Methods for treating neurological disorders

Abstract

Providing methods for treating neurological disorders. [Solution] The embodiments of this disclosure relate to compositions and methods useful for treating neurological diseases and disorders. In some embodiments, the disclosure provides a method for treating a neurological disease or disorder, comprising the administration of both a viral vector containing interfering nucleic acid (e.g., artificial miRNA) and a viral vector containing the CYP46A1 protein. In some embodiments, the disclosure provides a method for treating Huntington's disease, comprising the administration of both a viral vector containing interfering nucleic acid (e.g., artificial miRNA) targeting the huntingtin gene (HTT) and a viral vector containing the CYP46A1 protein. In some embodiments, the viral vector comprises a modified viral capsid for preferentially targeting cells, for example, in the CNS or PNS.

Description

[Technical Field]

[0001] Cross-references to related applications This application, based on U.S. Provisional Application No. 63 / 080,925 filed September 21, 2020, U.S. Provisional Application No. 63 / 121,152 filed December 3, 2020, U.S. Provisional Application No. 63 / 139,410 filed January 20, 2021, U.S. Provisional Application No. 63 / 140,440 filed January 22, 2021, and U.S. Provisional Application No. 63 / 180,407 filed April 27, 2021, asserts a benefit under Section 119(e) of the U.S. Patent Act, the contents of each of those applications, in whole, are incorporated herein by reference.

[0002] Technical field The techniques described herein relate to methods for treating neurological diseases or disorders, such as Huntington's disease. [Background technology]

[0003] background Huntington's disease (HD) is a destructive hereditary neurodegenerative disorder caused by the elongation of the CAG repeat region in exon 1 of the huntingtin gene. While the huntingtin protein (HTT) is expressed throughout the body, the polyglutamine elongation protein is particularly toxic to medium spiny neurons and their cortical connections in the striatum. Patients struggle with emotional symptoms, including depression and anxiety, as well as characteristic motor impairments and chorea. Currently, there is no cure for Huntington's disease, and treatment options are limited to improving disease symptoms. [Overview of the project] [Means for solving the problem]

[0004] Abstract One aspect provided herein describes a method for treating a neurological disorder or condition in a subject requiring treatment, the method comprising administering a therapeutically effective amount of (a) a nucleic acid encoding at least one miRNA and (b) a nucleic acid encoding the CYP46A1 protein to a subject having or at risk of developing a neurological disorder or condition.

[0005] In one embodiment, a composition or combination comprising (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs, and (b) an isolated nucleic acid encoding a CYP46A1 protein is described herein. In one embodiment, a composition or combination comprising (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat sequence (ITR) or a variant thereof, and (ii) a second region comprising a transgene encoding one or more miRNAs, and (b) an isolated viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein is described herein.

[0006] In one embodiment, a method for treating a neurological disorder or impairment in a subject requiring treatment is described herein, the method comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs, and (b) an isolated nucleic acid encoding a CYP46A1 protein to a subject having or at risk of developing a neurological disorder or impairment. In one embodiment, a method for treating a neurological disorder or impairment in a subject requiring treatment is described herein, the method comprising administering a therapeutically effective amount of a recombinant viral vector comprising (a)(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, and (b) a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein to a subject having or at risk of developing a neurological disorder or impairment.

[0007] In some embodiments, neurological disorders or conditions include Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma resulting from spinal cord and head injuries, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan syndrome, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorders, schizophrenia, drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, psychopathy, sleep disorders, pain disorders, and eating or weight disorders. In some embodiments, neurological disorders or conditions include central nervous system (CNS) disorders or conditions. In some embodiments, the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, amyotrophic lateral sclerosis, and Parkinson's disease.

[0008] In some embodiments, the CNS disease or disorder is Alzheimer's disease, and at least one miRNA contains a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and their disease-associated alleles.

[0009] In some embodiments, the CNS disease or disorder is Parkinson's disease, and at least one miRNA contains a seed sequence complementary to SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and their disease-associated alleles.

[0010] In some embodiments, the CNS disease is Huntington's disease, and at least one miRNA contains a seed sequence complementary to SEQ ID NO: 4, or at least one miRNA contains one of the sequences SEQ ID NOs: 6-17, 40-44, or 50-66 adjacent to the miRNA backbone sequence. In some embodiments, the CNS disease is Huntington's disease, and at least one miRNA contains one of the sequences SEQ ID NOs: 6-17, 40-44, or 50-66. In some embodiments, at least one of the miRNAs hybridizes with human huntingtin and inhibits its expression. In some embodiments, the subject includes a huntingtin gene having 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.

[0011] In some embodiments, the recombinant viral vector is selected from the group consisting of AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0012] In some embodiments, the recombinant viral vector containing (a) is the same as the recombinant viral vector containing (b). In some embodiments, the isolated nucleic acids of (a) and (b) are contained in separate recombinant viral vectors. In some embodiments, the isolated nucleic acids of (a) and (b) are contained in the same recombinant viral vector.

[0013] In some embodiments, (a) and (b) are administered at substantially the same time. In some embodiments, (a) and (b) are administered at different times. In some embodiments, the different times are separated by at least one minute, at least one hour, at least one day, at least one week, at least one month, at least one year, or longer. In some embodiments, (a) is administered before administration of (b). In some embodiments, (b) is administered before administration of (a). In some embodiments, administration of (a), (b), or (a) and (b) is repeated at least once.

[0014] In some embodiments, the transgene contains two miRNAs in tandem adjacent to an intron. In some embodiments, the adjacent introns are identical. In some embodiments, the adjacent introns are of the same species origin. In some embodiments, the adjacent introns are hCG introns.

[0015] In some embodiments, the transgene includes a promoter. In some embodiments, the promoter is a synapsin (Syn1) promoter or one of the promoters listed in Tables 10-13.

[0016] In some embodiments, one or more miRNAs are located in the untranslated region of the transgene. In some embodiments, the untranslated region is an intron. In some embodiments, the untranslated region is between the last codon of the protein-coding nucleic acid sequence and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence. In some embodiments, the untranslated region is the 5' untranslated region (5'UTR).

[0017] In some embodiments, the nucleic acid or viral vector further comprises a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof.

[0018] In some embodiments, the ITR variant lacks a functional terminal resolution site (TRS), and optionally the ITR variant is an ATRS ITR.

[0019] In some embodiments, administration results in delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the subject. In some embodiments, administration is by injection, optionally intravenous injection or intrastriatal injection.

[0020] In some embodiments, the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimera thereof. In some embodiments, the viral vector comprises a capsid protein derived from an AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimera thereof. In some embodiments, the capsid protein is an AAV9 capsid protein. In some embodiments, the viral vector is self-complementary AAV (scAAV). In some embodiments, the viral vector is formulated for delivery to the central nervous system (CNS).

[0021] In some embodiments of any of the aspects, the viral vector comprises a modified viral capsid.

[0022] In some embodiments of any of the aspects, the viral vector comprises a modification to the viral capsid.

[0023] In some embodiments of any of the aspects, the modification is a chemical modification, non-chemical modification or amino acid modification of the viral capsid.

[0024] In some embodiments of any of the aspects, at least one of the capsid modifications preferentially targets cells in the CNS or PNS.

[0025] In some embodiments of any of the aspects, the chemical modification comprises a chemically modified tyrosine residue modified to include a covalently linked monosaccharide or polysaccharide moiety.

[0026] In some embodiments of any of the aspects, the chemically modified tyrosine residue comprises a monosaccharide selected from galactose, mannose, N-acetylgalactosamine, cross-linked GalNac, and mannose-6-phosphate.

[0027] In some embodiments of any of the aspects, the chemical modification comprises a ligand covalently linked to a primary amino group of the capsid polypeptide via a -CSNH- bond.

[0028] In some embodiments of any of the aspects, the ligand comprises an arylene or heteroarylene radical covalently attached to the ligand.

[0029] In some embodiments of any of the aspects, the modified viral capsid is a chimeric capsid or a monomeric capsid.

[0030] In some embodiments of any of the aspects, the modified viral capsid is a monomeric capsid.

[0031] In some embodiments of any of the aspects, the modified viral capsid is a chimeric capsid or a monomeric capsid that further comprises the modification.

[0032] In some embodiments of the model, the modified viral capsid is an AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a mutant variant, chimera, mosaic, or rational haploid thereof.

[0033] In some embodiments of the model, the modification alters the antigenic profile of the modified viral capsid compared to the unmodified viral capsid.

[0034] In some embodiments of any of the models, the modified viral capsid may be used for repeated administration. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 is a schematic diagram showing the HD plasmid map of pJAL130-CYP46A1, 7314 bp. See, for example, SEQ ID NO: 111 and Table 16, which shows the ITR-ITR sequence of the CYP46 variant sequence (see, for example, SEQ ID NO: 110) from the plasmid.

[0036] [Figure 2] Figure 2 shows the intracranial in vivo distribution in sagittal section of the transgene GFP delivered by intraventricular (ICV) and intravenous (IV) injections: CNS-1 (e.g., see SEQ ID NO: 112), CNS-2 (e.g., see SEQ ID NO: 113), CNS-3 (e.g., see SEQ ID NO: 114), CNS-4 (e.g., see SEQ ID NO: 115), CNS-5 (e.g., see SEQ ID NO: 122), CNS-6 (e.g., see SEQ ID NO: 123), CNS-7 (e.g., see SEQ ID NO: 124), and CNS-8 (e.g., see SEQ ID NO: 125), as well as the transgene GFP under the control of the control promoter hSyn1 (e.g., see SEQ ID NO: 152). The scale bar is 1 mm.

[0037] [Figure 3A] Figures 3A and 3B show images of coronal brain sections. Figure 3A shows the intracranial biodistribution of transgene GFP in coronal sections under the control of CNS-1 (e.g., see SEQ ID NO: 112), CNS-2 (e.g., see SEQ ID NO: 113), CNS-3 (e.g., see SEQ ID NO: 114), and CNS-4 (e.g., see SEQ ID NO: 115) delivered by ICV. The scale bar is 1 mm. Figure 3B shows the intracranial biodistribution of transgene GFP in coronal sections under the control of CNS-5 (e.g., see SEQ ID NO: 122), CNS-6 (e.g., see SEQ ID NO: 123), CNS-7 (e.g., see SEQ ID NO: 124), and CNS-8 (e.g., see SEQ ID NO: 125) delivered by ICV, as well as the control promoter hSyn1 (e.g., see SEQ ID NO: 152). The scale bar is 1 mm. [Figure 3B] Figures 3A and 3B show images of coronal brain sections. Figure 3A shows the intracranial biodistribution of transgene GFP in coronal sections under the control of CNS-1 (e.g., see SEQ ID NO: 112), CNS-2 (e.g., see SEQ ID NO: 113), CNS-3 (e.g., see SEQ ID NO: 114), and CNS-4 (e.g., see SEQ ID NO: 115) delivered by ICV. The scale bar is 1 mm. Figure 3B shows the intracranial biodistribution of transgene GFP in coronal sections under the control of CNS-5 (e.g., see SEQ ID NO: 122), CNS-6 (e.g., see SEQ ID NO: 123), CNS-7 (e.g., see SEQ ID NO: 124), and CNS-8 (e.g., see SEQ ID NO: 125) delivered by ICV, as well as the control promoter hSyn1 (e.g., see SEQ ID NO: 152). The scale bar is 1 mm.

[0038] [Figure 4-1]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs. 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO. 152). Data were obtained by quantitative measurement of GFP staining intensity in 10 non-overlapping RGB images by marginal value analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification across separate brain regions while maintaining constant settings. Foreground immunostaining was defined by the mean of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactivity per field of view for each region of interest (n=3). With ICV delivery, expression is highest in the cortical and hippocampal brain regions. CNS1-8 (see, for example, SEQ ID NOs. 112-115 and 122-125) show higher expression in the hippocampus compared to the hSyn1 control. CNS-1 (see, for example, SEQ ID NO. 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1. [Figure 4-2]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs. 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO. 152). Data were obtained by quantitative measurement of GFP staining intensity in 10 non-overlapping RGB images by marginal value analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification across separate brain regions while maintaining constant settings. Foreground immunostaining was defined by the mean of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactivity per field of view for each region of interest (n=3). With ICV delivery, expression is highest in the cortical and hippocampal brain regions. CNS1-8 (see, for example, SEQ ID NOs. 112-115 and 122-125) show higher expression in the hippocampus compared to the hSyn1 control. CNS-1 (see, for example, SEQ ID NO. 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1. [Figure 4-3]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs. 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO. 152). Data were obtained by quantitative measurement of GFP staining intensity in 10 non-overlapping RGB images by marginal value analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification across separate brain regions while maintaining constant settings. Foreground immunostaining was defined by the mean of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactivity per field of view for each region of interest (n=3). With ICV delivery, expression is highest in the cortical and hippocampal brain regions. CNS1-8 (see, for example, SEQ ID NOs. 112-115 and 122-125) show higher expression in the hippocampus compared to the hSyn1 control. CNS-1 (see, for example, SEQ ID NO. 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1.

[0039] [Figure 5A]Figures 5A-5B show the tissue expression patterns for the faf1 and pitx3 genes, which were designed with CRE / proximal promoters derived from CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4. Figure 5A shows the expression pattern of the faf1 gene in mouse PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. faf1 is expressed in many PNS neurons. Figure 5B shows the expression pattern of the pitx3 gene in PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. pixt3 is expressed in sympathetic PNS neurons. faf1 is expressed in many PNS neurons; therefore, synthetic promoters containing a CRE or proximal promoter designed from the faf1 gene, e.g., CNS-5 and CNS-5_v2, are expected to have strong expression in the PNS. pitx3 is expressed in sympathetic PNS neurons; therefore, synthetic promoters containing a CRE designed from the pitx3 gene, e.g., CNS-2, CNS-3, or CNS-4, are expected to have expression in PNS sympathetic neurons. Similar analyses for lmx1b and pitx2 did not show expression in the PNS above the cutoff score for the analysis (trinization score less than 0.95; data not shown); therefore, CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not expected to be active in PNS neurons. [Figure 5B]Figures 5A-5B show the tissue expression patterns for the faf1 and pitx3 genes, which were designed with CRE / proximal promoters derived from CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4. Figure 5A shows the expression pattern of the faf1 gene in mouse PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. faf1 is expressed in many PNS neurons. Figure 5B shows the expression pattern of the pitx3 gene in PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. pixt3 is expressed in sympathetic PNS neurons. faf1 is expressed in many PNS neurons; therefore, synthetic promoters containing a CRE or proximal promoter designed from the faf1 gene, e.g., CNS-5 and CNS-5_v2, are expected to have strong expression in the PNS. pitx3 is expressed in sympathetic PNS neurons; therefore, synthetic promoters containing a CRE designed from the pitx3 gene, e.g., CNS-2, CNS-3, or CNS-4, are expected to have expression in PNS sympathetic neurons. Similar analyses for lmx1b and pitx2 did not show expression in the PNS above the cutoff score for the analysis (trinization score less than 0.95; data not shown); therefore, CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not expected to be active in PNS neurons.

[0040] [Figure 6A] Figure 6A shows the expression pattern of the HTT gene in a sagittal section from the brain of an adult mouse (obtained from the Allen Mouse brain atlas; mouse.brain-map.org). HTT (huntingtin) is highly expressed throughout the brain.

[0041] [Figure 6B] Figure 6B shows the expression pattern of the CYP46A1 gene in a coronal section of the brain of an adult mouse (obtained from the Allen Mouse brain atlas; mouse.brain-map.org). CYP46A1 is widely expressed in the brain.

[0042] [Figure 7A] Figure 7A shows the median GFP expression of synapsin-1 in neuroblastoma-derived SH-SY5Y cells compared to the control promoter CAG for the synthetic NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036, and the control promoter. NTC indicates untransfected cells. Data were collected from three biological replicates, each of which is the mean of two technical replicates. Error bars are standard errors.

[0043] [Figure 7B] Figure 7B shows the transfection efficiency in neuroblastoma-derived SH-SY5Y cells when transfected with synthetic NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036, or the control promoters synapsin-1 and CAG, operably linked to GFP. NTC indicates untransfected cells. Data were collected from three biological replicates, each of which is the mean of two technical replicates. Error bars are standard errors. GFP-positive% indicates the percentage of all cells that were GFP-positive. [Modes for carrying out the invention]

[0044] Detailed explanation Aspects of the present invention relate to the administration of both interfering RNA (e.g., miRNAs such as artificial miRNAs) and nucleic acids encoding the CYP46A1 protein, which are effective in reducing the expression of pathogenic genes in a subject when delivered to that subject. Accordingly, the methods and compositions described herein are useful in some embodiments for the treatment of neurological diseases or disorders. Treatment method

[0045] Methods for targeting and delivering nucleic acids and / or transgenes (e.g., inhibitory RNAs such as miRNAs, and / or nucleic acids encoding CYP46A1) are provided by this disclosure. The methods typically involve targeting an effective amount of nucleic acids / inhibitory nucleic acids encoding at least one interfering RNA capable of reducing the expression of a target gene, e.g., a pathogenic gene associated with a neurological disease or disorder (e.g., huntingtin (htt) protein), and a nucleic acid encoding CYP46A1. In some embodiments, one or both of the nucleic acids are provided in a viral vector and / or in viral particles, e.g., rAAV.

[0046] As used herein, “neurological disorder or condition” may mean any disease, disorder or condition affecting or relating to the nervous system, namely those affecting the central nervous system (brain and spinal cord) and the peripheral nervous system (PNS; e.g., peripheral nerves and cranial nerves), as well as the autonomic nervous system (its parts located in both the central and peripheral nervous systems). More than 600 neurological disorders have been identified in humans. In non-limiting examples, neurological disorders or conditions may include Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, Niemann-Pick disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma resulting from spinal and head injuries, eye diseases and disorders, Tay-Sachs disease, Rett syndrome, neuropathic pain, Resch-Nyhan syndrome, epilepsy, stroke, depression, bipolar disorder, persistent affective disorder, secondary mood disorders, schizophrenia, drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, psychosexial disorders, sleep disorders, painful disorders, and / or eating or weight disorders. In some embodiments, the neurological disease or disorder is a central nervous system (CNS) disease or disorder, such as Huntington's disease, Parkinson's disease, or Alzheimer's disease.

[0047] As used herein, “Huntington’s disease” or “HD” refers to a neurodegenerative disorder characterized by progressively worsening motor, cognitive, and behavioral changes caused by a trinucleotide repeat elongation (e.g., CAG, which is translated into a polyglutamine tract or polyQ tract) in the HTT gene, resulting in the production of a pathogenic mutant huntingtin protein (HTT, or mHTT).

[0048] As used herein, "HTT" or "huntingtin" refers to the gene that codes for the huntingtin protein. Normal huntingtin protein functions in nerve cells, and a normal HTT gene typically has approximately 7 to 35 CAG repeats at its 5' end. The HTT gene is often mutated in patients who have Huntington's disease or are at risk of developing it. In some embodiments, the mutant huntingtin protein accelerates the rate of neuronal cell death in certain areas of the brain. Generally, the severity of HD correlates with the size of the trinucleotide repeat elongation in the subject. For example, subjects with a CAG repeat region containing repeats between 36 and 39 are characterized as having “low penetrant” HD, while subjects with more than 40 repeats are characterized as having “full penetrant” HD. Therefore, in some embodiments, subjects who have HD or are at risk of having it have an HTT gene containing CAG repeats between approximately 36 and 39 (e.g., repeats of 36, 37, 38, or 39). In some embodiments, subjects who have HD or are at risk of having HD have an HTT gene containing 40 or more (e.g., 40, 45, 50, 60, 70, 80, 90, 100, 200, or more) CAG repeats. In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats develop HD earlier than subjects with fewer than 100 CAG repeats. In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats may develop HD symptoms before approximately 20 years of age and are referred to as having juvenile HD (also known as akinesiological-rigidity HD, or Westphal variant HD). The number of CAG repeats in the allele of the subject's HTT gene can be determined by any suitable modality known in the art. For example, nucleic acids (e.g., DNA) can be isolated from a target biological sample (e.g., blood), and the number of CAG repeats in 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.).The HTT gene sequence is known in several species, for example, the mRNA sequence (NCBI reference sequence: NM_002111.8, SEQ ID NO: 4) and protein sequence (NCBI reference sequence: NP_0021012.4, SEQ ID NO: 5) of human HTT (NCBI gene ID: 3064). Therefore, in some embodiments relating to the treatment of Huntington's disease, one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with HTT and / or reduce its expression.

