Aav treatment of huntington's disease

Abstract

To provide compositions and methods useful for treating Huntington' s disease.SOLUTION: In some embodiments, the disclosure provides interfering nucleic acids (e.g., artificial miRNAs) targeting the huntingtin gene (HTT) and methods of treating Huntington's disease using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application No. 62 / 398,487, filed September 22, 2016, and entitled "AAV Treatment of Huntington's Disease," the entire contents of which are incorporated herein by reference.

[0002] Federally supported research This invention was made with government support under NS038194 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] background Huntington's disease (HD) is a devastating inherited neurodegenerative disorder caused by an expansion of a CAG repeat region in exon 1 of the Huntington gene. While the Huntington protein (HTT) is expressed throughout the body, the polyglutamine-expanded protein is particularly toxic to medium spiny neurons in the striatum and their cortical connections. Patients suffer from emotional symptoms, including depression and anxiety, as well as characteristic movement disturbances and chorea. Currently, there is no cure for Huntington's disease; treatment options are limited to alleviating symptoms. Summary of the Invention

[0004] overview Aspects of the present disclosure relate to compositions and methods useful for treating Huntington's disease (HD). In some embodiments, inhibitory nucleic acids (e.g., miRNAs, such as artificial miRNAs) that specifically hybridize to and inhibit the expression of human Huntington (HTT) are provided.

[0005] Thus, in some aspects, the disclosure provides an isolated nucleic acid comprising or encoding a sequence set forth in any one of SEQ ID NOs: 2-10 or 21-22. In some embodiments, human huntingtin comprises a sequence as set forth in SEQ ID NO: 1. In some embodiments, the disclosure provides a nucleic acid (e.g., an 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: 1.

[0006] In some aspects, the present 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. In some embodiments, the sequence encoding each miRNA comprises a sequence set forth in any one of SEQ ID NOs: 2-10 adjacent to a sequence encoding a miRNA backbone sequence.

[0007] In some embodiments, each miRNA backbone sequence is a mir-155 backbone sequence, a mir-30 backbone sequence, or a mir-64 backbone sequence. In some embodiments, the transgene further comprises a nucleic acid sequence encoding a promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter or a U6 promoter.

[0008] In some embodiments, the transgene further comprises a nucleic acid sequence encoding a protein, hi 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).

[0009] In some embodiments, one or more miRNAs are located in the untranslated portion of the transgene. In some embodiments, the untranslated portion is an intron. In some embodiments, the untranslated portion is between the last codon of the nucleic acid sequence encoding the protein and the polyA tail sequence. In some embodiments, the untranslated portion is between the last nucleic acid base of the promoter sequence and the first base of the polyA tail sequence.

[0010] In some embodiments, the isolated nucleic acid further comprises a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. In some embodiments, the first or second ITR variant lacks a functional terminal release site (TRS), and optionally the ITR variant is a ΔTRS ITR.

[0011] In some embodiments, at least one of the miRNAs hybridizes to and inhibits expression of human huntingtin. In some aspects, the present disclosure provides a vector comprising an isolated nucleic acid as described by the present disclosure. In some embodiments, the vector is a plasmid.

[0012] In some aspects, the present disclosure provides a host cell comprising an isolated nucleic acid or vector as described by the present disclosure. In some aspects, the present disclosure provides a recombinant AAV (rAAV) comprising a capsid protein; and an isolated nucleic acid as described by the present disclosure.

[0013] In some embodiments, the capsid protein is an AAV9 capsid protein. In some embodiments, the capsid protein comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the rAAV is a self-complementary AAV (scAAV). In some embodiments, the rAAV is formulated for delivery to the central nervous system (CNS).

[0014] Aspects of the present disclosure relate to isolated nucleic acids that can reduce (e.g., inhibit) expression of pathogenic Huntington's disease and thus may be useful in treating Huntington's disease. Accordingly, in some aspects, the present disclosure provides methods of treating Huntington's disease in a subject in need thereof, the methods comprising administering a therapeutically effective amount of an isolated nucleic acid or rAAV as described by the present disclosure to a subject having or at risk of developing Huntington's disease.

[0015] In some embodiments, the subject comprises a Huntington gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. In some embodiments, the subject is under 20 years of age or is diagnosed with early-onset HD.

[0016] In some embodiments, administration results in delivery of the isolated nucleic acid or rAAV to the central nervous system (CNS) of the subject. In some embodiments, administration is via injection, optionally intravenous or intrastriatal injection.

[0017] In some aspects, the present disclosure provides an isolated nucleic acid comprising a transgene encoding one or more miRNAs, wherein the sequence of the transgene encoding each miRNA comprises a sequence set forth in SEQ ID NO: 7 or 8 adjacent to the miRNA backbone sequence.

[0018] In some embodiments, each miRNA backbone sequence is a mir-155 backbone sequence, a mir-30 backbone sequence, or a mir-64 backbone sequence. In some embodiments, the transgene comprises a promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter or a U6 promoter.

[0019] In some embodiments, the transgene comprises the sequence set forth in SEQ ID NO: 21 or 22. In some embodiments, the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs) or variants thereof. In some embodiments, the ITR variant lacks a functional terminal release site (TRS). In some embodiments, the ITR variant that lacks a TRS is a ΔTRS ITR.

[0020] In some aspects, the disclosure provides a vector comprising an isolated nucleic acid comprising a transgene encoding one or more miRNAs, wherein the sequence of the transgene encoding each miRNA comprises adjacent to the miRNA backbone sequence a sequence set forth in SEQ ID NO: 7 or 8. In some embodiments, the vector is a plasmid.

[0021] 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. In some embodiments, the transgene comprises a promoter. In some embodiments, the promoter is a chicken beta-actin (CBA) promoter or a U6 promoter.

[0022] In some embodiments, the transgene comprises the sequence set forth in SEQ ID NO: 21 or 22. In some embodiments, the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs) or variants thereof. In some embodiments, the ITR variant lacks a functional terminal release site (TRS). In some embodiments, the ITR variant that lacks a TRS is a ΔTRS ITR.

[0023] In some aspects, the present disclosure provides a recombinant AAV (rAAV) comprising a capsid protein; and an isolated nucleic acid comprising a transgene encoding one or more miRNAs, wherein the sequence of the transgene encoding each miRNA comprises a sequence set forth in SEQ ID NO: 7 or 8 adjacent to the miRNA backbone sequence.

[0024] In some embodiments, the transgene is flanked by full-length AAV ITR sequences, hi some embodiments, the transgene is flanked by full-length AAV ITRs and a ΔTRS ITR. In some embodiments, the capsid protein is an AAV9 capsid protein. In some embodiments, the capsid protein comprises the sequence set forth in SEQ ID NO:20.

[0025] In some aspects, the present disclosure provides methods of treating Huntington's disease in a subject in need thereof, the methods comprising administering a therapeutically effective amount of an rAAV as described herein (e.g., an rAAV comprising a transgene encoding one or more miRNAs) to a subject having Huntington's disease or at risk of developing Huntington's disease, wherein the sequence of the transgene encoding each miRNA comprises a sequence set forth in SEQ ID NO: 7 or 8 adjacent to the miRNA backbone sequence.

[0026] In some embodiments, the transgene of the isolated nucleic acid is flanked by full-length AAV ITR sequences, hi some embodiments, the transgene is flanked by full-length AAV ITRs and a ΔTRS ITR.

[0027] In some embodiments, the rAAV comprises an AAV9 capsid protein. In some embodiments, the subject comprises a Huntington gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. In some embodiments, the subject is under 20 years of age.

[0028] In some embodiments, administration is via injection. In some embodiments, the injection is intravenous or intrastriatal. [Brief explanation of the drawings]

[0029] Brief description of the drawings [Figure 1]Figure 1 shows HeLa cells transfected with a plasmid expressing mir-HTT-6433 (targeting human huntingtin). Forty-eight hours after transfection, cells were harvested and RNA was extracted for quantitative RT-PCR (qRT-PCR). The results indicate that mir-HTT-6433 reduces endogenous human huntingtin by up to 50%.

[0030] [Figure 2] Figure 2 shows mir-HTT-6433 packaged into an AAV9 vector and injected directly into the striatum of transgenic mice (Yac128 mice) expressing mutant human huntingtin. One month after injection, human huntingtin mRNA levels were measured by qRT-PCR. In one set of animals (n = 5), the levels of human huntingtin in the injected side were compared with those in the uninjected side. A significant (p = 0.0017) reduction in huntingtin mRNA was observed in the injected side. In a second set of animals (n = 5 / group), the levels of huntingtin mRNA in animals injected with mir-HTT-6433 were compared with those in animals that received only vehicle injections. A significant (p = 0.0004) reduction in huntingtin was also observed in these animals. In a third set of animals (n=5 / group), the levels of huntingtin mRNA in animals injected with mir-HTT-6433 were compared with those injected with AAV9-GFP. Huntington mRNA was also significantly reduced (p=0.0064) in these animals. In summary, the data indicate that mir-HTT-6433 reduces huntingtin mRNA by 50% in the brain in vivo.

[0031] [Figure 3]Figure 3 shows that transgenic mice expressing mutant human huntingtin were unilaterally injected with AAV9-mir-HTT-6433 or PBS. Six months after injection, the mice were tested on a balance beam. The data show that mice treated with mir-HTT-6433 showed a decreased time to cross the beam compared to HD mice treated with PBS.

[0032] [Figure 4] Figures 4A-4C show that artificial miRNAs targeting human huntingtin reduce huntingtin mRNA in cell culture and in vivo. Figure 4A shows the location of the target site on human huntingtin mRNA. Figure 4B shows HeLa cells transfected with a plasmid expressing an artificial miRNA targeting human huntingtin; huntingtin mRNA levels were measured by qPCR 48 hours later. Huntington expression was normalized to HPRT to account for well-to-well variations in cell number. Huntington expression is expressed relative to untreated / naive controls. Error bars represent standard error. Figure 4C shows candidate miRNAs selected for in vivo testing based on the cell culture results. The miRNAs were injected unilaterally into the striatum of mice. One month after injection, the striatum was harvested and GFP-positive tissue was dissected out. Data were normalized to HPRT and expressed relative to the GFP-only control.

[0033] [Figure 5]Figures 5A-5C show that expression of an artificial miRNA from the U6 promoter does not improve huntingtin silencing in the mouse striatum. Figure 5A shows data for AAV9 constructs expressing miRNAs from the CBA (pol II) and U6 promoters. The CBA-promoter-driven miRNA is located in the 3'-UTR of the GFP gene, while the construct containing the U6-promoter-driven artificial miRNA coexpresses GFP from a separate promoter. Figure 5B shows the relative amount of huntingtin mRNA in the injected striatum after injection of U6- and CBA-promoter-driven artificial miRNAs targeting sites 5155 (left) and 6433 (right). Data are expressed relative to the uninjected side. Figure 5C shows the relative amount of huntingtin mRNA in mice unilaterally injected with U6- and CBA-promoter-driven miRNAs targeting site 6433. Data are expressed relative to a group of mice injected with a GFP-expressing control vector.

[0034] [Figure 6] Figures 6A-6B show that long-term striatal expression of mir-HTT-6433 driven by the U6 promoter causes behavioral abnormalities. Figure 6A shows that 6 months after injection, mice injected with U6-promoter-driven mir-HTT-6433 failed to build nests. Images were taken 24 hours after placing new small nestlets in the cage. Figure 6B shows cage monitoring of Yac128 mice treated with PBS, CBA-mir-HTT-6433, or U6-mir-HTT-6433. The amount of time spent moving around the cage was recorded for 24 to 27 hours. The average time per hour was calculated by dividing the total amount of time by the number of hours recorded.

[0035] [Figure 7]Figures 7A-7B show that long-term expression of mir-HTT-6433 from the U6 promoter causes striatal atrophy. Figure 7A shows representative images of DARPP-32 staining on the injected side in Yac128 mice at 1 month (top) and 6 months (bottom) after injection. Figure 7B shows quantification of the DARPP-32-positive area 6 months after injection.

[0036] [Figure 8] Figures 8A-8D show that long-term expression of mir-HTT-6433 from the U6 promoter leads to sustained microglial activation. Figure 8A shows representative images of Iba1 staining on the injected side in Yac128 mice at 1 month (top) and 6 months (bottom) after injection. Images were taken at the injection site. Quantification of total body (Figure 8B), activated microglia (Figure 8C), and quiescent microglia (Figure 8D) at 6 months after injection is shown.

[0037] [Figure 9] Figures 9A-9C show that expression of miR155-based artificial miRNAs from the U6 promoter results in overexpression of the guide strand and other sequences targeting huntingtin. Figure 9A shows the starting positions of reads mapping to the artificial miRNA hairpin (mir-155 backbone) targeting huntingtin. Positions are reported relative to the mature strand, and reads are normalized to the total number of endogenous miRNAs mapped in each sample. The horizontal line represents the background level of the artificial miRNA found in the control sample. Figure 9B shows the relative quantification of mature miR-HTT (from the miR-155 backbone) by quantitative RT-PCR. Figure 9C shows the starting positions of reads mapping to the artificial miRNA targeting huntingtin, incorporated into the miR-30 backbone and expressed from the U6 promoter.

[0038] [Figure 10]Figures 10A-10B show that expression of mir-HTT-6433 preferentially reduces mRNAs with the target site. Figure 10A shows that mRNAs were separated into those containing canonical miRNA binding sites matching the artificial miRNA site (legend) and those without. In the group of mice injected with AAV-CbA-mir-HTT-6433, there was no difference between mRNAs with and without such sites. Figure 10B, in contrast, shows a shift toward suppression of mRNAs with perfect 8-mer sites in the AAV-U6-mir-HTT-6433 group.

[0039] [Figure 11] Figures 11A-11C show that reducing vector dose results in reduced spread and knockdown in the mouse striatum. Figure 11A shows GFP staining in the striatum of mice injected with vectors encoding both an artificial miRNA targeting huntingtin and EGFP. ImageJ was used to measure the percentage of striatum that was GFP-positive. Figure 11B shows quantitative RT-PCR measuring human huntingtin mRNA in the striatum of Yac128 mice. Figure 11C shows representative photographs of mice injected with three different doses of vectors encoding both an miRNA targeting huntingtin and EGFP. The data indicate that reducing vector dose results in reduced spread and knockdown.

[0040] [Figure 12]Figures 12A-12B show data indicating that Yacl28 mice exhibit a decline in their ability to cross the balance beam after injection of a U6-promoter-driven artificial miRNA targeting Huntington. Figure 12A shows that mice injected at 2-3 months took a clear increase in the time it took to cross the balance beam, and some of them were unable to cross at all. Figure 12B shows that Yacl28 mice injected at 7 months of age with either PBS or CBA-mir-HTT-6433 exhibited an age-related decline in beam performance. Injection with U6-mir-HTT-6433 (red dots) accelerated this decline.

[0041] [Figure 13] Figures 13A-13B show that C57BL / 6 mice exhibit an initial deterioration in balance beam performance after injection of a U6-promoter-driven artificial miRNA targeting Huntington. Figure 13A shows the amount of time it takes to cross the balance beam for control (naive) mice and mice injected with AAV-U6-mir-HTT-6433 and AAV-CbA-mir-HTT-6433. Figure 13B shows quantification of DARPP-32-positive striatal area in control (naive) mice and mice injected with AAV-U6-mir-HTT-6433 and AAV-CbA-mir-HTT-6433.

[0042] [Figure 14] Figures 14A-14B show that the distribution of endogenous miRNAs is largely unaffected after injection of mir-HTT-6433. Figure 14A shows the distribution in mice injected with AAV-CbA-mir-HTT-6433; levels of the mir-HTT-6433 guide and passenger strands are shown in green, and all endogenous miRNA species showing significant changes in mice injected with AAV-CbA-mir-HTT-6433 are shown in red. Figure 14B shows the distribution in mice injected with AAV-U6-mir-HTT-6433.

