ ID NO: 35); italic text indicates miHTT followers (e.g., nt 69-87 or nt 264-282 of SEQ ID NO: 35; see, for example, the reverse complement of SEQ ID NO: 1 used as a non-limiting example; reverse complements of any of SEQ ID NO: 2-SEQ ID NO: 24 may be used in place of the reverse complement of SEQ ID NO: 1 in SEQ ID NO: 35); bold italic text indicates miR30a rings (e.g., nt 90-104 or nt 285-299 of SEQ ID NO: 35); italic double-underlined text indicates miHTT guide chains (e.g., nt 107-126 or nt 302-321 of SEQ ID NO: 35; see, for example, SEQ ID NO: 1 used as a non-limiting example; SEQ ID Any of NO: 2-SEQ ID NO: 24 can be used in place of SEQ ID NO: 1 in SEQ ID NO: 35; and the bold double-underlined text indicates the 3' flanking sequence (e.g. nt130-191 or nt325-386 of SEQ ID NO: 35).

[0256]

[0257] In some embodiments of any aspect, the isolated nucleic acid or vector as described herein contains a polyadenylated region (e.g., a small polyA). In some embodiments of any aspect, the polyA as described herein contains SEQ ID NO: 36, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 36 and retaining the same function (e.g., polyadenylation) as SEQ ID NO: 36.

[0258] SEQ ID NO: 36, small polyA, 54nt,

[0259]

[0260] In some embodiments of any aspect, the isolated nucleic acid or vector (e.g., for testing miRNAs as described herein) contains luciferase as a reporter. In some embodiments of any aspect, the reporter (e.g., luciferase) described herein contains SEQ ID NO: 29, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 29 and retains the same amino acid sequence as SEQ ID NO: 29 (e.g., luciferase activity and luminescence).

[0261] In some embodiments in any aspect, the reporter (e.g., luciferase) described herein is encoded by a nucleic acid sequence comprising: SEQ ID NO: 28, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 28 and retaining the same function or an optimized form of the codons of SEQ ID NO: 28.

[0262] SEQ ID NO: 28, luciferase nucleic acid, 1653nt

[0263]

[0264] SEQ ID NO: 29, luciferase protein, 550 aa

[0265]

[0266]

[0267] In some embodiments of any aspect, the isolated nucleic acid or vector (e.g., for testing miRNA as described herein) comprises an HTT targeting sequence (i.e., a short nucleic acid sequence from the HTT gene). In some embodiments of any aspect, the HTT targeting sequence comprises SEQ ID NO: 30, or a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 30 and retaining the same function as SEQ ID NO: 30 (e.g., targeting or testing miRNA as described herein).

[0268] SEQ ID NO: 30, HTT-targeted seq (e.g., HTT-3'UTR / mutant), 417nt

[0269]

[0270] Nucleic acid molecules encoding the enzyme of the claimed invention can be introduced into one or more cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced using standard protocols, such as transformation by chemical transformation and electroporation, transduction, particle bombardment, etc. Expression of nucleic acid molecules encoding the enzyme of the claimed invention can also be achieved by integrating said nucleic acid molecules into the genome.

[0271] Reagent test kit

[0272] In some embodiments, the reagents described herein may be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. Kits may include one or more containers containing the components of this disclosure, along with instructions for use. Specifically, such kits may contain one or more reagents described herein, along with instructions describing the intended applications and proper use of these reagents. In some embodiments, the reagents in the kit may be pharmaceutical dosage forms and dosages suitable for a particular application and method of reagent administration. Kits for research purposes may contain appropriate concentrations or amounts of components to run a variety of experiments.

[0273] In some embodiments, this disclosure relates to a kit for generating rAAV, the kit comprising a container holding isolated nucleic acids comprising miRNAs, the miRNAs comprising or encoding a sequence as described in any one of SEQ ID NO: 1-SEQ ID NO: 24. In some embodiments, the kit further comprises a container holding isolated nucleic acids encoding an AAV capsid protein (e.g., AAV9 or AAVrh10 capsid protein).

[0274] The kit may be designed to facilitate use by researchers using the methods described herein, and the kit may take many forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions may be constructible or otherwise processable (e.g., forming an active form), for example by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, “instructions” may define the components that are described and / or promoted, and generally refers to written instructions on or associated with the packaging of this disclosure. Instructions may also include any oral or electronic instructions provided in any way that would make it clear to the user that the instructions are associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet and / or web-based communications, etc. Written instructions may be in the form prescribed by a government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, and may also reflect approval from the agency regarding the manufacture, use, or sale in animal contexts.

[0275] The kit may contain one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or separating and mixing samples, as well as instructions for application to a subject. The kit may include a container holding the reagents described herein. The reagents may be in liquid, gel, or solid (powder) form. The reagents may be aseptically prepared, packaged in syringes, and transported under refrigeration. Alternatively, it may be contained in vials or other containers for storage. A second container may contain other aseptically prepared reagents. Alternatively, the kit may include pre-mixed active agents transported in syringes, vials, tubes, or other containers.

[0276] Exemplary embodiments of the present invention will be described in more detail through the following examples. These embodiments are examples of the present invention, and those skilled in the art will recognize that the present invention is not limited to the exemplary embodiments described.

[0277] Immunomodulators

[0278] In some embodiments, the methods and compositions described herein further include administration of an immunomodulator. In some embodiments, the immunomodulator may be administered at, before, or after administration. In cases where the subject is re-administered with at least a second composition, the immunomodulator may be administered before, simultaneously with, or after the at least second administration.

[0279] In some embodiments, the immunomodulator is an immunoglobulin-degrading enzyme, such as IdeS, IdeZ, IdeS / Z, Endo S, or functional variants thereof. Non-limiting examples of such immunoglobulin-degrading enzymes and their uses are described in references to US 7,666,582, US 8,133,483, US 20180037962, US 20180023070, US20170209550, US 8,889,128, WO2010 / 057626, US 9,707,279, US 8,323,908, US20190345533, US 20190262434, and WO2020 / 016318, each of which is incorporated herein by reference in its entirety.

[0280] In some embodiments, the immunomodulator is a proteasome inhibitor. In some aspects, the proteasome inhibitor is bortezomib. In some aspects of the embodiments, the immunomodulator includes bortezomib and the anti-CD20 antibody rituximab. In other aspects of the embodiments, the immunomodulator includes bortezomib, rituximab, methotrexate, and intravenous immunoglobulin. Non-limiting examples of such references disclosing proteasome inhibitors and their combinations with rituximab, methotrexate, and intravenous immunoglobulin are described in US 10,028,993, US 9,592,247, and US 8,809,282, each of which is incorporated herein by reference in its entirety.

[0281] In alternative embodiments, the immunomodulator is an inhibitor of the NF-κB pathway. In some aspects of the embodiments, the immunomodulator is rapamycin or a functional variant. Non-limiting examples of the following references disclosing rapamycin and its uses are incorporated herein by reference in their entirety: US 10,071,114, US 20160067228, US 20160074531, US 20160074532, US 20190076458, US 10,046,064. In other aspects of the embodiments, the immunomodulator is a synthetic nanocarrier comprising an immunosuppressant. Non-limiting examples of references to immunosuppressants, immunosuppressants conjugated with synthetic nanocarriers, synthetic nanocarriers containing rapamycin and / or tolerogenic synthetic nanocarriers, their dosages, administration and uses are found in US20150320728, US 20180193482, US20190142974, US 20150328333, US20160243253, US 10,039,822, US 20190076522, US20160022650, US 10,441,651, US 10,420,835, US 20150320870, US 2014035636, US 10,434,088, US 10,335,395, US The following are described in US 20200069659, US 10,357,483, US 20140335186, US 10,668,053, US 10,357,482, US 20160128986, US 20160128987, US 20200038462, and US 20200038463, and each is incorporated herein by reference in its entirety.

[0282] In some embodiments, the immunomodulator is a synthetic nanocarrier (ImmTOR) containing rapamycin. TM Nanoparticles (Kishimoto et al., 2016, Nat Nanotechnol, 11(10):890-899; Maldonado et al., 2015, PNAS, 112(2):E156-165), as disclosed in US20200038463 and US Patent 9,006,254, are each incorporated herein in their entirety. In some embodiments, the immunomodulator is an engineered cell, such as an immune cell modified using the SQZ technology disclosed in WO2017192786 (which is incorporated herein by reference in its entirety).

[0283] In some embodiments, the immunomodulator is selected from the group consisting of: poly-ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, resiquimod, SRL172, viriosomes and other virus-like particles, YF-17D, VEGF Trap, R848, β-glucan, Pam3Cys, and QS21 stimulants of Aquila. In another further embodiment, the immunomodulator or adjuvant is poly-ICLC.

[0284] In some embodiments, the immunomodulator is a small molecule that inhibits the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and 2-aminopurine (a PKR inhibitor), and may also be administered in combination with a composition comprising at least one rAAV as disclosed herein. Some non-limiting examples of commercially available TLR signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available from INVIVOGEN). TM (Purchase). In addition, inhibitors of pattern recognition receptors (PRRs) (which are involved in innate immune signal transduction), such as 2-aminopurine, BX795, chloroquine, and H-89, may also be used in compositions and methods comprising at least one rAAV vector as disclosed herein for in vivo protein expression as disclosed herein.

[0285] In some embodiments, the rAAV vector having a modified viral capsid may also encode a negative regulator of innate immunity (e.g., NLRX1). Therefore, in some embodiments, the rAAV vector may optionally encode one or more, or any combination of, NLRX1, NS1, NS3 / 4A, or A46R. Furthermore, in some embodiments, compositions comprising at least one rAAV vector as disclosed herein may also contain a synthetic, modified RNA-encoded inhibitor of the innate immune system to avoid an innate immune response generated by tissues or a subject.

[0286] In some embodiments, the immunomodulator used in the administration methods disclosed herein is an immunosuppressant. As used herein, the term "immunosuppressive drug or agent" is intended to include agents that inhibit or interfere with normal immune function. Examples of immunosuppressants suitable for use with the methods disclosed herein include agents that inhibit the T-cell / B-cell co-stimulatory pathway (e.g., agents that interfere with T-cell and B-cell coupling via the CTLA4 and B7 pathways), as disclosed in U.S. Patent Publication No. 2002 / 0182211. In one embodiment, the immunosuppressant is cyclosporine A. Other examples include myophenylate mofetil, rapamycin, and anti-thymocyte globulin. In one embodiment, the immunosuppressive drug is administered in a composition comprising at least one rAAV carrier as disclosed herein, or may be administered as a single composition, but concurrently with, before, or after administration of a composition comprising at least one rAAV carrier according to the administration methods disclosed herein. The immunosuppressive drug is administered in a formulation compatible with the route of administration and at a dose sufficient to achieve the desired therapeutic effect to the subject. In some embodiments, the immunosuppressive drug is administered transiently for a sufficient duration to induce tolerance to the rAAV vector disclosed herein.