[0049] As used herein, “Alzheimer’s disease” or “AD” refers to a neurodegenerative disorder characterized by progressively worsening memory, disorientation, mood swings, and increasing difficulties with language, motivation, and self-care. Several genes, including amyloid precursor protein (APP; NCBI gene ID: 351), presenilin 1 (PSEN1; NCBI gene ID: 5663), presenilin 2 (PSEN2; NCBI gene ID: 5664), ATP-binding cassette subfamily A member 7 (ABCA7; NCBI gene ID: 10347), and sortilin-associated receptor 1 (SORL1; NCBI gene ID: 6653), may contribute to or increase the risk of AD. Sequences of such AD-associated genes are known in several species, and for example, human mRNA and protein sequences are available in the NCBI database using the provided gene ID numbers. These AD-related genes and others, as well as their AD-related alleles (e.g., mutants, SNPs, etc.), are publicly known in the art, e.g., Sims et al. Nature Neuroscience 2020 23:311-22; Bellenguez et al. al. Current Opinion in Neurobiology 2020 61:40-48; Tabuas-Pereira et al. 2020 Neurogenetics and Psychiatric Genetics 8:1-16; and further details in Chapter 15 of Porter et al. "Neurodegeneration and Alzheimer's Disease" 2019. These are described herein, and each of them is incorporated herein by reference in whole. Thus, in some embodiments relating to the treatment of Alzheimer's disease, one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with APP, PSEN1, PSEN2, ABCA7, and / or SORL1, and / or reduce their expression.

[0050] As used herein, “Parkinson’s disease” or “PD” refers to a neurodegenerative disorder characterized by progressively worsening tremors and rigidity, as well as increasing problems with balance, gait, and coordination. Synuclein alpha (SNCA; NCBI gene ID: 6622), leucine-rich repeat kinase 2 (LRRK2 / PARK8; NCBI gene ID 120892), glucosylceramidase beta (GBA1; NCBI gene ID 2629), parkin RBR E3 ubiquitin (PRKN; NCBI gene ID 5071), PTEN-inducible kinase 1 (PINK1; NCBI gene ID 65018), parkinsonism-related deglycase (DJ1 / PARK7; NCBI gene ID 11315), VPS35 retromer complex component (VPS35; NCBI gene ID 55737), eukaryotic translation initiation factor 4 gamma 1 (EIF4G1; NCBI gene ID 1981), DnaJ heat shock protein family member C13 (DNAJC13; NCBI gene ID Several genes, including 23317), coiled-coil helix-coiled-coil helix domain-containing 2 (CHCHD2; NCBI gene ID 51142), and / or ubiquitin C-terminal hydrolase L1 (UCHL1; NCBI gene ID 7345), may contribute to or increase the risk of PD. Sequences of such PD-related genes are known in several species, and for example, human mRNA and protein sequences are available in the NCBI database using the provided gene ID numbers.These PD-related genes and others, as well as their PD-related alleles (e.g., mutants, SNPs, etc.), are publicly known in the art, for example, D'Souza et al. Acta Neuropsychiatrica 2020 32:10-22; Sardi et al. Parkinsonism & Related Disorders 2019 59:32-38; Hardy et al. Current Opinion in Genetics & Development 2009 19:254-65; Ferreria et al. Neurologica 2017 135:273-84; Jain et al. Clinical Science 2005 109:355-64. Further details are found in Fagan et al. European Journal of Neurology 2017 24:561-e20; Campelo et al. Parkinson's Disease 2017 4318416; and in Chapter 15 of Porter et al. "Neurodegeneration and Alzheimer's Disease" 2019. Each of these is incorporated herein by reference in whole. Thus, in some embodiments relating to the treatment of Parkinson's disease, one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with and / or reduce the expression of SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, and / or GBA1.

[0051] The “effective amount” of a substance is an amount sufficient to produce the desired effect. In some embodiments, the effective amount of isolated nucleic acid is sufficient to transfect (or, in the context of rAAV-mediated delivery) a sufficient number of target cells in the target tissue of the subject. In some embodiments, the target tissue is central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, the effective amount of isolated nucleic acid (e.g., one that can be delivered via rAAV) may be sufficient to have a therapeutic benefit in the subject, for example, reducing the expression of a pathogenic gene or protein (e.g., HTT), extending the lifespan of the subject, or improving one or more symptoms of a disease in the subject (e.g., symptoms of Huntington's disease). The effective amount depends on various factors, such as the species, age, weight, health, and target tissue of the subject, and therefore may vary between subjects and tissues, as described elsewhere in this disclosure. Inhibitory RNA

[0052] In some embodiments, the Disclosure provides inhibitory nucleic acids, such as miRNAs, that specifically bind to (e.g., hybridize with) at least two consecutive bases (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) of a target, such as human huntingtin mRNA (e.g., SEQ ID NO: 4). In some embodiments, the Disclosure provides inhibitory nucleic acids, such as miRNAs, that specifically bind to (e.g., hybridize with) at least two consecutive bases (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) of exon 1 of human huntingtin mRNA (e.g., SEQ ID NO: 3). As used herein, “consecutive bases” means two or more nucleotide bases that are covalently bonded to each other (e.g., as part of a nucleic acid molecule) (e.g., one or more phosphodiester bonds). In some embodiments, at least one miRNA is approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 99%, or approximately 100% identical to a target, for example, two or more consecutive nucleotide bases of SEQ ID NOs. 3 or 4 (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). In some embodiments, the inhibitory RNA is a miRNA containing or encoding a sequence shown in any one of SEQ ID NOs. 6-17, 40-44, or 50-66.

[0053] In one embodiment, an inhibitory RNA that can be used for the treatment of a neurological disease or disorder is described herein. In some embodiments of any of the embodiments, the nucleic acid sequence of the inhibitory RNA includes one of sequences that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to at least one of the sequences of SEQ ID NOs: 6-17, 40-44, or 50-66, or SEQ ID NOs: 3 or 4, which maintain the same function (e.g., HTT inhibition).

[0054] In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a sequence shown in one of sequence numbers 6-17, 40-44, or 50-66. In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a sequence shown in one of sequence numbers 6-17, 40-44, or 50-66 adjacent to the miRNA backbone sequence.

[0055] In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a seed sequence complementary to one of sequence numbers 3, 4, 18-39, or 46-49. In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a seed sequence adjacent to the miRNA backbone sequence that is complementary to one of sequence numbers 3, 4, 18-39, or 46-49. In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a seed sequence substantially complementary to one of sequence numbers 3, 4, 18-39, or 46-49. In some embodiments, the vectors described herein include at least one miRNA, each miRNA containing a seed sequence adjacent to the miRNA backbone sequence that is substantially complementary to one of sequence numbers 3, 4, 18-39, or 46-49.

[0056] [Table 1]

[0057] [Table 2]

[0058] [Table 3-1] [Table 3-2]

[0059] In some embodiments, the miRNAs include SEQ ID NOs: 6 and 11, SEQ ID NOs: 7 and 12, SEQ ID NOs: 8 and 11, SEQ ID NOs: 8 and 16, SEQ ID NOs: 8 and 17, SEQ ID NOs: 9 and 14, or SEQ ID NOs: 10 and 15.

[0060] In some embodiments, the vector includes a pre-miRNA having the sequence of SEQ ID NO: 40 or 41. These pre-miRNAs include a scaffold containing SEQ ID NO: 8. The alternative first RNA sequences disclosed herein may be substituted for SEQ ID NO: 8 in either SEQ ID NO: 40 or 41.

[0061] In some embodiments, the vector includes a pri-miRNA having the sequence of SEQ ID NO: 42 or 43. The pri-miRNA of SEQ ID NO: 42 includes a scaffold containing SEQ ID NOs: 8 and 16. Alternative RNA sequences disclosed herein may be substituted for SEQ ID NOs: 8 and 16 in SEQ ID NO: 42. The pri-miRNAs of SEQ ID NOs: 43 and 44 include a scaffold containing SEQ ID NOs: 8 and 17. Alternative RNA sequences disclosed herein may be substituted for SEQ ID NOs: 8 and 17 in either SEQ ID NOs: 43 or 44.

[0062] [Table 4-1] [Table 4-2]

[0063] In some embodiments, the inhibitory nucleic acid may include one or more of SEQ ID NOs: 1-102 and / or 103-249 of International Patent Publication WO2017 / 201258. In some embodiments, the inhibitory nucleic acid may include one or more of the dual combinations selected from SEQ ID NOs: 1-249 of International Patent Publication WO2017 / 201258, provided in Tables 3-5 of International Patent Publication WO2017 / 201258. In some embodiments, the vector may include one or more of the pri-miRNAs provided in Table 9 of International Patent Publication WO2017 / 201258 or the pri-raiRNAs provided in Table 10. The contents of International Patent Publication WO2017 / 201258 are incorporated herein by reference in their entirety.

[0064] In some embodiments, the inhibitory nucleic acid may include one or more of sequence numbers 914-1013 and / or 1014-1160 of International Patent Publication WO2018 / 204803. In some embodiments, the inhibitory nucleic acid may include one or more of a dual combination selected from sequence numbers 914-1160 of International Patent Publication WO2018 / 204803, provided in Tables 4-6 of International Patent Publication WO2018 / 204803. The contents of International Patent Publication WO2018 / 204803 are incorporated herein by reference in their entirety.

[0065] In some embodiments, the inhibitory nucleic acid may include one or more of sequence numbers 916-1015 and / or 1016-1162 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may include one or more of sequence numbers 916-1015, 1016-1162, 1164-1332 and / or 1333-1501 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may include one or more of a dual combination selected from sequence numbers 916-1162 of International Patent Publication WO2018 / 204797 provided in Tables 4-6 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may include one or more of the dual combinations selected from Sequence IDs 1164-1501 of International Patent Publication WO2018 / 204797, provided in Table 9 of International Patent Publication WO2018 / 204797. The contents of International Patent Publication WO2018 / 204797 are incorporated herein by reference in their entirety.

[0066] In some embodiments, the inhibitory nucleic acid may target a sequence complementary or substantially complementary to a heterozygous SNP in the gene encoding the gain-of-function mutant huntingtin protein, for example, including such a sequence. In some embodiments, the SNP has an allele frequency of at least 10% in the sample population. In some embodiments, the SNP is located in a genomic site selected from the group consisting of RS362331, RS4690077, RS363125, RS363075, RS362268, RS362267, RS362307, RS362306, RS362305, RS362304, RS362303, and RS7685686. Such SNPs are described in more detail, for example, in U.S. Patent No. 9,343,943, which is incorporated herein by reference in its entirety. In some embodiments, the target sequence is one of SEQ ID NOs. 45–49. In some embodiments, the inhibitory nucleic acid sequence includes one or more of sequence numbers 50 to 61. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 50 and 51. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 52 and 53. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 54 and 55. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 56 and 57. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 58 and 59. In some embodiments, the inhibitory nucleic acid sequence is, for example, double-stranded and includes at least sequence numbers 60 and 61.

[0067] [Table 5]

[0068] [Table 6]

[0069] In some embodiments, inhibitory nucleic acids, such as miRNAs, can specifically hybridize to or target polymorphisms, mutants, or SNPs in one of the genes disclosed herein. Methods for selecting inhibitory nucleic acid sequences that target polymorphisms, such as SNPs, in the HTT gene are known in the art. For example, such methods are disclosed in U.S. Patents 8,679,750 and 7,947,658, each of which is incorporated herein by reference in whole. In some embodiments, the inhibitory nucleic acid may include one or more sequences, for example, sequence numbers 1 to 342 of U.S. Patents 8,679,750 or 7,947,658.

[0070] In some embodiments, the inhibitory nucleic acid may include one or more of SEQ ID NOs: 62-66.

[0071] [Table 7]

[0072] Further preferred sequences are, for example, U.S. Patent No. 7,951,934, Miniarikova et al. Molecular Therapy - Nucleic Acids 2015 5:e297; This is known from Kordasiweicz et al. Neuron 2012 74:1031-1044, and these Each of these is incorporated herein by reference in its entirety.

[0073] In some embodiments of the model, the inhibitory RNA (e.g., miRNA) binds to and / or targets the 5' untranslated region (UTR) of the target. In some embodiments of the model, the inhibitory RNA (e.g., miRNA) binds to and / or targets one or more exons of the target. In some embodiments of the model, the inhibitory RNA (e.g., miRNA) binds to and / or targets the 5'UTR, exon 1, CAG repeat, CAG5'-jumper, or CAG3'-jumper of HTT.

[0074] In some embodiments, the inhibitory RNA and / or vector does not contain any of the sequences of SEQ ID NOs. 67–73. In some embodiments, the inhibitory RNA and / or vector does not contain any sequence having sequence identity higher than 80%, higher than 85%, higher than 90%, higher than 95%, or higher than 98% with any of SEQ ID NOs. 67–73.

[0075] In some embodiments, the inhibitory RNA and / or vector does not contain any of the sequences of SEQ ID NOs. 67-73. In some embodiments, the inhibitory RNA and / or vector does not contain any of the sequences of SEQ ID NOs. 135-151. In some embodiments, the inhibitory RNA and / or vector does not contain any sequence that has sequence identity higher than 80%, higher than 85%, higher than 90%, higher than 95%, or higher than 98% with any of SEQ ID NOs. 67-73. In some embodiments, the inhibitory RNA and / or vector does not contain any sequence that has sequence identity higher than 80%, higher than 85%, higher than 90%, higher than 95%, or higher than 98% with any of SEQ ID NOs. 135-151.

[0076] In some embodiments, the inhibitory RNA and / or vector includes any of the sequences of SEQ ID NOs. 67–73. In some embodiments, the inhibitory RNA and / or vector includes a sequence having sequence identity higher than 80%, higher than 85%, higher than 90%, higher than 95%, or higher than 98% with any of the sequences of SEQ ID NOs. 67–73.

[0077] In some embodiments, the inhibitory RNA and / or vector includes any of the sequences of SEQ ID NOs. 67-73 or 135-151. In some embodiments, the inhibitory RNA and / or vector includes sequences having sequence identity higher than 80%, higher than 85%, higher than 90%, higher than 95%, or higher than 98% with any of the sequences of SEQ ID NOs. 67-73 or 135-151. See, for example, International Patent Application No. WO2021 / 127455, the contents of which are incorporated herein by reference in their entirety.

[0078] [Table 8]

[0079] Suitable sequences for use in inhibitory nucleic acids (e.g., miRNAs) targeting AD and / or PD-related targets are known in the art, see, for example, International Patent Publications WO2011 / 133890, WO2012 / 036433, WO2013 / 007874; U.S. Patent Application Publication US2016 / 0264965; U.S. Patents 7,829,694, 8,415,319, 10,125,363, and 10,011,835. The contents of the aforementioned references are incorporated herein by reference in their entirety.

[0080] In some embodiments of any aspect, the agent for treating a neurological disease or disorder is or comprises an inhibitory nucleic acid. In some embodiments of any aspect, the inhibitor of expression of a given gene may be an inhibitory nucleic acid. As used herein, “inhibitory nucleic acid” refers to a nucleic acid molecule that can inhibit the expression of a target, such as double-stranded RNA (dsRNA) or inhibitory RNA (iRNA).

[0081] Double-stranded RNA molecules (dsRNAs) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein may include RNA strands (antisense strands) having a region of 30 nucleotides or less, i.e., 15–30 nucleotides, generally 19–24 nucleotides, which is substantially complementary to the at least partially targeted mRNA transcript. The use of these iRNAs enables targeted degradation of the mRNA transcript, resulting in a reduction of the target's expression and / or activity.

[0082] As used herein, the term “iRNA” refers to an agent containing RNA (or a modified nucleic acid as described below herein) that mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of any aspect, the iRNAs described herein result in inhibition of the expression and / or activity of a target. In some embodiments of any aspect, contact with the inhibitor (e.g., iRNA) results in a reduction of the target mRNA level in the cell to a maximum of 100%, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, and 100% of the target mRNA level found in cells without the iRNA. In some embodiments of the model, administration of an inhibitor (e.g., iRNA) to a subject can result in a reduction of the target mRNA level in the subject by up to 100%, including at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, and 100% of the target mRNA level observed in the subject where the iRNA is absent.

[0083] In some embodiments of any aspect, the iRNA may be a dsRNA. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementarity region that is substantially complementary to, and generally fully complementary to, the target sequence. The target sequence may originate from the sequence of mRNA formed during the expression of the target, for example, this may extend to one or more intron boundaries. The other strand (the sense strand) contains a region that is complementary to the antisense strand so that the two strands hybridize and combine under favorable conditions to form a double-stranded structure. Generally, the double-stranded structure is inclusively between 15 and 30 base pairs in length, more generally between 18 and 25 base pairs, even more generally between 19 and 24 base pairs, and most generally between 19 and 21 base pairs. Similarly, the region complementary to the target sequence is inclusively between 15 and 30 base pairs in length, more generally between 18 and 25 base pairs, even more generally between 19 and 24 base pairs, and most commonly between 19 and 21 base pairs in length. In some embodiments of any of the embodiments, the dsRNA is inclusively between 15 and 20 nucleotides in length, and in other embodiments, the dsRNA is inclusively between 25 and 30 nucleotides in length. As those skilled in the art will recognize, the targeted region of the RNA targeted for cleavage is in most cases a larger RNA molecule, often a part of an mRNA molecule. Where applicable, the “part” of the mRNA target is a continuous sequence of mRNA targets of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNAs having double helix as short as about 9 base pairs can, under certain circumstances, mediate RNAi-directed cleavage. In most cases, the target is at least 15 nucleotides in length, preferably 15 to 30 nucleotides. Exemplary embodiments of the types of inhibitory nucleic acids include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art.

[0084] In some embodiments of any aspect, the inhibitory RNA is a miRNA. MicroRNAs (miRNAs) are small RNAs of 17-25 nucleotides that function as regulators of gene expression in eukaryotes. "MicroRNA" or "miRNA" is a small, non-coding RNA molecule that can mediate transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed as hairpin or stem-loop (e.g., having a self-complementary single-stranded skeleton) double-stranded structures, referred to as primary miRNAs (pri-miRNAs), which are enzymatically processed into pre-miRNAs (e.g., by Drosha, DGCR8, Pasha, etc.). The double-stranded structure includes a) a first RNA sequence in a complementarity region that is substantially complementary to the target sequence and generally fully complementary, and b) a second RNA sequence region complementary to the first RNA sequence strand so that the two sequences hybridize and combine under favorable conditions to form a double-stranded structure. The target sequence may originate from the mRNA sequence formed during the expression of the target, for example, it may extend to one or more intron boundaries. Generally, the double-stranded structure is inclusively between 15 and 30 base pairs in length, more generally between 18 and 25 base pairs, even more generally between 19 and 24 base pairs, and most commonly between 19 and 21 base pairs.

[0085] miRNAs are initially expressed in the nucleus as part of a longer primary transcript called primary miRNA (pri-miRNA). The length of pri-miRNA can vary. In some embodiments, pri-miRNAs are in the range of about 100 to about 5000 base pairs in length (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base pairs). In some embodiments, pri-miRNAs are longer than 200 base pairs (e.g., 2500, 5000, 7000, 9000, or longer).