[0043] [Figure 15] Figures 15A-15C show mRNA profiles in mice treated with mir-HTT-6433. Figure 15A shows that mice injected with AAV-CbA-mir-HTT-6433 show little change in mRNA expression; all genes showing significant p-values ​​are in green, and genes that remain significant after adjusting for multiple comparisons are in blue. Figure 15B shows that mice injected with AAV-U6-mir-HTT-6433 show more changes in mRNA expression compared to CBA. Figure 15C shows the mRNA profiles in mice treated with mir-HTT-6433; seven RNAs are significantly differentially expressed between the U6- and CbA-treated groups.

[0044] [Figure 16] Figure 16 shows data on the relative expression of human huntingtin (human htt) RNA in the middle caudate of a sheep model of Huntington's disease one month after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Data on htt expression levels in uninjected control sheep are also shown. Relative htt expression levels were normalized to ovine calnexin. Note: The mir155 backbone was used in each of the CBA and U6 constructs.

[0045] [Figure 17]Figure 17 shows data on the relative expression of human huntingtin (human htt) RNA in the middle putamen of a sheep model of Huntington's disease one month after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Data on expression levels in uninjected control sheep are also shown. Relative htt expression levels were normalized to ovine calnexin.

[0046] [Figure 18] Figure 18 shows data on the relative expression of human huntingtin (human htt) RNA in the medial mid-caudate region of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter") or an empty scAAV9 control vector. Data are shown for expression levels in the uninjected and injected sides. Relative htt expression levels were normalized to ovine calnexin.

[0047] [Figure 19] Figure 19 shows data on the relative expression of human huntingtin (human htt) RNA in the lateral side of the medial caudate in a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter") or an empty scAAV9 control vector. Data are shown for expression levels in the uninjected and injected sides. Relative htt expression levels were normalized to ovine calnexin.

[0048] [Figure 20]Figure 20 shows data on the relative expression of human huntingtin (human htt) RNA in the mid-caudate region of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Data on htt expression levels in uninjected control sheep are also shown. Relative htt expression levels were normalized to ovine calnexin.

[0049] [Figure 21] Figure 21 shows data on the relative expression of human huntingtin (human htt) RNA in the lateral medial putamen of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter") or an empty scAAV9 control vector. Data are shown for expression levels in the uninjected and injected sides. Relative htt expression levels were normalized to ovine calnexin.

[0050] [Figure 22] Figure 22 shows data on the relative expression of human huntingtin (human htt) RNA in the medial central putamen of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter") or an empty scAAV9 control vector. Data are shown for expression levels in the uninjected and injected sides. Relative htt expression levels were normalized to ovine calnexin.

[0051] [Figure 23]Figure 23 shows data on the relative expression of human huntingtin (human htt) RNA in the central putamen of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Data on htt expression in uninjected control sheep are also shown. Relative htt expression levels were normalized to ovine calnexin.

[0052] [Figure 24] Figure 24 shows data on the relative expression of human huntingtin (human htt) RNA in the anterior striatum of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Data on htt expression levels in uninjected control sheep are also shown. Relative htt expression levels were normalized to ovine calnexin.

[0053] [Figure 25] Figures 25A-25B show predicted hairpin structures of artificial miRNAs targeting human huntingtin. Figure 25A shows the predicted hairpin structure of mir-155-6433 (SEQ ID NO: 23). Figure 25B shows the predicted hairpin structure of mir-30-6433 (SEQ ID NO: 24).

[0054] [Figure 26]Figures 26A-26B show AAV vector delivery to the sheep brain. Figure 26A shows a schematic overview of a sheep brain dissected in the coronal plane so that the entire striatum was contained within a 4-6 mm block (top). The anterior block contains the anterior portion of the striatum undivided by the internal capsule (middle). The medial block targeted for injection has the putamen defined and the caudate shown below. Figure 26B shows AAV vector genomes in control (AAV9) and treated (AAV9miRHTT) animals. Vector genomes were measured by digital droplet PCR using genomic HPRT as a reference gene. Values ​​are plotted on a log scale.

[0055] [Figure 27] Figure 27 shows that artificial miRNA guide strands were quantified by digital droplet PCR. Relative miRNA levels were calculated by normalizing to let-7e* and plotted on a log scale. Samples with an RQN < 5 were excluded. The reported number is the total number of samples used for miRNA analysis that survived this quality threshold. P values ​​were calculated using a 2-way ANOVA with Tukey's correction for multiple testing.

[0056] [Figure 28]Figure 28 shows that scAAV9-anti-HTT-6433 reduces human mutant huntingtin mRNA in the striatum. Data shown are HTT mRNA signals normalized to human calnexin. Asterisks indicate significant differences in means between treatment groups (AAV9 or AAV9miRHTT) at p<0.03 in an unpaired t-test. U6-promoter-driven artificial miRNA significantly reduces human mutant HTT mRNA in the caudate and putamen at 1 month post-injection and in the putamen at 6 months post-injection. CBA-promoter-driven artificial miRNA reduces HTT mRNA in the caudate, putamen, and anterior striatum at 1 month post-injection and at 6 months post-injection in the caudate and putamen. The medial region of the caudate, lateral putamen, and anterior striatum were examined in the analysis.

[0057] [Figure 29] Figures 29A-29B show endogenous ovine htt mRNA and protein levels in sheep treated with AAV9 and AAV9miRHTT. Figure 29A shows that ovine htt mRNA levels were determined as described in the Methods and are expressed relative to ovine calnexin (Canx). Results from Study 2 are shown. There is no difference in endogenous ovine htt mRNA levels between the AAV9- and AAV9miRHTT-treated groups. Figure 29B shows endogenous ovine huntingtin and human mHTT protein levels detected in the putamen using anti-htt1-17 antibody (Ab1) 6 months after injection from Study 2. Western blot samples show signals for wild-type htt (arrows) and human mutant htt (arrowheads) for the injected and uninjected sides of the brains of four different sheep unilaterally injected with AAV9miRHTT. The graph shows the average signal of ovine htt and human mHTT determined by densitometry as a percentage of the injected side relative to the non-injected side. Note that treatment with AAV9miRHTT does not affect the level of endogenous ovine huntingtin protein, but significantly reduces the level of human mHTT. The asterisk indicates p=0.005 by unpaired t-test.

[0058] [Figure 30] Figure 30 shows that AAV9-miRHTT reduces human mutant huntingtin protein in the striatum. Western blot samples of putamen from Studies 1 and 2 show mutant HTT detected with antibody 3B5H10 and actin as a loading control (top). Graphs show the distribution and mean (horizontal bars) of individual values ​​for sheep treated with either AAV9 (control) or AAV9-miRHTT (bottom). Shown are results for different striatal regions (caudate, putamen, and anterior striatum) for Studies 1 and 2 and 1 and 6 months after injection. In Study 2, 6 months after injection, two areas (areas 1 and 2) were examined in each region. Asterisks indicate significant differences on the injection side between AAV9 and AAV9-miRHTT at p<0.05 based on an unpaired t-test.

[0059] [Figure 31] Figure 31 shows human mutant HTT levels detected by MSD assay at 1 and 6 months post-injection in Study 1 (U6 promoter) and Study 2 (CBA promoter). The graph shows the distribution and mean (horizontal bar) of individual values ​​for sheep treated with either AAV9 (control) or AAV9-miRHTT. Results are shown for various striatal regions (caudate, putamen, and anterior striatum). Asterisks * indicate significant differences on the injection side between AAV9 and AAV9-miRHTT at p<0.05 based on an unpaired t-test.

[0060] [Figure 32]Figures 32A-32B show that mHTT levels are unchanged in the cortex ipsilateral to the miRHTT-injected sheep striatum and the caudate putamen contralateral to the miRHTT-injected striatum. Figure 32A shows a graph indicating the mean levels of mHTT normalized to actin in the cortex ipsilateral to the miRHTT-injected striatum. Data are from Study 1, NS, 1 month and 6 months after injection, based on an unpaired t-test. Figure 32B shows a bar graph indicating the mean levels of mHTT normalized to actin in the caudate and putamen contralateral to the miRHTT-injected striatum. Data are from Study 1, NS, 1 month and 6 months after injection, based on an unpaired t-test. DETAILED DESCRIPTION OF THE INVENTION

[0061] Detailed Description Aspects of the present invention relate to certain interfering RNAs (e.g., miRNAs, such as artificial miRNAs) that, when delivered to a subject, are effective in reducing the expression of pathogenic Huntington's protein (HTT) in the subject. As a result, the methods and compositions described by this disclosure are useful, in some embodiments, for the treatment of Huntington's disease.

[0062] Methods for treating Huntington's disease Methods for delivering a transgene (e.g., an inhibitory RNA such as an miRNA) to a subject are provided by the present disclosure. The methods typically involve administering to the subject an effective amount of an rAAV containing an isolated nucleic acid encoding an interfering RNA capable of reducing expression of huntingtin (htt) protein, or a nucleic acid for expressing an inhibitory RNA capable of reducing expression of huntingtin protein.

[0063] In some aspects, the present disclosure provides inhibitory miRNAs that specifically bind to (e.g., hybridize to) at least two consecutive (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) bases of human huntingtin (e.g., SEQ ID NO: 1). As used herein, "consecutive bases" refers to two or more nucleotide bases that are covalently linked (e.g., by one or more phosphodiester bonds, etc.) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, at least one miRNA is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% identical to 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: 1. In some embodiments, the inhibitory RNA is an miRNA that comprises or is encoded by a sequence set forth in any one of SEQ ID NOs: 2-10.

[0064] As used herein, "Huntington's disease" or "HD" refers to a neurodegenerative disease characterized by gradually worsening motor, cognitive, and behavioral changes caused by a tri-nucleotide repeat expansion (e.g., CAG, which is translated into poly-glutamine or poly-Q tracts) in the HTT gene, which results in the production of a pathogenic mutant Huntington protein (HTT or mHTT). In some embodiments, the mutant Huntington protein accelerates the rate of neuronal cell death in certain regions of the brain. Generally, the severity of HD correlates with the size of the tri-nucleotide repeat expansion in a 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, a subject with HD or at risk of having HD has an HTT gene containing between about 36 and about 39 CAG repeats (eg, 36, 37, 38, or 39 repeats).

[0065] In some embodiments, subjects with HD or at risk of having HD have an HTT gene that contains 40 or more CAG repeats (e.g., 40, 45, 50, 60, 70, 80, 90, 100, 200 or more). In some embodiments, subjects with an HTT gene that contains more than 100 CAG repeats develop HD earlier than subjects with less than 100 CAG repeats. In some embodiments, subjects with an HTT gene that contains more than 100 CAG repeats may develop HD symptoms before the age of about 20 years, and are referred to as having early-onset HD (also referred to as akinetic-rigid HD or Westphal variant HD). The number of CAG repeats in a subject's HTT gene allele can be determined by any suitable modality known in the art. For example, nucleic acid (e.g., DNA) can be isolated from a subject's biological sample (e.g., blood), and the number of CAG repeats of the HTT allele can be determined by hybridization-based methods such as PCR or nucleic acid sequencing (e.g., Illumina sequencing, Sanger sequencing, SMRT sequencing, etc.).

[0066] An "effective amount" of a substance is an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or, in the context of rAAV-mediated delivery, infect) a sufficient number of target cells in a target tissue of a subject. In some embodiments, the target tissue is a central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, an effective amount of an isolated nucleic acid (e.g., that may be delivered via rAAV) may be an amount sufficient to have a therapeutic benefit in a subject, e.g., to reduce the expression of a pathogenic gene or protein (e.g., HTT), extend the lifespan of a subject, ameliorate one or more symptoms of a disease in a subject (e.g., symptoms of Huntington's disease), etc. The effective amount will depend on various factors, such as the species, age, weight, health status, and tissue being targeted of the subject. Thus, it may vary between subjects and tissues as described elsewhere in this disclosure.

[0067] Isolated nucleic acids In some aspects, the present disclosure provides isolated nucleic acids useful for reducing (e.g., inhibiting) the expression of human huntingtin (HTT). A "nucleic acid" sequence refers to a DNA sequence or an RNA sequence. In some embodiments, the proteins and nucleic acids of the present 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, e.g., by polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and gel separation; or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is one that is readily manipulable by recombinant DNA techniques well known in the art.

[0068] Thus, a nucleotide sequence contained in a vector (whose 5' and 3' restriction sites are known or whose polymerase chain reaction (PCR) primer sequences are disclosed) is considered isolated, while a nucleic acid sequence existing in its natural state in its natural host is not. An isolated nucleic acid may, but need not, be substantially purified. For example, a nucleic acid isolated within a cloning or expression vector is not pure, in that it may comprise a small percentage of the material in the cell in which it is present. However, such a nucleic acid, as the term is used herein, is isolated because it is one that can be readily manipulated by standard techniques known to those of skill in the art. As used herein with respect to a protein or peptide, the term "isolated" refers to a protein or peptide that has been separated from its natural environment or that has been artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0069] Those skilled in the art will also recognize that conservative amino acid substitutions can be made to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, the present disclosure utilizes sequence alterations that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art (such as those found in references summarizing such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). Conservative amino acid substitutions include substitutions made between amino acids within 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. Thus, conservative amino acid substitutions may be made in the amino acid sequences of the proteins and polypeptides disclosed herein.

[0070] The isolated nucleic acid of the present invention may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described herein comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. A "recombinant AAV (rAAV) vector" typically minimally comprises a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise one or more regions encoding one or more inhibitory RNAs (e.g., miRNAs), including nucleic acids targeting endogenous mRNAs of the subject, as disclosed elsewhere herein. The transgene may also comprise regions encoding, for example, proteins and / or expression control sequences (e.g., a polyA tail), as described elsewhere herein.

[0071] Generally, ITR sequences are approximately 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are acceptable. The ability to modify these ITR sequences is within the knowledge of the art. (See, for example, texts such as 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 employed in the present invention is a "cis-acting" plasmid containing a selected transgene sequence and associated regulatory elements flanked by 5' and 3' AAV ITR sequences containing the transgene. AAV ITR sequences 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, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., the first region) encoding an AAV2 ITR.

[0072] In some embodiments, the isolated nucleic acid further comprises a region comprising 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, 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" may refer to an AAV ITR containing a mutation (e.g., a sense mutation, such as a nonsynonymous mutation, or a missense mutation) that abolishes the function of the terminal resolution site (TRS) of the ITR, or may refer to a truncated AAV ITR (e.g., a ΔTRS ITR) that lacks a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors containing ITRs that lack a functional TRS produce self-complementary rAAV vectors, such as those described by McCarthy (2008) Molecular Therapy 16(10):1648-1656.

[0073] In addition to the key elements identified above for recombinant AAV vectors, the vectors also include conventional control elements operably linked to the transgene elements in a manner that permits its transcription, translation, and / or expression in cells transfected with the vector or infected with a virus produced by the present invention. As used herein, "operably linked" sequences encompass both expression control sequences adjacent to a gene of interest to regulate the gene of interest, as well as expression control sequences acting in trans or at a distance. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and, when desired, sequences that improve secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible and / or tissue-specific promoters, are known in the art and may be utilized.

[0074] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked such that expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frameshift mutations, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, and (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein.

[0075] Thus, a promoter region would be operably linked to a DNA sequence if it is capable of effecting transcription of that nucleic acid sequence (such that the resulting transcript is translated into a desired protein or polypeptide). Similarly, two or more coding regions are operably linked when their transcription from a common promoter results in the expression of two or more proteins translated in-frame. In some embodiments, operably linked coding sequences produce a fusion protein. In some embodiments, operably linked coding sequences produce a functional RNA (e.g., miRNA).