[0287] In any embodiment of the methods and compositions disclosed herein, the subject given the disclosed composition is also given an immunosuppressant. Various methods are known to induce immunosuppression of the immune response in patients given AAV. Methods known in the art include administering an immunosuppressant (e.g., a proteasome inhibitor) to a patient. One such proteasome inhibitor known in the art is bortezomib, disclosed, for example, in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15 / 796,137, both of which are incorporated herein by reference. In some embodiments, the immunosuppressant may be an antibody, including polyclonal, monoclonal, scfv, or other antibody-derived molecules capable of suppressing an immune response, for example, by eliminating or suppressing antibody-producing cells. In a further embodiment, the immunosuppressive element may be a short hairpin RNA (shRNA). In such embodiments, the coding region of the shRNA is contained within the rAAV cassette and is typically located downstream, at the 3' of the poly-A tail. shRNA may be targeted to reduce or eliminate the expression of immunostimulants, such as cytokines, growth factors (including transforming growth factor β1 and β2, TNF, and other publicly known immunostimulants)

[0288] The use of such immunomodulatory agents has facilitated the ability to administer multiple doses (e.g., multiple administrations) over months and / or years. This allows for the use of a variety of agents, such as rAAV vectors encoding multiple genes, or multiple administrations to subjects.

[0289] Exemplary embodiments of the present invention will be described in more detail through the following examples. These embodiments are examples of the present invention, and those skilled in the art will recognize that the present invention is not limited to the exemplary embodiments described.

[0290] definition

[0291] For convenience, the following provides the meanings of some terms and phrases used in this specification, embodiments, and appended claims. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. 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. If there is a significant difference between the use of terms in the art and their definitions provided herein, the definitions provided in this specification shall prevail.

[0292] For convenience, certain terms used herein, including those in this specification, embodiments, and appended claims, are collected herein.

[0293] The terms “decrease,” “reduced / reduction,” or “inhibit” are used herein to indicate a statistically significant reduction. In some embodiments, “decrease,” “reduction,” or “inhibit” generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “decrease” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction can preferably be lowered to a level that is acceptable as a normal range for individuals without a given disorder.

[0294] The terms “increased / increase,” “enhance,” or “activate” are used herein to refer to an increase of a statistically significant amount. In some implementations, the terms “increased,” “enhance,” or “activate” may indicate an increase of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% increase, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level. In the context of a biomarker or symptom, “increase” is a statistically significant increase with respect to that level.

[0295] As used herein, “subject” refers to a person or animal. Generally, animals are vertebrates, such as primates, rodents, livestock, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate (e.g., a person). The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0296] Preferably, the subject is a mammal. The mammal may be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans may be advantageously used as subjects representing an animal model of Huntington's disease. The subject may be male or female.

[0297] Subjects may be subjects who have been previously diagnosed with or identified as suffering from or having a condition (such as Huntington's disease) or one or more complications associated with such condition, and optionally have undergone treatment for Huntington's disease or one or more complications associated with Huntington's disease. Alternatively, subjects may be subjects who have not been previously diagnosed with Huntington's disease or one or more complications associated with Huntington's disease. For example, subjects may be subjects who exhibit one or more risk factors for Huntington's disease or one or more complications associated with Huntington's disease, or subjects who do not exhibit risk factors.

[0298] "Subjects in need of treatment for a specific condition" may be subjects who have the condition, have been diagnosed with the condition, or are at risk of developing the condition.

[0299] As used herein, the terms “protein” and “peptide” are used interchangeably to refer to a series of amino acid residues linked together by peptide bonds between adjacent α-amino and carboxyl groups. The terms “protein” and “peptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. “Protein” and “peptide” are generally used to refer to relatively large polypeptides, while the term “peptide” is generally used to refer to small polypeptides, but the usage of these terms overlaps in the art. When referring to gene products and fragments thereof, the terms “protein” and “peptide” are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologues, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs described above.

[0300] The variant amino acid or DNA sequence may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identity with the natural or reference sequence. For example, the degree of homology (percentage of identity) between the natural and mutant sequences can be determined by comparing the two sequences using commonly available, free computer programs (such as BLASTp or BLASTn with default settings) on the World Wide Web for this purpose.

[0301] Alterations to the natural amino acid sequence can be achieved using any of a variety of techniques known to those skilled in the art. For example, a mutation can be introduced at a specific locus by synthesizing an oligonucleotide containing the mutated sequence and flanking it with restrictive sites that allow it to be linked to a fragment of the natural sequence. After linking, the resulting reconstructed sequence encodes an analogue with the desired amino acid insertion, substitution, or deletion. Alternatively, site-specific mutagenesis procedures directed by oligonucleotides can be used to provide altered nucleotide sequences with specific codons changed according to the desired substitution, deletion, or insertion. Techniques for making such modifications are well-established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981) and U.S. Patents 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine ​​residue that does not participate in maintaining the correct conformation of the polypeptide may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, one or more cysteine ​​bonds may be added to the polypeptide to improve its stability or promote oligomerization.

[0302] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or the like. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be one strand of denatured double-stranded DNA. Alternatively, it may be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid may be DNA. In another aspect, the nucleic acid may be RNA. Suitable DNA may include, for example, genomic DNA or cDNA. Suitable RNA may include, for example, mRNA or miRNA.

[0303] In some embodiments of any aspect, peptides, nucleic acids, or cells as described herein may be engineered. As used herein, “engineered” refers to an aspect that has been manipulated by human hand. For example, a peptide is considered “engineered” when at least one aspect of it (e.g., its sequence) has been manipulated by human hand to differ from aspects that exist in nature. As is customary and as understood by those skilled in the art, progeny of engineered cells are generally still referred to as “engineered” even if the actual manipulation was performed on a previous entity.

[0304] In some embodiments of any aspect, the miRNA described herein is exogenous. In some embodiments of any aspect, the miRNA described herein is ectopic. In some embodiments of any aspect, the miRNA described herein is non-endogenous.

[0305] The term “exogenous” refers to a substance present in a cell that is not of its natural origin. When used herein, “exogenous” can refer to nucleic acids (e.g., nucleic acids encoding polypeptides) or polypeptides that have been introduced into a biological system (e.g., a cell or organism) in which they are not normally present through a human-assisted process, and it is desirable to introduce said nucleic acids or polypeptides into such cells or organisms. Alternatively, “exogenous” can refer to nucleic acids or polypeptides that have been introduced into a biological system (e.g., a cell or organism) in which they are present in relatively low amounts through a human-assisted process, and it is desirable to increase the amount of said nucleic acids or polypeptides in that cell or organism (e.g., to produce ectopic expression or levels). Conversely, the term “endogenous” refers to a substance native to a biological system or cell. As used herein, “ectopic” refers to a substance present in an unusual location and / or amount. Ectopic substances can be substances normally present in a given cell, but in much smaller amounts and / or present at different times. Ectopic also includes substances, such as polypeptides or nucleic acids that are not naturally present or expressed in a given cell in its natural environment.

[0306] In some embodiments, nucleic acids encoding repressive RNAs as described herein (e.g., SEQ ID NO: 1-SEQ ID NO: 24) are contained in a vector. In some aspects described herein, nucleic acid sequences encoding a given polypeptide or any module thereof as described herein are operatively ligated to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, vectors may be viral or non-viral. The term "vector" includes any genetic element capable of replication and transfer of a gene sequence into a cell when associated with appropriate control elements. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, viral particles, etc.

[0307] In some embodiments of any aspect, the vector is recombinant (e.g., it contains sequences derived from at least two different sources). In some embodiments of any aspect, the vector contains sequences derived from at least two different species. In some embodiments of any aspect, the vector contains sequences derived from at least two different genes, for example, it contains a fusion protein or a nucleic acid encoding an expression product, said nucleic acid being operatively linked to at least one non-natural (e.g., heterologous) genetic control element (e.g., promoter, repressor, activator, enhancer, response element, etc.).

[0308] In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, for example, the natural or wild-type sequence of the nucleic acid has been altered or engineered to include alternative codons, such that the altered or engineered nucleic acid encodes a polypeptide expression product identical to the natural / wild-type sequence, but will be transcribed and / or translated with improved efficiency in the desired expression system. In some embodiments of any aspect, the expression system is an organism (or cells obtained from such an organism) other than the source of the natural / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells (e.g., mouse, rodent, or human cells). In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in Escherichia coli (E. coli) cells.

[0309] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptides by a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is typically, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms (e.g., for expression in human cells and for cloning and amplification in a prokaryotic host).

[0310] As used herein, the term "viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding polypeptides as described herein, replacing non-essential viral genes. The vectors and / or particles can be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Various forms of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenovirus vectors, lentiviral vectors, retroviral vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0311] It should be understood that, in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vectors are additive. Using suitable additive vectors provides a method for maintaining high copy numbers of extrachromosomal DNA of the nucleotides of interest in a subject, thereby eliminating the potential impact of chromosomal integration.

[0312] As used herein, the terms “treat / treatment / treating” or “amelioration” refer to therapeutic treatment aimed at reversing, reducing, alleviating, suppressing, slowing, or stopping the progression or severity of symptoms associated with a disease or disorder (e.g., Huntington's disease). The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with Huntington's disease. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of the disease is reduced or stopped. That is, “treatment” includes not only improvement in symptoms or markers but also the cessation or at least slowing of the progression or worsening of symptoms compared to the expected outcome without treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, relieving or mitigating the disease state, reduction (whether partial or complete) and / or decrease in mortality, whether detectable or undetectable. The term “treatment” for a disease also includes providing relief (including palliative care) for the symptoms or side effects of the disease.

[0313] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be an artificial or engineered carrier, such as a carrier that does not exist in nature.

[0314] As used herein, the term "administration" means placing a compound disclosed herein into a subject by a method or route that causes at least partial delivery of the agent to a desired site. Pharmaceutical compositions comprising compounds disclosed herein may be administered via any appropriate route that produces an effective treatment in a subject. In some embodiments, administration includes physical activities of a person, such as injection, ingestion, application, and / or operation of a delivery device or machine. Such activities may be performed, for example, by a medical professional and / or the subject being treated.

[0315] As used herein, “contact” means any suitable manner for delivering or exposing a drug to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture media, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, contact includes human physical activities (e.g., injection; dispensing, mixing, and / or decanting); and / or operating a delivery device or machine.

[0316] The terms “statistically significant” or “significantly” refer to statistical significance and usually mean a difference of two standard deviations (2SD) or greater.

[0317] Except in operational examples or where otherwise specified, all figures used herein to indicate the amounts of components or reaction conditions should be understood to be modified by the term "about" in all cases. When used in conjunction with percentages, the term "about" may mean ±1%.

[0318] As used herein, the term "includes / contains" means that other elements may exist in addition to those defined in the present document. "Includes / contains" is used to indicate inclusion, not limitation.

[0319] The term "composed of" refers to the compositions, methods and their respective components as described herein, excluding any elements not listed in the description of the embodiments.

[0320] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements that do not substantially affect the basic, novel, or functional characteristics of that embodiment of the invention.

[0321] As used herein, the term "corresponding to" means an amino acid or nucleotide at a position listed in the first polypeptide or nucleic acid, or an amino acid or nucleotide equivalent to an amino acid or nucleotide listed in the second polypeptide or nucleic acid. Equivalent listed amino acids or nucleotides can be determined by comparing candidate sequences using homology procedures known in the art (e.g., BLAST).

[0322] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, wherein a first entity binds to a second target entity with greater specificity and affinity than it binds to a third, non-target entity. In some embodiments, specific binding may mean that the affinity of the first entity for the second target entity is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or greater than its affinity for the third, non-target entity. A reagent specific to a given target is a reagent that exhibits specific binding to that target under the assay conditions used.