[0086] Inside the nucleus, pri-miRNA is partially digested by the enzyme Drosha to form hairpin precursor miRNAs (pre-miRNAs) 65–120 nucleotides long, which are then transported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs, which are the active molecules. In animals, these shorter RNAs contain a 5' proximal "seed" region (2–8 nucleotides) which is thought to be the primary determinant of pair formation specificity for the 3' untranslated region (3'-UTR) of the miRNA's target mRNA. Pre-miRNAs, also characterized by a hairpin or stem-loop double-stranded structure, can also vary in length. In some embodiments, pre-miRNAs range in size from about 40 base pairs to about 500 base pairs in length. In some embodiments, pre-miRNAs range in size from about 50 to 100 base pairs in length. In some embodiments, the pre-miRNA is in a size range of approximately 50 to approximately 90 base pairs in length (e.g., approximately 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or approximately 90 base pairs).

[0087] Generally, pre-miRNAs are transported to the cytoplasm and enzymatically processed by Dicer, initially forming incomplete miRNAs / miRNAs. * Double-stranded, and then single-stranded, mature miRNA molecules are generated, which are then loaded into an 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 this disclosure contain sequences encoding pri-miRNA, pre-miRNA, or mature miRNA, including sequences shown in any one of SEQ ID NOs: 6–17, 40–44, or 50–66.

[0088] In the context of the present invention, a miRNA molecule or its equivalent or mimetic, or isomiR, may be a synthetic or natural or recombinant or mature miRNA or human miRNA or a part of a mature or human miRNA, or may be derived from a human miRNA as further defined in the part that provides the general definition. A human miRNA molecule is a miRNA molecule found in human cells, tissues, organs or bodily fluids (i.e., an endogenous human miRNA molecule). A human miRNA molecule may also be a human miRNA molecule derived from an endogenous human miRNA molecule by nucleotide substitution, deletion and / or addition. A miRNA molecule, or its equivalent or mimetic, may be a single-stranded or double-stranded RNA molecule. Preferably, the miRNA molecule, or its equivalent or mimetic, is 6 to 30 nucleotides long, preferably 12 to 30 nucleotides long, preferably 15 to 28 nucleotides long, and more preferably, the molecule has at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides long, or longer.

[0089] In a preferred embodiment, the miRNA molecule, or its equivalent, mime, or isomiR, contains at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule, or its equivalent, mime, or isomiR. Preferably, in this embodiment, the miRNA molecule, or its equivalent, mime, or isomiR, is 6 to 30 nucleotides long, and more preferably contains at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule or its equivalent. Even more preferably, the miRNA molecule, or its equivalent, mime, or isomiR, is 15 to 28 nucleotides long, and more preferably contains at least 6 of the 7 nucleotides present in the seed sequence, and even more preferably, the miRNA molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, or longer.

[0090] Therefore, a preferred miRNA molecule, or its equivalent or mime or isomiR, comprises at least six of the seven nucleotides present in sequence numbers 6-17, 40-44, or 50-66 or in the seed sequences identified therein, and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, or longer.

[0091] Delivery vehicles for miRNA include, but are not limited to, liposomes, polymer nanoparticles, viral systems, lipid or receptor-binding molecule conjugations, exosomes, and bacteriophages. See, for example, Baumann and Winkler, miRNA-based therapies: Strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents, Future Med Chem. 2014, 6(17): 1967-1984; U.S. Patent Nos. 8,900,627; 9,421,173; 9,555,060; and WO2019 / 177550, the contents of which are incorporated herein by reference in their entirety.

[0092] The microRNA sequence includes a “seed” region, i.e., the sequence of the region between positions 2 and 8 of the mature microRNA, which has complete Watson-Crick complementarity to the miRNA target sequence of the nucleic acid. In one embodiment, the viral genome may be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0093] The term substantial complementarity means that it is not necessary to have first and second RNA sequences that are completely complementary, or to have a first RNA sequence and a reference or target sequence (e.g., SEQ ID NO: 3 or 4) that are completely complementary. In one embodiment, substantial complementarity between an RNA sequence and a target consists of having no mismatches, having one mismatched nucleotide, or having two mismatched nucleotides. One mismatched nucleotide is understood to mean that one nucleotide does not base-pair with the target over the entire length of the RNA sequence that would base-pair with the target. No mismatches means that all nucleotides base-pair with the target, and two mismatches means that two nucleotides do not base-pair with the target.

[0094] miRNAs and / or transgenes containing one or more miRNAs may be provided in or include a scaffold sequence. As used herein, “scaffold” refers to the portion of the miRNA coding sequence outside the mature double-stranded structure. For example, a scaffold may include loops and / or stem regions. Thus, a scaffold is useful for generating, coding, and / or expressing the miRNAs described herein. The scaffolds used in the compositions and methods described herein may be, obtain, and / or be derived from endogenous and / or naturally occurring miRNA scaffolds, e.g., sequences of human miRNAs. In some embodiments, the scaffold sequences obtained for use in the compositions and methods described herein may be, obtain, and / or be derived from endogenous and / or naturally occurring miRNA scaffold sequences of miRNAs overexpressed in one or more NS and / or CNS diseases. nucleic acid

[0095] In some embodiments, the Disclosure provides isolated nucleic acids that are useful for reducing (e.g., inhibiting) the expression of a pathogenic gene (e.g., HTT) and / or encoding CYP46A1. “Nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of the Disclosure are isolated. As used herein, “isolated” means artificially produced. As used herein with respect to nucleic acids, “isolated” means (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, such as by cleavage and gel separation, or (iv) synthesized, e.g., by chemical synthesis. Isolated nucleic acids are readily manipulable by recombinant DNA techniques well known in the Art. Therefore, nucleotide sequences contained in vectors where the 5' and 3' restriction sites are known or polymerase chain reaction (PCR) primer sequences for them are disclosed are considered isolated, but nucleic acid sequences present in their native state in their natural host are not. Isolated nucleic acids may be substantially purified, but this is not required. For example, nucleic acids isolated within a cloning or expression vector may not be pure in the sense that they contain only a small percentage of the substance in the cell in which they exist. However, such nucleic acids are isolated, as the term is used herein because it is readily manipulable by standard techniques known to those skilled in the art. As used herein with respect to proteins or peptides, the term “isolated” refers to proteins or peptides that have been isolated from their natural environment or that have been artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0096] Those skilled in the art will also recognize that conservative amino acid substitutions may be made to provide a functionally equivalent variant or homolog of a capsid protein. In some embodiments, this disclosure encompasses sequence modifications resulting 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 on which the substitution is made. Variants are made according to methods for modifying polypeptide sequences known to those skilled in the art, e.g., references compiling such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. It can be prepared by: (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. Conservative amino acid substitutions can be made to the amino acid sequences of proteins and polypeptides disclosed herein.

[0097] 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 described herein includes a region (e.g., a first region) containing 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 in a capsid protein and administered to a subject and / or delivered to selected target cells. A “recombinant AAV (rAAV) vector” typically consists of a minimal transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may include one or more regions encoding one or more inhibitory RNAs (e.g., miRNAs) containing nucleic acids that target the endogenous mRNA of a subject, as disclosed elsewhere in this specification. The transgene may also include, as described elsewhere in this disclosure, a protein-coding region and / or an expression regulatory sequence (e.g., a poly-A tail).

[0098] Generally, ITR sequences are approximately 145 bp in length. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, but minor modifications of these sequences to some degree are permissible. The ability to modify these ITR sequences is within the scope of the skills of the art (e.g., Sambrook et al., "Molecular Cloning. A Laboratory Manual"). See also texts such as 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996). An example of such a molecule used in the invention is a “cis-acting” plasmid containing a transgene, where the selected transgene sequence and associated regulatory elements are adjacent to the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including the mammalian AAV types identified by the invention. In some embodiments, the isolated nucleic acid (e.g., rAAV vector) contains at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and their variants. In some embodiments, the isolated nucleic acid contains a region encoding the AAV2 ITR (e.g., a first region).

[0099] In some embodiments, the isolated nucleic acid further comprises regions containing a second AAV ITR (e.g., a second region, a third region, a fourth region, etc.). In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11, and their variants. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal segregation site (TRS). The term "lacking a terminal segregation site" may refer to an AAV ITR containing a mutation that disables the function of the terminal segregation site (TRS) of the ITR (e.g., a sense mutation such as a non-synonymous mutation, or a missense mutation), or a truncated AAV ITR lacking a nucleic acid sequence encoding a functional TRS (e.g., an ATRS ITR). While I do not wish to be bound to any particular theory, rAAV vectors containing ITRs lacking functional TRS are, for example, described in McCarthy (2008) Molecular Therapy 16(10): This invention generates self-complementary rAAV vectors as described by 1648-1656. In some embodiments of any of the shown models, at least one or more ITRs are less than 145 bp in length, for example, 130 bp in length.

[0100] In addition to the key elements identified above for recombinant AAV vectors, the vector also includes conventional regulatory elements operably ligated to the elements of the transgene in a manner that enables its transcription, translation, and / or expression in cells transfected with the vector or infected with a virus produced by the Invention. As used herein, “operably ligated” sequences include both expression regulatory sequences that are contiguous with the gene of interest and expression regulatory sequences that act trans or remotely to control the gene of interest. Expression regulatory sequences include appropriate transcription start sequences, stop sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of the encoded product. Several expression regulatory sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known and available in the Art.

[0101] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be operably ligated if they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. If it is desirable that the nucleic acid sequences be translated into functional proteins, two DNA sequences are said to be operably ligated if the induction of a promoter in the 5' regulatory sequence results in the transcription of the coding sequence, and the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the promoter region's ability to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably ligated to a nucleic acid sequence if the promoter region can result in the transcription of its DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operably ligated if they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that are translated in frame. In some embodiments, the operably linked coding sequence results in a fusion protein. In some embodiments, the operably linked coding sequence results in a functional RNA (e.g., miRNA).

[0102] In some embodiments, the disclosure provides isolated nucleic acids comprising a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more microRNAs (e.g., miRNAs).

[0103] It should be recognized that, in some embodiments, an isolated nucleic acid or vector (e.g., an rAAV vector) contains nucleic acid sequences encoding two or more (e.g., multiple, e.g., 2, 3, 4, 5, 10, or more) miRNAs. In some embodiments, each of the two or more miRNAs targets the same target gene (e.g., if each miRNA targets the HTT gene, then the isolated nucleic acid encoding three unique miRNAs) (e.g., hybridizes to it or specifically binds to it). In some embodiments, each of the two or more miRNAs targets a different target gene (e.g., hybridizes to it or specifically binds to it).

[0104] In some embodiments, the disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) encoding one or more artificial miRNAs. Where used herein, “artificial miRNA” or “amiRNA” means, for example, Eamens et al. (2014). As described in Methods Mol. Biol. 1062:211-224, miRNA and miRNA * The sequence (for example, the passenger strand of a miRNA double helix) directs the highly efficient RNA silencing of the target gene, corresponding to the amiRNA / amiRNA sequence. * This refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA skeleton that is a precursor miRNA capable of generating a functionally mature miRNA) that has been replaced by a sequence. For example, in some embodiments, the artificial miRNA includes a miR-155 pri-miRNA skeleton in which a sequence encoding a mature HTT-specific miRNA (e.g., any one of sequence numbers 6-17, 40-44, or 50-66) is inserted in place of the endogenous miR-155 mature miRNA coding sequence. In some embodiments, the miRNA described herein (e.g., the artificial miRNA) includes a miR-155 skeleton sequence, a miR-30 skeleton sequence, a miR-64 skeleton sequence, or a miR-122 skeleton sequence.

[0105] The region containing the transgene (e.g., the second region, third region, fourth region, etc.) may be placed at any suitable location on the isolated nucleic acid. The region may be placed at any untranslated portion of the nucleic acid, including, for example, an intron, or a 5' or 3' untranslated region.

[0106] In some cases, it may be desirable to position the regions (e.g., the second, third, and fourth regions) upstream of the first codon of the protein-coding nucleic acid sequence (e.g., the protein-coding sequence). For example, the region may be positioned between the first codon of the protein-coding sequence and 2000 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein-coding sequence and 1000 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein-coding sequence and 500 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein-coding sequence and 250 nucleotides upstream of the first codon. The region may be positioned between the first codon of the protein-coding sequence and 150 nucleotides upstream of the first codon. In some cases (e.g., when the transgene lacks a protein-coding sequence), it may be desirable to position the regions (e.g., the second, third, and fourth regions) upstream of the poly-A tail of the transgene. For example, the region may be located between the first base of the polyA tail and 2000 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 1000 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 500 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 250 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 150 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 100 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 50 nucleotides upstream of the first base. The region may be located between the first base of the polyA tail and 20 nucleotides upstream of the first base. In some embodiments, the region is located between the last nucleotide of the promoter sequence and the first nucleotide of the polyA tail sequence.

[0107] In some cases, the region may be located downstream of the last base of the polyA tail of the transgene. The region may be located between the last base of the polyA tail and 2000 nucleotides downstream of the last base. The region may be located between the last base of the polyA tail and 1000 nucleotides downstream of the last base. The region may be located between the last base of the polyA tail and 500 nucleotides downstream of the last base. The region may be located between the last base of the polyA tail and 250 nucleotides downstream of the last base. The region may be located between the last base of the polyA tail and 150 nucleotides downstream of the last base.

[0108] It should be recognized that when a transgene codes for two or more miRNAs, each miRNA may be placed in any suitable location within the transgene. For example, the nucleic acid encoding the first miRNA may be placed in an intron of the transgene, and the nucleic acid sequence encoding the second miRNA may be placed in another uncoding region (e.g., between the last codon of the protein-coding sequence and the first base of the polyA tail of the transgene).

[0109] In some embodiments, the transgene further comprises a nucleic acid sequence encoding one or more regulatory sequences (e.g., promoters). Regulatory sequences include appropriate transcription start sequences, stop sequences, promoter sequences and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of the encoded product. Numerous regulatory sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known and available in the art.

[0110] A "promoter" refers to a DNA sequence that is recognized by or introduced into a cell's synthetic mechanism, which is necessary to initiate the specific transcription of a gene. The terms "operatably positioned," "controlled," or "transcriptionally regulated" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and gene expression.

[0111] For protein-coding nucleic acids, polyadenylated sequences are generally inserted after the transgene sequence and before the 3'AAV ITR sequence. Useful rAAV constructs in this disclosure may also contain introns, which are 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 intra-sequence ribosome entry site (IRES). IRES sequences are used to generate two or more polypeptides from a single gene transcript. IRES sequences would be used to generate proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional, and many such sequences are available [e.g., Sambrook et al., and, for example, sections 3.18, 3.26, and 16.17]. References cited on pages 16 and 27, as well as Ausubel et al., Current Protocols in See Molecular Biology, John Wiley & Sons, New York, 1989. In some embodiments, the foot-and-mouth disease virus 2A sequence is contained within a polyprotein, which 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.). al., J Virology, November 1996; p. 8124-8127;Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, M). D 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; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11 : 1921- 1931.; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0112] Examples of constitutive promoters include, but are not limited to, the retroviral Roussarcoma virus (RSV) LTR promoter (with RSV enhancer as needed), the cytomegalovirus (CMV) promoter (with CMV enhancer as needed) [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 EF1a promoter [Invitrogen]. In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is the U6 promoter.

[0113] Inducible promoters allow for the regulation of gene expression, which can be regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as the presence of an acute phase, a specific differentiation state of the cell, or only in replicating cells. Inducible promoters and inducible systems are available from various commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)); and the tetracycline inhibitory system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline-inducible systems (Gossen et al., Science, 268: 1766-1769 (1995), and Harvey et al., Curr. Opin. Chem. Biol., See also 2:512-518 (1998), RU486-derived system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) ) and rapamycin-derived systems (Magari et al., J. Clin. Invest., 100:2865-2872) (1997)) is one example. Further types of inductive promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation states of cells, or only in replicating cells.

[0114] In another embodiment, a native promoter is used for the transgene. A native promoter may be preferred when it is desired that the expression of the transgene should mimic native expression. A native promoter may be used when the expression of the transgene must be regulated temporally, developmentally, in a tissue-specific manner, or in response to a specific transcriptional stimulus. In further embodiments, other native expression regulatory elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression. In some embodiments, regulatory sequences confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters, as will be apparent to those skilled in the art, 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); and the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24: 185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11: 654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161: 1063-8 (1998)); immunoglobulin heavy chain promoter; T Neuronal promoters such as cell receptor α-chain promoters and neuron-specific enolase (NSE) promoters (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), and neuronal filament light chain gene promoters (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991), and neuron-specific VGF genes Examples include the gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). Other NS-specific promoters intended for use in the Method and Composition are those described in Japanese Patent Application No. GB2013940.8, filed September 4, 2020, and No. GB2005732.9, filed April 20, 2020, which are incorporated herein by reference in their entirety. In some embodiments, the NS-specific promoter is a promoter of Table 10, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters of Table 10. In some embodiments, the NS-specific promoter is a promoter of Table 10, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters of Table 10, and retaining the NS-specific promoter activity of the promoters of Table 10.

[0115] Examples of CNS-specific promoters intended for use in the Method and Composition include those described in International Patent Application PCT / GB2021 / 050939, filed on April 19, 2021, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the CNS-specific promoter is a promoter of the promoters in Tables 11-13, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters in Tables 11-13. In some embodiments, the CNS-specific promoter is a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters in Tables 11-13 and retaining the CNS-specific promoter activity of the promoters in Tables 11-13.

[0116] In some embodiments, the nucleic acid comprises one or more CREs. In some embodiments, the nucleic acid comprises one or more NS-specific CREs or CNS-specific CREs. In some embodiments, the nucleic acid comprises one or more CREs from Tables 13-15, or CREs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the CREs from Tables 13-15. In some embodiments, the CRE is a CRE that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the CREs from Tables 13-15 and retains the activity of the CREs from Tables 13-15.

[0117] In some embodiments, the CRE may include one or more CREs known in the Art. For example, in one embodiment, one or more CREs may be selected from Sequence IDs 19-24, 27, 28, 37, and 38 of Japanese Patent Application GB2013940.8, filed on 4 September 2020. For example, in one embodiment, one or more CREs may be selected from Sequence IDs 1-8 of WO2019 / 199867A1, Sequence IDs 1-7 of WO2020 / 076614A1, and Sequence IDs 25-51, 177-178, and 188 of WO2020 / 097121. The aforementioned references are incorporated herein by reference in their entirety.

[0118] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8]

[0119] [Table 11-1] [Table 11-2] [Table 11-3] Table 11-4 Table 11-5 Table 11-6

[0120] Table 12

[0121] Table 13

[0122] Table 14-1 Table 14-2

[0123] Table 15-1 Table 15-2

[0124] Aspects of this disclosure relate to isolated nucleic acids comprising two or more promoters (e.g., 2, 3, 4, 5, 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., miRNA), it may be desirable to use a first promoter sequence (e.g., a first promoter sequence operably ligated to the protein-coding region) to drive the expression of the protein-coding region, and to use a second promoter sequence (e.g., a second promoter sequence operably ligated to the inhibitory RNA-coding region) to drive the expression of the inhibitory RNA-coding region. Generally, the first and second promoter sequences may be the same or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter driving the expression of the protein-coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include the U6 and HI promoter sequences. In some embodiments, the second promoter sequence (e.g., a promoter sequence that drives the expression of inhibitory RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of polII promoter sequences include the T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the polIII promoter sequence drives the expression of an inhibitory RNA (e.g., miRNA) coding region. In some embodiments, the polII promoter sequence drives the expression of a protein-coding region.