[0076] In some aspects, the present disclosure provides an isolated nucleic acid comprising a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more microRNAs (e.g., miRNAs). A "microRNA" or "miRNA" is a small, non-coding RNA molecule capable of mediating post-transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed as double-stranded structures, such as hairpins or stem-loops (e.g., with a self-complementary single-stranded backbone), referred to as primary miRNAs (pri-miRNAs), which are enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.), to form pre-miRNAs. The length of a pri-miRNA can vary. In some embodiments, a pri-miRNA ranges in length from about 100 base pairs to about 5,000 base pairs (e.g., about 100, about 200, about 500, about 1,000, about 1,200, about 1,500, about 1,800, or about 2,000 base pairs). In some embodiments, the pri-miRNA is greater than 200 base pairs in length (eg, 2500, 5000, 7000, 9000, or more base pairs in length).

[0077] Pre-miRNAs are also characterized by a hairpin or stem-loop duplex structure, but can vary in length. In some embodiments, pre-miRNAs range in size from about 40 base pairs to about 500 base pairs. In some embodiments, pre-miRNAs range in size from about 50 to 100 base pairs. In some embodiments, pre-miRNAs range in size from about 50 to about 90 base pairs (e.g., 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).

[0078] Generally, pre-miRNAs are transported into the cytoplasm and enzymatically processed by Dicer to first generate an incomplete miRNA / miRNA* duplex and then a single-stranded mature miRNA molecule, which is then loaded into the RNA-induced silencing complex (RISC). Typically, mature miRNA molecules range in size from about 19 to about 30 base pairs in length. In some embodiments, mature miRNA molecules are 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 length. In some embodiments, the isolated nucleic acids of the present disclosure comprise a sequence encoding a pri-miRNA, a pre-miRNA, or a mature miRNA comprising a sequence set forth in any one of SEQ ID NOS: 2-10 or 21-22.

[0079] It should be understood that an isolated nucleic acid or vector (e.g., an rAAV vector) in some embodiments comprises a nucleic acid sequence encoding more than one miRNA (e.g., a plurality of miRNAs, such as 2, 3, 4, 5, 10, or more). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes to or specifically binds to) the same target gene (e.g., an isolated nucleic acid encoding three unique miRNAs, where each miRNA targets the HTT gene). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes to or specifically binds to) a different target gene.

[0080] In some aspects, the present 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 an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone, a precursor miRNA capable of generating a functional mature miRNA), in which the miRNA and miRNA* (e.g., the passenger strand of an miRNA duplex) sequences are replaced with corresponding amiRNA / amiRNA* sequences that direct highly efficient RNA silencing of targeted 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, but a sequence encoding a mature HTT-specific miRNA (e.g., any one of SEQ ID NOS: 2-10) is inserted into the backbone in place of the endogenous miR-155 mature miRNA coding sequence. In some embodiments, a miRNA (eg, an artificial miRNA) as described by the present disclosure comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a mir-64 backbone sequence, or a miR-122 backbone sequence.

[0081] The region containing the transgene (e.g., the second, third, fourth, etc. region) may be located in any suitable location in 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.).

[0082] In some cases, it may be desirable to locate the region upstream of the first codon of a nucleic acid sequence encoding a protein (e.g., a protein-coding sequence) (e.g., a second region, a third region, a fourth region, etc.). 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.

[0083] In some cases (e.g., when the transgene lacks a protein-coding sequence), it may be desirable to locate the region upstream of the poly-A tail of the transgene (e.g., the second, third, fourth, etc. region). For example, the region may be located between the first base of the poly-A tail and 2000 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 1000 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 500 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 250 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 150 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 100 nucleotides upstream of that first base. The region may be located between the first base of the poly-A tail and 50 nucleotides upstream of that 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.

[0084] 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 between the last base of the poly-A tail and a position 2000 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 1000 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 500 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 250 nucleotides downstream of the last base. The region may be between the last base of the poly-A tail and a position 150 nucleotides downstream of the last base.

[0085] It should be understood that in cases where a transgene encodes more than one miRNA, each miRNA may be located in any suitable location within the transgene. For example, the nucleic acid encoding a first miRNA may be located in an intron of the transgene, and the nucleic acid sequence encoding a second miRNA may be located in a separate 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).

[0086] In some embodiments, the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., a promoter, etc.). Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and, if desired, sequences that improve secretion of the encoded product. Numerous expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be used.

[0087] A "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery and required to initiate the specific transcription of a gene. The phrases "operably located," "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.

[0088] In nucleic acids encoding proteins, a polyadenylation sequence is generally inserted after the transgene sequence and before the 3'AAV ITR sequence. rAAV constructs useful in the present disclosure may also contain an intron, preferably 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-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). IRES sequences are used to produce more than one polypeptide from a single gene transcript. IRES sequences may be used to produce proteins containing more than one polypeptide chain. The selection of these and other common vector elements is conventional, and many such sequences are available (see, e.g., Sambrook et al., and references cited therein, e.g., pages 3.18, 3.26, and 16.17, 16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989).

[0089] In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the 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 2A sequences has previously been 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; pp. 864-873; and Halpin, C et al., The Plant Journal, 1999; pp. 453-459; de Felipe, P et al., Gene Therapy, 1999; pp. 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; pp. 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001;8:811-817).

[0090] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (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 a U6 promoter.

[0091] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological states (e.g., acute phase, specific differentiation state of cells), or only during cell replication. Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Numerous other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), and the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)). (1998)), the 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)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only during replicating cells.

[0092] In another embodiment, the native promoter will be used for the transgene. The native promoter may be preferred when it is desired that the expression of the transgene mimics the native expression. The native promoter may be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic the native expression.

[0093] In some embodiments, the regulatory sequence confers tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequence binds 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: the liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, alpha-myosin heavy chain (a-MHC) promoter, or cardiac troponin T (cTnT) promoter.

[0094] Other exemplary promoters include the beta-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 bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); the immunoglobulin heavy chain promoter; the T cell receptor α-chain promoter, and neuronal promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), as would be apparent to one skilled in the art.

[0095] Aspects of the present disclosure relate to isolated nucleic acids comprising more than one promoter (e.g., two, three, four, five, or more promoters). For example, in the context of a construct having a transgene comprising a first region encoding a protein and a second region encoding an inhibitory RNA (e.g., an miRNA), it may be desirable to drive expression of the protein-coding region using a first promoter sequence (e.g., a first promoter sequence operably linked to the protein-coding region) and to drive expression of the inhibitory RNA-coding region with a second promoter sequence (e.g., a second promoter sequence operably linked to the inhibitory RNA-coding region). Generally, the first promoter sequence and the second promoter sequence can be the same promoter sequence or different promoter sequences.

[0096] In some embodiments, the first promoter sequence (e.g., a promoter driving expression of a protein coding region) is an RNA polymerase III (pol III) promoter sequence. Non-limiting examples of pol III promoter sequences include U6 and H1 promoter sequences. In some embodiments, the second promoter sequence (e.g., a promoter sequence driving expression of an inhibitory RNA) is an RNA polymerase II (pol II) promoter sequence. Non-limiting examples of pol II promoter sequences include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the pol III promoter sequence drives expression of an inhibitory RNA (e.g., miRNA) coding region. In some embodiments, the pol II promoter sequence drives expression of a protein coding region.

[0097] In some embodiments, the nucleic acid comprises a transgene encoding a protein. The protein can be a therapeutic protein (e.g., a peptide, protein, or polypeptide useful for treating or preventing a pathology in a mammalian subject) or a reporter protein. In some embodiments, the therapeutic protein is polyglutamine-binding peptide 1 (QBP1), PTD-QBP1, ED11, C4 intrabody, VL12.3 intrabody, MW7 intrabody, Happ1 antibody, Happ3 antibody, mEM48 intrabody, certain monoclonal antibodies (e.g., 1C2), and peptide P42 and variants thereof, useful for treating or preventing Huntington's disease, e.g., as described in Marelli et al. (2016) Orphanet Journal of Rare Disease 11:24; doi: 10.1186 / s13023-016-0405-3. In some embodiments, the therapeutic protein is a wild-type Huntington protein (e.g., a Huntington protein having a polyQ repeat region containing fewer than 36 repeats).

[0098] Although not wishing to be bound by any particular theory, allele-specific silencing of mutant huntingtin (HTT) can provide an improved safety profile for subjects compared with non-allele-specific silencing (for example, silencing both wild-type and mutant HTT alleles).This is because the expression and function of wild-type HTT are preserved in cells.The present invention relates to the inventors' recognition and belief that the isolated nucleic acid and vector incorporating one or more inhibitory RNA (for example, miRNA) sequences that target HTT gene in a non-allele-specific manner, while promoting the expression of hardened wild-type HTT gene (the wild-type HTT gene that is not targeted by miRNA), can achieve the increased expression of wild-type HTT and the knockdown of mutant HTT (for example, in CNS tissue).

[0099] Generally, the nucleic acid sequence encoding the endogenous wild-type or mutant HTT mRNA and the nucleic acid sequence of the transgene encoding the "hard" wild-type HTT mRNA are sufficiently different so that the "hard" wild-type HTT transgene mRNA is not targeted by one or more inhibitory RNAs (e.g., miRNAs). This can be achieved, for example, by introducing one or more silent mutations into the HTT transgene sequence (so that it encodes a protein having the same but different nucleic acid sequence as the endogenous wild-type HTT gene). In this case, the exogenous mRNA can be referred to as "hard." Alternatively, the inhibitory RNA (e.g., miRNA) can target the 5' and / or 3' untranslated regions of the endogenous wild-type HTT mRNA. These 5' and / or 3' regions in the transgene mRNA are then removed or replaced so that the transgene mRNA is not targeted by one or more inhibitory RNAs.

[0100] Reporter sequences (e.g., nucleic acid sequences encoding reporter proteins) that may be provided in a transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When linked to regulatory elements that drive their expression, reporter sequences provide signals that can be detected by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays (including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry). For example, if the marker sequence is a LacZ gene, the presence of the signal-transmitting vector is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-transmitting vector can be visually measured by color or light production in a luminometer. Such reporters can be useful, for example, to verify tissue-specific targeting capabilities of nucleic acids and tissue-specific promoter regulatory activity.

[0101] Recombinant adeno-associated virus (rAAV) In some aspects, the present disclosure provides isolated AAV. As used herein with respect to AAV, the term "isolated" refers to an AAV that has been artificially produced or obtained. Isolated AAV may be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV." Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities, such that the rAAV nuclease and / or transgene is specifically delivered to one or more predetermined tissue(s). The AAV capsid is an important factor in determining these tissue-specific targeting capabilities. Thus, rAAVs having a capsid appropriate for the tissue to be targeted can be selected.

[0102] Methods for obtaining recombinant AAVs with desired capsid proteins are well known in the art. (See, e.g., 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 the AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector 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, virion proteins 1 to 3 (designated VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, when translated, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the function of the capsid protein is 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.

[0103] In some embodiments, the AAV capsid protein is of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAV9, and AAV10. In some embodiments, the AAV capsid protein is of a serotype derived from a non-human primate, such as the AAVrh8 serotype. In some embodiments, the AAV capsid protein is of the AAV9 serotype. In some embodiments, the AAV capsid protein comprises the sequence set forth in SEQ ID NO:20.

[0104] The components to be cultured in a host cell for packaging the rAAV vector into an AAV capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components using methods known to those skilled in the art. Most preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may also be under the control of a constitutive promoter.

[0105] Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and selected other component(s) under the control of one or more inducible promoters. For example, stable host cells derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) can be generated, which contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated by those skilled in the art.

[0106] In some embodiments, the disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a protein (e.g., a wild-type huntingtin protein, optionally a "hard" wild-type huntingtin protein). In some embodiments, the disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell further comprises a cryopreservation agent.

[0107] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to produce the rAAV of the present disclosure may be delivered to a packaging host cell using any suitable genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0108] In some embodiments, recombinant AAV may be produced using a triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes).

[0109] Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19, 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 entireties. Accessory function vectors encode nucleotide sequences for cellular functions (i.e., "accessory functions") on which non-AAV-derived viruses and / or AAVs depend for replication. Accessory functions include those functions required for AAV replication, but also include, without limitation, those moieties involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0110] In some aspects, the present disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell; a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Numerous transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory 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 exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into suitable host cells.

[0111] "Host cell" refers to any cell that harbors or is capable of harboring a substance of interest. Often, the host cell is a mammalian cell. Host cells may be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNA involved in the production of recombinant AAV. The term encompasses the progeny of the original transfected cell. Thus, as used herein, "host cell" may refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutation.

[0112] As used herein, the term "cell line" refers to a group of cells that can grow and divide continuously or for a long period in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes in karyotype may occur during the storage or transfer of such clonal populations. Therefore, the cells derived from the cell line referred to may not be strictly identical to the ancestral cell or culture, and the cell line referred to encompasses such variants.

[0113] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that leads to the transcription of a biologically active polypeptide or the production of a nucleic acid such as a biologically active RNA.

[0114] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that can replicate and transfer genetic sequences between cells when linked to appropriate control elements. Thus, the term encompasses cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated as those vectors in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery and required to initiate the specific transcription of a gene.

[0115] The phrases "operably located," "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 a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to produce a biologically active polypeptide product or functional RNA (e.g., guide RNA) from the transcribed gene.

[0116] The foregoing methods for packaging a recombinant vector into the desired AAV capsid to produce the rAAV of the present disclosure are not intended to be limiting, and other suitable methods will be apparent to those of skill in the art.

[0117] In some embodiments, any one or more thymidine (T) or uridine (U) nucleotides in the sequences provided herein (including those provided in the Sequence Listing) may be replaced with any other nucleotide suitable for base pairing with an adenosine nucleotide (e.g., Watson-Crick base pairing). 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 suitable for replacement with a uridine (U) nucleotide, or vice versa.

[0118] Mode of administration The rAAV of the present disclosure can be delivered to a subject in a composition according to any suitable method known in the art.For example, the rAAV is preferably suspended in a physiologically compatible carrier (i.e., in a composition), and can be administered to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human animal of primate order (for example, macaque).In some embodiments, the host animal does not include a human.

[0119] Delivery of rAAV to a mammalian subject can be, for example, by intramuscular injection or by administration into the mammalian subject's bloodstream. Administration into the bloodstream can be 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 arts (a method that essentially allows the artisan to isolate a limb from the systemic circulation prior to administration of rAAV virions). A variant of the isolated limb perfusion technique described in U.S. Pat. No. 6,177,403 can also be employed by those skilled in the art to administer virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissues.

[0120] Moreover, in some instances, it may be desirable to deliver virions to the CNS of a subject. "CNS" refers to all cells and tissues of the vertebrate brain and spinal cord. Thus, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAV may be delivered directly to the CNS or brain using neurosurgical techniques known in the art, such as by injection into the ventricular region and into the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule (such as by stereotactic injection), with a needle, catheter, or related device (see, e.g., 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).

[0121] In some embodiments, the rAAV as described in the present disclosure is administered by intravenous injection. In some embodiments, the rAAV is administered by intracerebral injection. In some embodiments, the rAAV is administered by intrathecal injection. In some embodiments, the rAAV is administered by intrastriatal injection. In some embodiments, the rAAV is administered by intracranial injection. In some embodiments, the rAAV is delivered by cisternal injection. In some embodiments, the rAAV is delivered by lateral ventricle injection.

[0122] Aspects of the present disclosure relate to compositions comprising a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises a sequence set forth in any one of SEQ ID NOS: 2-10. In some embodiments, the nucleic acid further comprises an AAV ITR. In some embodiments, the rAAV comprises an rAAV vector represented by a sequence set forth in any one of SEQ ID NOS: 16-19, or a portion thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0123] Compositions of the present disclosure may include 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 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs (each carrying one or more different transgenes).

[0124] Suitable carriers can be easily selected by those skilled in the art, taking into account the indications for which rAAV is intended. For example, one suitable carrier includes saline, which may be formulated 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 selection of a carrier is not a limitation of the present disclosure.