[0323] Unless the context clearly indicates otherwise, the singular terms “a / an” and “the” include the plural objects referred to. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and”. Although methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation “eg” originates from the Latin *exempli gratia* and is used herein to denote a non-limiting instance. Therefore, the abbreviation “eg” is synonymous with the term “for example”.

[0324] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0325] Unless otherwise defined herein, the scientific and technical terms used in connection with this application should have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, and therefore can be varied. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (Eds.), WWNorton&Company, 2016 (ISBN0815345054, 978-0815345053); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (editor) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (editor), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (editor), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Colligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737); WO 2018 / 057855A; US 10,457,940; their contents are incorporated herein by reference in their entirety.

[0326] In any of the embodiments described herein, the disclosure does not relate to processes for cloning humans, processes for modifying the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic characteristics of animals that may cause suffering to animals and have no substantial medical benefit to humans or animals, and the animals produced by such processes.

[0327] Other terms are defined within the description of various aspects of this invention herein.

[0328] For purposes of description and disclosure, all patents and other publications (including references, granted patents, published patent applications, and co-pending patent applications) cited herein in their entirety are expressly incorporated herein by reference, for example, the methodologies described in such publications may be used in conjunction with the techniques described herein. These publications are provided only because they were published prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventor was not entitled to such prior disclosure by virtue of a prior invention or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0329] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order or may perform functions substantially simultaneously. The teachings of this disclosure provided herein can be suitably applied to other procedures or methods. The various embodiments described herein may be combined to provide further embodiments. If desired, aspects of this disclosure may be modified to incorporate the components, functions, and concepts of the foregoing references and applications to provide further embodiments of this disclosure. These and other changes may be made to this disclosure based on the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0330] Specific elements of any of the foregoing embodiments may be combined with or substituted for elements in other embodiments. Furthermore, while advantages relating to certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of this disclosure.

[0331] The techniques described herein are further illustrated by the following embodiments, which should in no way be construed as making further limitations.

[0332] Some implementations of the techniques described herein can be defined according to any of the following numbered paragraphs:

[0333] 1. An isolated nucleic acid, said isolated nucleic acid comprising:

[0334] a. A first region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and

[0335] b. A second region comprising a transgene encoding one or more miRNAs, wherein each miRNA comprises a seed sequence complementary to SEQ ID NO: 25.

[0336] 2. An isolated nucleic acid, said isolated nucleic acid comprising:

[0337] a. A first region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and

[0338] b. A second region comprising a transgene encoding one or more miRNAs, wherein each miRNA is encoded by a sequence comprising any one of SEQ ID NO: 1-SEQ ID NO: 22 with a miRNA backbone sequence side-attached.

[0339] 3. The isolated nucleic acid as described in paragraph 1 or 2, wherein the transgene comprises two miRNAs or two precursor miRNAs in tandem, the two miRNAs or two precursor miRNAs being flanked by introns.

[0340] 4. The isolated nucleic acid as described in paragraph 3, wherein the side-connected introns are identical.

[0341] 5. The isolated nucleic acid as described in paragraph 3, wherein the side-connected introns are from the same species.

[0342] 6. The isolated nucleic acid as described in paragraph 3, wherein the side-connected intron is an hCG intron.

[0343] 7. The isolated nucleic acid as described in any of paragraphs 1-6, wherein the transgene contains a promoter.

[0344] 8. The isolated nucleic acid as described in paragraph 7, wherein the promoter is the synaptic protein (Syn1) promoter.

[0345] 9. The isolated nucleic acid as described in any of paragraphs 1-8, wherein the transgene further encodes a protein.

[0346] 10. The isolated nucleic acid as described in paragraph 9, wherein the protein is CYP46A1.

[0347] 11. The isolated nucleic acid as described in any of paragraphs 1-10, wherein the one or more miRNAs are located in the untranslated portion of the transgene.

[0348] 12. The isolated nucleic acid as described in paragraph 11, wherein the untranslated portion is an intron.

[0349] 13. The isolated nucleic acid as described in paragraph 11, wherein the untranslated portion is located between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence, or between the last nucleotide base of the promoter sequence and the poly-A tail sequence.

[0350] 14. The isolated nucleic acid as described in any of paragraphs 1-13, wherein the isolated nucleic acid further comprises a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

[0351] 15. The isolated nucleic acid as described in any of paragraphs 1-14, wherein the ITR variant lacks a functional terminal resolution site (TRS), optionally wherein the ITR variant is an ATRS ITR.

[0352] 16. The isolated nucleic acid as described in any of paragraphs 1-15, wherein at least one of the miRNAs hybridizes with human huntingtin protein (e.g., SEQ ID NO: 25) and inhibits the expression of human huntingtin protein (e.g., SEQ ID NO: 25).

[0353] 17. A vector comprising isolated nucleic acids as described in any one of paragraphs 1-16.

[0354] 18. The vector as described in paragraph 17, wherein the vector is a plasmid.

[0355] 19. A host cell comprising isolated nucleic acid as described in any one of paragraphs 1-16 or a vector as described in paragraph 17 or 18.

[0356] 20. A recombinant AAV (rAAV), said recombinant AAV comprising:

[0357] a. capsid protein; and

[0358] b. Nucleic acids isolated as described in any of paragraphs 1-16.

[0359] 21. rAAV as described in paragraph 20, wherein the capsid protein is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13, or AAVrh10 capsid protein, or any chimera thereof.

[0360] 22. rAAV as described in paragraph 20 or 21, wherein the capsid protein is AAVrh10 capsid protein.

[0361] 23. The rAAV as described in any of paragraphs 20-22, wherein the rAAV is a self-complementary AAV (scAAV).

[0362] 24. The rAAV as described in any of paragraphs 20-22, wherein the rAAV is formulated for delivery to the central nervous system (CNS).

[0363] 25. An isolated nucleic acid, said isolated nucleic acid encoding any one of the sequences described in SEQ ID NO: 1-SEQ ID NO: 22.

[0364] 26. A composition comprising isolated nucleic acids as described in any one of paragraphs 1-16.

[0365] 27. A composition comprising a carrier as described in paragraph 17 or 18.

[0366] 28. A composition comprising rAAV as described in any one of paragraphs 20-24.

[0367] 29. A method for treating Huntington's disease in a subject in need, the method comprising administering to a subject who has Huntington's disease or is at risk of developing Huntington's disease a therapeutically effective amount of an isolated nucleic acid as described in any of paragraphs 1-16, rAAV as described in any of paragraphs 20-24, or a composition as described in any of paragraphs 25-28.

[0368] 30. The method as described in paragraph 29, wherein the subject comprises a huntingtin protein gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

[0369] 31. The method as described in paragraph 29 or 30, wherein the subject is less than 20 years old.

[0370] 32. The method as described in any of paragraphs 29-31, wherein the administration causes the isolated nucleic acid or rAAV to be delivered to the central nervous system (CNS) of the subject.

[0371] 33. The method as described in any of paragraphs 29-32, wherein the administration is performed by injection, optionally intravenous injection or intrastriatal injection.

[0372] 34. The method as described in any of paragraphs 29-33, wherein the administration is performed via a catheter or related device.

[0373] 35. The method as described in paragraph 34, the method further comprising the step of diagnosing the subject as having Huntington's disease or at risk of developing Huntington's disease prior to administration.

[0374] 36. The method as described in paragraph 34, the method further comprising the step of receiving, prior to administration, the results of a assay that diagnoses the subject with Huntington's disease or is at risk of developing Huntington's disease.

[0375] Example

[0376] Example 1

[0377] One aspect described herein is a repressive RNA that can be used to treat Huntington's disease. In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with at least one of SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9 and retaining the same function (e.g., HTT inhibition) as SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9.

[0378] This article describes constructs containing artificial miRNAs (see example...). Figure 1The pEMBL-D(+)-Syn1-hCG intron is a control vector containing a blank human chorionic gonadotropin (hCG) intron (hCGin; see, for example, SEQ ID NO: 34) and driven by a synaptic protein promoter (see, for example, SEQ ID NO: 31-SEQ ID NO: 32). Two copies of a control miRNA precursor (random sequence or non-functional mutation) are inserted into the hCGin in the vector pEMBL-D(+)-Syn1-hCGin-2x control pre-miR. Two copies of an artificial pre-miR (see, for example, SEQ ID NO: 35; perfectly matched to the 3'-UTR targeting sequence, containing approximately 100-150 bp flanked upstream and downstream sequences) are cloned between the hCG introns. Two copies of the artificial miRNA sequence are inserted into the human chorionic gonadotropin (hCG) intron, which cleaves the insert to form a precursor of the miRNA. The pre-miRNA is a precursor with a hairpin loop construct. Pre-miRNAs are translated into the cytoplasm with the aid of export protein 5 (Exp5) and Ran-GTP. These miRNA precursors are further processed into mature miRNAs with the help of the nuclear enzyme Drosha and the ribonuclease III of Dicer in the cytoplasm (e.g., Dicer cleaves the precursors into mature miRNAs, which can be approximately 20-22 bp). The vector pEMBL-D(+)-Syn1-CYP46A1-hCGin-2x artificial pre-miRNA is a combo construct that can simultaneously generate CYP46A1 and artificial miRNA. To determine whether the pre-miRNA can be processed into mature miRNAs and combined with an HTT targeting sequence containing CAG amplification (which is perfectly complementary to mature miRNAs), the HTT targeting sequence is inserted after the luciferase gene. Due to packaging size limitations, a small poly A is used in the construct.

[0379] The following sequences are known in the art: pEMBL; synaptic promoter (Syn1); ITR (e.g., from AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10); hCG intron; small polyA; CYP46A1; luciferase; and / or HTT targeting sequence (e.g., HTT-3'UTR / mutant).

[0380] Synaptic protein-1 (Syn1) is a member of the synaptic protein gene family. Synaptic proteins encode neuronal phosphoproteins that are associated with the cytoplasmic surface of synaptic vesicles. Family members are characterized by common protein domains, and they are involved in the regulation of synapsis and neurotransmitter release, suggesting potential roles in several neuropsychiatric disorders. Syn1 plays a role in the regulation of axonogenesis and synapsis. Syn1 protein functions as a substrate for several different protein kinases, and phosphorylation may play a role in the regulation of this protein in nerve endings. Mutations in this gene may be associated with X-linked disorders with primary neuronal degeneration, such as Rett syndrome. Transcript variants encoding different isoforms of alternatively spliced ​​transcripts have been identified. In some implementations of any aspect, the Syn1 promoter may comprise the human promoter Syn1 (see, for example, the Syn1 promoter associated with NCBI reference number NG_008437.1 Ref Seq Gene Range 5001-52957; NM_006950.3; NP_008881.2; NM_133499.2; NP_598006.1; see, for example, SEQ ID NO: 31-SEQ ID NO: 32).