[0125] In some embodiments, the nucleic acid includes a transgene encoding a protein. The protein may be a therapeutic protein (e.g., a peptide, protein, or polypeptide useful for treating or preventing a disease condition in a mammalian subject) or a reporter protein. In some embodiments, the protein is CYP46A1. In some embodiments, the protein is human CYP46A1. In some embodiments, the protein encodes SEQ ID NO: 2, or a protein containing SEQ ID NO: 2. In some embodiments, the protein encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the therapeutic protein is useful for treating or preventing Huntington's disease and is, for example, polyglutamine-binding peptide 1( described in Marelli et al. (2016) Orphanet Journal of Rare Disease 11:24; doi: 10.1186 / s l3023-016-0405-3). These include QBP1), PTD-QBP1, ED11, C4 intrabody, VL12.3 intrabody, MW7 intrabody, Happ1 antibody, Happ3 antibody, mM48 intrabody, certain monoclonal antibodies (e.g., 1C2) and peptide P42, as well as their variants. In some embodiments, the therapeutic protein is wild-type huntingtin protein (e.g., huntingtin protein having a PolyQ repeat region containing fewer than 36 repeats). CYP46A1

[0126] Cholesterol 24-hydroxylase is a neuronal enzyme encoded by the CYP46A1 gene. It converts cholesterol to 24-hydroxycholesterol and plays a crucial role in cholesterol efflux from the brain (Dietschy, JM). (et al., 2004). Brain cholesterol is essentially produced in situ but cannot be broken down, and the intact blood-brain barrier restricts the direct transport of cholesterol from the brain (Dietschy, JM et al., 2004). 24-Hydroxycholesterol is located in the plasma membrane and It can cross the blood-brain barrier and reach the liver where it is broken down.

[0127] CYP46A1 is neuroprotective in cellular models of HD (see, for example, WO2012 / 049314). Furthermore, a reduction in CYP46A1 mRNA is observed in the striatum, a more vulnerable brain structure in the R6 / 2 transgenic HD mouse model.

[0128] During the early stages of AD, 24-hydroxycholesterol concentrations are high in the CSF and peripheral circulation. In the later stages of AD, 24-hydroxycholesterol concentrations may decrease, reflecting neuronal loss (Kolsch, H. et al., 2004). CYP46A1 is expressed around the amyloid core of senile plaques in the brains of AD patients. (Brown, J., 3rd et al., 2004).

[0129] Agonism of cholesterol 24-hydroxylase encoded by CYP46A1 has provided a significant reduction in neuropathology and improvement in cognitive impairment in mouse models of CNS diseases. For example, co-expression of CYP46A1 with ExpHtt in a Huntington's disease model promoted a strong and significant reduction in ExpHtt aggregate formation (58% vs. 27.5%) (WO2012 / 049314) (see also International Patent Publication WO2009 / 034127, which is incorporated herein by reference in its entirety). The methods described herein relate to agonism of CYP46A1 in combination with the administration of miRNAs targeting certain other targets. For example, the methods may relate to the administration of a viral vector for the treatment of a neurological disease or disorder, where the vector expresses CYP46A1 in cells of the central nervous system.

[0130] In some embodiments, a viral vector for treating a neurological disease or disorder is described herein, wherein the vector comprises a nucleic acid encoding cholesterol 24-hydroxylase. In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2. In some embodiments, the viral vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with respect to SEQ ID NO: 2. In some embodiments, the viral vector comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with respect to SEQ ID NO: 1. In some embodiments, the viral vector comprises the sequence of SEQ ID NO: 1. In some embodiments, the viral vector may be an adeno-associated virus (AAV) vector.

[0131] Further descriptions of CYP46A1 and its therapeutic uses (e.g., for Alzheimer's disease, ALS, and ataxia) are described in the Art, for example, in WO2012 / 049314, WO2009 / 034127, WO2018 / 138371, and WO2020 / 089154. The sequences, methods, and compositions described therein can be used in the methods and compositions described herein. The aforementioned references are incorporated herein by reference in their entirety. The term “gene” means a polynucleotide containing at least one open reading frame that can encode a particular polypeptide or protein after transcription or translation.

[0132] The term "coding sequence" or "sequence that codes for a specific protein" refers to a nucleic acid sequence that, when controlled by appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into polypeptides in vitro or in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coded sequences may include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.

[0133] The cDNA sequence for CYP46A1 is disclosed in Genbank access number NM_006668 (SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2. The present invention relates to the use of nucleic acid constructs comprising the sequence of SEQ ID NO: 1 or a variant thereof for the treatment of neurological diseases or disorders. Variants include, for example, naturally occurring variants resulting from inter-individual allele mutations (e.g., polymorphisms), alternative splicing forms, etc. The term variant also includes CYP46A1 gene sequences of other origins or from other organisms. The variant is preferably substantially homologous to SEQ ID NO: 1 and / or 2, i.e., typically exhibits at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95% nucleotide sequence identity with SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid construct has at least 95% sequence identity with SEQ ID NO: 1 and includes a sequence that retains the activity of SEQ ID NO: 1 or 2 (e.g., the ability to convert cholesterol to 24-hydroxycholesterol). Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the sequence (or its complementary strand) defined above under stringent hybridization conditions. Typical stringent hybridization conditions include a temperature above 30°C, preferably above 35°C, more preferably above 42°C, and / or a salinity of less than about 500 mM, preferably less than 200 mM. Hybridization conditions can be adjusted by those skilled in the art by modifying the temperature, salinity, and / or the concentrations of other reagents such as SDS, SSC, etc.

[0134] Exemplary CYP46A1 variants intended for use herein are provided in SEQ ID NOs: 109 and 110. In some embodiments, the viral vector includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 109. In some embodiments, the viral vector includes a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with respect to SEQ ID NO: 109. In some embodiments, the viral vector includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with respect to the sequence of SEQ ID NO: 110.

[0135] In one embodiment, a composition is provided herein comprising an isolated nucleic acid having a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 110. In one embodiment, a composition is provided herein comprising a recombinant viral vector having an isolated nucleic acid having a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 110. In some embodiments, the isolated nucleic acid encoding the CYP46A1 protein has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 mutations compared to SEQ ID NO: 1. In some embodiments, the mutations have at least one nucleic acid deletion and / or addition and / or substitution compared to the sequence shown in SEQ ID NO: 1. Mutations may result in, for example, the removal of bacterial sequences and / or alternative leading frames and / or CpGs and / or restriction enzyme sites. In some embodiments, the aforementioned compositions can be used, for example, in the absence of administered miRNAs for treating neurological diseases or disorders as described herein. In various embodiments, the aforementioned compositions can be used, for example, in the presence of administered miRNAs for treating neurological diseases or disorders as described herein. In some embodiments, a recombinant viral vector containing the isolated nucleic acid shown in SEQ ID NO: 110, for example recombinant AAV, is administered to a subject requiring treatment in order to express the CYP46A1 protein and / or to treat a neurological disease or disorder as described herein.In some embodiments, a recombinant viral vector, for example recombinant AAV, containing an isolated nucleic acid sequence that is at least 80% identical to SEQ ID NO: 110, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical, is administered to a subject in need of treatment to express the CYP46A1 protein and / or to treat a neurological disorder or disability as described herein. In some embodiments, a recombinant viral vector, for example recombinant AAV, containing an isolated nucleic acid sequence that is at least 80% identical to SEQ ID NO: 111, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical, is administered to a subject in need of treatment to express the CYP46A1 protein and / or to treat a neurological disorder or disability as described herein.

[0136] [ka] [ka]

[0137] [ka] [ka] [ka] vector

[0138] While we do not wish to be bound by any particular theory, allele-specific silencing of a pathogenic gene, e.g., mutant huntingtin (HTT), may provide an improved safety profile in a subject compared to non-allele-specific silencing (e.g., silencing both wild-type and mutant HTT alleles), since wild-type expression and function are conserved in the cell. For example, aspects of the present invention relate to our recognition and understanding that isolated nucleic acids and vectors incorporating one or more inhibitory RNA (e.g., miRNA) sequences that target the HTT gene in a non-allele-specific manner while driving the expression of a hardened wild-type HTT gene (a wild-type HTT gene not targeted by miRNA) can achieve, for example, concomitant mutant HTT knockdown in CNS tissues having increased wild-type HTT expression. Generally, the nucleic acid sequences of endogenous wild-type and mutant HTT mRNA, as well as the nucleic acid sequences of the transgene encoding "hardened" wild-type HTT mRNA, are sufficiently different that the mRNA of the "hardened" wild-type HTT transgene 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, resulting in a gene that encodes the same protein as the endogenous wild-type HTT gene but has a different nucleic acid sequence. In this case, the exogenous mRNA can be referred to as "hardened." Alternatively, inhibitory RNAs (e.g., miRNAs) can target the 5' and / or 3' untranslated regions of the endogenous wild-type HTT mRNA. These 5' and / or 3' regions can then be removed or replaced in the transgene mRNA so that the transgene mRNA is not targeted by one or more inhibitory RNAs.

[0139] Reporter sequences that may be provided in a transgene (e.g., nucleic acid sequences encoding reporter proteins) 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 well known in the art. When related to regulatory elements that drive their expression, the reporter sequence provides a signal detectable by conventional means including enzymatic assays, radiographic assays, colorimetric assays, fluorescence assays or other spectroscopic assays, fluorescently labeled cell sorting assays, and immunoassays including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemical tests. For example, if the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by an assay for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-carrying vector may be visually measured by the generation of color or light in an luminescence photometer. Such reporters may be useful, for example, for verifying the tissue-specific targeting ability of nucleic acids and their tissue-specific promoter-modulating activity. Recombinant adeno-associated virus (rAAV).

[0140] In some embodiments, the vector is adeno-associated virus (AAV) or recombinant AAV. In some embodiments, this disclosure provides isolated AAV. As used herein with respect to AAV, the term “isolated” refers to artificially generated or obtained AAV. Isolated AAV may be generated using a recombinant method. Such AAV is referred to herein as “recombinant AAV.” Recombinant AAV (rAAV) preferably has tissue-specific targeting ability, so that the nuclease and / or transgene of the rAAV is delivered specifically to one or more given tissues. The AAV capsid is an important element in determining these tissue-specific targeting abilities. Therefore, it is possible to select an rAAV that has a capsid suitable for targeting a particular tissue.

[0141] Methods for obtaining recombinant AAV having a desired capsid protein are well known in the art (see, for example, US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the method involves culturing a host cell containing a nucleic acid sequence encoding the AAV capsid protein; a functional rep gene; a recombinant AAV vector comprising an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper function to enable packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. AAV comprises three capsid proteins, virion proteins 1-3 (named 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, during translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid protein are to protect the viral genome, deliver the genome, and interact with the host. In some embodiments, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0142] In some embodiments, recombinant AAV (rAAV) comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV 2G9, AAV 2.5G9, AAV9, and AAV10. In some embodiments, the recombinant AAV capsid (rAAV) protein is of a serotype derived from a non-human primate, for example, the AAVrh10 serotype. In some embodiments, rAAV is AAV PhP.eB or AAV PhP.B, as described in U.S. Publications and U.S. Registered Patents US20170166926A1, US9585971, US10301360, US9957303, US10202425, US10519198, US20190292230A1, and US20200087353A1, which are incorporated herein by whole reference. In some embodiments, rAAV includes an AAV comprising surface-bound peptides, such as PB5-3, PB5-5, and PB5-14, as described in International Publication WO201912635, which is incorporated herein by whole reference. In some embodiments, rAAV is the AAV9 serotype. In some embodiments, rAAV is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 serotype, or a chimera thereof. In some embodiments, rAAV comprises a capsid protein derived from serotype AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 2G9, AAV 2.5G9, AAV rh8, AAV rh10, AAV rh74, AAV10, or AAV11, or a chimera thereof. In certain embodiments, rAAV comprises a chemically modified capsid disclosed in WO2017 / 212019, for example, in which a mannose ligand is chemically coupled to AAV2. The rAAV having a chemically modified capsid disclosed in WO2017 / 212019 is incorporated herein by reference in its entirety.In further embodiments, rAAVs include AAV capsid proteins of the present invention that may be polyploid (also referred to as singular, rational singular, or rational polyploid) in that they may contain VP1, VP2, and VP3 capsid proteins derived from two or more AAV serotypes in a single AAV virion as described in PCT / US18 / 22725, PCT / US2018 / 044632, or US10,550,405, which are incorporated by reference. In some embodiments, rAAVs include capsid proteins selected from the AAV serotypes listed in Table 17.

[0143] [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6] [Table 17-7] [Table 17-8] [Table 17-9] [Table 17-10] [Table 17-11] [Table 17-12] Table 17-13 Table 17-14 Table 17-15 Table 17-16 Table 17-17

[0144] The components cultured in host cells for packaging the rAAV vector within the AAV capsid may be provided to the host cells in trans. Alternatively, one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper function) 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 contains the required components under the control of an inductive promoter. However, the required components may also be under the control of a constitutive promoter. Examples of suitable inductive and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, a selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inductive promoters. For example, a stable host cell can be created that originates from 293 cells (containing El helper function under the control of a constitutive promoter) but contains rep and / or cap proteins under the control of an inductive promoter. Yet another stable host cell can be created by those skilled in the art. In some embodiments, the disclosure relates to host cells containing nucleic acids comprising a coding sequence encoding a protein (e.g., wild-type huntingtin protein, optionally, “hardened” wild-type huntingtin protein). In some embodiments, the disclosure relates to compositions comprising the host cells described above. In some embodiments, the compositions comprising the host cells further comprise a cryopreservative.

[0145] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to generate the rAAV of this disclosure may be delivered to host cells to be packaged using any suitable genetic element (vector). The selected genetic element may be delivered by any preferred method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. For example, Sambrook et al. See, al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for preparing rAAV vilions are well known, and the selection of a preferred method is not an limitation of this disclosure. For example, see K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,7 Please refer to issue 45.

[0146] In some embodiments, recombinant AAV may be generated using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is generated by transfecting host cells with a recombinant AAV vector (containing the transgene), an AAV helper function vector, and an accessory function vector, which are packaged into AAV particles. The AAV helper function vector encodes “AAV helper function” sequences (i.e., rep and cap), which function in trans for generative AAV replication and capsid formation. Preferably, the AAV helper function vector supports efficient AAV vector generation without creating any detectable wild-type AAV virions (i.e., AAV virions containing the functional rep and cap genes). Non-limiting examples of vectors suitable for use according to this disclosure include the pHLP19 vector described in U.S. Patent No. 6,001,650 and the pRep6cap6 vector described in U.S. Patent No. 6,156,303, both of which are incorporated herein by reference in their entirety. Accessory function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (i.e., “accessory functions”) on which AAV replication depends. Accessory functions include, but are not limited to, those required for AAV replication, including regions involved in AAV gene transcription, step-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and activation of AAV capsid assembly. Virus-based accessory functions may originate from any known helper viruses, such as adenoviruses, herpesviruses (other than herpes simplex virus type 1), and vacciniaviruses.

[0147] In some embodiments, this disclosure provides transfected host cells. The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell is “transfected” when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are generally known in the art. For example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, See New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197. Using such techniques, one or more exogenous nucleic acids, such as nucleotide insertion vectors and other nucleic acid molecules, can be introduced into suitable host cells.

[0148] “Host cell” refers to any cell that harbors or is capable of harboring the substance of interest. Host cells are often mammalian cells. Host cells may be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNA associated with the production of recombinant AAV. The term includes offspring of the transfected original cell. Therefore, “host cell” may also refer to a cell transfected with an exogenous DNA sequence, as used herein. It is understood that offspring of a single parental cell may not necessarily be completely identical to the original parent in morphology or in terms of gene or DNA complement as a whole, due to natural, accidental, or planned mutations.

[0149] As used herein, the term “cell line” refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in the karyotype during the preservation or transfer of such a clonal population. Thus, cells derived from a cell line referred to may not be exactly identical to the ancestral cells or culture, and the cell line referred to may include such variants.

[0150] As used herein, the term “recombinant cell” refers to a cell into which an exogenous DNA segment, such as a DNA segment, has been introduced, resulting in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0151] As used herein, the term “vector” includes any genetic element, such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, etc., which are replicable when associated with appropriate regulatory elements and can transfer gene sequences between cells. Therefore, the term includes cloning and expression vehicles, as well as viral vectors. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which an additional DNA segment is ligated. Another type of vector is a viral vector into which an additional DNA segment is ligated to a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Also, certain vectors can direct the expression of genes into which they are functionally linked. Such vectors are referred to herein as “expression vectors.” Generally, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Hereinafter, “plasmid” and “vector” are used interchangeably, as plasmids are the most commonly used form of vectors. However, the present invention is intended to include other forms of expression vectors such as viral vectors that perform equivalent functions (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0152] Cloning vectors are those that can spontaneously replicate or be incorporated into the genome of a host cell, further characterized by one or more endonuclease restriction sites, which can be cleaved in a deterministic manner and ligated so that the desired DNA sequence retains its ability for the new recombinant vector to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur multiple times, as the plasmid increases its copy number within a host cell such as a host bacterium, or only once per host before the host regenerates through mitosis. In the case of phages, replication can occur actively during the lysis phase or passively during the lysogenous phase.

[0153] An expression vector is one in which a desired DNA sequence can be inserted by restriction and ligation, and as a result, it can be operably joined to a regulatory sequence and expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for use in the identification of cells, which may be transformed or transfected with the vector, or untransformed or untransfected. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes (e.g., β-galactosidase, luciferase, or alkaline phosphatase) whose activity is detectable by standard assays known in the art, and genes (e.g., green fluorescent protein) that visually affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques. In certain embodiments, the vectors used herein are capable of self-replication and expression of structural gene products present in the DNA segments to which they are operably joined.

[0154] In some embodiments, useful vectors are intended to be those vectors in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter. When it is desirable that the coding sequence be translated into a functional protein, the two DNA sequences are said to be operably conjugated if the induction of a promoter at the 5' regulatory sequence results in the transcription of the coding sequence, and the nature of the conjugation between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the promoter region's ability to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, the promoter region would be operably conjugated to the coding sequence if the promoter region can result in the transcription of its DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide.

[0155] A “promoter” refers to a DNA sequence that is recognized by or introduced into a cellular synthetic mechanism, which is necessary to initiate the specific transcription of a gene. When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, its expression can be directed using various transcriptional regulatory sequences (e.g., promoter / enhancer sequences). A promoter may be a native promoter, i.e., the promoter of the gene in its intrinsic context, which provides normal regulation of gene expression. In some embodiments, a promoter may be constitutive, i.e., the promoter is unregulated and allows for the continued transcription of the gene in question. Various conditional promoters, e.g., promoters controlled by the presence or absence of a molecule, may also be used.

[0156] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally, they may include, as needed, 5' non-transcriptional and 5' non-translating sequences involved in the initiation of transcription and translation, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5' non-transcriptional regulatory sequences include a promoter region containing a promoter sequence for transcriptional control of the operably conjugated gene. The regulatory sequences may also optionally include enhancer sequences or upstream activator sequences. The vectors of the present invention may optionally include 5' leader sequences or signal sequences. The selection and design of appropriate vectors are within the skill and discretion of those skilled in the art.

[0157] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. See reference. Cells are genetically engineered by introducing heterologous DNA (RNA) into them. This heterologous DNA (RNA) is placed under the activatable control of transcription elements that enable the expression of the heterologous DNA in the host cell.

[0158] The terms “operatably positioned,” “controlled,” or “transcriptionally controlled” mean that the promoter is in the correct position and orientation with respect to the nucleic acid to control RNA polymerase initiation and gene expression. The term “expression vector or construct” means any kind of gene construct containing nucleic acid that can transcribe part or all of a nucleic acid coding sequence. In some embodiments, expression involves, for example, the transcription of nucleic acid to produce a biologically active polypeptide product or functional RNA (e.g., guide RNA) from the transcribed gene.

[0159] The foregoing method for packaging a recombinant vector into a desired AAV capsid to generate the rAAV of this disclosure is not intended to limit, and other suitable methods will be apparent to those skilled in the art.