[0125] Optionally, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier(s). Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0126] rAAV is administered in an amount sufficient to transfect the cells of desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects.Conventional and pharmaceutically acceptable administration routes include, but are not limited to, direct delivery to selected organs (for example, intraportal delivery to the liver), oral, inhalation (including nasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral and other parenteral administration routes.Administration routes can also be combined as desired.

[0127] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., a dose unit in genome copies per kilogram body weight (GC / kg), will vary based on several factors, including, but not limited to: the route of administration of the rAAV virions, the gene or RNA expression level required to achieve the therapeutic effect, the particular disease or disorder being treated, and the gene or RNA product stability. One of skill in the art can readily determine the rAAV virion dose range for treating a patient with a particular disease or disorder based on the aforementioned factors as well as other factors well known in the art.

[0128] The effective amount of rAAV is sufficient to infect animals and target desired tissues.In some embodiments, the effective amount of rAAV is sufficient to produce a stable somatic transgenic animal model.The effective amount mainly depends on factors such as the species, age, weight, health status, and tissue to be targeted of the subject, and therefore may vary between animals and tissues.For example, the effective amount of rAAV is generally about 10 9 From 10 16 In some cases, the amount of the solution containing up to about 10 genome copies ranges from about 1 ml to about 100 ml. 11 ~10 13 Dosage between 10 and 20 copies of the rAAV genome is appropriate. 12 or 10 13rAAV genome copies are effective in targeting CNS tissues, and in some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0129] In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than every other week (e.g., once every 2 calendar weeks). In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, a dose of rAAV is administered to a subject no more than once per 6 calendar months. In some embodiments, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in leap years).

[0130] In some embodiments, the rAAV composition specifically comprises rAAV at a high concentration (e.g., at a concentration of 10 13 The rAAV is formulated to reduce aggregation of AAV particles in compositions present at concentrations of 1000 uAAV / ml or higher. Methods for reducing rAAV aggregation are well known in the art and include, for example, adding detergents, adjusting pH, adjusting salt concentrations, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the entire contents of which are incorporated herein by reference.)

[0131] The formulation of pharmaceutically acceptable excipient and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for use with the specific compositions described herein in various treatment regimens.

[0132] Typically, these preparations may contain at least about 0.1% or more of the active compound, but the percentage of the active ingredient(s) may of course vary, and may conveniently be between about 1 or 2% and about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of active compound in each therapeutically useful composition may be adjusted so that a suitable dosage is obtained in any unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, administration route, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical preparations. In light of this, various dosages and treatment regimens may be desired.

[0133] In some situations, it may be desirable to deliver a rAAV-based therapeutic construct in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities described in U.S. Patent Nos. 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver the rAAV. In some embodiments, the preferred mode of administration is via portal vein injection.

[0134] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0135] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0136] For administration of injectable aqueous solutions, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermic injection fluid or injected at the proposed injection site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0137] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV into an appropriate solvent with various other ingredients listed herein (if required), followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any desired additional ingredients, from a previously sterile-filtered solution thereof.

[0138] The rAAV compositions disclosed herein may also be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the protein's free amino groups) and acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are conveniently administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like.

[0139] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0140] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. may be used for the introduction of compositions of the present disclosure into suitable host cells. In particular, transgenes delivered by rAAV vectors may be formulated for delivery either encapsulated in lipid particles, liposomes, vesicles, nanospheres, nanoparticles, etc.

[0141] Such formulations may be preferred for the introduction of 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 circulation half-times have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for the preparation of liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0142] Liposomes have been successfully used with numerous cell types that are normally resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints typical of viral-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the efficacy of liposome-mediated drug delivery have been completed.

[0143] Liposomes are formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters ranging from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain an aqueous solution in their core.

[0144] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally encapsulate substances stably and reproducibly. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0145] In addition to the delivery methods described above, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and is described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and efficiency of drug penetration into and through the circulatory system. Other contemplated drug delivery alternatives are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (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).

[0146] Kits and Related Compositions In some embodiments, the agents described herein may be assembled into pharmaceutical, diagnostic, or research kits to facilitate their use in therapeutic, diagnostic, or research applications. The kits may include one or more containers housing the components of the present disclosure and instructions for use. Specifically, such kits may include one or more agents described herein along with instructions describing the intended application and suitable use of these agents. In some embodiments, the agents in the kits may be in pharmaceutical formulations and dosages suitable for specific applications and methods of administering the agents. Kits for research purposes may contain components in concentrations or amounts appropriate for performing various experiments.

[0147] In some embodiments, the disclosure relates to a kit for producing rAAV, the kit comprising a container housing an isolated nucleic acid comprising an miRNA comprising or encoded by a sequence set forth in any one of SEQ ID NOs: 2-10. In some embodiments, the kit further comprises a container housing an isolated nucleic acid encoding an AAV capsid protein, e.g., an AAV9 capsid protein.

[0148] Kits may be designed to facilitate researchers' use of the methods described herein and may take a number of forms. Each of the components of the kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), where applicable. In some cases, some of the compositions may be constitutable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" may define instructional and / or promotional components, typically accompanied by written instructions on or associated with the packaging of the present disclosure. Instructions may also encompass any oral or electronic instructions, such as audiovisual (e.g., videotape, DVD, etc.), internet, and / or web-based communications, etc., provided in any manner that would clearly identify the instructions to the user as being related to the kit. The written instructions may be in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, and these instructions may also reflect approval by the agency of the manufacture, use, or sale for animal administration.

[0149] The kit may contain any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or for isolating, mixing, and applying a sample to a subject. The kit may include a container containing an agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may hold another agent that is sterilely prepared. Alternatively, the kit may include an active agent that is premixed and shipped in a syringe, vial, tube, or other container.

[0150] Exemplary embodiments of the present invention will be described in more detail by the following examples. These embodiments are exemplary of the present invention, and those skilled in the art will recognize that they are not limited to the exemplary embodiments.

[0151] example Example 1: Materials and Methods Cell culture and screening assays HeLa cells were maintained in DMEM high glucose (ThermoFisher) containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. 24 h before transfection, cells were cultured at 0.8–1.0 × 10 6 Cells were seeded into 6-well plates at 1000 x g / well. On the day of transfection, growth medium was replaced with 1.6 ml of Opti-MEM (ThermoFisher). Plasmids were transfected using 2 μl / well of DharmaFECT Duo (Dharmacon). Each well received 0.6 μg of plasmid DNA. 48 hours after transfection, cells were harvested, and total RNA was extracted using the MirVana RNA isolation kit. cDNA was produced using 1 μg of RNA / reaction with oligo-dT and Superscript III (Invitrogen). Huntington mRNA was measured using a TaqMan assay (ThermoFisher). Relative levels of huntingtin mRNA were calculated using the ΔΔC(T) method with human hypoxanthine-guanine phosphoribosyltransferase (HPRT) as the housekeeping gene.

[0152] Mouse housing, injections and maintenance YAC128 and wild-type FVB mice were obtained. Mice were bred on the FVB background by mating wild-type male mice with YAC128 female mice. The resulting heterozygous YAC128 and wild-type mice were maintained on a 12:12 light-dark schedule with free access to food and water. Genotypes were confirmed by PCR on DNA extracted from tail snips or ear punches. Mice were injected with the selected AAV directly into the striatum by means of a small animal stereotaxic SAS-4100 (ASI Instruments, Warren, MI) assisted by a UMPC3 or UMPC4 microinjector (World Precision Instruments, Sarasota, FL). Mice were anesthetized with 284 mg / kg tribromoethanol and positioned stereotaxically. Surgery was performed using the previous procedure, measuring 1.0 mm anterior and 2.0 mm lateral, and lowering the 33-gauge needle 3.0 mm into the striatum as the zero point. The pump was set to deliver 3.0 μl at a rate of 125 nl / min. After injection, mice were allowed to recover on a heating pad and then returned to their cages in the home area.

[0153] tissue extraction At the appropriate time points, mice were sacrificed, and tissues were extracted for RNA analysis or immunohistochemistry. For RNA extraction, mice were anesthetized and killed by cervical dislocation. Brains were removed, and the striatum was dissected. When available, GFP expression was used to guide dissection so that only GFP-positive tissue was analyzed. Tissues were immediately placed in RNALater (Ambion). They were then stored frozen at -80°C. At the end of the experiment, mice intended for immunohistochemistry were deeply anesthetized and perfused intracardially with saline, followed by 4% paraformaldehyde. Samples were fixed overnight in cold 2% paraformaldehyde and then stored in phosphate-buffered saline. Coronal sections were prepared by slicing 40-micron sections on a Leica VT1000s vibratome.

[0154] Mouse behavior Beam walking: Mice were trained to walk across a beam (beam size). After training, the mice were recorded as they walked from one end of the beam to the other. Three trials were recorded per mouse. Based on the recordings, the time it took for the mouse to walk from the mark at one end of the beam to the other was measured.

[0155] Home cage activity: Mice were placed singly in an automated home cage genotyping scanning system (Clever Sys, Inc., Reston VA) for 26 hours. To calculate the average hourly activity time, the first hour of data was removed while the mice habituated to the new environment; the total time spent crossing was then calculated as (total recording time - 1 hour).

[0156] Immunohistochemistry and quantification Fixed tissue sections were blocked with 3% hydrogen peroxide for 3 minutes and then incubated with 0.5% Triton-X for 20 minutes. Immunohistochemistry was performed using Laboratories Elite ABC kit reagents with rabbit or mouse antibodies against DARPP32 (Abcam ab40801; 1:10,000 dilution), Iba1 (Wako 019-19741; 1:1,000 dilution), GFP (Life Technologies G10362; 1:1,000 dilution), and NeuN (EMD Millipore MAB377; 1:1,000 dilution). Sections were stained with diaminobenzidine for 2 minutes using the Metal Enhanced DAB Substrate Kit (Pierce).

[0157] Small RNA library cloning and analysis Total RNA was extracted using the MirVana RNA Isolation Kit. Size selection of 18-30 nucleotide RNA was performed on a 15% denaturing polyacrylamide gel using 5 μg of total RNA. After size selection, small RNAs were ethanol precipitated and ligated to a pre-adenylated 3'-adapter (5'-rAppTGGAATTCTCGGGTGCCAAGG / ddC / -3'; SEQ ID NO: 11). The ligation product was annealed to an RT primer (5'-CCTTGGCACCCGAGAATTCCA-3'; SEQ ID NO: 12) and ligated to a 5'-adapter (RNA: 5'-GUUCAGAGUUCUACAGUCCGACGAUC-3'; SEQ ID NO: 13). Reverse transcription was performed using AMV Reverse Transcriptase Mix (NEB) and PCR amplified using AccuPrime Pfx DNA Polymerase (Invitrogen) with one universal primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3'; SEQ ID NO: 14) and one barcode primer (5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA-3'; SEQ ID NO: 15). Libraries were sequenced and mapped to the mm9 and AAV genomes. miRNA species were classified based on the position of the 5' end mapping relative to the miRNA hairpin; therefore, each species consists of all small RNAs with a common seed sequence. The 3' end was not considered in species assignment. Differential expression of endogenous miRNAs was analyzed using the edgeR package.

[0158] mRNA library cloning and analysis RNA was extracted as described above. Libraries were constructed by standard methods. Reads were mapped using toPHat2, and differential expression was calculated using the deseq2 package.

[0159] Sheep experiment Transgenic sheep models of Huntington's disease (e.g., transgenic sheep expressing the pathogenic human Huntington protein) were injected with either scAAV9-CBA-mir-HTT (containing miRNA 6433 in a mir-155 backbone), scAAV-U6-mir-HTT (containing miRNA 6433 in a mir-155 backbone), or an empty scAAV9 control vector. The sheep were sacrificed either 1 or 6 months after injection. Tissue and nucleic acid samples were prepared and analyzed by quantitative PCR and immunohistochemistry.

[0160] Example 2: Mice in vivo experiments Design and selection of Huntington-targeting artificial miRNAs Nine sequences targeting human huntingtin mRNA (Table 1, Figure 1) were tested. A schematic diagram showing the location of the nine targeting sequences is provided in Figure 4A. Two copies of the artificial miRNA were tandemly cloned into a backbone based on endogenous miRNA-155 or miR-30, and the entire artificial miRNA was inserted into the 3'-UTR of EGFP (Figure 5A, upper panel). The predicted hairpin structures of the miR-155- and miR-30-based anti-HTT miRNAs are shown in Figures 25A-25B, respectively. The resulting plasmids were transfected into HeLa cells. After 48 hours, cells were harvested, and the levels of endogenous huntingtin mRNA were measured by quantitative RT-PCR (qRT-PCR). Three of the nine artificial miRNAs reduced huntingtin by more than 50% (Figures 1-2 and 4B-4C).

[0161] Table 1: Huntington mir targets [Table 1]

[0162] Three sequences from the initial screening were selected for in vivo experiments. Artificial miRNAs based on known siRNAs (E1.4) were also tested. Candidate sequences were packaged into self-complementary AAV9 vectors and injected directly into the striatum of transgenic mice (Yac128 mice) expressing human huntingtin with a stretch of approximately 128 polyglutamine coding repeats. One month later, the distribution of AAV9 and expression of the GFP reporter were assessed at three different doses. At the highest dose, GFP staining was present throughout the striatum (Figure 11A), and human huntingtin mRNA was significantly reduced in mice treated with either AAV9-CβA-anti-HTT-6433 (p=0.006) or AAV9-CβA-anti-HTT-5155 (p=0.013, Figure 4C; using the mir-155 backbone). Reducing the dose of the vector reduced GFP expression (Figure 11A), and at a 1:10 dilution, no significant reduction in human huntingtin mRNA was achieved (Figure 11B). Figure 11C provides exemplary photographs of mice injected with vectors encoding both the miRNA and huntingtin targeting EGFP at three different doses.

[0163] Expression of artificial miRNAs from the U6 promoter does not improve huntingtin silencing A single copy of the most potent miRNA (HTT-6433) was cloned into an AAV9 vector under the control of the U6 promoter (Figure 5A, bottom panel). Mice were unilaterally injected with either the first two copies of AAV9-CBA-anti-HTT-6433 (containing SEQ ID NO: XX), AAV9-CBA-anti-HTT-5155, AAV9-U6-anti-HTT-6433 (containing SEQ ID NO: XX), or AAV9-U6-anti-HTT-5155. One month later, striatum was harvested, GFP expression was confirmed, and huntingtin mRNA levels were measured by qRT-PCR. No significant differences in knockdown were observed between mice treated with AAV9-U6-anti-HTT and AAV9-CBA-anti-HTT, regardless of the artificial miRNA sequence. In both AAV9-U6-anti-HTT-6433 and AAV9-CBA-anti-HTT-6433-treated mice, the amount of huntingtin mRNA in the injected side was approximately 50% of that in the uninjected side (Fig. 5B ).

[0164] It should be noted that using the contralateral (non-injected) side as a control for each animal reduces animal-to-animal variability. In mice unilaterally injected with AAV9-GFP, a small number of GFP-positive neurons were occasionally observed on the contralateral side, indicating that some virus was transmitted to the non-injected side. Therefore, using the contralateral side as a control may underestimate knockdown. To eliminate the potential confounding effect of using the contralateral side as a control, the experiment was repeated using a group of animals injected with PBS only as a control. The data showed that both AAV9-U6-anti-HTT-6433 and AAV9-CBA-mir-HTT-6433 reduced huntingtin mRNA by approximately 50% (Figure 5C).