[0381] CYP46A1 is a member of the cytochrome P450 enzyme superfamily. Cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and the synthesis of cholesterol, steroids, and other lipids. This endoplasmic reticulum protein is expressed in the brain, where it converts cholesterol to 24S-hydroxycholesterol. Although cholesterol cannot cross the blood-brain barrier, 24S-hydroxycholesterol can be secreted in the brain into circulation and returned to the liver for catabolism. In some embodiments in any aspect, CYP46A1 may comprise human CYP46A1 (see, for example, NCBI ref number NG_007963.1 RefSeqGene Range 4881-47884; NM_006668.2; NP_006659.1; see, for example, SEQ ID NO: 26-SEQ ID NO: 27). CYP46A1 is the rate-limiting enzyme for cholesterol degradation and has neuroprotective properties in Huntington's disease (see, for example, Boussicault et al., CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease, Brain, March 2016, 139(Pt3):953-70; Kacher et al., CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease, Brain, August 1, 2019; 142(8):2432-2450; the contents of each are incorporated herein by reference in their entirety).

[0382] Non-limiting examples of miRNAs disclosed herein include SEQ ID NO: 1 or SEQ ID NO: 4-SEQ ID NO: 9.

[0383]

[0384] In some embodiments of any aspect, the miRNA comprises a sequence that is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive bases of the sequence to which the miRNA backbone sequence is side-attached. In some embodiments of any aspect, the miRNA comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutively complementary sequences of an untranslated region (e.g., 5'UTR, 3'UTR), exon, CAG repeat, or CAG crossover region (e.g., CAG 5' crossover, CAG 3' crossover) associated with the HTT (see, for example, NCBI Gene ID: 3064; SEQ ID NO: 25; see, for example, Table 1 or Table 2).

[0385] Table 2: Location of target sequences

[0386]

[0387] SEQ ID NO: 25 Huntington protein mRNA (Homo sapiens); NCBI Ref. Seq NM002111.8 (see, for example, NG_009378.1 Ref Seq Gene Range 5001-174286, exemplary HTT gene)

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396] Example 2

[0397] AAV-mediated artificial miRNAs and their application in Huntington's disease (HD)

[0398] This article describes artificial miRNAs that can be used to treat HD (see example). Figures 2-3 Several screenings were conducted to identify and test artificial miRNAs. In general, the process of screening artificial miRNAs for HD included: (1) designing and synthesizing artificial miRNAs (e.g., 24 artificial miRNA constructs; see, for example, Table 1, SEQ ID NO: 1-SEQ ID NO: 24, Figure 1 , Figures 5-6 , Figure 8A , Figure 19 The first in vitro screening includes: (2) in vitro co-transfection of plasmids (e.g., screening for 24 miRs by plasmid co-transfection in 293 cells; see example). Figure 7 , Figure 8B , Figures 16-18 (3) Perform AAVRH10-mediated infection in vitro using the first 5 candidates from step (2) (e.g., using the CMV promoter; see example). Figures 10-14 , Figures 20-21 (3) and / or (4) use the first 2-3 candidates from step (3) for AAVrh10-mediated infection driven by a neuron-specific promoter (e.g., hSyn1 promoter with optional CYP46A1 co-expression) to test the efficiency of miRs in vitro. (5) A second in vivo screening includes AAVrh10-mediated in vivo treatment, including testing AAVrh10-mediated artificial miRNAs in transgenic (Tg) mice (e.g., Hu-128 or B6CBA-R6 / 2). (6) The efficiency and safety of the artificial miRs and their combination with CYP46A1 are then evaluated. See, for example Figure 4 , Figure 9 , Figure 15 .

[0399] Phase I: Screening for artificial miRNAs located in Regions I-III

[0400] Artificial miRNAs located in regions I-III include miHTT-H1 (SEQ ID NO: 1), miHTT-H2 (SEQ ID NO: 2), miHTT-H3 (SEQ ID NO: 3), miHTT-H4 (SEQ ID NO: 4), miHTT-H5 (SEQ ID NO: 5), miHTT-H6 (SEQ ID NO: 6), miHTT-H7 (SEQ ID NO: 7), miHTT-H8 (SEQ ID NO: 8), miHTT-H9 (SEQ ID NO: 9), or miHTT-H10 (SEQ ID NO: 10); see Table 1 for example.

[0401] The process of screening for artificial miRNAs located in regions I-III for HD includes: (1) designing and synthesizing artificial miRNAs (e.g., 9-10 artificial miRNA constructs; see, for example, Table 1, SEQ ID NO: 1-SEQ ID NO: 10, Figure 8A The first in vitro screening includes: (2) in vitro co-transfection of plasmids (e.g., screening for 9-10 miRs by co-transfection of plasmids in 293 cells; see example). Figure 8B (3) Perform AAVRH10-mediated infection in vitro using the first 5 candidates from step (2) (e.g., using the CMV promoter; see example). Figures 10-14 (3) and / or (4) perform AAVrh10-mediated infection driven by a neuron-specific promoter (e.g., hSyn1 promoter with optional CYP46A1 co-expression) using the first two candidates from step (3) (e.g., miR-H2 and miR-H5) to test the efficiency of the miR in vitro. (5) A second in vivo screening includes AAVrh10-mediated in vivo treatment, including testing the artificial miRNA mediated by AAVrh10 in Tg mice (e.g., Hu-128 or B6CBA-R6 / 2). (6) The efficiency and safety of the artificial miR with or without CYP46A1 are then evaluated. See, for example Figure 9 The experiments described in this article identified the following artificial miRNAs as particularly effective: miHTT-H2 (SEQ ID NO: 2), miHTT-H4 (SEQ ID NO: 4), or miHTT-H5 (SEQ ID NO: 5); see, for example... Figures 8A-8B , Figures 11A-11B , Figures 13-14 In particular, such as Figure 13 and Figure 14As described, miHTT-H2, H4, and H5 effectively downregulated HTT expression in the U87 neural cell line compared to HTT expression using a blank vector (without miRNA) or other tested miRNAs (e.g., miHTT-H1 or miHTT-H3).

[0402] Phase II: Screening for artificial miRNAs located in regions IV-V

[0403] miRNAs located in regions IV-V include miHTT-H11 (SEQ ID NO: 11), miHTT-H12 (SEQ ID NO: 12), miHTT-H13 (SEQ ID NO: 13), miHTT-H14 (SEQ ID NO: 14), miHTT-H15 (SEQ ID NO: 15), miHTT-H16 (SEQ ID NO: 16), miHTT-H17 (SEQ ID NO: 17), miHTT-H18 (SEQ ID NO: 18), miHTT-H19 (SEQ ID NO: 19, miR-137), miHTT-H20 (SEQ ID NO: 20, miR-455), miHTT-H21 (SEQ ID NO: 21, miR-216), or miHTT-H22 (SEQ ID NO: 22, miR-27a); see Table 1 for example. The following miRs can be used as positive controls in Phase I: miHTT-H2 (SEQ ID NO: 2), miHTT-H4 (SEQ ID NO: 4), or miHTT-H5 (SEQ ID NO: 5).

[0404] The process of screening for miRNAs located in regions IV-V for HD includes: (1) designing and synthesizing artificial miRNAs (e.g., 12 artificial miRNA constructs; see, for example, Table 1, SEQ ID NO: 11-SEQ ID NO: 22, Figure 19 The first in vitro screening includes: (2) in vitro co-transfection of plasmids (e.g., screening for 12 miRs by co-transfection of plasmids in 293 cells; see example). Figures 16-18 (3) Perform AAVRH10-mediated infection in vitro using the first 5 candidates from step (2) (e.g., miHTT-H14; miHTT-H15; miHTT-H17; miHTT-H19; and miHTT-H21) (e.g., using the CMV promoter; see example). Figures 20-21(3) and / or (4) use the first 2-3 candidates from step (3) for AAVrh10-mediated infection driven by a neuron-specific promoter (e.g., hSyn1 promoter with optional CYP46A1 co-expression) to test the efficiency of miRs in vitro. (5) A second in vivo screening includes AAVrh10-mediated in vivo treatment, including testing artificial miRNAs mediated by AAVrh10 in Tg mice (e.g., Hu-128). (6) The efficiency and safety of artificial miRs with or without CYP46A1 combination are then evaluated. See, for example Figure 15 The experiments described in this article identified the following artificial miRNAs as particularly effective: miHTT-H14 (SEQ ID NO: 14), miHTT-H15 (SEQ ID NO: 15), and miHTT-H17 (SEQ ID NO: 17), as well as human-expressed miRNAs miHTT-H19 (SEQ ID NO: 19; miR-137) and miHTT-H21 (SEQ ID NO: 21; miR-216); see, for example Figures 17-19 .

[0405] miR-137, miR-455, miR-216, and miR-27a (e.g., miHTT-H19-H22, SEQ ID NO: 19-SEQ ID NO: 22) are examples of human-expressed miRNAs used to test their efficacy in downregulating HTT. The inhibitory activity of these sequences against HTT was previously unknown. As shown herein, miR-137 (miHTT-H19, SEQ ID NO: 19) and miR-216 (miHTT-H21, SEQ ID NO: 21) are two particularly potent candidates targeting the HTT 3'-UTR.

[0406] miR-137 (see, for example, miHTT-H19, SEQ ID NO: 19) is located on human chromosome 1p22 and has been suggested to act as a tumor suppressor in several cancer types, including colorectal cancer, squamous cell carcinoma, and melanoma, through cell cycle control. The regulation of neural stem cell proliferation and differentiation in mouse embryonic stem cells, as well as neuronal maturation, is illustrated, including the regulation of dendritic length, branching points, endpoints, and dendritic spine density in neurons derived from adult mouse hippocampal neural progenitor cells and mouse fetal hippocampal neurons. Diseases associated with miR455 include endometrial serous adenocarcinoma and Pettigrew syndrome.

[0407] miR-455 (see, for example, miHTT-H20, SEQ ID NO: 20) is located on human chromosome 9q32. Diseases associated with miR-455 include serous adenocarcinoma of the endometrium and Pettigrew syndrome. miR-216 (see, for example, miHTT-H21, SEQ ID NO: 21) is located on human chromosome 2p16.1. Diseases associated with miR-216 include pancreatic ductal adenocarcinoma and microvascular complications of diabetes. miR-27a (see, for example, miHTT-H22, SEQ ID NO: 22) is located on human chromosome 19p13.12. Diseases associated with miR27a include leukemia and gastric cancer. miR-27a was used as a positive control in this paper and is reported to reduce the aggregation of mutant HTT in vitro; see, for example, Ban et al., Biochemical and Biophysical Research Communications 488(2), 2017, 316-321.

[0408] Phase III: Testing for additional artificial miRNAs located in region V against miRNAs identified in Phase II and Phase III.

[0409] Additional artificial miRNAs located in region V include miR-451a (SEQ ID NO: 23) or miR-155 (SEQ ID NO: 24); see, for example, Table 1. miR-451a (SEQ ID NO: 23) is located on human chromosome 17q11.2. miR-451 regulates the drug transporter P-glycoprotein and may promote resistance to the chemotherapeutic drug paclitaxel. Diseases associated with miR451A include glioma susceptibility and gastric cancer. miR-155 (see, for example, SEQ ID NO: 24) is located on human chromosome 21q21.3. In vivo and exogenous molecular control of miR-155 expression may inhibit malignant growth, viral infection, and promote the progression of cardiovascular disease. Diseases associated with miR155 include metastatic large B-cell lymphoma and pancreatic ductal adenocarcinoma. See, for example, U.S. Patent 10,767,180, for its discussion of these additional artificial miRNAs miR-451a and miR-155, the contents of which are incorporated herein by reference in their entirety. Without wishing to be bound by theory, it is expected that at least one or more miRNAs, as disclosed herein (e.g., in Table 1), will be better at inhibiting the target gene (e.g., HTT) compared to the inhibitory efficiency of miR-451a or miR-155 targeting the same target gene.