[0160] In some embodiments, one or more thymidine (T) nucleotides or uridine (U) nucleotides in any sequence provided herein, including the sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing with an adenosine nucleotide (e.g., via Watson-Crick type base pairing). For example, in some embodiments, one or more thymidine (T) nucleotides in any sequence provided herein, including the sequence provided in the sequence listing, may be suitably replaced with uridine (U) nucleotides, and vice versa.

[0161] In some embodiments of any of the aspects described herein, nucleic acids (e.g., miRNAs) are chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein are based on methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. These may be synthesized and / or modified by the following: Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, reverse linking, etc.), 3' terminal modifications (conjugation, DNA nucleotide, reverse linking, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that form base pairs with a wide repertoire of partners, base removal (baseless nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacements, and (d) skeleton modifications, including modifications or replacements of phosphodiester linkages. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids that contain a modified skeleton or do not contain natural internucleoside linkages. Nucleic acids having a modified skeleton include, in particular, those that do not have a phosphorus atom in the skeleton. For the purposes of this specification and as may be referenced in the art, modified nucleic acids that do not have a phosphorus atom in their internucleoside skeleton can also be considered as oligonucleosides. In some embodiments of any aspect, the modified nucleic acid has a phosphorus atom in its internucleoside skeleton.

[0162] Modified nucleic acid skeletons may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'-alkylenephosphonates and chiralphosphonates, phosphinates, phosphoramides including 3'-aminophosphoramides and aminoalkylphosphoramides, thionophosphoramides, thionoalkylphosphonates, thionoalkylphosphotriesters, as well as boranophosphates having the usual 3'-5' linkage, their 2'-5' linked analogues, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Modified nucleic acid skeletons that do not contain a phosphorus atom have skeletons formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatomic or heterocyclic nucleoside linkages. These include morpholino linkages (partially formed from the sugar portion of a nucleoside); siloxane skeletons; sulfide, sulfoxide, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; others having mixed N, O, S, and CH2 constituent parts, as well as hetero Examples include oligonucleotides having an atomic skeleton, and in particular those having --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [wherein the formula the native phosphodiester skeleton is represented as --O--P--O--CH2--].

[0163] In other nucleic acid mimetic compounds, both the sugar and nucleoside linkages of the nucleotide unit, i.e., the backbone, are replaced with novel groups. The base unit is maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, in particular, an aminoethylglycine backbone. The nucleic acid bases are retained and bond directly or indirectly to the aza nitrogen atom of the amide portion of the backbone.

[0164] Nucleic acids can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety contains an extra crosslink connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-end conformation. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol. Canc. Ther. 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0165] Modified nucleic acids may also contain one or more substituted sugar moieties. The nucleic acids described herein may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Exemplary preferred modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are 1 to about 10. In some embodiments of any aspect, the nucleic acid comprises at the 2' position one of the following: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of the nucleic acid, or group for improving the pharmacodynamic properties of the nucleic acid, and other substituents having similar properties. In some embodiments of the model, modifications include 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE as described in the following examples herein, and 2'dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as also described in the following examples herein.

[0166] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in nucleic acids, particularly at the 3' position of the sugar in the 3'-terminal nucleotide, or at the 5' position of the 2'-5' ligated dsRNA and the 5' position of the 5'-terminal nucleotide. Nucleic acids may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0167] Nucleic acids may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. Other synthetic and native nucleic acid bases may include (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine. Certain of these nucleic acid bases are particularly useful for increasing the binding affinity of inhibitory nucleic acids, which is a feature of the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitution has been shown to increase the double-strand stability of nucleic acids by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and more specifically, 2'- These are exemplary base substitutions when combined with O-methoxyethyl sugar modifications. In some embodiments of any of the models, the modified nucleic acid bases may include d5SICS and dNAM, which are non-exclusive examples of non-natural nucleic acid bases that can be used separately or together as base pairs (e.g., Leconte et. al. J. Am. Chem. Soc. 2008, 130, 7, 2336-2343; Malyshev et. al. PNAS. 2012. 109 (30) 12005-12010). (See [reference]). In some embodiments of any aspect, the oligonucleotide tag (e.g., Oligopaint) includes any modified nucleic acid base known in the art, i.e., any nucleic acid base modified from unmodified and / or natural nucleic acid bases.

[0168] The preparation of the modified nucleic acids, skeletons, and nucleic acid bases described above is well known in the art.

[0169] Another modification of nucleic acids, which is a feature of the present invention, involves chemical linkage to the nucleic acid to one or more ligands, moieties, or conjugates that enhance the activity, cell distribution, pharmacokinetic properties, or cell uptake of the nucleic acid. Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et (al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, e.g., di-hexadecyl -rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654;Shea et al., Nucl. Acids Res., 1990, 18:3777-3783 ), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides (& Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine as well. Other examples include lipid portions such as the hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0170] In some embodiments of the model, the vector is pEMBL. In some embodiments of the model, the vector is pEMBL-D(+)Syn1. In some embodiments of the model, the vector is pEMBL-D(+)Syn1-hCG intron only. In some embodiments of the model, the vector is pEMBL-D(+)Syn1-hCGin-2x controlled pre-miR. In some embodiments of the model, the vector is pEMBL-D(+)Syn1-hCGin-2x artificial pre-miR. In some embodiments of the model, the vector is pEMBL-D(+)Syn1-CYP46A1-hCGin-2x artificial pre-miR. In some embodiments of the model, the vector is pEMBL-D(+)Syn1-luc-HTT-3'UTR / mutant. In some embodiments of the model, the vector comprises at least one of the following: at least one (e.g., two) ITRs; a Syn1 promoter; at least one (e.g., two) hCG introns; at least one (e.g., two) copies of premiR (e.g., regulatory pre-miR; artificial pre-miR; SEQ ID NOs. 6-17, 40-44, or 50-66); small polyA; CYP46A1; luciferase; an HTT-targeting sequence; and / or an HTT-3'UTR / mutant. In some embodiments, the vector comprises a neuron-specific synthetic promoter selected from Tables 10-13, and / or a CRE selected from Tables 13-15. In certain embodiments of the model, the miRNA targets the wild-type HTT allele. In other embodiments of the model, the miRNA targets the mutant HTT allele. In yet another embodiment, the miRNA targets both the wild-type and mutant HTT alleles. In yet another embodiment, the miRNA targets any HTT mRNA.

[0171] In some embodiments, one or more of the recombinantly expressed genes may be incorporated into the cell's genome.

[0172] The nucleic acid molecule encoding the enzyme of the invention described in the claims can be introduced into one or more cells using methods and techniques standard in the art. For example, the nucleic acid molecule can be introduced by standard protocols such as chemical transformation and transformation including electroporation, transduction, and particulate gun. The expression of the nucleic acid molecule encoding the enzyme of the invention described in the claims may also be achieved by incorporating the nucleic acid molecule into a genome.

[0173] In some embodiments, the promoter is a synapsin (Syn1) promoter (see, for example, SEQ ID NO: 152). In one embodiment, the promoter contains a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 152, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical. In one embodiment, a composition is provided herein comprising a recombinant viral vector containing a promoter that contains a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 152, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical.

[0174] [ka] [ka]

[0175] In one embodiment, a composition is provided herein comprising an isolated nucleic acid containing a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to sequence number 111. In one embodiment, a composition is provided herein comprising a recombinant viral vector containing an isolated nucleic acid containing a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to sequence number 111. In some embodiments, the vector (e.g., rAAV) comprises a promoter (e.g., a synthetic nervous system-specific promoter; see, for example, Tables 10-13) or a fragment thereof, and / or an enhancer, and / or a cis-modulatory element (CRE; see, for example, Tables 13-15) that replaces the promoter and / or enhancer of sequence number 111. In some embodiments, the vector (e.g., rAAV) comprises an isolated nucleic acid containing a sequence that is at least 80% identical to SEQ ID NO: 110, e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical, and further comprises a promoter (e.g., a synthetic nervous system-specific promoter; see e.g., Tables 10-13) or a fragment thereof, and / or an enhancer, and / or a cis-regulating element (CRE; see e.g., Tables 13-15). In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the promoter is an ACTB proximal promoter. In some embodiments, the vector further comprises an intron. In some embodiments, the intron comprises an ACTB intron / chimeric ACTB-HBB2 intron. See e.g., SEQ ID NO: 111, Table 16. In some embodiments, the compositions described above can be used, for example, in the absence of administered miRNA to treat neurological diseases or disorders described herein. In various embodiments, the aforementioned compositions can be used, for example, in the presence of administered miRNAs for treating neurological disorders or disorders as described herein.In some embodiments, a recombinant viral vector, such as recombinant AAV, containing an isolated nucleic acid sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 111, is administered to a subject in need of treatment to express the CYP46A1 protein and / or to treat a neurological disorder or disability as described herein. In some embodiments, a recombinant viral vector, such as recombinant AAV, containing an isolated nucleic acid sequence of SEQ ID NO: 111, is administered to a subject in need of treatment to express the CYP46A1 protein and / or to treat a neurological disorder or disability as described herein, wherein the CMV enhancer and / or the ACTB proximal promoter and / or the chimeric ACTB-HBB2 intron of SEQ ID NO: 111 is replaced by one or more synthetic nervous system-specific promoters or fragments thereof selected from Tables 10-13 and / or enhancers and / or cis-regulatory elements (CREs) selected from Tables 13-15.

[0176] An ITR-ITR sequence of 4036 bp, sequence number 111, containing the CYP46A1 variant sequence (see, for example, sequence number 110).

[0177] The text in bold (for example, nucleotides (nt) 1-130 of sequence number 111) indicates the left ITR.

[0178] Italicized text (for example, nt182-436 of sequence number 111) indicates an enhancer.

[0179] The bold, italicized text (for example, nt550-804 in sequence number 111) indicates the promoter.

[0180] Text with a double underline (for example, nt824~1892 in sequence number 111) indicates an intron.

[0181] The bold, double-underlined text (for example, nt1966~3465 in sequence number 111) shows the code sequence (CDS) of the CYP46A1 variant sequence (see, for example, sequence number 110).

[0182] Italicized, double-underlined text (for example, nt3629~3853 in sequence number 111) indicates poly(A).

[0183] Bold, italicized, double-underlined text (for example, nt3907-4036 in sequence number 111) indicates right ITR. [ka] [ka]

[0184] [Table 16]

[0185] In one embodiment, a composition is provided herein comprising an isolated nucleic acid having a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to sequence number 153. In one embodiment, a composition is provided herein comprising a recombinant viral vector having an isolated nucleic acid having a sequence that is at least 80% identical to, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to sequence number 153. In some embodiments, the vector (e.g., rAAV) comprises a promoter (e.g., a synthetic nervous system-specific promoter; see, for example, Tables 10-13) or a fragment thereof, and / or an enhancer, and / or a cis-modulatory element (CRE; see, for example, Tables 13-15) that replaces the promoter and / or enhancer of sequence number 153. [ka]

Chem.

Chem.

[0186] Some of the aspects provided herein are nucleic acid sequences shown in SEQ ID NO: 153 and are used for producing rAAV lacking bacterial sequences. In some embodiments, rAAV is produced from a plasmid DNA template, as shown in SEQ ID NO: 111, for example. In some embodiments, rAAV is produced from a linear double-stranded DNA with closed ends, as shown in SEQ ID NO: 153 or SEQ ID NO: 111, for example. Modified capsid

[0187] In one embodiment, the capsids described herein are further modified to increase tropism for the CNS. Compositions are provided herein that include a modified viral capsid containing a payload, wherein the payload includes a regulatory sequence and a nucleic acid sequence adjacent to an inverted terminal repeat (ITR) targeting a central nervous system disorder, and the modification is a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the payload includes (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs, and (b) an isolated nucleic acid encoding the CYP46A1 protein. In some embodiments, the nucleic acid sequence of the payload includes an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the payload includes an isolated nucleic acid encoding the CYP46A1 protein.

[0188] Further provided herein are compositions comprising (a) a first modified viral capsid containing a first payload, and (b) at least a second modified viral capsid containing a second payload, wherein the payloads contain a nucleic acid sequence adjacent to a regulatory sequence and an inverted terminal repeat (ITR) targeting central nervous system disorders, the first and at least second modified viral capsids are the same, the first and second payloads are different, and the modification is chemical, non-chemical, or amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload contains an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload contains an isolated nucleic acid encoding the CYP46A1 protein.

[0189] Further provided herein are compositions comprising (a) a first modified capsid containing a first payload, and (b) at least a second modified capsid containing a second payload, wherein the payloads contain a nucleic acid sequence adjacent to a regulatory sequence and an inverted terminal repeat (ITR) targeting central nervous system disorders, the first and at least second modified capsids may be different, the first and second payloads may be the same or different, and the modification may be a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload contains an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload contains an isolated nucleic acid encoding the CYP46A1 protein.

[0190] In certain embodiments, the modified viral capsid includes modifications that result in its preferred targeting of the CNS or PNS. For example, the modified viral capsid has increased tropism to the CNS and / or decreased tropism to at least a second site, e.g., the liver. Preferred targeting of the CNS does not preclude targeting of other sites, but rather indicates that it is more highly targeted to the CNS compared to other sites.

[0191] In one embodiment, the modified viral capsid includes modifications that result in its targeting of the CNS or PNS. For example, a modification to a capsid that typically targets a non-CNS site (e.g., the liver) can be redirected to immediately target both CNS and non-CNS sites. In such embodiments, CNS targeting does not need to be preferred.

[0192] In one embodiment, the modification to the capsid is an amino acid modification, such as the deletion, insertion, or substitution of an amino acid. In one embodiment, the amino acid modification increases the tropism to the CNS or PNS. In one embodiment, the amino acid modification targets the capsid modified to the CNS or PNS.

[0193] In one embodiment, the modified viral capsid has, consists of, or is essentially composed of a nucleic acid sequence whose contents are 90% identical to Sequence IDs 1-4 of U.S. Patent Application No. 16 / 511,913, which is incorporated herein by reference in its entirety. This U.S. Patent Application describes a chimeric AAV capsid sequence exhibiting a dominant tropism towards oligodendrocytes, which can be used to create an AAV vector for transduction into oligodendrocytes in the target CNS.

[0194] In one embodiment, the modified viral capsid is an AAV capsid protein comprising one or more amino acid substitutions, where the substitutions introduce a new glycan-binding site to the AAV capsid protein. In some embodiments, the amino acid substitutions are located at amino acids 266, 463–475, and 499–502 in AAV2, or at the corresponding amino acid positions in AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV10. Such AAV capsid proteins are further described, for example, in U.S. Patent Application No. 16 / 110,773, which is incorporated herein by reference in its entirety.

[0195] In one embodiment, the modified viral capsid is an AAV capsid protein comprising, consisting of, or essentially being an AAV 2.5 capsid protein (Sequence ID 1 of International Patent Application PCT / US2020 / 029493; the contents thereof are incorporated herein by reference in their entirety) that includes one or more amino acid substitutions introducing a new glycan-binding site. Such amino acid substitutions may allow the capsid to target neurons and glial cells, such as astrocytes. In embodiments of the capsid protein, capsid, viral vector and method described in International Patent Application PCT / US2020 / 029493, one or more amino acid substitutions include A267S, SQAGASDIRDQSR464-476SX1AGX2SX3X4X5X6QX7R (where X1-7 may be any amino acid), and EYSW 500-503EX8X9W (where X8-9 may be any amino acid). In embodiments of the capsid proteins, capsids, viral vectors and methods described herein, X1 is V or a conserved substitution thereof; X2 is P or a conserved substitution thereof; X3 is N or a conserved substitution thereof; X4 is M or a conserved substitution thereof; X5 is A or a conserved substitution thereof; X6 is V or a conserved substitution thereof; X7 is G or a conserved substitution thereof; X8 is F or a conserved substitution thereof; and / or X9 is A or a conserved substitution thereof. In embodiments of the capsid proteins, capsids, viral vectors and methods described herein, X1 is V, X2 is P, X3 is N, X4 is M, X5 is A, X6 is V, X7 is G, X8 is F, and X9 is A, where the new glycan-binding site is a galactose-binding site. Such AAV capsid proteins are further described, for example, in International Patent Application No. PCT / US2020 / 029493, which is incorporated herein by reference in its entirety.

[0196] In one embodiment, for example, as described in U.S. Patent Application No. 16 / 956,306, the modified viral capsid is an AAV capsid protein particle containing a surface-bound peptide, where the peptide bound to the surface of the AAV particle is Angiopep-2, GSH, HIV-1 TAT(48-60), ApoE(159-167)2, leptin-30(61-90), THR, PB5-3, PB5-5, PB5-14, or any combination thereof, the contents of which are incorporated herein by reference in their entirety. Such an AAV capsid crosses the blood-brain barrier to enable, for example, the delivery of a payload.

[0197] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein), where the VP3 region of the capsid protein is modified (e.g., substitution of tyrosine residues with non-tyrosine residues and / or substitution of threonine residues with non-threonine residues) compared to the wild-type AAV1 capsid protein (e.g., Sequence ID No. 1 of U.S. Patent Application No. 16 / 565,191; the contents of which are incorporated herein by reference in their entirety). One or more of Y705, Y731, and T492, or each of them; one or more of Y436, Y693, and Y719, or each of them, of the wild-type AAV5 capsid protein (e.g., SEQ ID NO. 2 of U.S. Patent Application No. 16 / 565,191); or one or more of Y705, Y731, and T492, or each of them, of the wild-type AAV6 capsid protein (e.g., SEQ ID NO. 3 of U.S. Patent Application No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0198] In one embodiment, the modified viral capsid is a Y-to-F (tyrosine to phenylalanine) modification or a T-to-V (threonine to valine) modification in the VP3 region of the capsid, one or more of Y705F, Y731F, and T492V of the wild-type AAV1 capsid protein (e.g., Sequence ID No. 16 / 565,191), or each of them; or the wild-type AAV5 capsid protein (e.g., Sequence ID No. 16 / The AAV capsid proteins (e.g., AAV1, AAV5, or AAV6 capsid proteins) contain one or more of Y436F, Y693F, and Y719F of SEQ ID NO: 2 of Patent No. 565,191, or each of them; or one or more of Y705F, Y731F, and T492V of the wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191), or each of them, in the corresponding positions. Such AAV capsids target neurons and astrocytes.

[0199] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein), where the VP3 region of the capsid protein is modified (e.g., substitution of tyrosine residues with non-tyrosine residues, and / or substitution of threonine residues with non-threonine residues) to Y705, Y731, and Y705 of the wild-type AAV1 capsid protein (e.g., Sequence ID No. 16 / 565,191) One or more of T492, or each of them; one or more of Y436, Y693, and Y719 of the wild-type AAV5 capsid protein (e.g., SEQ ID NO. 2 of U.S. Patent Application No. 16 / 565,191), or each of them; or one or more of Y705, Y731, and T492 of the wild-type AAV6 capsid protein (e.g., SEQ ID NO. 3 of U.S. Patent Application No. 16 / 565,191), or each of them, at the corresponding position. Such AAV capsids target neurons and astrocytes.

[0200] In one embodiment, the modified viral capsid is a Y-to-F (tyrosine to phenylalanine) modification or a T-to-V (threonine to valine) modification in the VP3 region of the capsid protein, one or more of Y705F, Y731F, and T492V of the wild-type AAV1 capsid protein (e.g., Sequence ID No. 16 / 565,191); or wild-type AAV5 capsid protein (e.g., Sequence ID No. The AAV capsid proteins (e.g., AAV1, AAV5, or AAV6 capsid proteins) contain one or more of Y436F, Y693F, and Y719F, or each of them, as in the wild-type AAV6 capsid protein (e.g., SEQ ID NO. 3, U.S. Patent Application No. 16 / 565,191), or each of them, in the corresponding positions. Such AAV capsids target neurons and astrocytes.