[0165] Long-term striatal expression of mir-HTT-6433 from the U6 promoter is toxic in mice AAV9-U6-anti-HTT-6433 or AAV9-CBA-anti-HTT-6433 was unilaterally injected into the striatum of Yacl28 mice. Six months after injection, mice injected with AAV9-U6-anti-HTT-6433 were observed not building nests, and some exhibited a hyperactive phenotype. To demonstrate these abnormalities, the small nests in each cage were replaced. After 24 hours, the new small nests in the AAV9-U6-anti-HTT-6433 mice were unused, while mice injected with PBS and AAV9-CBA-anti-HTT-6433 built nests, as expected (Figure 6A). Mice were recorded for 24 hours using a home cage monitoring system. AAV9-U6-anti-HTT-6433-treated mice spent significantly more time moving around their home cage than PBS- or AAV9-CBA-anti-HTT-6433-treated mice (Fig. 6B).

[0166] The average time it took the mice to cross the balance beam was also measured. For this test, the mice were required to complete the beam crossing three times. On average, AAV9-CBA-anti-HTT-6433-treated mice tended to cross faster than mice that received only PBS injections (Figures 3 and 12A). Of the four remaining mice in the AAV9-U6-anti-HTT-6433 group, two were unable to cross successfully, either jumping or falling off the beam (Figure 12A). In a second experiment performed on aged Yacl28 mice (7 months old), an age-related increase in the time it took to cross the beam was observed. This increase was present in both naive mice and mice treated with AAV9-CBA-anti-HTT-6433, and was enhanced in mice treated with AAV9-U6-anti-HTT-6433 (Figure 12B).

[0167] Neuropathological findings correlated with the behavioral results described above. On the injected side, AAV9-U6-anti-HTT-6433 mice exhibited enlarged ventricles, loss of DARPP-32-positive neurons, and striatal atrophy (Figures 7A-7B). In the remaining striatum, they exhibited increased Iba1 staining (Figure 8A, bottom panel), an increase in total activated microglia, and a decrease in the number of resting microglia (Figures 8B-8D). Wild-type C57Bl / 6 and FVB mice were injected with the same vector to assess the effects of U6-driven miR to determine whether toxicity in this context depended on the presence of mutant Huntington protein. In FVB mice, the effects were similar to those in Yac128 mice, with rapid degeneration of the balance beam and severely enlarged ventricles. However, in C57BL6 mice, the effects were present but less pronounced. Although the time to cross the balance beam initially increased in the U6 cohort, there were no significant differences between groups at the end of the study (Fig. 13A). Striatal atrophy was less severe in C57BL6 mice (Fig. 13B).

[0168] Expression of an artificial huntingtin-targeting miRNA from the U6 promoter results in overexpression of huntingtin-targeting small RNAs Groups of mice were unilaterally injected with scAAV9 vectors expressing artificial miRNAs from the U6 and CβA promoters. Small RNAs produced 2 weeks after injection were cloned and sequenced. In both groups, 96% of sequences mapping to the AAV genome mapped to the expected small RNA products, with only a small percentage representing incorrect Dicer or Drosha cleavage. In the group injected with the U6 promoter-driven artificial miRNA, the Huntingtonin targeting sequence dominated the sequencing results, accounting for half (50%) of all mappable sequences, whereas in mice injected with the CBA vector, only 5% of sequences matched the vector-encoded small RNA (Figure 9A). Thus, potentially, small RNAs with alternative seed sequences, including sense strands and translation slippage products, may be present at levels comparable to those of functional endogenous miRNAs (Figure 9A). The relative abundance of Huntingtonin targeting small RNAs was measured by qPCR to confirm overexpression of the Huntingtonin targeting sequence. Expression of small RNAs was observed to be 150-250-fold higher than in mice injected with the U6 promoter-driven construct (Figure 9B).

[0169] The endogenous miRNA-30 sequence is commonly used as a scaffold for artificial miRNAs. To determine whether this scaffold-derived isomir profile was more favorable, we embedded the anti-HTT-6433 sequence in the miR-30 backbone and injected it into 10-week-old Yac128 mice (Figure 9C). The miR-30 scaffold produced mature artificial miRNAs at levels comparable to those produced by the CβA promoter (Figure 9A) and reduced human huntingtin expression by nearly 50%. Unlike the miR-155 scaffold, the miR-30 scaffold produced mature sense (passenger) strands at levels comparable to those of the antisense (guide) strands. The combination of the CβA promoter and the miR-155 backbone produced only the intended antisense strand above background (Figure 9A).

[0170] Although more than half of the reads in the samples could be mapped to AAV-encoded artificial miRNAs, overexpression of the artificial miRNA targeting Huntington had minimal effect on the distribution of endogenous miRNAs (Figures 14A-14B). Indeed, the greatest differences were observed when comparing the injected group with the non-injected (contralateral) side, suggesting that the injection itself generates local changes in the miRNA profile. This may reflect a local immune response to the injury that may resolve over time.

[0171] Expression of an artificial Huntington-targeting miRNA from the U6 promoter disrupts the expression of multiple mRNAs We performed RNA sequencing analysis of the striatum of mice treated with either AAV9-U6-anti-HTT-6433 or AAV9-CBA-anti-HTT-6433 to investigate the consequences of overexpression of huntingtin-targeting miRNAs. Two weeks after injection, the striatal mRNA profiles of the injected and non-injected sides were compared. The data show that there were few significant differences in mice treated with CBA-mirHTT-6433 (Figure 14A). In mice treated with AAV9-U6-anti-HTT-6433, both increased and decreased mRNAs were observed in response to treatment (Figure 14B). When the profiles of AAV9-U6-anti-HTT-6433 and AAV9-CBA-anti-HTT-6433 were compared at 2 weeks, only eight mRNAs were observed to be significantly differentially expressed between the two groups.

[0172] RNAs were classified according to the presence or absence of predicted target sites for the artificial miRNA, and the change in cumulative distribution of mRNAs with or without target sites was plotted. In mice injected with AAV-U6-6433, we observed a small shift toward downregulation of genes containing perfect 8-mer target sites in their 3'-UTRs that matched the most abundant AAV-derived small RNA species (Figure 10B). This shift was not evident in mice injected with AAV-CBA-6433 (Figure 10A) or any of the other AAV-derived small RNA species (Figures 15A-15C).

[0173] Example 3: Sheep in vivo experiments A sheep model of human Huntington's disease was used in this example. Briefly, transgenic sheep expressing human huntingtin (human htt) protein were generated. The sheep were intrastriatally injected with either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector. Each construct contains a single copy of the anti-huntingtin mir-6433 sequence (SEQ ID NO: 7) inserted into the mir-155 backbone located within the intron between the CBA promoter or U6 promoter and the β-globin polyadenylation sequence, respectively. The constructs used to generate the rAAV administered in this experiment are set forth in SEQ ID NOs: 18 (scAAV9 CBA-mir-HTT) and 19 (scAAV9 U6-mir-HTT).

[0174] The sheep were sacrificed either 1 or 6 months after injection. Tissue and nucleic acid samples were prepared and analyzed by quantitative PCR and immunohistochemistry.

[0175] The data show that injection of mir-HTT expressed under the U6 promoter resulted in reduced htt expression in both the mid-caudate and mid-putamen of sheep at one month compared to uninjected and empty scAAV9-injected control mice. Figure 16 shows data on the reduction of human htt expression in the mid-caudate of sheep one month after injection of scAAV9 U6-mir-HTT. Figure 17 shows data on the reduction of human Huntington's expression in the mid-putamen of a sheep model one month after injection of scAAV9 U6-mir-HTT. It should be noted that, unlike the mouse model described in the previous example, mir-HTT expressed from the U6 promoter was not toxic in the sheep model of Huntington's disease.

[0176] The data show that at 6 months, injection of mir-HTT expressed under the CBA promoter resulted in reduced htt expression in both the mid-caudate and mid-putamen of sheep compared to uninjected and empty scAAV9-injected control mice.

[0177] Figures 18 and 19 show data related to the reduction of human htt expression in the medial (Figure 18) and lateral (Figure 19) midcaudate of sheep. The data show that mir-HTT expressed from the CBA promoter causes a reduction in htt expression in the injected side of the brain compared to the uninjected side of the brain and empty scAAV9-injected control mice. The effect of mir-HTT expression from the CBA and U6 promoters on htt silencing in the midcaudate was also compared 6 months after injection. The data show that mir-HTT expressed from either the U6 or CBA promoter resulted in a reduction in human htt expression in the midcaudate compared to uninjected and empty scAAV9-injected control animals (Figure 20).

[0178] Figures 21 and 22 show data on the reduction of human htt expression in the lateral (Figure 21) and medial (Figure 22) medial putamen of sheep 6 months after injection. The data show that mir-HTT expressed from the CBA promoter causes a reduction in human htt expression in the injected side of the brain compared to the uninjected side of the brain and the uninjected side of empty scAAV9-injected control mice. The effect of mir-HTT expression from the CBA and U6 promoters on htt silencing in the medial caudate was also compared 6 months after injection. The data show that mir-HTT expressed from either the U6 or CBA promoter caused a reduction in human htt expression in the medial putamen compared to uninjected and empty scAAV9-injected control animals (Figure 23).

[0179] Figure 24 shows the relative expression of human huntingtin (human htt) RNA in the anterior striatum of a sheep model of Huntington's disease 6 months after intrastriatal injection of either scAAV9 CBA-mir-HTT ("CBA promoter"), scAAV9 U6-mir-HTT ("U6 promoter"), or an empty scAAV9 control vector.

[0180] Example 4: Artificial miRNA reduces human mutant Huntington throughout the striatum in a transgenic sheep model of Huntington's disease Animals and animal procedures Merino sheep were used in this example. Prior to administration of anesthesia, animals were fasted overnight for approximately 8 hours. Pre-operative physical characteristics of the animals were obtained, including heart rate, respiratory rate, body temperature, and weight. Baseline samples of serum (5 ml) and CSF were collected.

[0181] The study was conducted in two parts using two different cohorts of sheep. For the first study, 41 transgenic animals (21 wether rams and 20 ewe ewes) approximately 8 months of age were injected with 300 μl of self-complementary AAV9 (scAAV9) vectors for a total of 3 × 10 12 1 x 10 genome copies 13 The vector was injected unilaterally at a titer of 10 ...

[0182] A curvilinear incision was made using a #15 scalpel to expose the anterior anterior section. Once the anterior anterior section was identified, a 3-4 mm burr hole was placed 10 mm rostral to the anterior anterior section and 11 mm lateral to the midline using an electric drill. A convection-enhanced delivery (CED) cannula (MRI Interventions, Irvine, CA) was secured to a manipulator and primed with the agent to be injected to remove air from the line. The dura was opened using a #11 scalpel with a 1.5 mm incision, and the CED camera was advanced 25 mm from the dura surface to the target depth. An outer cannula (1.65 mm) sealed the dural incision and prevented SCF leakage during perfusion. Perfusion began 5 min after cannulation, allowing the tissue around the tip to stabilize. The perfusion rate was set at 3.33 μl / min until a total volume of 300 μl was injected. Ten minutes after the infusion was complete, the cannula was slowly removed and a bone wax plug was used to repair the skull and prevent CSF leakage. The wound was cleaned with saline and closed using 3.0 Vicryl sutures. Standard anesthesia wake-up and recovery procedures were followed.

[0183] Postoperative MRI was performed to measure gadolinium diffusion. One animal from the first study was excluded from the following surgery because gadolinium could not be visualized during imaging, and a second animal from the second study was excluded because gadolinium appeared to be primarily present in the ventricles. After surgery, animals were kept under observation for three days and housed indoors for five days. They were then moved to an outdoor paddock and an outdoor enclosure for the remainder of the study. Animals were visually monitored for signs of anxiety and behavioral changes throughout the study. Two animals suffered surgical complications, resulting in partial limb paralysis. This was thought to be due to the positioning of the anesthetized animals. One animal was anesthetized initially, and one was transferred from the 6-month to the 1-month cohort. Animals were weighed periodically throughout the postinjection period, and cerebrospinal fluid, blood, and serum samples were collected and stored for further analysis.

[0184] For cell counts and differentials, blood was collected via jugular venipuncture into potassium EDTA blood collection tubes (lavender top; LT), and a complete blood study was performed using a CBE differential. For clinical chemistry, blood was collected via venipuncture into serum collection tubes (red top; RT). Samples were submitted for multiple biochemical analysis (MBA).

[0185] One and six months after injection, the animals were harvested for either histological or biochemical analysis. The animals were transferred to the operating table and placed in ventral recumbency while approximately 6 mL of CSF was collected. The animals were then repositioned in dorsal recumbency. The carotid artery was exposed and cannulated 4 cm from the tip of the cannula. The jugular vein was exposed, and 200–500 U heparin / kg was injected into the jugular vein. Five minutes after heparin administration, the sheep were euthanized by intravenous injection of lethabarb (325 mg pentobarbital sodium / ml) at 1 ml / 2 kg body weight. An infusion pump was primed with cold 9% NaCl and connected to the carotid artery cannula. The animals were perfused with approximately 8 L of cold 9% NaCl at a pressure of 500 mmHg. For histology, the infusion was switched to 8 L of 4% paraformaldehyde at 500 mmHg pressure. Brains and livers were extracted. Tissues were post-fixed in 4% paraformaldehyde for 24 hours at 4°C and transferred to 30% sucrose in 1× phosphate-buffered saline for a minimum of 14 days at 4°C.

[0186] For RNA, protein, and DNA assays, sheep were perfused with cold 9% NaCl as described above. Peripheral tissue collection was performed in the following order: liver, adrenal glands, ovaries (if applicable), muscle, and heart. Cross-contamination was prevented by using different instruments and cleaning the autopsy surface with 10% bleach and 70% ethanol. Organs were removed from the body, and samples were collected using a 3 mm biopsy punch. A total of 10 samples were collected from each organ; two samples were immediately frozen in liquid nitrogen, and eight samples were later stored for RNA at 4°C for 24 hours (300 μl of RNA for liver, muscle, and heart samples, and 500 μl of RNA for adrenal and ovary samples).

[0187] The brain was removed from the skull using a circular saw and rongeur. After extraction, the brain was weighed and placed ventrally in a custom-made Plexiglas brain matrix. Nine cuts were made in the brain, including the entire striatum in four 6 mm blocks. The first cut was made posterior to the olfactory bulb attachment (approximately 18 mm from the beginning of the matrix), and four subsequent cuts were made at 6 mm intervals. The striatum was divided into four 6 mm blocks from posterior to anterior: 2p (posterior), 2m1 (medial 1), 2m2 (medial 2), and 2a (anterior). Striatal dissections were performed in the following order: 2p, 2m1, 2a. The striatum of the right (non-injected) hemisphere was dissected first in all blocks, and scalpels were exchanged between hemispheres. Dissections were performed in a Petri dish on dry ice, with care taken to remove as much white matter as possible from the striatal tissue. Once dissected, striatal sections (caudate and putamen) were divided in half; the central section (closest to the midline of the block) was later stored in 1 ml of RNA at 4°C, and the lateral sections were immediately frozen in liquid nitrogen. Striatal dissections for the 6-month cohort in the CBA study were performed in a manner that generated four striatal samples from both the caudate and putamen. The dorsal sections (both medial and lateral) were immediately frozen in liquid nitrogen, and the ventral sections (medial and lateral) were later stored in 1 ml of RNA at 4°C. RNA was later harvested after 24 hours, and samples were stored at -80°C.

[0188] The 2m2 block was covered with OCT and frozen in 2-methylbutane and a dry ice bath. The remaining 2a, 2m1, and 2p blocks were frozen in the same manner. Ten cortical samples were harvested from each block in a dorsal-to-ventral manner; two were frozen in liquid nitrogen, and eight were later stored in 1 ml of RNA at 4°C.

[0189] Sectioning of tissue for histological analysis Prior to tissue sectioning for histological analysis, the striatum was isolated from the brain, gently covered with OCT, and stored at -20°C for 24 hours. 40 μm-thick coronal sections were cut through the entire striatum using a Reichert-Jung Tetrander sliding microtome. Sections were stored in 0.01% sodium azide in 1× phosphate-buffered saline at 4°C.