[0410] The following artificial miRs from phase I and II can be tested for the additional miRNAs mentioned above: miHTT-H2 (SEQ ID NO: 2), miHTT-H4 (SEQ ID NO: 4), miHTT-H5 (SEQ ID NO: 5), miHTT-H14 (SEQ ID NO: 14), miHTT-H15 (SEQ ID NO: 15), miHTT-H17 (SEQ ID NO: 17), miHTT-H19 (SEQ ID NO: 19; miR-137), or miHTT-H21 (SEQ ID NO: 21; miR-216).

[0411] The procedure for testing additional artificial miRNAs for HD based on artificial miRNAs identified in Phase I and II includes: (1) designing and synthesizing artificial miRNAs (e.g., two artificial miRNA constructs; see, for example, Table 1, SEQ ID NO: 23-SEQ ID NO: 24, Figure 6 The first in vitro assay includes: (2) in vitro co-transfection with plasmids (e.g., screening for two miRs by co-transfection with plasmids in 293 cells); (3) in vitro AAVRH10-mediated infection (e.g., using the CMV promoter); and / or (4) AAVRH10-mediated infection driven by a neuron-specific promoter (e.g., the hSyn1 promoter with optional CYP46A1 co-expression) to test the efficiency of the miRs in vitro. (5) The second in vivo assay includes AAVRH10-mediated in vivo treatment, including testing artificial miRNAs mediated by AAVrh10 in Tg mice (e.g., Hu-128 or B6CBA-R6 / 2). (6) The efficiency and safety of the artificial miRs with or without CYP46A1 are then evaluated.

[0412] Furthermore, the efficacy of the additional miRNAs was compared with that of artificial miRNAs identified in phases I and II (e.g., miHTT-H2 (SEQ ID NO: 2), miHTT-H4 (SEQ ID NO: 4), and miHTT-H5 (SEQ ID NO: 5), miHTT-H14 (SEQ ID NO: 14), miHTT-H15 (SEQ ID NO: 15), miHTT-H17 (SEQ ID NO: 17), miHTT-H19 (SEQ ID NO: 19; miR-137), or miHTT-H21 (SEQ ID NO: 21; miR-216)). See, for example... Figure 4Not wishing to be bound by theory, it is expected that at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 21 may exhibit increased efficiency and / or efficacy (e.g., in reducing the level or activity of HTT mRNA or protein) compared to SEQ ID NO: 23 or SEQ ID NO: 24.

[0413] Selection of artificial miRNAs for different HTT mouse models

[0414] Table 3: HTT mouse models (“√” indicates that the region is present in the mouse model; therefore, miRNAs targeting each specified region can be used in the specified mouse model).

[0415]

[0416] The transgenic mouse model Hu128 has a full-size knock-in of the human HTT gene, including the 5' untranslated region (5'-UTR) and the 3'-UTR (see example). Figure 6 (and Table 3). Other transgenic mouse models (e.g., B6CBA-R6 / 2(CAG 120+ / -5), B6CBA-Tg(HDexon1)62Gpb / 3J, B6CBA-R6 / 2(CAG 160+ / -5), or B6CBA-Tg(HDexon1)62Gpb / 1J) contain a 1kb 5'-UTR of human HTT, exon I, and 260bp introns, which are inserted into another gene (e.g., Gm12695, chromosome 4, chr4:96,409,585-96,414,930).