[0201] In one embodiment, amino acid modification allows the modified capsid to avoid neutralizing antibodies, for example, against the same serotype viral vector. In another embodiment, amino acid modification allows the modified capsid to be used for repeated administration, for example, the modification allows the capsid to have a therapeutic effect upon re-administration.

[0202] In one embodiment, the modified viral capsid is a chimeric capsid. As used herein, "chimeric" capsid protein means an AAV capsid protein (e.g., one or more of VP1, VP2, or VP3) modified by substitution of one or more amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) in the amino acid sequence of the capsid protein compared to the wild type, and by insertion and / or deletion of one or more amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) in the amino acid sequence compared to the wild type. In some embodiments, a complete or partial domain, functional region, epitope, etc., derived from one AAV serotype, in any combination, can be used to replace the corresponding wild-type domain, functional region, epitope, etc., from different AAV serotypes to generate the chimeric capsid protein of the present invention. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a considerable number of chimeric capsid proteins that may be included in the capsids of the present invention are described in the literature and herein.

[0203] In one embodiment, the modified viral capsid is a singular capsid. As used herein, the term “singular AAV” means the AAVs described in International Application WO2018 / 170310 or U.S. Application US2018 / 037149, which are incorporated herein by reference in their entirety. In some embodiments, the virion population is a singular AAV population capable of constructing virion particles, where at least one viral protein derived from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins required to form virion particles capable of encapsulating the AAV genome. For each viral protein present (VP1, VP2, and / or VP3), the proteins are of the same type (e.g., all AAV2 VP1). In one example, at least one of the viral proteins is a chimeric viral protein, and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric, while only VP3 is not. For example, only viral particles composed of VP1 / VP2 derived from chimeric AAV2 / 8 (the N-terminus of AAV2 and the C-terminus of AAV8) pair with VP3 derived from AAV2, or only chimeric VP1 / VP2 28m-2P3 (the N-terminus of AAV8 without a mutation in the VP3 start codon and the C-terminus of AAV2) pair with VP3 derived from AAV2. In another embodiment, only VP3 is chimeric, while VP1 and VP2 are not. In yet another embodiment, at least one of the viral proteins is derived from a completely different serotype. For example, only chimeric VP1 / VP2 28m-2P3 pairs with VP3 derived solely from AAV3. In yet another example, no chimeric proteins exist.

[0204] In some embodiments of the technology described herein, the modified viral capsid includes one or more modifications, such as chemical modifications, non-chemical modifications, or amino acid modifications to the capsid. Such modifications can, for example, among other things, modify the tissue tropism or cell tropism of the modified capsid.

[0205] The modifications can directly change the properties of the capsid, including biochemical properties such as receptor binding, such that the modification itself can change the behavior of the capsid or enable further modifications, such as the attachment of ligands that in turn modify the behavior of the capsid in a desired manner.

[0206] In one embodiment, chemical modification of cysteine residues that can occur naturally or be introduced by genetic modification of the capsid polypeptide coding sequence enables covalent attachment of ligands via disulfide bond formation (see, e.g., WO2005 / 106046, which is incorporated herein by reference).

[0207] A variety of ligands are contemplated, including, but not limited to, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by target cells (see, e.g., WO2000 / 002654, which is incorporated herein by reference).

[0208] WO2015 / 062516, the content of which is also incorporated herein by reference, describes the insertion of amino acids containing azide groups by genetic modification of the capsid gene, followed by chemical conjugation of ligands via the azide groups.

[0209] Modification of AAV capsid tropism by glycosylation or chemical conjugation of sugar moieties is described by Horowitz et al., Bioconjugate Chem. 22: 529-532 (2011). This approach and similar approaches are contemplated for the modification of capsids described herein.

[0210] In other embodiments, coating of the viral capsid with a polymer such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl) methacrylamide (pHPMA) is specifically considered. Such modifications can, for example, reduce specific and nonspecific interactions with non-target tissues.

[0211] In other embodiments, carbodiimide coupling is specifically considered. See, for example, Joo et al. ACS Nano 5, title "Enhanced Real-time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates" (2011). I want to be treated that way.

[0212] In other embodiments, the viral capsid can be modified, for example, as described in WO2017 / 212019, and also see U.S. National Phase USSN16 / 308,740, the contents of which are incorporated herein by reference, respectively. The method described therein couples the viral capsid to a ligand via a bond containing the -CSNH- and aromatic moieties. Genetically modified viral capsids can be further modified by this method, but the modifications described therein do not require genetic modification of the viral capsid. Ligands described therein include, for example, targeting agents, steric shielding agents to avoid neutralizing antibody interactions, labeling agents, or magnetic agents. Targeting ligands described therein include, for example, cell type-specific ligands, proteins, monosaccharides or polysaccharides, steroid hormones, RGD motif peptides (e.g., Arg-Gly-Asp, a cell adhesion motif that mimics cell adhesion proteins and can bind to integrins), vitamins, and small molecules.

[0213] In one embodiment, the chemical modification of the present invention is the modification described in International Patent Application No. PCT / EP2017 / 064089, which is incorporated herein by reference in its entirety.

[0214] In one embodiment, the chemical modification of the present invention is the modification described in International Patent Application No. PCT / EP2020 / 069554, which is incorporated herein by reference in its entirety.

[0215] In one embodiment, the capsid has at least one chemically modified tyrosine residue in the capsid, where the chemically modified tyrosine residue is given by formula (I): [ka]

[0216] [In the formula,

[0217] - X1 is [ka] Selected from the group consisting of,

[0218] - Ar is the aryl or heteroaryl portion, which is substituted as needed. It belongs to them.

[0219] In one embodiment, the capsid is given by formula (Ia): [ka]

[0220] [In the formula,

[0221] - Xi and Ar are as defined herein above,

[0222] - The spacer is a group for linking the "Ar" group to the functional part "M", which is in the form of a chemical chain containing up to 1000 carbon atoms, preferably including heteroatoms and / or cyclic parts as needed.

[0223] - n is either 0 or 1,

[0224] - M is a functional moiety that includes a stereochemical agent, labeling agent, cell type-specific ligand, or drug moiety. It has at least one chemically modified tyrosine residue.

[0225] In one embodiment, Xi is of formula (a), and / or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl.

[0226] In one embodiment, the capsid is given by formula (Ic): [ka]

[0227] [In the formula,

[0228] - X2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)-, or -(C=S)-NH-, preferably -(C=O)-NH- or -(C=O)-O-,

[0229] - X2 is located at the para, meta, or ortho position of the phenyl group, preferably at the para position.

[0230] - Spacers, n, and M are as defined herein above. It has at least one chemically modified tyrosine.

[0231] In one embodiment, the "spacer," if present, is selected from the group consisting of saturated or unsaturated linear or branched C2-C40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-hydroxypropyl)methacrylamide), polylactic acid-co-glycolic acid (PLGA), alkyldiamine polymers, and combinations thereof, as well as / or

[0232] "M" comprises or consists of cell-type targeted ligands, preferably monosaccharides or polysaccharides, hormones including steroid hormones, peptides, for example, RGD peptide (e.g., Arg-Gly-Asp, a cell adhesion motif that mimics cell adhesion proteins and can bind to integrins), muscle-targeting peptides (MTP) or Angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof, for example, antigen-binding fragments (Fab), Fab' (an antigen-binding fragment further containing a free sulfhydryl group), and VHH, single-chain variable fragments (ScFv), spiegelmer, peptide aptamers, vitamins, and drugs, for example, cell-type targeted ligands selected from cannabinoid receptor 1 (CB1) and / or cannabinoid receptor 2 (CB2) ligands.

[0233] In one embodiment, the "spacer" (if present) is selected from the group consisting of linear or branched C2-C20 alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyldiamine polymers, and combinations thereof, wherein the polymer has 2-20 monomers, and / or, "M" comprises or consists of transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor 13FGF, one or more monosaccharides or polysaccharides including galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNac or mannose-6-phosphate, MTP selected from SEQ ID NOs: 1-7, and a cell type-specific ligand derived from a protein selected from vitamins, such as folic acid.

[0234] In one embodiment, the capsid further comprises at least one additional chemically modified amino acid residue, which, unlike a tyrosine residue, is preferably of formula (V): [ka]

[0235] [In the formula,

[0236] - N * This is the nitrogen atom of the amino group of an amino acid residue, such as a lysine residue or an arginine residue.

[0237] - Ar, spacer, n, and M have the same definitions as Ar, spacer, n, and M in formula (II) of claim 2. It has an amino group that has been chemically modified with the base.

[0238] In one embodiment, the capsid is incubated with a chemical reagent having a reactive group selected from aryldiazonium and 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moieties, under conditions that contribute to the reaction of the reactive group with tyrosine residues present in the capsid to form a covalent bond.

[0239] In one embodiment, the capsid is incubated with the chemical reagent of formula VId, and then converted to formula Ic [ka] At least one chemically modified tyrosine residue is obtained in the capsid. Administration

[0240] The rAAVs of this disclosure may be delivered to a subject in a composition according to any suitable method known in the art. For example, rAAV, preferably suspended in a physiologically suitable carrier (i.e., in a composition), may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., a macaque). In some embodiments, the host animal does not include humans.

[0241] Delivery of rAAV to a mammalian subject may be, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream by isolated limb perfusion, a technique well known in the art of surgery, which essentially allows a person skilled in the art to separate the limb from the systemic circulation before administration of the rAAV virion. A variant of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 may also be used by a person skilled in the art to administer the virion into the blood vessels of the isolated limb to potentially enhance transduction into muscle cells or tissues. In certain specific cases, it may also be desirable to deliver the virion to the CNS of the subject. "CNS" means all cells and tissues of the brain and spinal cord of vertebrates. Therefore, this term includes, but is not limited to, nerve cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. Recombinant AAVs may be delivered directly to the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule using needles, catheters, or associated devices, for example, by injection into the ventricular region to the CNS or brain, and by stereotactic injection, using neurosurgical techniques known in the art (see, for example, Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, the rAAVs described herein are delivered intravenously. It is administered by intracerebral injection. In some embodiments, rAAV is administered by intracerebral injection. In some embodiments, rAAV is administered by subarachnoid injection. In some embodiments, rAAV is administered by intrastriatal injection. In some embodiments, rAAV is delivered by intracranial injection. In some embodiments, rAAV is delivered by cisterna magna injection. In some embodiments, rAAV is delivered by lateral ventricle injection into the cerebrum.

[0242] Delivery of the composition to a mammalian subject may be by any known means of delivery to a desired site, such as the CNS. It may be desirable for the composition to be delivered to the CNS of the subject. "CNS" means all cells and tissues of the brain and spinal cord of vertebrates. Therefore, this term includes, but is not limited to, nerve cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. Any composition described herein may be delivered directly to the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule using needles, catheters, or associated devices, for example, by injection into the ventricular region, or by stereotactic injection, using neurosurgical techniques known in the art (see, for example, Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000). In some embodiments, the compositions described herein are administered by intravenous injection. In some embodiments, the compositions described herein are administered by intraspinal injection. In some embodiments, the compositions described herein are administered by intraventricular injection. In some embodiments, the compositions are administered by intracerebral injection. In some embodiments, the compositions are administered by subarachnoid injection. In some embodiments, the compositions are administered by intrastriatal injection. In some embodiments, the compositions are delivered by intracranial injection. In some embodiments, the compositions are delivered by cisterna magna injection. In some embodiments, the compositions are delivered by lateral ventricle injection into the cerebrum.

[0243] The CNS can include, but is not limited to, specific regions of the CNS, neural pathways, somatosensory systems, visual systems, auditory systems, nerves, neuroendocrine systems, neurovascular systems, neurotransmitter systems, and dural and meningeal systems.

[0244] Exemplary regions of the CNS include, but are not limited to, the cerebrospinal fluid; medulla oblongata; medullary pyramidal body; olivary body; inferior olivary nucleus; rostral ventrolateral medullary area; caudal ventrolateral medullary area; nucleus tractus solitarius (nucleus of the tractus solitarius); respiratory center - respiratory group, dorsal respiratory group; ventral respiratory group or sustained inspiratory center; Prebetzinger complex; Botzinger complex; retrotrapezoidal nucleus; posterior nucleus of the facial nerve; posterior nucleus ambiguous; paranucleus ambiguous; median parareticular nucleus; giant cellular reticular nucleus; para Facial girdle; cuneate nucleus; gracile nucleus; periglossal nucleus; internucleus; anterior nucleus; subglossal nucleus; area posterior; medullary cranial nuclei; inferior salivary nucleus; nucleus ambiguus; dorsal nucleus of the vagus nerve; hypoglossal nucleus; chemoreceptor trigger zone; hindbrain; pons; pontine nuclei; pontine cranial nuclei; principal or pontine nucleus of the trigeminal sensory nucleus; motor nucleus for the trigeminal nerve; abducens nucleus (VI); facial nucleus (VII); vestibulocochlear nucleus (vestibular and cochlear nuclei) (VIII); superior salivary nucleus; pontine tegmentum; pons Mitigation center (Barlington nucleus); locus coeruleus; peduncle-pontine nuclei; dorsolateral tegmental nucleus; tegmentopontine reticular nucleus; insertion nucleus; paracerebellar peduncle region; medial parabrachial nucleus; lateral parabrachial nucleus; parabrachial-inferior nucleus (Kerriker-Fuse nucleus); pontine respiratory group; superior olivary complex; medial superior olivary; lateral superior olivary; medial nucleus of the trapezoid; paramedian pontine reticular formation; parcellous reticular nucleus; caudal pontine reticular nucleus; cerebellar peduncle; superior cerebellar peduncle; middle cerebellar peduncle; inferior cerebellar peduncle; fourth ventricle; cerebellar vermis of the cerebellum; small Hemispheres; anterior lobe; posterior lobe; flocculonodular lobe; cerebellar nuclei; fastigial nucleus; insertion nucleus; globose nucleus; embolus nucleus; dentate nucleus; midbrain (middle head); tectum, tetracumulus; inferior colliculus; superior colliculus; pretectal area; tegmentum, periaqueductal gray matter; rostral interstitial nucleus of medial longitudinal fasciculus; midbrain reticular formation; dorsal raphe nucleus; red nucleus; ventral tegmental area; paracerebellar peduncle pigment nucleus; paranisigma nucleus; rostral tegmental nucleus; caudal linear nucleus; rostral linear nucleus of the raphe; interfascicular nucleus; substantia nigra; parenchyma compacta; parenchyma reticularis; interpeduncle nucleus; cerebral peduncle Peduncle; cerebral peduncle (Crus cerebri); cranial nerve nuclei of the midbrain; oculomotor nucleus (III); Edinger-Westphal nucleus; trochlear nucleus (IV); mesencephalic duct (cerebrospinal aqueduct, Sylvian aqueduct); forebrain (prosencephalon); diencephalon; suprathalamus; pineal gland; habenula; stria medullaris; thalamic cord; third ventricle; subcomisticular organ; thalamus; anterior nuclei; anterior ventral nucleus (also known as ventral nucleus) Anterior nucleus); Anterior dorsal nucleus; Anterior medial nucleus; Neoblastic group; Middle dorsal nucleus; Median nucleus group; Parastring nucleus; Connective nucleus; Rhombus nucleus; Intralaminar nuclei group; Median central nucleus; Parafascicular nucleus; Paracentral nucleus; Lateral central nucleus; Lateral nuclei group; Dorsolateral nucleus; Posterolateral nucleus; Pillow; Ventral nuclei group, Anterior ventral nucleus; Lateral ventral nucleus; Posterior ventral nucleus; Medial posterolateral nucleus; Ventral posteromedial nucleus; Posterior thalamus; Medial geniculate body; Lateral geniculate body; Reticular nucleus of the thalamus; Hypothalamus (limbic system) (HPA axis); Anterior medial region of the preoptic area; Medial preoptic nucleus INAH 1; INAH 2; INAH 3; INAH 4; median preoptic nucleus; suprachiasmatic nucleus; paraventricular nucleus of the hypothalamus; supraoptic nucleus (mainly); anterior hypothalamic nucleus; lateral area; part of preoptic area; lateral preoptic nucleus; anterior part of lateral nucleus; part of supraoptic nucleus; other nuclei of preoptic area; median preoptic nucleus; periventricular preoptic nucleus; medial area of ​​the prominence; dorsomedial hypothalamic nucleus; ventromedial nucleus; arcuate nucleus; lateral area prominence of the lateral nucleus; lateral prominence nucleus; posteromedial area mammillary nucleus (part of mammillary body); posterior nucleus; posterior part of lateral area of ​​lateral nucleus; surface median prominence; mammillary body; pituitary stalk (infundibular organ); optic chiasm; subfornix organ; periventricular nucleus; gray area; prominence nucleus; mammillary nucleus prominence; prominence; mammillary nucleus; ventral area of ​​the thalamus (HPA axis); subthalamic nucleus; zone of uncertainty; pituitary gland (HPA axis); neurogenic pituitary gland; intermediate lobe; adenohypophysis; telencephalon (cerebrum); cerebral hemispheres; white matter; center of semioval; corona radiata; internal capsule; external capsule; polar capsule; subcortex; hippocampus (medial temporal lobe); dentate gyrus; Ammon's horn (CA area); Ammon's horn area 1 (CA1); Ammon's horn area 2 (CA2); Ammon's horn area 3 (CA3); Ammon's horn area 4 (CA4); amygdala (limbic system) (limbic lobe); central nucleus (autonomic nervous system); medial nucleus (accessory olfactory system); cortical nucleus and medial basal ganglia (main olfactory system); lateral nucleus and basolateral nucleus (frontotemporal system); amygdala extension; bed nucleus of the stria terminalis; claustrum; basal ganglia; striatum, dorsal striatum (also known as Neostriatum); Putamen; Caudate nucleus; Ventral striatum; Nucleus accumbens; Olfactory tubercle; Globus pallidus (together with the putamen to form the lenticular nucleus); Ventral globus pallidus; Subthalamic nucleus; Forebrain basal base; Anterior perforated mass; Mass nominum; Basal ganglia; Broca's diagonal zone; Septal nucleus; Medial septal nucleus; Plate endoscopy; Vesicular organs of the plate endoscopy; Olfactory brain (paleocortex); Olfactory bulb; Olfactory tract; Anterior olfactory nucleus; Piriform cortex; Anterior commissure; Uncinate; Periamygdala cortex; Cerebral cortex (neocortex); Frontal lobe; Primary motor cortex (Precentral gyrus, M1); Accessory motor area; Premotor cortex; Prefrontal cortex; Orbitofrontal cortex; Dorsolateral prefrontal cortex; Superior frontal gyrus; Middle frontal gyrus; Inferior frontal gyrus; Brodmann's areas: 4, 6, 8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, 47;Parietal lobe cortex: primary somatosensory cortex (S1); secondary somatosensory cortex (S2); posterior parietal lobe cortex; postcentral gyrus (primary somatosensory cortex); Brodmann areas 1, 2, 3 (primary somatosensory cortex); 5, 7, 23, 26, 29, 31, 39, 40; occipital lobe cortex: primary visual cortex (V1), V2, V3, V4, V5 / MT; lateral occipital gyrus; Brodmann area 17 (V1, primary visual cortex); 18, 19; temporal lobe cortex: primary auditory cortex (A1); secondary auditory cortex Quality (A2); inferior temporal cortex; posterior inferior temporal cortex; superior temporal gyrus; middle temporal gyrus; inferior temporal gyrus; entorhinal cortex; perinasal cortex; parahippocampal gyrus; fusiform gyrus; Brodmann areas: 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, 42; insular cortex; anterior cingulate cortex; posterior cingulate cortex; posterior corpus callosum; gray zone; subgenital area 25; Brodmann areas 23, 24; 26, 29, 30 (posterior corpus callosum); 31, and 32 are examples.