[0190] Vector cloning and rAAV9 production For the first study, the test vector consisted of the endogenous mir155 backbone (AAV9-U6-miR HTT The first vector contained a U6 promoter driving an artificial miRNA based on the CMV enhancer and CBA promoter, an intron, and miRNA-155-based artificial miRNA (AAV9-CBA-miRNA). This artificial miRNA targets human huntingtin, but not sheep. A chimeric cytomegalovirus enhancer / chicken beta-actin (CBA) promoter-driven chimeric intron was included to improve AAV packaging. The control vector (AAV9) contained only the empty CBA promoter and intron. For the second study, the test vector contained the CMV enhancer and CBA promoter, an intron, and miRNA-155-based artificial miRNA (AAV9-CBA-miRNA). HTT ) was included.

[0191] For packaging, the rAAV vector plasmid, packaging plasmid, and adenovirus helper plasmid were co-transfected into HEK293 cells. The packaging plasmid expresses regulatory and AAV9 capsid proteins and directs the excision, replication, and packaging of the recombinant genome from the rAAV vector plasmid into AAV virions. Recombinant viruses were purified by standard CsCl gradient sedimentation and desalted by dialysis.

[0192] Analysis of huntingtin mRNA levels RNA levels in the subsequently stored RNA samples were analyzed using a branched DNA assay (bDNA). Samples were processed according to the manufacturer's guidelines for preparing tissue homogenates from tissues subsequently stored with RNA (Affymetrix eBioscience, Quantigene® Sample Processing Kit). Homogenized samples were analyzed according to the manufacturer's guidelines for the bDNA assay (QuantiGene® 2.0 Reagent System). Samples were analyzed using probes to detect human huntingtin (Human HD, SA-50339 from Quantigene), sheep huntingtin (Sheep Huntingin, SF-10586 from Quantigene), and sheep calnexin (SF-10622 from Quantigene) as a housekeeping gene. Assay results were measured using a Tecan Infinite M1000 PRO luminometer (integration time set to 200 ms).

[0193] Analysis of miR-Htt levels Biopsy punches (2 mm) were sampled from the lateral caudate and medial putamen from frozen blocks. RNA extraction was performed using the TRIzol manufacturer's guidelines (Ambion) with some modifications. After phase separation during TRIzol extraction, the aqueous phase was transferred to an RNA Clean & Concentrator (Zymo) column and the protocol was followed. RNA was stored at -80°C until analysis. RNA quantity and concentration were measured using a Fragment Analyzer (Advanced Analytical Technologies Inc.). RNA was diluted to 20 ng / μl immediately before analysis. Artificial miRNA guide strands were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Cat# 4366596, Thermo Scientific), 2 μl of RNA, and a guide strand-specific stem-loop primer (ThermoFisher custom assay targeting UAAGCAUGGAGCUAGCAGGCU (SEQ ID NO: 25) or assay id 002407, let7e*) according to the manufacturer's instructions. The ddPCR reaction was set up using 5 μl of RT product, 1× concentration of miR-Htt assay, and 0.3× concentration of let-7e* assay to allow for multiplexing. Droplets were generated using a QX200 Droplet Generator (Cat# 1864002, Biorad) and monitored for a positive signal after end-point PCR amplification (40 cycles). HTT The relative expression of miRs HTT The concentration of let-7e* was determined by calculating the ratio between the absolute concentrations of let-7e* and let-7e*.

[0194] Vector genome distribution Genomic DNA was extracted from samples immediately frozen in liquid nitrogen using the Gentra Puregene Tissue kit (Qiagen). Genomic DNA concentrations were determined using a NanoDrop ONE kit. cMeasurements were performed using a spectrophotometer. Droplet Digital PCR (ddPCR, Biorad) was performed according to the manufacturer's recommendations using 50 ng of DNA as input and TaqMan assays to detect the vector-specific CB and U6 promoters and the HPRT reference gene. Results are expressed as vector genomes per diploid genome (vg / dg).

[0195] Analysis of Huntington protein levels - Mesoscale Detection Assay (MSD) for mHTT Striatal samples were homogenized in a buffer consisting of 10 mM HEPES, 250 mM sucrose, 1 mM EDTA, and protease inhibitors (Roche) and sonicated for 10 seconds at 10% amplitude. Protein concentration was measured using a Bradford assay. A 96-well QuicPlex standard plate (MSD) was coated with rabbit monoclonal anti-HTT proline 1220 domain antibody (D7F7, Cell Signaling, 1:250) in PBS overnight at 4°C. The plate was washed 3 times for 10 minutes with PBST (PBS + 0.05% Tween 20) and blocked with 3% bovine serum albumin (BSA) in PBS for 2 hours at room temperature. After 3 x 10 min washes with PBST, technical duplicate samples containing 20 µg of protein in 25 µL of homogenization buffer or blank (homogenization buffer) were dispensed onto the plate and incubated overnight at 4 °C on an orbital shaker. The plate was washed 3 x 10 min in PBST and incubated in the secondary / detection antibody mix as follows: for detection of mHTT, mouse monoclonal anti-polyQ antibody MW1 (DSHB) was mixed with anti-mouse SulfoTag detection antibody (MSD) at 1 µg / mL of each antibody in 1% BSA in PBS. 30 µL of the detection antibody mix was applied per well and incubated for 3 h at RT on an orbital shaker. The plate was washed 3 x 10 min in PBST, and 150 µL of 2x Read Buffer (MSD) was applied per well immediately before reading on a QuickPlex SQ120 (MSD).

[0196] Western blotting Tissue pieces were removed from the frozen block and homogenized on ice in 200 μl 10 mM HEPES pH 7.2, 250 mM sucrose, 1 mM EDTA, and a protease inhibitor tablet (mini, complete, EDTA-free Roche #11836170001). Samples were sonicated for 10 seconds, and protein concentrations were determined using the Bradford method (BioRad #500-0006). Equal concentrations of protein (25 μg) were separated by SDS-PAGE on a 3-8% Tris-Acetate gel (Life Technologies #EA03785BOX) and transferred to nitrile cellulose using a TransBlot Turbo (BioRad). Blots were blocked for 1 hour in 5% nonfat dry milk in Tris-buffered saline + 0.1% Tween-20 (TBST) and then incubated overnight in primary antibody blocking solution at 4°C. The primary antibodies used were anti-polyQ (MW1, Coriell, 1:500 or 3B5H10, Sigma, 1:1000), anti-huntingtin (MAB2166, EMD Millipore, 1:1000 or Ab1, DiFiglia et al., 1995, 1:1000), anti-DARPP32 (#ab40801, Abcam, 1:10,000), anti-actin (A4700, Sigma, 1:1000), and anti-spectrin (MAB1622, EMD Milliopore, 1:4000). Blots were washed in TBST, incubated in peroxidase-conjugated secondary antibodies diluted 1:5000 in blocking solution for 1 hour at room temperature, washed in TBST, and incubated in SuperSignal West Pico Chemiluminescent Substrate (Pierce #34080). Images were acquired using a CCD imaging system (Alpha Innotech) and a Hyperfilm ECL (GE Healthcare). Densitometry was performed on digital images using ImageJ software (NIH). Statistical analysis was performed using an unpaired t-test, and results were expressed as the mean value of the injected side.

[0197] Immunohistochemistry for DARPP32, NeuN, and Iba1 To quantify DARPP32-positive cells, every 20th section was incubated in 3% hydrogen peroxide in 1x PBS for 3 minutes, 0.5% Triton-X-100 for 20 minutes, and then 1.5% normal goat serum (Vector Labs, S-1000) in 1x PBS for 4 hours. Sections were incubated overnight at 4°C in anti-DARPP32 (AbCam, ab40801, 1:1,000 dilution) in 1.5% normal goat serum. Sections were then incubated in biotinylated goat anti-rabbit IgG antibody (Vector Labs, AP-1000, 1:200 dilution) in 1x PBS for 10 minutes. Sections were then incubated with 2% Elite A and 2% Elite B reagents from the Vectastain Elite ABC Kit (Vector Labs, PK-6100) in 1x PBS for 5 minutes. DARPP32-positive cells were visualized using a Metal Enhanced DAB kit (ThermoFisher Scientific, 34065). Sections were incubated in 1x 3,3'-diaminobenzidine in stable peroxide buffer.

[0198] To quantify NeuN-positive cells, every 20th section was incubated in 3% hydrogen peroxide in 1x PBS for 3 minutes, 0.5% Triton-X-100 for 20 minutes, and then 1.5% normal goat serum (Vector Labs, S-1000) in 1x PBS overnight at 4°C. Sections were incubated in anti-NeuN (Chemicon, MAB377, 1:1,000 dilution) in 1.5% normal goat serum for 1 hour at 4°C. Sections were then incubated in fluorescent AF594 goat anti-mouse IgG (ThermoFisher Scientific, A-11005, 1:2,000 dilution) for 40 minutes to visualize NeuN-positive cells.

[0199] To quantify Iba1-positive cells, every 20th section was incubated for 1 hour in a solution of 5% normal goat serum (Vector Labs, S-1000), 1% bovine serum albumin (Sigma, A-3059), 0.2% Triton-X-100, and 0.03% hydrogen peroxide in 1x PBS. Sections were incubated overnight at 4°C in anti-Iba1 (Wako Chemicals, 019-19741, 1:1,000 dilution) and 1% bovine serum albumin (Sigma, A-3059) in 5% normal goat serum (Vector Labs, S-1000). Sections were then incubated for 10 minutes in biotinylated goat anti-rabbit IgG antibody (Vector Labs, AP-1000, 1:200 dilution) in 1x PBS. Sections were incubated for 5 min with 2% Elite A and 2% Elite B reagents from the Vectastain Elite ABC Kit (Vector Labs, PK-6100) in 1× PBS. The reaction was visualized by incubating sections in 1× 3′,3-diaminobenzidine in stable peroxide buffer using a Metal Enhanced DAB kit (ThermoFisher Scientific, 34605).

[0200] Quantification of DARPP32- and Iba1-positive cells in the left and right hemispheres of the brain was performed by taking images of each section (20x for DARPP32 and 40x for Iba1) using a Nikon Eclipse E600 microscope. To consistently capture images across different sections, the first image was captured at the medial dorsal edge of the striatum, and the stage was shifted 0.5 cm ventrally. Once at the ventral edge, the stage was shifted 0.5 cm laterally and 0.5 cm dorsally until 10 images were captured. Random numbers were assigned to each image to eliminate bias in quantifying cells. Cells were counted using ImageJ software (NIH).

[0201] Quantification of NeuN-positive cells was performed using a Nikon Eclipse E600 with a Chiu Technical Corporation Mercury 100-W lamp at 60×. The stereological method used to capture the DARPP32 and Iba1 images was also used to quantify NeuN-positive cells.

[0202] The areas of the striatum, caudate, and putamen for each section were measured by manually cycling the DARPP32-stained area on the injected and non-injected sides of every 20th section (29–35 sections per side per animal) using ImageJ software (NIH). The observer was blinded to the conditions. The total volume for each region was determined by multiplying the area by the number of sections across the slide (20) and the section thickness (40 microns), and adding them together for each animal. Statistical analysis was performed using Microsoft Excel, paired and unpaired t-tests, with an N = 3 or 4 animals per group.

[0203] Vector genome and miRNA distribution after injection We observed expanded murine Huntington mRNA in a knock-in model of HD and silencing of human mHTT transgene mRNA in a transgenic mouse model of HD. In this example, two cohorts of HD sheep (Study 1 and Study 2) were unilaterally injected into the striatum. In Study 1, scAAV9-U6-miR HTT In Study 2, sheep were injected with scAAV9-CBA-miRHTT (AAV9miRHTT) or scAAV9-CBA-empty (AAV9), in which a non-coding stuffer sequence is inserted between the promoter and polyA signal, at 8-9 months of age. HTT or scAAV9-CBA-empty (AAV9). HTT Patients were collected at 1 month or 6 months after administration.

[0204] Genome copies were determined in a subset of regions (Figure 26A) by droplet digital PCR (ddPCR, Figure 26B). Genome copies were highest in the caudate and putamen compared with the uninjected side, with the highest levels in the scAAV9-U6-miRHTT-treated group 1 and 6 months after injection. Small amounts of vector genome were present in the cortex and liver and were undetectable in the adrenal gland (Figure 26B).

[0205] RNA quantity was measured using a fragment analyzer, which generates a score called the RNA Quality Number (RQN). The RQN is generated by analyzing electropherograms and integrates multiple different measures of RNA integrity, such as the peak sharpness and baseline ratio between the 28S and 18S ribosomes. Scores generally range from 0 (completely degraded) to 10. Samples with a score greater than 5 were used to analyze the levels of artificial miR guide strands. Two animals from Study 1 and two animals from Study 2 were excluded from the analysis due to low RQN scores. The levels of artificial miRNA guide strands were measured using ddPCR and normalized to endogenous let7e* (Figure 27). The relative abundance of the artificial miR antisense strand was 3.5-1000-fold higher in the injected side than in the non-injected side and was higher at 1 month compared to 6 months after injection. The miRNA guide strand was expressed as an AAV9-miR HTT was detected at low levels on the side opposite to the injection.

[0206] scAAV9-miR HTT Single long-term administration of acetaminophen reduces human mutant huntingtin mRNA in the caudate and putamen HTT mRNA in the anterior and central striatum was measured using a branched DNA (bDNA) assay that specifically recognizes human, but not ovine, HTT mRNA. This assay does not require RNA isolation, and all samples are included in the analysis. One month after injection, the anterior striatum, closest to the injection in the central striatum, was significantly higher than the scAAV9-U6-miR. HTT(Study 1) reduced human HTT mRNA by more than 50% in both the caudate and putamen (Figure 28). No significant silencing was detected in the anterior striatum, which is further away from the injection site (Figure 28). Six months after injection, mRNA silencing was evident in the caudate (Figure 28). scAAV9-CβA-miR HTT In cohort (Study 2), significant silencing of HTT mRNA occurred in the central caudate, central putamen, and anterior striatum one month after injection (Figure 28), and in the central caudate and parts of the central putamen six months after injection. The anterior striatum showed no significant decline at six months. There was no significant silencing of endogenous ovine HTT miRNA (Figure 29A).

[0207] Western blot assay and electrochemiluminescence (MSD assay) confirmed the activity of scAAV9-miR HTT reduces human mutant Huntington protein in the caudate and putamen HTT protein was detected by Western blot (Figure 30) and electrochemiluminescence (Meso Scale Discovery (MSD, Figure 30)) in the same sample preparations. In Study 1, mHTT protein was detected by Western blot (Figure 30) using the 3BH510 antibody, which preferentially detects mHTT (mutant HTT) compared to wild-type HTT. scAAV9-U6-miR HTT in the caudate, putamen, and anterior striatum after 1 month of treatment with miR-1, and in the putamen after 6 months of treatment. HTT In Study 2 (Figure 30, bottom panel), there was a significant reduction in mHTT protein compared to treatment with AAV9 lacking scAAV9-CβA-miR. HTT Treatment significantly silenced mHTT in the caudate one month after injection, and in the caudate, putamen, and anterior striatum six months after injection.

[0208] The results of the SMD assay using MW1 for detection showed that scAAV9-U6-miR HTTTreatment (Study 1) significantly reduced mHTT protein levels in the caudate, putamen, and anterior striatum at 1 and 6 months after treatment. HTT significantly silenced mHTT protein in the caudate one month after injection, and in the caudate, putamen, and anterior striatum six months after treatment (Figure 31). These results indicate good agreement between the results of the MSD assay (Figure 31) and those obtained by Western blot assay (Figure 30).