[0417] Therefore, in order to test a specific miR, the selected mouse model should contain the target region of HTT (see example). Figures 5-6 (See Tables 1 and 3). As a non-limiting example, the Hu128 transgenic mouse model with full-size human HTT gene knock-in can be used to test any miR in target regions I-V (e.g., SEQ ID NO: 1-SEQ ID NO: 24). As another non-limiting example, other transgenic mouse models with a 1kb 5'-UTR of human HTT, exon I, and 260bp intron (e.g., B6CBA-R6 / 2 or B6CBA-Tg(HDexon1) strain) can be used to test any miR in target regions I-III (e.g., SEQ ID NO: 1-SEQ ID NO: 10). sequence list <110> AskBio Inc. <120> Methods for treating Huntington's disease <130> 046192-096790WOPT <150> 62 / 951,582 <151> 2019-12-20 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 1 gctgctgctg ctgctgctgc 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 2 tgctgctgct gctgctgctg 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 3 ggcggcggcg gcggcggcgg 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 4 tgctggaagg acttgaggga 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> 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sequence <220> <223> Synthetic polynucleotides <400> twenty one taatctcttt actgatataa 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty two tcagcagtgt tatttcttac 20 <210> twenty three <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty three aaaccgttac cattactgag tt 22 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> twenty four aaatcgctga tttgtgtagt c 21 <210> 25 <211> 13498 <212> DNA <213> Homo sapiens <400> 25 gctgccggga cgggtccaag atggacggcc gctcaggttc tgcttttacc tgcggcccag 60 agccccattc attgccccgg tgctgagcgg cgccgcgagt cggcccgagg cctccgggga 120 ctgccgtgcc gggcgggaga ccgccatggc gaccctgga aagctgatga aggccttcga gtccctcaag tccttccagc gtcctcagc gtcctcagc gtcctcagc 240 gcagcagcag cagcagcagc aacagccgcc accgccgccg ccgccgccgc cgcctcctca 300 gcttcctcag ccgccgccgc aggcacagcc gctgctgcct cagccgcagc cgcccccgcc 360 gccgccccg ccgccccg gcccggctgt ggctgaggag ccgctgcacc 420 agaactttca gctaccaaga aagaccgtgt gaatcattgt ctgacaatat gtgaaaacat agtggcacag tctgtcagaa attctccaga atttcagaaa cttctgggca tcgctatgga actttttctg ctgtgcagtg atgacgcaga gtcagatgtc aggatggtgg ctgacgaatg cctcaacaaa gttatcaaag ctttgatgga ttctaatctt ccaaggttac agctcgagct ctaagga attaaaaaga atggtgcccc tcggagtttg cgtgctgccc tgtggaggtt 720 tgctgagctg gctcacctgg ttcggcctca gaaatgcagg ccttacctgg tgaaccttct 780 gcctgcctg actcgaacaa gcaagagacc cgaagaatca gtccaggaga ccttggctgc 840 agctgttccc aaaattatgg cttcttttgg caattttgca aatgacaatg aaattaaggt 900 tttgttaaag gccttcatag cgaacctgaa gtcaagctcc cccaccattc ggcggacagc 960 ggctggatca gcagtgagca tctgccagca ctcaagaagg acacaatatt tctatagttg 1020 gctactaaat gtgctcttag gcttactcgt tcctgtcgag gatgaacact ccactctgct 1080 gattcttggc gtgctgctca ccctgaggta tttggtgccc ttgctgcagc agcaggtcaa 1140 ggacacaagc ctgaaaggca gcttcggagt gacaaggaaa gaaatggaag tctctccttc 1200 tgcagagcag cttgtccagg tttatgaact gacgttacat catacacagc accaagacca 1260 caatgttgtg accggagccc tggagctgtt gcagcagctc ttcagaacgc ctccacccga 1320 gcttctgcaa accctgaccg cagtcggggg cattgggcag ctcaccgctg ctaaggagga 1380 gtctggtggc cgaagccgta gtgggagtat tgtggaactt atagctggag ggggttcctc 1440 atgcagccct gtcctttcaa gaaaacaaaa aggcaaagtg ctcttaggag aagaagaagc 1500 cttggaggat gactctgaat cgagatcgga tgtcagcagc tctgccttaa cagcctcagt 1560 gaaggatgag atcagtggag agctggctgc ttcttcaggg gtttccactc cagggtcagc 1620 aggtcatgac atcatcacag aacagccacg gtcacagcac acactgcagg cggactcagt 1680 ggatctggcc agctgtgact tgacaagctc tgccactgat ggggatgagg aggatatctt 1740 gagccacagc tccagccagg tcagcgccgt cccatctgac cctgccatgg acctgaatga 1800 tgggacccag gcctcgtcgc ccatcagcga cagctcccag accaccaccg aagggcctga 1860 ttcagctgtt accccttcag acagttctga aattgtgtta gacggtaccg acaaccagta 1920 tttgggcctg cagattggac agccccagga tgaagatgag gaagccacag gtattcttcc 1980 tgatgaagcc tcggaggcct tcaggaactc ttccatggcc cttcaacagg cacatttatt 2040 gaaaaacatg agtcactgca ggcagccttc tgacagcagt gttgataaat ttgtgttgag 2100 agatgaagct actgaaccgg gtgatcaaga aaacaagcct tgccgcatca aaggtgacat 2160 tggacagtcc actgatgatg actctgcacc tcttgtccat tgtgtccgcc ttttatctgc 2220 ttcgtttttg ctaacagggg gaaaaaatgt gctggttccg gacagggatg tgagggtcag 2280 cgtgaaggcc ctggccctca gctgtgtggg agcagctgtg gccctccacc cggaatcttt 2340 cttcagcaaa ctctataaag ttcctcttga caccacggaa taccctgagg aacagtatgt 2400 ctcagacatc ttgaactaca tcgatcatgg agacccacag gttcgaggag ccactgccat 2460 tctctgtggg accctcatct gctccatcct cagcaggtcc cgcttccacg tgggagattg 2520 gatgggcacc attagaaccc tcacaggaaa tacattttct ttggcggatt gcattccttt 2580 gctgcggaaa acactgaagg atgagtcttc tgttacttgc aagttagctt gtacagctgt 2640 gaggaactgt gtcatgagtc tctgcagcag cagctacagt gagttaggac tgcagctgat 2700 catcgatgtg ctgactctga ggaacagttc ctattggctg gtgaggacag agcttctgga 2760 aacccttgca gagattgact tcaggctggt gagctttttg gaggcaaaag cagaaaactt 2820 acacagaggg gctcatcatt atacagggct tttaaaactg caagaacgag tgctcaataa 2880 tgttgtcatc catttgcttg gagatgaaga ccccagggtg cgacatgttg ccgcagcatc 2940 actaattagg cttgtcccaa agctgtttta taaatgtgac caaggacaag ctgatccagt 3000 agtggccgtg gcaagagatc aaagcagtgt ttacctgaaa cttctcatgc atgagacgca 3060 gcctccatct catttctccg tcagcacaat aaccagaata tatagaggct ataacctact 3120 accaagcata acagacgtca ctatggaaaa taacctttca agagttattg cagcagtttc 3180 tcatgaacta atcacatcaa ccaccagagc actcacattt ggatgctgtg aagctttgtg 3240 tcttctttcc actgccttcc cagtttgcat ttggagttta ggttggcact gtggagtgcc 3300 tccactgagt gcctcagatg agtctaggaa gagctgtacc gttgggatgg ccacaatgat 3360 tctgaccctg ctctcgtcag cttggttccc attggatctc tcagcccatc aagatgcttt 3420 gattttggcc ggaaacttgc ttgcagccag tgctcccaaa tctctgagaa gttcatgggc 3480 ctctgaagaa gaagccaacc cagcagccac caagcaagag gaggtctggc cagccctggg 3540 ggaccgggcc ctggtgccca tggtggagca gctcttctct cacctgctga aggtgattaa 3600 catttgtgcc cacgtcctgg atgacgtggc tcctggaccc gcaataaagg cagccttgcc 3660 ttctctaaca aacccccctt ctctaagtcc catccgacga aaggggaagg agaaagaacc aggagaacaa gcatctgtac cgttgagtcc caagaaaggc agtgaggcca gtgcagcttc 3840. 3840. 3840. 3840. 3840. 3840. 3840. 3840. 3840. 3840 ctatcatctt ccttcatacc tcaaactgca tgatgtcctg aaagctacac acgctaacta caaggtcacg ctggatcttc agaacagcac ggaaaagttt ggagggtttc tccgctcagc cttggatgtt ctttctcaga tactagagct ggccacactg caggacattg ggaagtgtgt tgaagagatc ctaggatacc tgaaatcctg ctttagtcga gaccaatga tggcaactgt ttgtgttcaa caattgttga agactctctt tggcacaaac ttggcctccc agtttgatgg 4140 cttatcttcc aaccccagca agtcacaagg ccgagcacag cgccttggct cctccagtgt gaggccaggc ttgtaccact actgcttcat ggccccgtac acccacttca cccaggccct 4260 cgctgacgcc agcctgagga acatggtgca ggcggagcag gagacgaca cctcgggatg gtttgatgtc ctccagaaag tgtctaccca gttgaagaca aacctcacga gtgtcacaaa gaaccgtgca gataagaatg ctattcataa tcacattcgt ttgtttgaac ctcttgttat 4440 aaaagcttta aaacagtaca cgactacaac atgtgtgcag ttacagaagc aggttttaga 4500 tttgctggcg cagctggttc agttacgggt taattactgt cttctggatt cagatcaggt 4560 gttattggc tttgtattga aacagtttga atacattgaa gtgggccagt tcagggaatc 4620 agaggcaatc attccaaaca tctttttctt cttggtatta ctatcttatg aacgctatca 4680 ttcaaaacag atcattggaa ttcctaaaat cattcagctc tgtgatggca tcatggccag 4740 tggaaggaag gctgtgacac atgccatacc ggctctgcag cccatagtcc acgacctctt 4800 tgtattaaga ggaacaaata aagctgatgc aggaaaagag cttgaaaccc aaaaagaggt 4860 ggtggtgtca atgttactga gactcatcca gtaccatcag gtgttggaga tgttcattct 4920 tgtcctgcag cagtgccaca aggagaatga agacaagtgg aagcgactgt ctcgacagat 4980 agctgacatc atcctcccaa tgttagccaa acagcagatg cacattgact ctcatgaagc 5040 ccttggagtg ttaaatacat tatttgagat ttggcccct tcctccctcc gtccggtaga 5100 catgctttta cggagtatgt tcgtcactcc aaacacaatg gcgtccgtga gcactgttca 5160 actgtggata tcgggaattc tggccatttt gagggttctg atttcccagt caactgaaga 5220 tattgttctt tctcgtattc aggagctctc cttctctccg tatttaatct cctgtacagt 5280 attaatagg ttaagagatg gggacagtac ttcaacgcta gaagaacaca gtgaagggaa 5340 acaaataaag aatttgccag aagaaacatt ttcaaggttt ctattacaac tggttggtat 5400 tcttttagaa gacattgtta caaaacagct gaaggtggaa atgagtgagc agcaacatac 5460 tttctattgc caggaactag gcacactgct aatgtgtctg atccacatct tcaagtctgg 5520 aatgttccgg agaatcacag cagctgccac taggctgttc cgcagtgatg gctgtggcgg 5580 cagtctctac accctggaca gcttgaactt gcgggctcgt tccatgatca ccacccacccc 5640 ggccctggtg ctgctctggt gtcagatact gctgcttgtc aaccacaccg actaccgctg 5700 gtgggcagaa gtgcagcaga ccccgaaaag acacagtctg tccagcacaa agttacttag 5760 tccccagatg tctggagaag aggaggattc tgacttggca gccaaacttg gaatgtgcaa 5820 tagagaata gtacgaagag gggctctcat tctcttctgt gattatgtct gtcagaacct 5880 ccatgactcc gagcacttaa cgtggctcat tgtaaatcac attcaagatc tgatcagcct 5940 ttcccacag cctccagtac aggacttcat cagtgccgtt catcggaact ctgctgccag 6000 cggcctgttc atccaggcaa ttcagtctcg ttgtgaaaac ctttcaactc caccatgct 6060 gaaaaaact cttcagtgct tggaggggat ccatctcagc cagtcgggag ctgtgctcac 6120 gctgtatgtg gacaggcttc tgtgcacccc tttccgtgtg ctggctcgca tggtcgacat 6180 ccttgcttgt cgccgggtag aaatgcttct ggctgcaaat ttacagagca gcatggccca 6240 gttgccaatg gaagaactca acagaatcca ggaatacctt cagagcagcg ggctcgctca 6300 gagacaccaa aggctctatt ccctgctgga caggtttcgt ctctccacca tgcaagactc 6360 acttagtccc tctctctcag tctcttccca cccgctggac ggggatgggc acgtgtcact 6420 ggaaacgtg agtccggaca aagactggta cgttcatctt gtcaaatccc agtgttggac 6480 caggtcagat tctgcactgc tggaaggtgc agagctggtg aatcggattc ctgctgaaga 6540 tatgaatgcc ttcatgatga actcggagtt caacctaagc ctgctagctc catgcttaag 6600 cctagggatg agtgaaattt ctggtggcca gaagagtgcc ctttttgaag cagcccgtga 6660 ggtgactctg gcccgtgtga gcggcaccgt gcagcagctc cctgctgtcc atcatgtctt 6720 ccagcccgag ctgcctgcag agccggcggc ctactggagc aagttgaatg atctgtttgg 6780 ggatgctgca ctgtatcagt ccctgcccac tctggcccgg gcctggcac agtacctggt 6840 ggtggtctcc aaactgccca gtcatttgca ccttcctcct gagaaagaga aggacattgt 6900 gaaattcgtg gtggcaaccc ttgaggccct gtcctggcat ttgatccatg agcagatccc 6960 gctgagtctg gatctccagg cagggctgga ctgctgctgc ctggccctgc agctgcctgg 7020 cctctggagc gtggtctcct ccacagagtt tgtgacccac gcctgctccc tcatctactg 7080 tgtgcacttc atcctggagg ccgttgcagt gcagcctgga gagcagcttc ttagtccaga 7140 aagaaggaca aataccccaa aagccatcag cgaggaggag gaggaagtag atccaaacac 7200 acagaatcct aagtatatca ctgcagcctg tgagatggtg gcagaaatgg tggagtctct 7260 gcagtcggtg ttggccttgg gtcataaaag gaatagcggc gtgccggcgt ttctcacgcc 7320 attgctaagg aacatcatca tcagcctggc ccgcctgccc cttgtcaaca gctacacacg 7380 tgtgccccca ctggtgtgga agcttggatg gtcacccaaa ccgggagggg attttggcac 7440 agcattccct gagatccccg tggagttcct ccaggaaaag gaagtcttta aggagttcat 7500 ctaccgcatc aacacactag gctggaccag tcgtactcag tttgaagaaa cttgggccac 7560 cctccttggt gtcctggtga cgcagcccct cgtgatggag caggaggaga gcccaccaga 7620 agaagacaca gagaggaccc agatcaacgt cctggccgtg caggccatca cctcactggt 7680 gctcagtgca atgactgtgc ctgtggccgg caacccagct gtaagctgct tggagcagca 7740 gccccggaac aagcctctga aagctctcga caccaggttt gggaggaagc tgagcattat 7800 cagagggatt gtggagcaag agattcaagc aatggtttca aagagagaga atattgccac 7860 ccatcattta tatcaggcat gggatcctgt cccttctctg tctccggcta ctacaggtgc 7920 cctcatcagc cacgagaagc tgctgctaca gatcaacccc gagcgggagc tggggagcat 7980 gagctacaaa ctcggccagg tgtccataca ctccgtgtgg ctggggaaca gcatcacacc 8040 cctgagggag gaggaatggg acgaggaaga ggaggaggag gccgacgccc ctgcaccttc 8100 gtcaccaccc acgtctccag tcaactccag gaaacaccgg gctggagttg acatccactc 8160 ctgttcgcag tttttgcttg agttgtacag ccgctggatc ctgccgtcca gctcagccag 8220 gaggaccccg gccatcctga tcagtgaggt ggtcagatcc cttctagtgg tctcagactt 8280 gttcaccgag cgcaaccagt ttgagctgat gtatgtgacg ctgacagaac tgcgaagggt 8340 gcacccttca gaagacgaga tcctcgctca gtacctggtg cctgccacct gcaaggcagc 8400 tgccgtcctt gggatggaca aggccgtggc ggagcctgtc agccgcctgc tggagagcac 8460 gctcaggagc agccacctgc ccagcagggt tggagccctg cacggcgtcc tctatgtgct 8520 ggagtgcgac ctgctggacg acactgccaa gcagctcatc ccggtcatca gcgactatct 8580 cctctccaac ctgaaaggga tcgcccactg cgtgaacatt cacagccagc agcacgtact 8640 ggtcatgtgt gccactgcgt tttacctcat tgagaactat cctctggacg tagggccgga 8700 attttcagca tcaataatac agatgtgtgg ggtgatgctg tctggaagtg aggagtccac 8760 cccctccatc atttaccact gtgccctcag aggcctggag cgcctcctgc tctctgagca 8820 gctctcccgc ctggatgcag aatcgctggt caagctgagt gtggacagag tgaacgtgca 8880 cagcccgcac cgggccatgg cggctctggg cctgatgctc acctgcatgt acacaggaaa 8940 ggagaaagtc agtccgggta gaacttcaga ccctaatcct gcagcccccg acagcgagtc 9000 agtgattgtt gctatggagc gggtatctgt tctttttgat aggatcagga aaggctttcc 9060 ttgtgaagcc agagtggtgg ccaggatcct gccccagttt ctagacgact tcttcccacc 9120 ccaggacatc atgaacaaag tcatcggaga gtttctgtcc aaccagcagc cataccccca 9180 gttcatggcc accgtggtgt ataaggtgtt tcagactctg cacagcaccg ggcagtcgtc 9240 catggtccgg gactgggtca tgctgtccct ctccaacttc acgcagaggg ccccggtcgc 9300 catggccacg tggagcctct cctgcttctt tgtcagcgcg tccaccagcc cgtgggtcgc 9360 ggcgatcctc ccacatgtca tcagcaggat gggcaagctg gagcaggtgg acgtgaacct 9420 tttctgcctg gtcgccacag acttctacag acaccagata gaggaggagc tcgaccgcag 9480 ggccttccag tctgtgcttg aggtggttgc agccccagga agcccatatc accggctgct 9540 gacttgttta cgaaatgtcc acaaggtcac cacctgctga gcgccatggt gggagagact 9600 gtgaggcggc agctggggcc ggagcctttg gaagtctgcg cccttgtgcc ctgcctccac 9660 cgagccagct tggtccctat gggcttccgc acatgccgcg ggcggccagg caacgtgcgt 9720 gtctctgcca tgtggcagaa gtgctctttg tggcagtggc caggcaggga gtgtctgcag 9780 tcctggtggg gctgagcctg aggccttcca gaaagcagga gcagctgtgc tgcaccccat 9840 gtgggtgacc aggtcctttc tcctgatagt cacctgctgg ttgttgccag gttgcagctg 9900 ctcttgcatc tgggccagaa gtcctccctc ctgcaggctg gctgttggcc cctctgctgt 9960 cctgcagtag aaggtgccgt gagcaggctt tgggaacact ggcctgggtc tccctggtgg 10020 ggtgtgcatg ccacgccccg tgtctggatg cacagatgcc atggcctgtg ctgggccagt 10080 ggctgggggt gctagacacc cggcaccatt ctcccttctc tcttttcttc tcaggattta 10140 aaatttaatt