[0245] Exemplary neural pathways include, but are not limited to, the superior longitudinal fasciculus, arcuate fasciculus; uncinate fasciculus; perforating tracts; thalamocortical radiations; corpus callosum; anterior commissure; amygdala efferent pathway; interthalamic pons; posterior commissure; habenary commissure; fornix; mammillolar tegmental fasciculus; fasciculus; incertohypothalamic pathway; cerebral peduncle; medial forebrain fasciculus; medial longitudinal fasciculus; myoclonal triangle; tractus solitarius; major dopaminergic pathways from dopaminergic cell populations; mesocortical pathways; mesolimbic pathways; nigrostriatal pathway; infundibular pathway; serotonergic pathways of the raphe nuclei; norepinephrine pathways of the locus coeruleus and other noradrenergic cell populations; epinephrine pathways from adrenergic cell populations; glutamate and acetylcholine pathways from the mesopontin nucleus Cholinergic pathways; motor / descending fibers; extrapyramidal tract; pyramidal tract; corticospinal tract; or cerebrospinal fibers; lateral corticospinal tract; anterior corticospinal tract; corticopontine tract fibers; frontopontine fibers; temporalopontine fibers; corticomedulla tract; corticomesencephalic tract; tectospinal tract; interstitial nucleus spinal tract; rubrospinal tract; rubroolivary tract; olivorocerebellar tract; olivorospinal tract; vestibulospinal tract; lateral vestibulospinal tract; medial vestibulospinal tract; reticulospinal tract; lateral raphenucleus spinal tract; alpha system; and gamma system.

[0246] Examples of somatosensory systems include, but are not limited to, the gracile fasciculus, cuneate fasciculus, medial lemniscus, spinothalamic tract, lateral spinothalamic tract, anterior spinothalamic tract, spinomesencephalic tract, spinocerebellar tract, olivospinal tract, and spinoreticular tract.

[0247] Examples of visual systems, though not limited to them, include the optic tract, optic radiation, and retinohypothalamic tract.

[0248] Examples of auditory systems, though not limited to them, include the stria medullaris of the fourth ventricle; the trapezoidal body; and the lateral lemniscus.

[0249] Examples of nerves, though not limited to them, include brainstem cranial nerve endings (0); olfactory nerve (I); optic nerve (II); oculomotor nerve (III); trochlear nerve (IV); trigeminal nerve (V); abducens nerve (VI); facial nerve (VII); vestibulocochlear nerve (VIII); glossopharyngeal nerve (IX); vagus nerve (X); accessory nerve (XI); and hypoglossal nerve (XII).

[0250] Examples of neuroendocrine systems, though not limited to them, include hypothalamic-pituitary hormones; HPA axis; HPG axis; HPT axis; and GHRH-GH.

[0251] Examples of neurovascular systems, though not limited to them, include the middle cerebral artery, posterior cerebral artery, anterior cerebral artery, vertebral artery, basilar artery, Circle of Willis (arterial system), blood-brain barrier, glymphatic system, venous system, and periventricular organs.

[0252] Exemplary neurotransmitter systems: noradrenergic system; dopamine system; serotonin system; cholinergic system; GABA; neuropeptides, opioid peptides; endorphins; enkephalins; dynorphins; oxytocin; and substance P.

[0253] Exemplary dural-meningeal systems include, but are not limited to, the cerebrospinal fluid barrier; meningeal dura mater; arachnoid mater; pia mater; epidural space; subdural space; subarachnoid space, interarachnoid septum; superior cisternus; cisternus of the terminal plate; crossing cisternus; interpeduncular cisternus; pontine cisternus; cisterna magna; spinal subarachnoid space; ventricular system; cerebrospinal fluid; third ventricle; fourth ventricle; horn fasciculus of the lateral ventricle; anterior horn; body of the lateral ventricle; inferior horn; posterior horn, calcaneal spur; and subventricular zone.

[0254] In one embodiment, AAV is administered to the PNS. "PNS" refers to the nerves and ganglia outside the brain and spinal cord. The primary function of the PNS is to connect the CNS to the limbs and organs, and essentially acts as a relay between the brain and spinal cord and the rest of the body. Unlike the CNS, the PNS is not protected by the spinal column and skull or by the blood-brain barrier, and is therefore vulnerable to, for example, toxins and mechanical injury.

[0255] The PNS is divided into the somatic nervous system and the autonomic nervous system. In the somatic nervous system, cranial nerves, along with the retina, are part of the PNS, with the exception of the optic nerve (cranial nerve II). The second cranial nerve is not a true peripheral nerve, but is a pathway of the diencephalon. Cranial ganglia originate from the CNS. However, the axons of the remaining 10 cranial nerves extend beyond the brain and are therefore considered part of the PNS. The autonomic nervous system exerts involuntary control over smooth muscles and glands. The connections between the CNS and organs allow the system to be in two distinct functional states: sympathetic and parasympathetic.

[0256] The somatic nervous system is under voluntary control and transmits signals from the brain to peripheral organs such as muscles. The sensory nervous system is part of the somatic nervous system and transmits signals from senses such as taste and touch (including fine and crude touch) to the spinal cord and brain. The autonomic nervous system is a "self-regulating" system that affects the function of organs outside of voluntary control, such as heart rate, or the function of the digestive system.

[0257] The PNS can be described in various sections, including the cervical spinal nerves (C1-C4). The first four cervical spinal nerves, C1-C4, divide and recombine to give rise to various nerves that function in the neck and occipital region. The spinal nerve C1 is called the suboccipital nerve, which provides motor innervation to the muscles at the base of the skull. C2 and C3 form many of the nerves in the neck, providing both sensory and motor control. These include the greater occipital nerve, which provides sensation to the occipital region, and the lesser occipital, greater auricular, and minor auricular nerves, which provide sensation to the area behind the ear. The phrenic nerve is a nerve essential for our survival, arising from nerve roots C3, C4, and C5. It supplies the thoracic diaphragm and enables breathing. If the spinal cord is severed above C3, spontaneous breathing is impossible. Brachial plexus (C5-T1). The last four cervical spinal nerves C5-C8, and the first thoracic spinal nerve T1, combine to form the brachial plexus (or plexus brachialis), a tangled network of nerves that divides, joins, and rejoins to form the nerves that assist the upper limbs and upper back. Although the brachial plexus can appear tangled, it is highly organized, predictable, and shows little variation among individuals. The lumbosacral plexus (L1-Co1). The anterior portions of the lumbar, sacral, and coccygeal nerves form the lumbosacral plexus, with the first lumbar nerve frequently joined by a branch from the 12th thoracic plexus. For the purposes of explanation, this plexus is usually divided into three parts: the lumbar plexus, the sacral plexus, and the pudendal plexus. The autonomic nervous system. Exemplary examples of the autonomic nervous system include the sympathetic, parasympathetic, and enteric nervous systems.

[0258] In one embodiment, administration results in the delivery of a modified capsid to a target CNS or PNS. In one embodiment, administration results in the delivery of a payload to a target CNS or PNS. In one embodiment, administration results in the delivery of a modified viral capsid to a CNS or PNS cell population. In one embodiment, administration results in the delivery of a payload to a CNS or PNS cell population. Examples of CNS cell populations include, but are not limited to, neurons, oligodendrocytes, astrocytes, microglia, ependymal cells, radial glial cells, and pituitary cells. Those skilled in the art can identify specific CNS cell populations by, for example, evaluating cell populations for known cell markers using standard techniques. In one embodiment, administration results in the delivery of a modified capsid to a cell type originating in the CNS, e.g., cells originating in the CNS but spreading away from it, e.g., nerves. In one embodiment, administration results in the delivery of a payload to a cell type originating in the CNS, e.g., cells originating in the CNS but spreading away from it, e.g., nerves.

[0259] In one embodiment, when the composition of the present invention is administered locally to the CNS or PNS, for example via a catheter, cannula, etc., the administration results in a distribution of the composition extending at least 0.5 inches from the administration site. In one embodiment, the administration results in a distribution of the composition extending at least 1 inch, at least 1.5 inches, at least 2 inches, at least 2.5 inches, at least 3 inches, at least 3.5 inches, at least 4 inches, at least 4.5 inches, at least 5 inches, at least 5.5 inches, at least 6 inches, at least 6.5 inches, at least 7 inches, at least 7.5 inches, at least 8 inches, at least 8.5 inches, at least 9 inches, at least 9.5 inches, at least 10 inches, or wider from the administration site. In other words, the modified viral capsid composition is detectable in cells (i.e., cells it transduces) located at least 0.5 inches, at least 1 inch, at least 1.5 inches, at least 2 inches, at least 2.5 inches, at least 3 inches, at least 3.5 inches, at least 4 inches, at least 4.5 inches, at least 5 inches, at least 5.5 inches, at least 6 inches, at least 6.5 inches, at least 7 inches, at least 7.5 inches, at least 8 inches, at least 8.5 inches, at least 9 inches, at least 9.5 inches, at least 10 inches, or more from the administration site.

[0260] In one embodiment, when the composition of the present invention is administered topically to the CNS or PNS, for example via a catheter, cannula, etc., the administration results in the expression of a modified capsid, viral vector, and / or payload in at least one cell type of the CNS or PNS. In one embodiment, when the composition of the present invention is administered topically to the CNS or PNS, for example via a catheter, cannula, etc., the administration results in the expression of a modified capsid, viral vector, and / or payload in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more cell types of the CNS or PNS. In a particular embodiment, at least two cell types are adjacent to each other in the CNS or PNS. Alternatively, at least the cell types do not need to be adjacent to each other.

[0261] Aspects of the present disclosure relate to compositions comprising recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises a sequence shown in any one of SEQ ID NOs: 6-17, 40-44, or 50-66. In some embodiments, the nucleic acid further comprises an AAV ITR. In some embodiments, the ITR is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 ITR. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The compositions of the present disclosure may comprise an rAAV alone or in combination with one or more other viruses (e.g., a second rAAV having one or more different transgenes encoding). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs, each having one or more different transgenes.

[0262] Suitable carriers can be readily selected by those skilled in the art, taking into account the indications for which rAAV applies. For example, one suitable carrier is 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 choice of carrier is not limited to this disclosure.

[0263] If necessary, the compositions of this disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers. 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.

[0264] rAAV is transfected into cells of the desired tissue and administered in an amount sufficient to provide adequate levels of gene transfer and expression without excessive adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to selected organs (e.g., intra-portal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Routes of administration may be combined as desired. In some embodiments, all or at least one of the nucleic acid sequences disclosed herein are delivered via a non-viral DNA construct containing at least one DD-ITR. For example, one or more of the nucleic acids described herein can be delivered using the non-viral DNA construct described in WO2019 / 246554, which is incorporated herein by reference in its entirety.

[0265] The unit of the dose of rAAV virions required to achieve a particular "therapeutic effect", e.g., the dose in terms of genome copies per kilogram of body weight (GC / kg), is not limited, but varies based on several factors including the route of rAAV virion administration, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of ordinary skill in the art can readily determine the dosage range of rAAV virions for treating a patient having a particular disease or disorder based on the aforementioned factors and other factors well known in the art.

[0266] An effective amount of rAAV is an amount sufficient to target, infect, and target the desired tissue in an animal. In some embodiments, an effective amount of rAAV is an amount sufficient to generate a stable somatic transgenic animal model. The effective amount depends primarily on factors such as the species, age, weight, health, and tissue to be targeted of the subject, and thus can vary between animals and tissues. For example, an effective amount of rAAV is generally in the range of about 1 ml to about 100 ml of a solution containing from about 10 9 ~10 16 genome copies. In some cases, a dosage between about 10 11 ~10 13 rAAV genome copies is appropriate. In certain embodiments, 10 12 or 10 13 rAAV genome copies are effective to target CNS tissue. In some cases, stable transgenic animals are generated by multiple doses of rAAV.

[0267] In some embodiments, the dose of rAAV is administered to the subject at least once per calendar day (e.g., a 24-hour period). In some embodiments, the dose of rAAV is administered to the subject at least once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the dose of rAAV is administered to the subject at least once per calendar week (e.g., 7 calendar days). In some embodiments, the dose of rAAV is administered to the subject at least once every two weeks (e.g., once every two calendar weeks). In some embodiments, the dose of rAAV is administered to the subject at least once per calendar month (e.g., once every 30 calendar days). In some embodiments, the dose of rAAV is administered to the subject at least once every 6 calendar months. In some embodiments, the dose of rAAV is administered to the subject at least once per calendar year (e.g., 365 days or 366 days in a leap year).

[0268] In some embodiments, the rAAV composition is particularly characterized by the presence of high concentrations of rAAV (e.g., -10). 13 When the GC / ml or higher is present, the formulation is designed to reduce the aggregation of AAV particles in the composition. Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of surfactants, pH adjustment, and salt concentration adjustment (see, for example, Wright FR, et al., Molecular Therapy (2005) 12, 171-178). (This content is incorporated herein by reference.)

[0269] Formulations of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as the development of suitable administration and treatment regimens is for the use of the specific compositions described herein in various treatment regimens.

[0270] Typically, these formulations may contain at least about 0.1% or more of the active compound, but the percentage of the active ingredient may, of course, vary and, conveniently, may be between about 1% or 2% and about 70% or 80% or more of the total formulation weight or volume. The amount of the active compound in each therapeutically useful composition can be prepared in such a way that a suitable dosage can be obtained at any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are contemplated by those skilled in the art preparing such pharmaceutical formulations, so that various dosing and treatment regimens may be desirable.

[0271] In certain circumstances, it is desirable to deliver therapeutic constructs based on rAAV in the suitably formulated pharmaceutical compositions disclosed herein by subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, or by oral, intraperitoneal, or inhalation. In some embodiments, rAAV may be delivered using the administration modalities described in U.S. Patents 5,543,158, 5,641,515 and 5,399,363 (each incorporated herein by reference in its entirety). In some embodiments, the preferred mode of administration is by portal vein injection.

[0272] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under normal storage and use conditions, these preparations contain preservatives that prevent microbial growth. In many cases, this form is sterile and fluid enough to be readily injectable. It must be stable under manufacturing and storage conditions and must be protected against microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. For example, suitable fluidity can be maintained by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0273] For the administration of aqueous solutions for injection, for example, the solution may be preferably buffered as needed, and the liquid diluent is first isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection solution, or injected into the planned site of injection (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Several variations in dosage will inevitably arise depending on the host's condition. In any case, the person responsible for administration will determine the appropriate dose for each individual host.

[0274] Sterile injectable solutions are prepared by incorporating active rAAV, along with various other components listed herein, in the required amounts in a suitable solvent, and subsequently sterilizing by filtration, if necessary. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a base dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any additional desired components from a pre-sterilized filtered solution.

[0275] The rAAV compositions disclosed herein may also be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is compatible with the drug formulation and administered in a manner in which such an amount is therapeutically effective. The formulations are readily administered in various dosage forms, such as injectable solutions or drug-release capsules.

[0276] As used herein, “carrier” includes all kinds of solvents, dispersions, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarders, 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. Co-active ingredients may also be incorporated into the composition. The term “pharmaceutically acceptable” means molecular entities and compositions that, when administered to a host, do not produce an allergic reaction or similar adverse reaction.

[0277] Delivery vehicles, such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc., may be used for the introduction of the compositions of this disclosure into suitable host cells. In particular, the transgene delivered by the rAAV vector may be formulated for delivery by being encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles, etc.

[0278] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulating half-life have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patents No. 5,567,434; No. 5,552,157; ​​No. 5,565,213; No. 5,738,868 and No. 5,795,587).

[0279] Liposomes have been successfully used in several cell types that are typically resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints typical of virus-based delivery systems. Liposomes are effectively used to introduce genes, drugs, radiotherapy agents, viruses, transcription factors, and allosteric effectors into various cultured cell lines and animals. Furthermore, several successful clinical trials investigating the efficacy of liposome-mediated drug delivery have been completed.

[0280] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also known as multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayer vesicles (SUVs) with a diameter in the range of 200 to 500 Å, containing an aqueous solution in the core.

[0281] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally capture substances in a stable and renewable manner. To avoid side effects resulting from intracellular polymer overloading, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are intended for use.

[0282] In addition to the delivery methods described above, the following techniques are also intended as alternative methods for delivering rAAV compositions to the host: Sonophoresis (i.e., ultrasound) has been used as a device to enhance the rate and efficiency of drug penetration into and through the circulatory system, as described in U.S. Patent No. 5,656,016. Other intended alternatives for drug delivery include 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. Patents No. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0283] In some embodiments, the methods described herein relate to treating subjects having or diagnosed with a neurological disorder or condition, such as Huntington's disease, with the nucleic acids described herein. Subjects having a neurological disorder or condition, such as Huntington's disease, may be identified by a physician using current methods for diagnosing such diseases and disorders. For example, symptoms and / or complications of Huntington's disease that characterize these conditions and aid in diagnosis include, but are not limited to, depression and anxiety, and are accompanied by characteristic motor impairments and chorea. Tests that may aid in the diagnosis of Huntington's disease include, but are not limited to, genetic testing. A family history of Huntington's disease may also be helpful in determining whether a subject may have Huntington's disease or in making a diagnosis of Huntington's disease.

[0284] The compositions and methods described herein can be administered to subjects having or diagnosed with a neurological disorder or impairment. In some embodiments, the methods described herein include administering an effective amount of the compositions described herein, for example, the nucleic acids described herein, to a subject to alleviate symptoms of a neurological disorder or impairment. As used herein, “alleviate symptoms” means improving any condition or symptom associated with a neurological disorder or impairment. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or higher, as measured by any standard technique.

[0285] Effective dose, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the minimum effective dose and / or maximum tolerated dose. Dosage may vary depending on the dosage form used and the route of administration utilized. The therapeutic effective dose can be initially estimated from cell culture assays. Dosage may also be formulated in animal models to achieve a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dosage can be monitored by suitable bioassays, such as assays for neuronal degradation or functionality, among other things. Dosage is determined by a physician and, if necessary, can be adjusted to match the observed effect of the treatment. Immunomodulator

[0286] In some embodiments, the methods and compositions for treating neurological disorders or conditions described herein further include administering an immunomodulator. In some embodiments, the immunomodulator may be administered at the time of administration of the rAAV vector, before administration of the rAAV vector, or after administration of the rAAV vector.

[0287] In some embodiments, the immunomodulator is an immunoglobulin-degrading enzyme such as IdeS, IdeZ, IdeS / Z, Endo S, or functional variants thereof. Non-limiting examples of reference literature for such immunoglobulin-degrading enzymes and their uses are described in US Nos. 7,666,582, US No. 8,133,483, US No. 20180037962, US No. 20180023070, US No. 20170209550, US No. 8,889,128, WO2010057626, US No. 9,707,279, US No. 8,323,908, US No. 20190345533, US No. 20190262434, and WO2020016318, each of which is incorporated by reference in whole.

[0288] In some embodiments, the immunomodulator is a proteasome inhibitor. In certain embodiments, the proteasome inhibitor is bortezomib. In some embodiments, the immunomodulator comprises bortezomib and the anti-CD20 antibody rituximab. In other embodiments, the immunomodulator comprises bortezomib, rituximab, methotrexate, and intravenous gamma globulin. Non-limiting examples of such references disclosing proteasome inhibitors, as well as their combinations with rituximab, methotrexate, and intravenous gamma globulin, are described in US No. 10,028,993, US No. 9,592,247, and US No. 8,809,282, each of which is incorporated by reference in whole.

[0289] In alternative embodiments, the immunomodulator is an inhibitor of the NF-κB pathway. In certain aspects of the embodiments, the immunomodulator is rapamycin or a functional variant. Non-limiting examples of references disclosing rapamycin and its use are US Nos. 10,071,114, US Nos. 20160067228, US Nos. 20160074531, US Nos. 20160074532, US Nos. 20190076458 and US Nos. 10,046,064, which are incorporated herein by reference in their entirety. In other aspects of the embodiments, the immunomodulator is a synthetic nanocarrier containing an immunosuppressant. Immunosuppressants, immunosuppressants coupled to synthetic nanocarriers, synthetic nanocarriers including rapamycin, and / or immunotolerogenic synthetic nanocarriers, non-limiting examples of reference literature for their doses, administration and use include US20150320728, US20180193482, US20190142974, US20150328333, US20160243253, US10,039,822, US20190076522, US20160022650, US10,441,651, US10 These are listed in US20,835, US20150320870, US2014035636, US10,434,088, US10,335,395, US20200069659, US10,357,483, US20140335186, US10,668,053, US10,357,482, US20160128986, US20160128987, US20200038462, and US20200038463, each of which is incorporated by reference as a whole.