[0209] Table 2: Mean percentage reduction of mutant huntingtin protein by Western blot and MSD assay in Studies 1 and 2. Human mutant huntingtin protein was measured by Western blot using anti-htt polyQ antibody 3B5H10 in Study 1, and antibody 3B5H10, which does not recognize ovine HTT, MAB2166 (anti-HTT 443-456) (Reid et al., 2013), and anti-polyQ monoclonal antibody MW1 in Study 2. In the MSD assay, MW1 was used as the detection antibody. This table reports the mean percentage reduction of mHTT for the caudate, putamen, and anterior striatum. The percentage reduction was calculated by dividing the mean signal for the injected side in AAV9miRHTT-treated sheep by the mean signal for the injected side in AAV9-only-treated animals.

[0210] [Table 2]

[0211] Because antibodies detecting mHTT can have different sensitivities, two other antibodies, MAB2166 and MW1, for detecting human mHTT protein by Western blot were included in Study 2 (Table 2). In HD transgenic sheep, MAB2166 recognizes only human huntingtin and not ovine HTT. MW1 preferentially recognizes the expanded polyglutamine region in HTT and was also used to detect mHTT in the MSD assay. Table 2 compares the average percentage reduction in mHTT detected by Western blot using three anti-mHTT antibodies (3B5H10, 2166, and MW1) and by the MSD assay using MW1 in Studies 1 and 2. All three antibodies in Western blot analysis detected significant reductions in mHTT (49%–81%) in multiple neostriatal regions in Study 2. The results of mHTT reduction by the MSD assay were consistent when two samples from the same striatal region were used in Study 2. Also noteworthy is the comparison of the Western blot results with the MSD assay using MW1 in Study 2. There was good agreement in the magnitude of mHTT reduction between these two different methods of mHTT detection.

[0212] Western blot analysis revealed that AAV9-miR HTT The cortex overlying the striatum injected with AAV9-miR showed no reduction in mHTT protein levels compared to the AAV9-injected cortex (Figure 32A). HTT It was detected in the caudate and putamen contralateral to the injected striatum, and these regions showed no reduction in mHTT protein levels by Western blot analysis (Figure 32B).

[0213] AAV9-miR against human HD genes HTTTo investigate whether treatment with miRs affected the levels of endogenous ovine HTT, the levels of human transgenic mHTT and endogenous ovine HTT were directly compared using Western blot analysis, which takes advantage of the differential migration of the two proteins on SDS-PAGE (Figure 29B). Western blot analysis using Ab1 antibody, which recognizes htt1-17, demonstrated that endogenous ovine HTT, but not human mHTT, was upregulated by miRs. HTT It was shown that the α-glucan-1-phosphate dehydrogenase (αGD)-induced thrombin production was not reduced by treatment with α-gDD (Figure 29B).

[0214] DARPP32-labeled neurons and striatal volume are not affected by miRNA treatment To investigate the safety of AAV vector injection, immunohistochemistry for DARPP32, a marker for medium spiny neurons, was performed and the number of DARPP32-positive cells was counted. HTT There was no significant difference between the cell counts of the treatment groups and the AAV9-treated group (Table 3), and there was no significant difference between the treatment groups in the number of cells stained for NeuN, a neuronal marker. Striatal volume was determined using measurements of the cross-sectional area of ​​the striatum in DARPP32-labeled sections and was found to be unchanged after miRNA treatment compared to controls (Table 4).

[0215] Table 3: Number of DARPP32 and Neu N positive cells. Data were analyzed by one-tailed t-test (injected vs. non-injected side). Six months after injection, significant differences were observed between the injected and non-injected sides only in WT sheep injected with AAV9-CBA-miRHTT. By paired t-test. 1 p=0.002. [Table 3]

[0216] Table 4: Striatal volumes. Volumes were determined from cross-sectional areas of 29–35 40 μm sections per side per animal, N=3 sheep per group. Using a paired t-test 1 p=0.01, 2 p=0.03,3 p=0.02. [Table 4]

[0217] Direct injection with scAAV9 results in a transient increase in activated microglia We investigated the immunohistochemical localization of Iba1, a protein that localizes to microglia and is upregulated upon activation. Labeled cells were identified based on morphology as either resting or activated microglia (Table 5). scAAV9-U6-miR HTT Injection of the corresponding control vector increased the number of activated microglia at the injection site 1 month after injection, but 6 months after injection, the injected and non-injected sides were indistinguishable. In a second study, microglial responses were examined only at the study endpoint (6 months), at which point there were no significant differences between groups. This finding suggests that the transient increase in activated microglia is independent of the AAV cargo and can occur with either the vector or surgery alone.

[0218] Table 5: Classification based on the number and morphology of IBA1-positive cells. Statistical analysis was performed by paired t-test (injected side vs. non-injected side). A significant increase in activated microglia on the injected side compared to the non-injected side was observed after AAV9-U6-miR HTT HD sheep injected with ( 1 p = 0.01) and WT sheep injected with AAV9 at 6 months ( 2 p=0.006). A significant decrease in microglia was observed in the AAV9 ( 3 p=0.05) and AAV9-U6-miR HTT ( 4 p=0.04) at 1 month in HD sheep injected with both IFN-γ and IFN-γ, and a significant increase in total microglia was found in HD sheep injected with AAV9 at 6 months in Study 2 ( 4p=0.04). All analyses were performed by paired t-test, comparing the injected side with the non-injected side.

[0219] [Table 5]

[0220] scAAV9-miR HTT Treatment does not affect blood counts, electrolytes, or liver and kidney function Blood samples were collected four times: at baseline (before treatment), 28 (or 30), 90, and 180 days after treatment. Complete blood counts, electrolytes, and liver and kidney function tests were performed (Table 6). None of these measurements indicated significant differences in AAV9-miR activity. HTT No changes were found between injected sheep and controls. Furthermore, there was no change in weight at these time points.

[0221] Table 6: Clinical pathology and complete blood counts for all sheep [Table 6-1]

[0222] [Table 6-2]

[0223] array >SEQ ID NO: 1 Huntington mRNA; NCBI Ref. Seq NM_002111.8)

[0224] >Sequence number 2 TAAATGTGCCTGTTGAAGGGC

[0225] >Sequence number 3 AAGAGGTGCAGAGTCATCATC

[0226] >SEQ ID NO:4 TTCTGGAGGACATCAAACCAT

[0227] >Sequence number 5 TGAACTGGCCCACTTCAATGT

[0228] >SEQ ID NO:6 TTCCATTGGCAACTGGGCCAT

[0229] >SEQ ID NO:7 TAAGCATGGAGCTAGCAGGCT

[0230] >Sequence number 8 TAGCGTTGAAGTACTGTCCCC

[0231] SEQ ID NO:9 TTGAGGCAGCAGCGGCTGTGC

[0232] >SEQ ID NO: 10 TTCATCAGCTTTTCCAGGGTC

[0233] >SEQ ID NO: 11 TGGAATTCTCGGGTGCCAAGG

[0234] >SEQ ID NO: 12 CCTTGGCACCCGAGAATTCCA

[0235] >SEQ ID NO: 13 GUUCAGAGUUCUACAGUCCGACGAUC

[0236] >SEQ ID NO: 14 AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA

[0237] >SEQ ID NO: 15 CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA

[0238] >SEQ ID NO: 16 LOCUS dsCB-GFP-mir155- 5483 bp DNA circular SYN 11-OCT-2012 DEFINITION Ligation of 6433 into dsCB-GFP-mirFlank-ployA* ACCESSION dsCB-GFP-mir155- KEYWORDS . SOURCE Unknown. ORGANISM Unknown Unclassified. REFERENCE 1 (bases 1 to 5483) AUTHORS Self JOURNAL Unpublished. COMMENT SECID / File created by SciEd Central, Scientific & Educational Software COMMENT SECNOTES|Vector molecule: dsCB-GFP-mirFlank-ployA* Fragment ends: BsmBI Fragment size: 5419 Insert molecule: 6433 Fragment ends: Fragment size: 64 FEATURES Location / Qualifiers misc_feature 662..767 / gene=“mutated ITR” / SECDrawAs=“Region” misc_feature 814..1093 / gene=“CMV enhancer” / SECDrawAs=“Region” misc_feature 870..899 / gene=“tentative for” / SECDrawAs=“Region” misc_feature 1100..1126 / gene=“Probe” / SECDrawAs=“Region” misc_feature 1100..1369 / gene=“B-Actin promotor” / product=“Chicken" / SECDrawAs=“Region” misc_feature complement (1168..1190) / gene=“rev” / SECDrawAs=“Region” misc_feature 1435..1465 / gene=“SV40_late_19s_int” / SECDrawAs=“Region” misc_feature 1435..1531 / gene=“modSV40_late_16s_int” / SECDrawAs=“Region” CDS 1605..2314 / gene=“GFP'” / SECDrawAs=“Gene” misc_feature 2341..2357 / gene=“'MCS” / SECDrawAs=“Region” misc_feature 2372..2395 / gene=“5'miR Flank'” / SECDrawAs=“Region” misc_feature 2460..2504 / gene=“3'miR Flank” / SECDrawAs=“Region” misc_feature 2573..2699 / gene=“Poly A signal” / product=“Rabbit globin poly A" / SECDrawAs=“Region” misc_feature complement (2788..2917) / gene=“3' ITR” / SECDrawAs=“Region” CDS 3680..4537 / gene=“Amp(R)” / SECDrawAs=“Gene” ORIGIN

[0239] 1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattct 61 aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg 121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 541 aatattaacg cttacaattt aaatatttgc ttatacaatc ttcctgtttt tggggctttt 601 ctgattatca accggggtac atatgattga catgctagtt ttacgattac cgttcatcgc 661 cctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt 721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggaat tcacgcgtgg 781 atctgaattc aattcacgcg tggtacctct ggtcgttaca taacttacgg taaatggccc 841 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 901 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 961 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 1021 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 1081 gcagtacatc tactcgaggc cacgttctgc ttcactctcc ccatctcccc cccctcccca 1141 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcgggggg 1201 ggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 1261 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 1321 cggcggcgg ggcggcccta taaaaagcga agcgcgcgg gggcgggagc gggatcagcc 1381 accgcggtgg cggcctagag tcgacgagga actgaaaaac cagaaagtta actggtaagt 1441 ttagtctttt tgtcttttat ttcaggtccc ggatccggtg gtggtgcaaa tcaaagaact 1501 gctcctcagt ggatgttgcc tttacttcta ggcctgtacg gaaggtttac ttctgctcta 1561 aaagctgcgg aattgtaccc gcggccgatc caccggtcgc caccatggtg agcaagggcg 1621 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1681 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 1741 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1801 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1861 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 1921 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 1981 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2041 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2101 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2161 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2221 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2281 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagcgttt 2341 ccggcgacgg tgctagcgtc gaccagtgga tcctggaggc ttgctgaagg ctgtatgctg 2401 taagcatgga gctagcaggc tgttttggcc actgactgac agcctgctct ccatgcttac 2461 aggacacaag gcctgttact agcactcaca tggaacaaat ggcccagatc tggccgcact 2521 cgaaaacggg ccctctagac tcgaggacgg ggtgaactac gcctgaggat ccgatctttt 2581 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta 2641 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg 2701 aagcaattcg ttgatctgaa tttcgaccac ccataatacc cattaccctg gtagataagt 2761 agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct 2821 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 2881 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg 2941 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 3001 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 3061 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg 3121 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3181 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3241 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3301 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3361 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 3421 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 3481 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 3541 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 3601 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 3661 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 3721 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 3781 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 3841 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 3901 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 3961 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4021 catgacagta agaaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4081 4141 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 4201 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4261 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 4321 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 4381 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 4441 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaatagaca 4501 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 4561 atatatactt tagattgatt taaaacttca ttttaattt aaaaggatct aggtgaagat 4621 cctttttgat aatctcatga ccaaaatccc ttaacgtgag tttcgttcc actgagcgtc 4681 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 4741 ctgcttgcaa acaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 4801 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 4861 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 4921 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 4981 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5041 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5101 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5161 cagggtcgga acaggagagc gcacgagggga gcttccaggg ggaaacgcct ggtatcttta 5221 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5281 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 5341 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 5401 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 5461 agtgagcgag gaagcggaag agc

[0240] >SEQ ID NO: 17 / / LOCUS pdsU6-Mir-htt-64 5686 bp DNA circular SYN 17-SEP-2013 DEFINITION Ligation of 6433 into pU6-miRNAFlank-GFP* ACCESSION pdsU6-Mir-htt-64 KEYWORDS . SOURCE Unknown. ORGANISM Unknown Unclassified. REFERENCE 1 (bases 1 to 5686) AUTHORS Self JOURNAL Unpublished. COMMENT SECID / File created by SciEd Central, Scientific & Educational Software COMMENT SECNOTES|Vector molecule: pU6-miRNAFlank-GFP* Fragment ends: BsmBI Fragment size: 5622 Insert molecule: 6433 Fragment ends: Fragment size: 64 FEATURES Location / Qualifiers misc_feature 662..767 / gene=“mutated ITR” / SECDrawAs=“Region” misc_feature 777..1041 / gene=“U6 promotor” / SECDrawAs=“Region” misc_signal 1041..1041 / gene=“Pol III Start” / product=“Transcriptional Start” / SECDrawAs=“Label” CDS 1042..1065 / gene=“5' miR Flank'” / SECDrawAs=“Gene” CDS 1130..1175 / gene=“miR 3' Flank” / SECDrawAs=“Gene” misc_signal 1176..1181 / gene=“Pol III term” / product=“pol III terminator” / SECDrawAs=“Label” misc_feature 1199..1478 / gene=“CMV enhancer” / SECDrawAs=“Region” misc_feature 1255..1284 / gene=“tentative for” / SECDrawAs=“Region” misc_feature 1485..1754 / gene=“B- Actin promotor” / product=“Chicken” / SECDrawAs=“Region” misc_feature 1485..1511 / gene=“Probe” / SECDrawAs=“Region” misc_feature complement (1553..1575) / gene=“rev" / SECDrawAs=“Region” misc_feature 1820..1916 / gene=“modSV40_late_16s_int” / SECDrawAs=“Region” misc_feature 1820..1850 / gene=“SV40_late_19s_int” / SECDrawAs=“Region” CDS 1990..2699 / gene=“GFP'” / SECDrawAs=“Gene” misc_feature 2726..2737 / gene=“'MCS'” / SECDrawAs=“Region” misc_feature 2776..2902 / gene=“Poly A signal” / product=“Rabbit globin poly A” / SECDrawAs=“Region” misc_feature complement (2991..3120) / gene=“3' ITR” / SECDrawAs=“Region” CDS 3883..4740 / gene=“Amp(R)” / SECDrawAs=“Gene” ORIGIN

[0241] 1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattct 61 aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg 121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 541 aatattaacg cttacaattt aaatatttgc ttatacaatc ttcctgtttt tggggctttt 601 ctgattatca accggggtac atatgattga catgctagtt ttacgattac cgttcatcgc 661 cctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt 721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggaat tctataaagg 781 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 841 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata 901 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 961 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 1021 tgtggaaagg acgaaacacc gcctggaggc ttgctgaagg ctgtatgctg taagcatgga 1081 gctagcaggc tgttttggcc actgactgac agcctgctct ccatgcttac aggacacaag 1141 gcctgttact agcactcaca tggaacaaat ggcccttttt tctagtggta cctctggtcg 1201 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 1261 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 1321 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 1381 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 1441 tgaccttatg ggactttcct acttggcagt acatctactc gaggccacgt tctgcttcac 1501 tctccccatc tcccccccct ccccaccccc aattttgtat ttattattt tttaattatt 1561 ttgtgcagcg atggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 1621 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 1681 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 1741 gcggcgggcg ggagcgggat cagccaccgc ggtggcggcc tagagtcgac gaggaactga 1801 aaaaccagaa agttaactgg taagtttagt cttttgtct tttatttcag gtcccggatc 1861 cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct 1921 gtacggaagt gttacttctg ctctaaaagc tgcggaattg tacccgcggc cgatccaccg 1981 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 2041 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 2101 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 2161 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac 2221 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 2281 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 2341 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 2401 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 2461 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 2521 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 2581 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 2641 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 2701 tacaagtaaa gcggccctag cgtttccggc gacggtgcta gactcgagga cggggtgaac 2761 tacgcctgag gatccgatct ttttccctct gccaaaaatt atggggacat catgaagccc 2821 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 2881 aattttttgt gtctctcact cggaagcaat tcgttgatct gaatttcgac cacccataat 2941 acccattacc ctggtagata agtagcatgg cgggttaatc attaactaca aggaacccct 3001 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 3061 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 3121 ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 3181 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 3241 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc 3301 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 3361 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 3421 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3481 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 3541 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 3601 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 3661 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 3721 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 3781 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 3841 taaccctgat aaatgcttca samaattga aaaaggaaga gtatgagtat tcaacatttc 3901 cgtgtcgccc ttatccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 3961 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4021 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4081 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4141 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4201 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4261 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4321 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4381 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4441 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4501 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4561 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 4621 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 4681 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 4741 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 4801 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 4861 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 4921 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 4981 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5041 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 5101 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5161 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5221 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5281 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5341 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5401 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5461 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5521 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 5581 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 5641 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagc

[0242] >Accession Number 18 LOCUS pCVscAsaq+-mir64 5155 bp DNA circular SYN 17-SEP-2013 DEFINITION Ligation of 6433 into pCVscAsaq+-mirFlank* ACCESSION pCVscAsaq+-mir64 KEYWORDS . SOURCE Unknown. ORGANISM Unknown Unclassified. REFERENCE 1 (bases 1 to 5155) AUTHORS Self JOURNAL Unpublished. COMMENT SECID / File created by SciEd Central, Scientific & Educational Software COMMENT SECNOTES|Vector molecule: pCVscAsaq+-mirFlank* Fragment ends: BsmBI Fragment size: 5090 Insert molecule: 6433 Fragment ends: Fragment size: 64 FEATURES Location / Qualifiers misc_feature 1..105 / gene=“ITR” / SECDrawAs=“Region” misc_feature 182..449 / gene=“CMV” / product=“CMV enhancer” / SECDrawAs=“Region” CDS 448..753 / gene=“CB promotor” / product=“Promotor Eukaryotic” / SECDrawAs=“Gene” CDS 754..1819 / gene=“Intron” / product=“Intron” / SECDrawAs=“Gene” CDS 1820..1839 / gene=“MCS” / SECDrawAs=“Gene” misc_feature 1843..1847 / gene=“''MCS” / SECDrawAs=“Region” misc_feature 1862..1885 / gene=“5'miR Flank'” / SECDrawAs=“Region” misc_feature 1950..1994 / gene=“3'miR Flank” / SECDrawAs=“Region” CDS 2002..2128 / gene=“RBG pA” / product=“PolyA Signal” / SECDrawAs=“Gene” misc_feature complement (2002..2128) / gene=“RBG\pA” / SECDrawAs=“Info only” misc_feature 2139..2281 / gene=“3'ITR” / SECDrawAs=“Region” CDS 2317..2509 / gene=“lacZ” / SECDrawAs=“Gene” CDS 2510..2965 / gene=“f1 ori” / SECDrawAs=“Gene” misc_feature 3097..3957 / gene=“bla-AmpR” / SECDrawAs=“Region” misc_feature 4117..4731 / gene=“rep-pMB1” / SECDrawAs=“Region” ORIGIN

[0243] 1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg 121 aagatcaatt caattcacgc gtcgacattg attattgact agctctggtc gttacataac 181 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 241 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 301 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc 361 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac 421 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 481 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 541 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 601 cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 661 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 721 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg 781 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc 841 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat 901 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt 961 tgtgcggggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg 1021 tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg 1081 ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc 1141 tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg 1201 ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc 1261 ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg 1321 gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg 1381 ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc 1441 cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt 1501 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg 1561 tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt 1621 ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga 1681 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac 1741 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg 1801 tctcatcatt ttggcaaaga attcatcgat accgtcgacg atctagcgtc gaccagtgga 1861 tcctggaggc ttgctgaagg ctgtatgctg taagcatgga gctagcaggc tgttttggcc 1921 actgactgac agcctgctct ccatgcttac aggacacaag gcctgttact agcactcaca 1981 tggaacaaat ggcccagatc cgatcttttt ccctctgcca aaaattatgg ggacatcatg 2041 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg 2101 tgttggaatt ttttgtgtct ctcactcgat cagatctgag gaacccctag tgatggagtt 2161 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg 2221 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc 2281 cccccccccc ccccccccct gcattctaga gagctccaat tcgccctata gtgagtcgta 2341 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 2401 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 2461 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc 2521 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 2581 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 2641 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 2701 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 2761 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 2821 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 2881 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 2941 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 3001 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 3061 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 3121 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 3181 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 3241 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 3301 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 3361 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 3421 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 3481 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 3541 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 3601 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 3661 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 3721 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 3781 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 3841 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 3901 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 3961 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 4021 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 4081 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 4141 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4201 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4261 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4321 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4381 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4441 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4501 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4561 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4621 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 4681 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 4741 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 4801 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 4861 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 4921 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 4981 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 5041 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 5101 acaggaaaca gctatgacca tgattacgcc agatttaatt aaggccttaa ttagg

[0244] >Fashion 19 LOCUS U6-mir6433-BGHpA 5223 bp DNA circular SYN 12-SEP-2013 DEFINITION Ligation of dsAAV CB MCS** into U6-MiRBA-6433-GFP** ACCESSION U6-mir6433-BGHpA KEYWORDS . SOURCE Unknown. ORGANISM Unknown Unclassified. REFERENCE 1 (bases 1 to 5223) AUTHORS Self JOURNAL Unpublished. COMMENT SECID / File created by SciEd Central, Scientific & Educational Software COMMENT SECNOTES|Vector molecule: U6-MiRBA-6433-GFP** Fragment ends: blunt and EagI Fragment size: 4954 Insert molecule: dsAAV CB MCS** Fragment ends: EagI and blunt Fragment size: 269 FEATURES Location / Qualifiers misc_feature 662..767 / gene=“mutated ITR” / SECDrawAs=“Region” misc_feature 777..1041 / gene=“U6 promotor” / SECDrawAs=“Region” misc_signal 1041..1041 / gene=“Pol III Start” / product=“Transcriptional Start” / SECDrawAs=“Label” CDS 1042..1065 / gene=“5' miR Flank'” / SECDrawAs=“Gene” CDS 1130..1175 / gene=“miR 3' Flank” / SECDrawAs=“Gene” misc_signal 1176..1181 / gene=“Pol III term” / product=“pol III terminator” / SECDrawAs=“Label” misc_feature 1199..1478 / gene=“CMV enhancer” / SECDrawAs=“Region” misc_feature 1255..1284 / gene=“tentative for” / SECDrawAs=“Region” misc_feature 1485..1511 / gene=“Probe" / SECDrawAs=“Region” misc_feature 1485..1754 / gene=“B-Actin promotor” / product=“Chicken” / SECDrawAs=“Region” misc_feature complement (1553..1575) / gene=“rev” / SECDrawAs=“Region” misc_feature 1820..1850 / gene=“SV40_late_19s_int” / SECDrawAs=“Region” misc_feature 1820..1916 / gene=“modSV40_late_16s_int” / SECDrawAs=“Region” misc_feature 2034..2230 / gene=“BGHpA” / SECDrawAs=“Region” misc_feature 2263..2274 / gene=“'MCS'” / SECDrawAs=“Region” misc_feature 2313..2439 / gene=“Poly A signal” / product=“Rabbit globin poly A” / SECDrawAs=“Region” misc_feature complement (2528..2657) / gene=“3' ITR” / SECDrawAs=“Region” CDS 3420..4277 / gene=“Amp(R)” / SECDrawAs=“Gene” ORIGIN

[0245] 1 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattct 61 aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg 121 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 181 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 241 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 301 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 361 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 421 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataagga ttttgccgat 481 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaaca 541 fathercg cttacaattt fatheratttgc ttatacaatc ttcctgtttt tggggctttt 601 ctgattatca accggggtac atatgattga catgctagtt ttacgattac cgttcatcgc 661 cctgcgcgct cgctcgctca ctgaggccgc ccggggcaaag cccggggcgtc gggcgacctt 721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggaat tctaaagg 781 tcgggcagga agggccta tttcccatga ttccttcata tttgcatata cgatacaagg 841 ctgttaga gataattaga attaatttga ctgtaacac aaagatatta gtacaaaata 901 cgtgacgtag aaagtaataa tttcttgggt agttgcagt tttaaatta tgttttaaaa 961 tggactatca tatgcttacc gtaacttgaa agtattcga tttcttggct ttatatatct 1021 tgtggaaagg acgaaacacc gcctggaggc ttgctgaagg ctgtatgctg tagcatgga 1081 gctagcaggc tgttttggcc actgactgac agcctgctct ccatgcttac agcacaag 1141 gcctgttact agcactcaca tggaacaaat ggcccttttt tctagtggta cctctggtcg 1201 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 1261 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 1321 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa 1381 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca 1441 tgaccttatg ggactttcct acttggcagt acatctactc gaggccacgt tctgcttcac 1501 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt 1561 ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg 1621 cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct 1681 ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc 1741 gcggcgggcg ggagcgggat cagccaccgc ggtggcggcc tagagtcgac gaggaactga 1801 aaaaccagaa agttaactgg taagtttagt ctttttgtct tttatttcag gtcccggatc 1861 cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct 1921 gtacggaagt gttacttctg ctctaaaagc tgcggaattg tacccgcggc cgcgtttaaa 1981 ccctgcaggt ctagaaagct tatcgatacc gtcgactaga gctcgctgat cagcctcgac 2041 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 2101 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 2161 gagtaggtgt cattctattc tgggggtgg ggtggggcag gacagcaagg gggaggattg 2221 ggagaacaat agcagggtac aagtaaagcg gccctagcgt ttccggcgac ggtgctagac 2281 tcgaggacgg ggtgaactac gcctgaggat ccgatctttt tccctctgcc aaaaattatg 2341 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca 2401 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aagcaattcg ttgatctgaa 2461 tttcgaccac ccataatacc cattaccctg gtagataagt agcatggcgg gttaatcatt 2521 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 2581 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 2641 agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgttt acaacgtcgt 2701 gactgggaaa acctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 2761 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 2821 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 2881 cgcagcgtga ccgctacact tgccagcgcc ctagcgccg ctcctttcgc tttcttccct 2941 tccttctg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctccttta 3001 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 3061 tcacgtagtg ggccatcgcc ctgatagacg gttttcgcc ctttgacgtt ggagtccacg 3121 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 3181 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 3241 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt 3301 ttcgggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3361 3421 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3481 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 3541 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 3601 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 3661 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 3721 ttgagtactc accagtcaca gaaagcatc ttacggatgg catgacagta agaattat 3781 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 3841 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 3901 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 3961 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4021 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4081 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 4141 gcggtatcat tgcagcactg gggccagatg gtaagccctc cgtatcgta gttatctaca 4201 cgacggggag tcaggcaact atggatgaac gaatagaca gatcgctgag ataggtgcct 4261 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 4321 taaaacttca ttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4381 ccaaaatccc ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca 4441 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4501 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 4561 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 4621 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 4681 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 4741 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 4801 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 4861 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 4921 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 4981 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5041 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 5101 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 5161 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 5221 agc

[0246] >SEQ ID NO: 20 AAV9 capsid protein MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0247] >SEQ ID NO: 21 GCCTGGAGGCTTGCTGAAGGCTGTATGCTGTAAGCATGGAGCTAGCAGGCTGTTTTGGCCACTGACTGACAGCCTGCTCTCTAGCTTACAGGACACAAGGCCTGTTACTAGCACTCACATAACAAATGGCCCTTTT

[0248] >Seq. ID 22 GCTCGAGTGAGCGCAGCCTGCTAGCTCCATGCTTACTGTAAAGCCACCAGATGGGTAAGCATGGAGCTAGCAGGCTTCGCCTACTAGTTTT

Claims

1. (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof; and (ii) a second region comprising a transgene encoding one or more miRNAs; 1. An isolated nucleic acid comprising: The isolated nucleic acid, wherein each miRNA comprises a seed sequence complementary to SEQ ID NO:

1.

2. (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof; and (ii) a second region comprising a transgene encoding one or more miRNAs; 1. An isolated nucleic acid comprising: The isolated nucleic acid, wherein each miRNA is encoded by a sequence comprising a sequence set forth in any one of SEQ ID NOs: 2 to 10 adjacent to the miRNA backbone sequence.

3. (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof; and (ii) a second region comprising a transgene encoding one or more miRNAs; 1. An isolated nucleic acid comprising: The isolated nucleic acid, wherein each miRNA comprises a sequence 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 set forth in SEQ ID NO:1 adjacent to the miRNA backbone sequence.

4. The isolated nucleic acid of claim 2 or 3, wherein each miRNA backbone sequence is a mir-155 backbone sequence, a mir-30 backbone sequence, or a mir-64 backbone sequence.

5. The isolated nucleic acid of any one of claims 1 to 4, wherein the transgene comprises a promoter.

6. 6. The isolated nucleic acid of claim 5, wherein the promoter is a chicken beta-actin (CBA) promoter or a U6 promoter.

7. The isolated nucleic acid of any one of claims 1 to 6, wherein the transgene further encodes a protein.

8. The isolated nucleic acid of any one of claims 1 to 7, wherein the one or more miRNAs are located in an untranslated portion of the transgene.

9. The isolated nucleic acid of claim 7, wherein the untranslated portion is an intron.

10. 9. The isolated nucleic acid of claim 8, wherein the untranslated portion is between the last codon of the nucleic acid sequence encoding the protein and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence.

11. 11. The isolated nucleic acid of any one of claims 1 to 10, further comprising a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

12. 12. The isolated nucleic acid of any one of claims 1 to 11, wherein the ITR variant lacks a functional terminal release site (TRS), optionally the ITR variant is a ΔTRS ITR.

13. The isolated nucleic acid according to any one of claims 1 to 12, wherein at least one of the miRNAs hybridizes to and inhibits expression of human huntingtin (eg, SEQ ID NO: 1).

14. A vector comprising the isolated nucleic acid of any one of claims 1 to 13.

15. The vector of claim 14 , wherein the vector is a plasmid.

16. A host cell comprising an isolated nucleic acid according to any one of claims 1 to 13 or a vector according to claim 14 or 15.

17. (i) a capsid protein; and (ii) An isolated nucleic acid according to any one of claims 1 to 13. A recombinant AAV (rAAV), comprising:

18. 18. The rAAV of claim 17, wherein the capsid protein is an AAV9 capsid protein.

19. 19. The rAAV of claim 17 or 18, wherein the capsid protein comprises the sequence set forth in SEQ ID NO:

20.

20. The rAAV according to any one of claims 17 to 19, wherein the rAAV is a self-complementary AAV (scAAV).

21. The rAAV of any one of claims 17 to 20, wherein the rAAV is formulated for delivery to the central nervous system (CNS).

22. 22. A method for treating Huntington's disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of an isolated nucleic acid of any one of claims 1-13 or an rAAV of any one of claims 17-21 to a subject having or at risk of developing Huntington's disease.

23. 23. The method of claim 22, wherein the subject comprises a Huntington gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

24. 24. The method of claim 22 or 23, wherein the subject is under 20 years of age.

25. The method of any one of claims 22-24, wherein administration results in delivery of the isolated nucleic acid or rAAV to the central nervous system (CNS) of the subject.

26. The method of any one of claims 22 to 25, wherein administration is via injection, optionally intravenous or intrastriatal injection.

27. An isolated nucleic acid encoding a sequence set forth in any one of SEQ ID NOs:2-10 or 21-22.

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