atatcagtaa agagattaat tttaacgtaa ctctttctat gcccgtgtaa 10200 agtatgtgaa tcgcaaggcc tgtgctgcat gcgacagcgt ccggggtggt ggacagggcc 10260 cccggccacg ctccctctcc tgtagccact ggcatagccc tcctgagcac ccgctgacat 10320 ttccgttgta catgttcctg tttatgcatt cacaaggtga ctgggatgta gagaggcgtt 10380 agtgggcagg tggccacagc aggactgagg acaggccccc attatcctag gggtgcgctc 10440 acctgcagcc cctcctcctc gggcacagac gactgtcgtt ctccacccac cagtcaggga 10500 cagcagcctc cctgtcactc agctgagaag gccagccctc cctggctgtg agcagcctcc 10560 actgtgtcca gagacatggg cctcccactc ctgttccttg ctagccctgg ggtggcgtct 10620 gcctaggagc tggctggcag gtgttgggac ctgctgctcc atggatgcat gccctaagag 10680 tgtcactgag ctgtgttttg tctgagcctc tctcggtcaa cagcaaagct tggtgtcttg 10740 gcactgttag tgacagagcc cagcatccct tctgcccccg ttccagctga catcttgcac 10800 ggtgacccct tttagtcagg agagtgcaga tctgtgctca tcggagactg ccccacggcc 10860 ctgtcagagc cgccactcct atccccaggc caggtccctg gaccagcctc ctgtttgcag 10920 gcccagagga gccaagtcat taaaatggaa gtggattctg gatggccggg ctgctgctga 10980 tgtaggagct ggatttggga gctctgcttg ccgactggct gtgagacgag gcaggggctc 11040 tgcttcctca gccctagagg cgagccaggc aaggttggcg actgtcatgt ggcttggttt 11100 ggtcatgccc gtcgatgttt tgggtattga atgtggtaag tggaggaaat gttggaactc 11160 tgtgcaggtg ctgccttgag acccccaagc ttccacctgt ccctctccta tgtggcagct 11220 ggggagcagc tgagatgtgg acttgtatgc tgcccacata cgtgaggggg agctgaaagg 11280 gagcccctcc tctgagcagc ctctgccagg cctgtatgag gcttttccca ccagctccca 11340 acagaggcct cccccagcca ggaccacctc gtcctcgtgg cggggcagca ggagcggtag 11400 aaaggggtcc gatgtttgag gaggccctta agggaagcta ctgaattata acacgtaaga 11460 aaatcaccat tccgtattgg ttgggggctc ctgtttctca tcctagcttt ttcctggaaa 11520 gcccgctaga aggtttggga acgaggggaa agttctcaga actgttggct gctccccacc 11580 cgcctcccgc ctcccccgca ggttatgtca gcagctctga gacagcagta tcacaggcca 11640 gatgttgttc ctggctagat gtttacattt gtaagaaata acactgtgaa tgtaaaacag 11700 agccattccc ttggaatgca tatcgctggg ctcaacatag agtttgtctt cctcttgttt 11760 acgacgtgat ctaaaccagt ccttagcaag gggctcagaa caccccgctc tggcagtagg 11820 tgtcccccac ccccaaagac ctgcctgtgt gctccggaga tgaatatgag ctcattagta 11880 aaaatgactt cacccacgca tatacataaa gtatccatgc atgtgcatat agacacatct 11940 ataattttac acacacacct ctcaagacgg agatgcatgg cctctaagag tgcccgtgtc 12000 ggttcttcct ggaagttgac tttccttaga cccgccaggt caagttagcc gcgtgacgga 12060 catccaggcg tgggacgtgg tcagggcagg gctcattcat tgcccactag gatcccactg 12120 gcgaagatgg tctccatatc agctctctgc agaagggagg aagactttat catgttccta 12180 aaaatctgtg gcaagcaccc atcgtattat ccaaattttg ttgcaaatgt gattaatttg 12240 gttgtcaagt tttgggggtg ggctgtgggg agattgcttt tgttttcctg ctggtaatat 12300 cgggaaagat tttaatgaaa ccagggtaga attgtttggc aatgcactga agcgtgtttc 12360 tttcccaaaa tgtgcctccc ttccgctgcg ggcccagctg agtctatgta ggtgatgttt 12420 ccagctgcca agtgctcttt gttactgtcc accctcattt ctgccagcgc atgtgtcctt 12480 tcaaggggaa aatgtgaagc tgaaccccct ccagacaccc agaatgtagc atctgagaag 12540 gccctgtgcc ctaaaggaca cccctcgccc ccatcttcat ggagggggtc atttcagagc 12600 cctcggagcc aatgaacagc tcctcctctt ggagctgaga tgagccccac gtggagctcg 12660 ggacggatag tagacagcaa taactcggtg tgtggccgcc tggcaggtgg aacttcctcc 12720 cgttgcgggg tggagtgagg ttagttctgt gtgtctggtg ggtggagtca ggcttctctt 12780 gctacctgtg agcatccttc ccagcagaca tcctcatcgg gctttgtccc tcccccgctt 12840 cctccctctg cggggaggac ccgggaccac agctgctggc cagggtagac ttggagctgt 12900 cctccagagg ggtcacgtgt aggagtgaga agaaggaaga tcttgagagc tgctgaggga 12960 ccttggagag ctcaggatgg ctcagacgag gacactcgct tgccgggcct gggcctcctg 13020 ggaaggaggg agctgctcag aatgccgcat gacaactgaa ggcaacctgg aaggttcagg 13080 ggccgctctt cccccatgtg cctgtcacgc tctggtgcag tcaaaggaac gccttcccct 13140 cagttgtttc taagagcaga gtctcccgct gcaatctggg tggtaactgc cagccttgga 13200 ggatcgtggc caacgtggac ctgcctacgg agggtgggct ctgacccaag tggggcctcc 13260 ttgtccaggt ctcactgctt tgcaccgtgg tcagagggac tgtcagctga gcttgagctc 13320 ccctggagcc agcagggctg tgatgggcga gtcccggagc cccacccaga cctgaatgct 13380 tctgagagca aagggaagga ctgacgagag atgtatattt aattttttaa ctgctgcaaa 13440 cattgtacat ccaaattaaa ggaaaaaaat ggaaaccatc aaaaaaaaaa aaaaaaaa 13498 <210> 26 <211> 1502 <212> DNA <213> Homo sapiens <400> 26 tgagccccgg gctgctgctg ctcggcagcg ccgtcctgct cgccttcggc ctctgctgca ccttcgtgca ccgcgctcgc agccgctacg agcacatccc cggccgccg cggcccagtt 120 tccttctagg acacctcccc tgcttttgga aaaaggatga ggttggtggc cgtgtgctcc 180 aagatgtgtt tttggattgg gctaagaagt atggacctgt tgtgcgggtc aacgtcttcc 240 acaaaacctc agtcatcgtc acgagtcctg agtcggttaa gaagttcctg atgtcaacca agtacaacaa ggactccaag atgtaccgtg cgctccagac tgtgtttggt gagagactct tcggccaagg cttggtgtcc gaatgcaact atgagcgctg gcacaagcag cggagagtca 420 tagcctggc cttcagccgg agctccttgg ttagcttaat ggaaacattc aacgagaagg ctgagcagct ggtggagatt ctagaagcca aggcagatgg gcagacccca gtgtccatgc 540 aggacatgct gacctacacc gccatggaca tcctggccaa ggcagctttt gggatggaga ccagtatgct gctgggtgcc cagaagcctc tgtcccaggc agtgaaactt atgttggagg 660 gaatcactgc gtcccgcaac actctggcaa agttcctgcc aggagagg aagcagctcc gggaggtccg ggagagcatt cgcttcctgc gccaggtggg cagggactgg gtccagcgcc 780 gccgggaagc cctgaagagg ggcgaggagg ttcctgccga catcctcaca cagattctga 840 aagctgaaga gggagcccag gacgacgagg gtctgctgga caacttcgtc accttcttca 900 ttgctggtca cgagacctct gccaaccact tggcgttcac agtgatggag ctgtctcgcc 960 agccagagat cgtggcaagg ctgcaggccg aggtggatga ggtcattggt tctaagaggt 1020 acctggattt cgaggacctg gggagactgc agtacctgtc ccaggtcctc aaagagtcgc 1080 tgaggctgta cccaccagca tggggcacct ttcgcctgct ggaagaggag accttgattg 1140 atggggtcag agtccccggc aacaccccgc tcttgttcag cacctatgtc atggggcgga 1200 tggacacata ctttgaggac ccgctgactt tcaaccccga tcgcttcggc cctggagcac 1260 ccaagccacg gttcacctac ttccccttct ccctgggcca ccgctcctgc atcgggcagc 1320 agtttgctca gatggaggtg aaggtggtca tggcaaagct gctgcagagg ctggagttcc 1380 ggctggtgcc cgggcagcgc ttcgggctgc aggagcaggc cacactcaag ccactggacc 1440 ccgtgctgtg caccctgcgg ccccgcggct ggcagcccgc acccccacca cccccctgct 1500 ga 1502 <210> 27 <211> 500 <212> PRT <213> Homo sapiens <400> 27 Met Ser Pro Gly Leu Leu Leu Leu Gly Ser Ala Val Leu Leu Ala Phe 1 5 10 15 Gly Leu Cys Cys Thr Phe Val His Arg Ala Arg Ser Arg Tyr Glu His 20 25 30 Ile Pro Gly Pro Pro Arg Pro Ser Phe Leu Leu Gly His Leu Pro Cys 35 40 45 Phe Trp Lys Lys Asp Glu Val Gly Gly Arg Val Leu Gln Asp Val Phe 50 55 60 Leu Asp Trp Ala Lys Lys Tyr Gly Pro Val Val Arg Val Asn Val Phe 65 70 75 80 His Lys Thr Ser Val Ile Val Thr Ser Pro Glu Ser Val Lys Lys Phe 85 90 95 Leu Met Ser Thr Lys Tyr Asn Lys Asp Ser Lys Met Tyr Arg Ala Leu 100 105 110 Gln Thr Val Phe Gly Glu Arg Leu Phe Gly Gln Gly Leu Val Ser Glu 115 120 125 Cys Asn Tyr Glu Arg Trp His Lys Gln Arg Arg Val Ile Asp Leu Ala 130 135 140 Phe Ser Arg Ser Ser Leu Val Ser Leu Met Glu Thr Phe Asn Glu Lys 145 150 155 160 Ala Glu Gln Leu Val Glu Ile Leu Glu Ala Lys Ala Asp Gly Gln Thr 165 170 175 Pro Val Ser Met Gln Asp Met Leu Thr Tyr Thr Ala Met Asp Ile Leu 180 185 190 Ala Lys Ala Ala Phe Gly Met Glu Thr Ser Met Leu Leu Gly Ala Gln 195 200 205 Lys Pro Leu Ser Gln Ala Val Lys Leu Met Leu Glu Gly Ile Thr Ala 210 215 220 Ser Arg Asn Thr Leu Ala Lys Phe Leu Pro Gly Lys Arg Lys Gln Leu 225 230 235 240 Arg Glu Val Arg Glu Ser Ile Arg Phe Leu Arg Gln Val Gly Arg Asp 245 250 255 Trp Val Gln Arg Arg Arg Glu Ala Leu Lys Arg Gly Glu Glu Val Pro 260 265 270 Ala Asp Ile Leu Thr Gln Ile Leu Lys Ala Glu Glu Gly Ala Gln Asp 275 280 285 Asp Glu Gly Leu Leu Asp Asn Phe Val Thr Phe Phe Ile Ala Gly His 290 295 300 Glu Thr Ser Ala Asn His Leu Ala Phe Thr Val Met Glu Leu Ser Arg 305 310 315 320 Gln Pro Glu Ile Val Ala Arg Leu Gln Ala Glu Val Asp Glu Val Ile 325 330 335 Gly Ser Lys Arg Tyr Leu Asp Phe Glu Asp Leu Gly Arg Leu Gln Tyr 340 345 350 Leu Ser Gln Val Leu Lys Glu Ser Leu Arg Leu Tyr Pro Pro Ala Trp 355 360 365 Gly Thr Phe Arg Leu Leu Glu Glu Glu Thr Leu Ile Asp Gly Val Arg 370 375 380 Val Pro Gly Asn Thr Pro Leu Leu Phe Ser Thr Tyr Val Met Gly Arg 385 390 395 400 Met Asp Thr Tyr Phe Glu Asp Pro Leu Thr Phe Asn Pro Asp Arg Phe 405 410 415 Gly Pro Gly Ala Pro Lys Pro Arg Phe Thr Tyr Phe Pro Phe Ser Leu 420 425 430 Gly His Arg Ser Cys Ile Gly Gln Gln Phe Ala Gln Met Glu Val Lys 435 440 445 Val Val Met Ala Lys Leu Leu Gln Arg Leu Glu Phe Arg Leu Val Pro 450 455 460 Gly Gln Arg Phe Gly Leu Gln Glu Gln Ala Thr Leu Lys Pro Leu Asp 465 470 475 480 Pro Val Leu Cys Thr Leu Arg Pro Arg Gly Trp Gln Pro Ala Pro Pro 485 490 495 Pro Pro Pro Cys 500 <210> 28 <211> 1653 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 28 atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga 60 accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120 gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc 180 gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240 tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300 gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt 360 tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420 aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga 480 tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540 600 tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg 660 catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720 gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780 cgagtcgtct taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac 840 aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg 900 attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct 960 aaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag ccaaggatat 1020 gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080 gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140 acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt 1200 tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260 ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct 1320 ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa 1380 caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt 1440 cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500 tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560 gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620 aaggccaaga agggcggaaa gatcgccgtg taa 1653 <210> 29 <211> 550 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 29 Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 40 45 Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ile Ala Val 545 550 <210> 30 <211> 417 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 30 acgcagcagc agcagcagca gcacagcagc agcagcagca gcagaccagc agcagcagca 60 gcagacccgc cgccgccgcc gccgccactc cctcaagtcc ttccagcaac cagcagcagc 120 agcagcaaca actgtgcccc gccccggcct cgacccctcc acggccccgc cccgactgaa 180 aacatagtgg cacagtacct ggaaaagctg atgaaggcac ccccagcctc cccacccctc 240 acattttaat gaaaccaggg taacttatat cagtaaagag attaacccta ggacagatct 300 actccggaac gagctcacga ctctagatca taatcagcca taccacattt gtagaggttt 360 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatg 417 <210> 31 <211> 477 <212> DNA <213> Homo sapiens <400> 31 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 60 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 120 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 180 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 240 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 300 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 360 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 420 aggagtcgtg tcgtgcctga gagcgcagtc gaggcgcgcc gagctcggat cctgacg 477 <210> 32 <211> 448 <212> DNA <213> Homo sapiens <400> 32 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 60 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 120 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 180 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 240 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 300 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 360 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 420 aggagtcgtg tcgtgcctga gagcgcag 448 <210> 33 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide <400> 33 gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt 60 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 120 tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg 180 tgggaggtct atataagcag agct 204 <210> 34 <211> 635 <212> DNA <213> Homo sapiens <400> 34 ctagcacatc gatacggtac ccaccgatat tatttgcccg atggtatccc cgtttacagg 60 taagaagatc tggcgcgcct cactagtacc tcgagattac gaagatatct tacctgagtc 120 gacaccctag gacagatctt ccggactggg caccttccac ctccttccag gcaatcactg 180 gcatgagaag gggcagacca gtgtgagctg tggaaggacg cctctttctg gaggagtgtg 240 acccccagta agcttcacgt ggggcagttc ctgagggtgg ggatctgaaa tgttggggta 300 tctcaggtcc ctcgggctgt ggggtgggct ctgaaaggca ggtgtccggg tggtgggtcc 360 tgaataggag atgccgggaa gggtctctgg gtctttgtgg gtggtgtacc ctgggggatg 420 ggaaggccgg ggctcagggc tgtggtctca ggcccgggtg aagcagtgtc cttgtccggt 480 taccctgcag ggcggcttcg tctgggttcc gtttatccgg gcaaaccggg cccgcgactc 540 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca 600 cacctccccc tgaacctgaa acataaaatg aatgc 635 <210> 35 <211> 386 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide <400> 35 agattacttc ttcaggttaa cccaacagaa ggctaaagaa ggtatattgc tgttgacagt 60 gagcgacgca gcagcagcag cagcagctag tgaagccaca gatgtagctg ctgctgctgc 120 tgctgcgctg cctactgcct cggacttcaa ggggctactt taggagcaat tatcttgttt 180 actaaaactg aagatatctt acttcttcag gttaacccaa cagaaggcta aagaaggtat 240 attgctgttg acagtgagcg acgcagcagc agcagcagca gctagtgaag ccacagatgt 300 agctgctgct gctgctgctg cgctgcctac tgcctcggac ttcaaggggc tactttagga 360 gcaattatct tgtttaactaa aactga 386 <210> 36 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 36 aggcctaata aagagctcag atgcatcgat cagagtgtgt tggttttttg tgtg 54

Claims

1. An isolated nucleic acid, said isolated nucleic acid comprising: a. A first region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR); and b. a second region comprising a transgene encoding one or more copies of a miRNA, wherein, The miRNA targets the 5' CAG junction region of the human huntingtin (HTT) gene and is encoded by a sequence comprising the sequence described in SEQ ID NO: 4, which is side-attached with the miRNA backbone sequence, wherein the human huntingtin (HTT) mRNA is as shown in SEQ ID NO:

25.

2. The isolated nucleic acid of claim 1, wherein, The transgene contains two miRNAs or two precursor miRNAs in tandem, with introns attached to the sides of the two miRNAs or two precursor miRNAs.

3. The isolated nucleic acid of claim 2, wherein, The introns connected to the sides are identical.

4. The isolated nucleic acid of claim 2, wherein, The lateral introns are from the same species.

5. The isolated nucleic acid of claim 2, wherein, The side-connected intron is the hCG intron.

6. The isolated nucleic acid according to any one of claims 1-5, wherein, The genetic modification contains a promoter.

7. The isolated nucleic acid as described in claim 6, wherein, The promoter is the synaptic protein (Syn1) promoter.

8. The isolated nucleic acid according to any one of claims 1-5, wherein, The genetic modification further encodes a protein.

9. The isolated nucleic acid of claim 8, wherein, The protein in question is CYP46A1.

10. The isolated nucleic acid according to any one of claims 1-5, wherein, The one or more copies of the miRNA are located in the untranslated portion of the transgene.

11. The isolated nucleic acid as described in claim 10, wherein, The untranslated portion is an intron.

12. The isolated nucleic acid as described in claim 10, wherein, The untranslated portion is located between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence, or between the last nucleotide base of the promoter sequence and the poly-A tail sequence.

13. The isolated nucleic acid according to any one of claims 1-5, wherein the nucleic acid further comprises a third region, the third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR).

14. The isolated nucleic acid as described in claim 13, wherein, The ITR lacks a functional terminal resolution site (TRS).

15. The isolated nucleic acid as described in claim 14, wherein, The ITR is ATRS ITR.

16. The isolated nucleic acid according to any one of claims 1-5, wherein, The second region contains two copies of miRNA.

17. A vector comprising the isolated nucleic acid as described in any one of claims 1-16.

18. The carrier as claimed in claim 17, wherein, The vector is a plasmid.

19. A host cell comprising the isolated nucleic acid as described in any one of claims 1-16 or the vector as described in claim 17 or 18.

20. A recombinant AAV (rAAV), said recombinant AAV comprising: a. Capsid protein; and b. The isolated nucleic acid as described in any one of claims 1-19.

21. The rAAV as described in claim 20, wherein, The capsid protein is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10 capsid protein, or any chimera thereof.

22. The rAAV as described in claim 20, wherein, The capsid protein is AAVrh10 capsid protein.

23. The rAAV as claimed in any one of claims 20-22, wherein, The rAAV is a self-complementary AAV (scAAV).

24. The rAAV as claimed in any one of claims 20-22, wherein, The rAAV is configured for delivery to the central nervous system (CNS).

25. An isolated nucleic acid, said isolated nucleic acid encoding the sequence described in SEQ ID NO:

4.

26. A composition comprising the isolated nucleic acid as described in any one of claims 1-16.

27. A composition comprising the carrier as described in claim 17 or 18.

28. A composition comprising rAAV as claimed in any one of claims 20-24.

29. Use of a composition comprising the isolated nucleic acid as described in any one of claims 1-16 or 25, a composition comprising rAAV as described in any one of claims 20-24, or a composition as described in any one of claims 26-28 in the preparation of a medicament for use in a subject in need of Huntington's disease.

30. The use as described in claim 29, wherein, The subjects included the huntingtin gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

31. The use as described in claim 29 or 30, wherein, The subjects were under 20 years of age and diagnosed with adolescent Huntington's disease (HD).

32. The use as described in claim 29 or 30, wherein, Administration of the composition enables the isolated nucleic acid or rAAV to be delivered to the central nervous system (CNS) of the subject.

33. The use as described in claim 29 or 30, wherein, The composition is administered by injection.

34. The use as described in claim 33, wherein, The composition is administered via intravenous or intrastriatal injection.

35. The use as described in claim 29 or 30, wherein, The composition is administered via a catheter or related device.

36. The use as described in claim 35, wherein, Prior to administration, the subjects were diagnosed with Huntington's disease or were at risk of developing Huntington's disease.

37. The use as described in claim 29 or 30, wherein, The miRNA hybridizes and inhibits the expression of human huntingtin protein (HTT), wherein the mRNA of the human huntingtin protein (HTT) is as shown in SEQ ID NO: 25.

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