[0290] In some embodiments, the immunomodulator is a synthetic nanocarrier containing rapamycin (ImmTOR® nanoparticles) disclosed in US Patent No. 20200038463 and US Patent No. 9,006,254 (Kishimoto, et al., 2016, Nat Nanotechnol, 11(10): 890-899; Maldonado, et al., 2015, PNAS, 112(2): E156-165), and Each of these is incorporated herein in whole. In some embodiments, the immunomodulator is an engineered cell, for example, an immune cell modified using the SQZ technique disclosed in WO2017192786, which is incorporated herein in whole by reference.

[0291] In some embodiments, the immunomodulator is poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide The immunomodulator or adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, reciquimod, SRL172, virosoms and other virus-like particles, YF-17D, VEGF traps, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon. In another further embodiment, the immunomodulator or adjuvant is poly-ICLC.

[0292] In some embodiments, the immunomodulator is a small molecule that inhibits the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and 2-aminopurine (a PKR inhibitor), and can be administered in combination with a composition comprising at least one rAAV disclosed herein. Some non-limiting examples of commercially available TLR signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available from INVIVOGEN®). In addition, inhibitors of pattern recognition receptors (PRRs) (involved in innate immune signaling), such as 2-aminopurine, BX795, chloroquine, and H-89, can also be used in compositions and methods comprising at least one rAAV vector disclosed herein for in vivo protein expression disclosed herein.

[0293] In some embodiments, the rAAV vector may also encode a negative regulator of innate immunity, such as NLRX1. Therefore, in some embodiments, the rAAV vector may optionally encode one or more of NLRX1, NS1, NS3 / 4A, or A46R, or any combination thereof. In addition, in some embodiments, a composition comprising at least one rAAV vector disclosed herein may also include a synthetically modified RNA encoding an inhibitor of the innate immune system to evade the innate immune response induced by a tissue or subject.

[0294] In some embodiments, the immunomodulator for use in the administration methods disclosed herein is an immunosuppressant. As used herein, the term “immunosuppressant” is intended to include pharmaceuticals that inhibit or interfere with normal immune function. Examples of immunosuppressants suitable for the methods disclosed herein include agents that inhibit T-cell / B-cell costimulation pathways, such as agents that interfere with T-cell and B-cell coupling via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Publication No. 2002 / 0182211. In one embodiment, the immunosuppressant is cyclosporine A. Other examples include myophenylate mofetil, rapamycin, and antithymocyte globulin. In one embodiment, the immunosuppressant is administered in a composition comprising at least one rAAV vector disclosed herein, or in separate compositions, which may be administered simultaneously with, before, or after, the administration of the composition comprising at least one rAAV vector, according to the administration methods disclosed herein. Immunosuppressants are administered in formulations suitable for the route of administration and in doses sufficient to achieve the desired therapeutic effect. In some embodiments, immunosuppressants are administered transiently for a period of time sufficient to induce tolerance to the rAAV vector disclosed herein.

[0295] In any embodiment of the methods and compositions disclosed herein, subjects administered with the rAAV vector or rAAV genome disclosed herein are also administered with an immunosuppressant. Various methods are known to result in immunosuppression of the immune response in patients administered with AAV. Methods known in the art include administering an immunosuppressant, such as a proteasome inhibitor, to the patient. For example, one such proteasome inhibitor known in the art, disclosed in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15 / 796,137, both incorporated herein by reference, is bortezomib. In some embodiments, the immunosuppressant may be an antibody comprising a polyclonal antibody, a monoclonal antibody, scfv, or other antibody-derived molecule, which can suppress the immune response, for example, by eliminating or suppressing antibody-producing cells. In further embodiments, the immunosuppressive element may be a small hairpin RNA (shRNA). In such embodiments, the coding region of the shRNA is contained in the rAAV cassette and is generally located downstream of the 3' of the poly-A tail. shRNAs can be targeted to reduce or eliminate the expression of immunostimulants such as cytokines and growth factors (including transforming growth factors β1 and β2, TNF, and others known).

[0296] The use of such immunomodulators facilitates the ability for individuals to use multiple doses (e.g., multiple administrations) over months and / or years. This allows for the use of multiple drugs, e.g., multiple genes encoding rAAV vectors, or multiple administrations to a subject, as discussed below. kit

[0297] In one embodiment, the disclosure relates to a nucleic acid or recombinant viral vector comprising (i) one or more inhibitory nucleic acids (e.g., miRNAs) and (ii) a nucleic acid encoding a CYP46A1 protein. In one embodiment, the disclosure relates to a combination of (i) one or more inhibitory nucleic acids (e.g., miRNAs) and (ii) a nucleic acid encoding a CYP46A1 protein. In the combination of (i) and (ii), two or more elements may be provided in a mixture or a single formulation. Alternatively, two or more elements may be provided in separate formulations packaged or provided as a set or kit.

[0298] The agents described herein, for example, viral vectors, may, in some embodiments, be assembled into a pharmaceutical kit or a diagnostic kit or research kit to facilitate their use in therapeutic, diagnostic, or research applications. The kit may include one or more containers containing the components of this disclosure and instructions for use. Specifically, such a kit may include one or more agents described herein, together with instructions describing the intended application and the proper use of these agents. In certain embodiments, the agents in the kit may be present in a pharmaceutical formulation and may be in dosages suitable for a particular application and method of administering the agent. A research kit may contain components in concentrations or amounts suitable for performing a variety of experiments.

[0299] In some embodiments, the Disclosure relates to a kit for generating rAAV, where the kit is a) Isolated nucleic acids containing miRNAs, for example, miRNAs containing, or encoding, the sequence shown in any one of sequence numbers 6-17, 40-44, or 50-66, or miRNAs containing a seed sequence complementary to sequence numbers 4, 18-39, or 46-49; b) A recombinant viral vector comprising isolated nucleic acids containing a transgene encoding one or more miRNAs, for example, wherein each miRNA contains a seed sequence complementary to SEQ ID NO: 4, or each miRNA contains a sequence adjacent to the miRNA backbone sequence, represented by one of SEQ ID NOs: 6-17, 40-44, or 50-66. Recombinant viral vectors; c) Recombinant viral vectors containing isolated nucleic acids encoding the CYP46A1 protein; and / or d) A recombinant viral vector comprising nucleic acids containing a transgene encoding one or more miRNAs, For example, a recombinant viral vector in which each miRNA contains a seed sequence complementary to SEQ ID NO: 4, or each miRNA contains a sequence shown in one of SEQ ID NOs: 6-17, 40-44, or 50-66 adjacent to the miRNA backbone sequence; and nucleic acid encoding the CYP46A1 protein The present invention relates to a kit comprising a container for containing one or more of the following. In some embodiments, the kit further comprises a container for containing an isolated nucleic acid encoding an AAV capsid protein, for example, the AAV9 capsid protein.

[0300] Kits may be designed to facilitate researchers' use of the methods described herein and may take many forms. Each of the components of a kit may be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), where applicable. In certain cases, parts of the components may be constitutable or otherwise processable (e.g., into an active form) by the addition of a suitable solvent or other kind (e.g., water or cell culture medium), which may or may not be provided with the kit. Where used herein, “instructions” may define components of instructions and / or promotions, and typically relate to written instructions for or relating to the packaging of this disclosure. Instructions may also include any oral or electronic instructions provided in any format that clearly recognizes to the user that the instructions relate to a kit, e.g., audiovisual communication (e.g., videotape, DVD, etc.), internet communication, and / or web-based communication. The written instructions may be in a form prescribed by the government agency that regulates the manufacture, use, or sale of the pharmaceutical or biological product, and these instructions may also reflect approval by the agency for manufacture, use, or sale for administration to animals.

[0301] The kit may contain 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 and mixing samples, and for application to a subject. The kit may include a container for containing the drug described herein. The drug may be in liquid, gel, or solid (powder) form. The drug may be sterilely prepared, packaged in syringes, and transported under refrigeration. Alternatively, it may be contained in vials or other containers for storage. A second container may contain other sterilely prepared drugs. Alternatively, the kit may contain active drugs that are premixed and transported in syringes, vials, tubes, or other containers.

[0302] Exemplary embodiments of the present invention are described in more detail by the following examples. These embodiments are illustrative of the present invention, and those skilled in the art will recognize that they are not limited to the exemplary embodiments. definition

[0303] For convenience, the meanings of some terms and phrases used herein, in the examples and in the appended claims are provided below. Unless otherwise specified or implied by the context, the following terms and phrases have the meanings provided below. The definitions are provided to help describe specific embodiments and are not intended to limit the invention described in the claims, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they would ordinarily understood by those skilled in the art to which the invention pertains. In the event of any apparent conflict between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0304] For convenience, certain terms used herein in this specification, the examples, and the appended claims are set forth herein.

[0305] The terms “reduce,” “reduced,” “reduce,” or “inhibit” are all used herein to mean a reduction of a statistically significant amount. In some embodiments, “reduce,” “reduce,” or “reduce” or “inhibit” typically mean a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or drug), and may include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater reductions. As used herein, “reduce” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction may preferably be a decrease to a level that is acceptable as being within the normal range for an individual without a given disorder.

[0306] The terms “increased,” “increased,” “enhanced,” or “activated” are all used herein to mean an increase of a statistically significant amount. In some embodiments, the terms “increased,” “increased,” “enhanced,” or “activated” may mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% compared to a reference level, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level, or any increase between 2 times and 10 times or a larger multiple. In the context of markers or symptoms, “increase” is a statistically significant increase of such a level.

[0307] As used herein, “subject” means human or animal. Typically, animals are vertebrates such as primates, rodents, domestic animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, and wolves, bird species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0308] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals can be advantageously used as subjects representing animal models of Huntington's disease. The subject may be male or female.

[0309] The subjects may be those who have been previously diagnosed with, or have been identified as having, a condition requiring treatment (e.g., Huntington's disease) or one or more complications associated with such a condition, and who, if necessary, have already received treatment for the condition or one or more complications associated with the condition. Alternatively, the subjects may also not have been previously diagnosed with the condition or one or more complications associated with the condition. For example, a subject may exhibit one or more risk factors for the condition or one or more complications associated with the condition, or may not exhibit any risk factors.

[0310] A person who "requires treatment" for a specific condition may be someone who has that condition, has been diagnosed with that condition, or is at risk of developing that condition.

[0311] As used herein, the terms “protein” and “polypeptide” are used interchangeably to refer to a set of amino acid residues linked to one another by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms “protein” and “polypeptide” refer to polymers of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used in reference to relatively large polypeptides, the term “peptide” is often used in reference to smaller polypeptides; however, the use of these terms overlaps in the art. The terms “protein” and “polypeptide” are used interchangeably herein when referring to gene products and their fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0312] A variant amino acid or DNA sequence may be identical to the native or reference sequence to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher. The degree of homology (percentage of identity) between a native sequence and a mutant sequence can be determined, for example, by comparing the two sequences using a free computer program commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn using default settings).

[0313] Modification of the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at a specific locus, for example, by synthesizing an oligonucleotide containing a mutant sequence adjacent to a restriction site that allows ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, a modified nucleotide sequence having a specific codon altered by the required substitution, deletion, or insertion can be provided using oligonucleotide-directed site-specific mutagenesis procedures. Techniques for making such modifications are very well established, for example, Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462. Examples of such disclosures are included herein by reference. Any cysteine ​​residues that do not participate in maintaining the proper conformation of the polypeptide can also be replaced with serine in general to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine ​​bonds can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0314] As used herein, the terms “nucleic acid” or “nucleic acid sequence” refer to any molecule, preferably a polymer molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one embodiment, the nucleic acid can be DNA. In another embodiment, the nucleic acid can be RNA. Suitable DNAs may include, for example, genomic DNA or cDNA. Suitable RNAs may include, for example, mRNA and miRNA.

[0315] In some embodiments of any of the features described herein, the polypeptides, nucleic acids, or cells described herein can be engineered. As used herein, “engineered” refers to an embodiment that has been manipulated by human hands. For example, a polypeptide is considered “engineered” if at least one embodiment of the polypeptide, for example, its sequence, has been manipulated by human hands to be different from the embodiment in which it exists naturally. As is common practice and as understood by those skilled in the art, the offspring of an engineered cell are typically still referred to as “engineered” even if the actual manipulation has been previously performed on the entity.

[0316] A variant amino acid or DNA sequence may be identical to the native or reference sequence to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher. The degree of homology (percentage of identity) between a native sequence and a mutant sequence can be determined, for example, by comparing the two sequences using a free computer program commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn using default settings).

[0317] In some embodiments of the model, the miRNAs described herein are exogenous. In some embodiments of the model, the miRNAs described herein are ectopic. In some embodiments of the model, the miRNAs described herein are not endogenous.

[0318] The term “exogenous” refers to a substance present in a cell other than its native origin. As used herein, “exogenous” may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide introduced into a biological system, such as a cell or organism, by a process involving human intervention, where the nucleic acid or polypeptide is not normally found and the aim is to introduce it into such a cell or organism. Alternatively, “exogenous” may refer to a nucleic acid or polypeptide introduced into a biological system, such as a cell or organism, by a process involving human intervention, where the aim is to increase the amount of nucleic acid or polypeptide in the cell or organism, for example, to create ectopic expression or a specific level. In contrast, the term “endogenous” refers to a substance that is native to a biological system or cell. As used herein, “ectopic” refers to a substance found in an unusual location and / or quantity. An ectopic substance may be one that is normally found in a given cell but is found in very low amounts and / or at different times. Ectopic also includes substances such as polypeptides or nucleic acids that are not found naturally or are not expressed in a given cell in its natural environment.

[0319] As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or nonviral. The term “vector” encompasses any genetic element that is replicable when associated with appropriate regulatory elements and can transfer a gene sequence into a cell. Examples of vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, chromosomes, viruses, and virions.

[0320] In some embodiments of any aspect, the vector is recombinant and, for example, comprises a sequence originating from at least two different origins. In some embodiments of any aspect, the vector comprises a sequence originating from at least two different species. In some embodiments of any aspect, the vector comprises a sequence originating from at least two different genes and, for example, comprises a nucleic acid encoding an expression product operably linked to a fusion protein or at least one non-native (e.g., heterologous) genetic regulatory element (e.g., promoter, suppressor, activator, enhancer, response element, etc.).

[0321] In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, for example, the native or wild-type sequence of the nucleic acid sequence is modified or engineered to include alternative codons so that the modified or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence but is transcribed and / or translated with improved efficiency in the desired expression system. In some embodiments of any aspect, the expression system is an organism other than the source of the native / wild-type sequence (or cells obtained from such an organism). In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells, e.g., mice, mouse cells, or human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any of the features, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in E. coli cells.

[0322] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptides from a sequence ligated to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often heterogeneous to the cell, though not necessarily so. An expression vector may contain additional elements; for example, an expression vector may have two replication systems, thus enabling it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification.

[0323] As used herein, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. Viral vectors may contain nucleic acids encoding polypeptides described herein, instead of non-essential viral genes. Vectors and / or particles may be used for the purpose of transferring any nucleic acid into cells, either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0324] It should be understood that the vectors described herein can be combined with other suitable compositions and treatments in some embodiments. In some embodiments, the vector is an episome. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of integration into the chromosome.

[0325] As used herein, the terms “to treat,” “treatment,” “to treat,” or “improve” refer to a therapeutic treatment whose purpose is to reverse, reduce, improve, inhibit, slow or halt the progression or severity of a disease or disorder, for example, a condition associated with Huntington's disease. The term “to treat” includes reducing or mitigating at least one adverse effect or symptom of a condition, disease or disorder. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is “effective” if the progression of the disease is reduced or interrupted. That is, “treatment” includes not only improvement of symptoms or markers but also cessation, or at least slowing, the progression or worsening of symptoms compared to what would be expected in the absence of the treatment. Beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, whether detectable or undetectable; a decrease in the severity of the disease; a stable (i.e., non-worsening) state of the disease; a delay or slowing of disease progression; improvement or mitigation of the disease state; remission (whether partial or whole); and / or a reduction in mortality. The term “treatment” of the disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

[0326] As used herein, the term “pharmaceutical composition” refers to an active agent in combination with a pharmaceutically acceptable carrier, e.g., a carrier commonly used in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean these compounds, materials, compositions, and / or dosage forms that, within the bounds of reasonable medical judgment, are suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are appropriate for a reasonable benefit / risk ratio. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticles, and / or ointment. In some embodiments of any aspect, the pharmaceutically acceptable carrier may be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found in nature.

[0327] As used herein, the term “administer” refers to the placement of a compound disclosed herein onto a subject by a method or route that results in at least partial delivery of the drug to a desired site. A pharmaceutical composition comprising a compound disclosed herein may be administered by any suitable route that results in effective treatment on the subject. In some embodiments, administration includes physical human activity, such as injection, ingestion, application, and / or operation of a delivery device or machine. Such activity may be performed, for example, by a healthcare professional and / or the subject being treated.

[0328] As used herein, “contact” refers to any suitable means for delivering or exposing a drug to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to a cell culture medium, perfusion, injection, or other delivery methods well known to those skilled in the art. In some embodiments, contact includes physical human activity, such as injection; dispensing, mixing, and / or decanting; and / or operation of a delivery device or machine.

[0329] The terms "statistically significant" or "significantly significant" refer to statistical significance, which generally means a difference of two standard deviations (2SD) or greater.

[0330] Except as in the operating examples, or where otherwise shown, all numbers representing quantities of ingredients or reaction conditions used herein should be understood to be modified in all examples by the term “approximately.” When used in conjunction with percentages, the term “approximately” may mean ±1%.

[0331] As used herein, the term “comprising” means that, in addition to the defined element expressed, other elements may also be present. The use of “comprising” indicates inclusion rather than limitation.

[0332] The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding all elements not listed in the description of the embodiments.

[0333] As used herein, the term "consisting essentially of" refers to these elements required for a given embodiment. The term allows for the presence of additional elements that do not substantially affect the basic and novel or functional features of the embodiments of the invention.

[0334] As used herein, the term "corresponding to" refers to an amino acid or nucleotide that is equivalent to an amino acid or nucleotide at a listed position in the first polypeptide or nucleic acid, or an amino acid or nucleotide listed in the second polypeptide or nucleic acid. Equivalent listed amino acids or nucleotides can be determined by alignment of candidate sequences using homology programs known in the art, such as the degree of BLAST.

[0335] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells, and / or particles in which a first entity binds to a second entity, targeting that entity with higher specificity and affinity than it would if it bound to a third, non-target entity. In some embodiments, specific binding may refer to the affinity of the first entity to a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or higher than its affinity to a third, non-target entity. A reagent specific to a given target is a reagent that exhibits specific binding to that target under the conditions of the assay being used.

[0336] The singular terms “a,” “an,” and “the” refer to multiple objects unless the context otherwise explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context otherwise explicitly indicates otherwise. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. The abbreviation “e.g.” (eg) is derived from the Latin “exempli gratia” (for example) and is used herein to indicate a non-restrictive example. Thus, the abbreviation “e.g. (eg)” is synonymous with the term “for example.”

[0337] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each member of each group may be referred to and described in the claims individually or in any combination with other members of that group or other elements found herein. One or more members of a group may be included in or removed from a group for convenience and / or patentability reasons. In the event of any such inclusion or removal, this specification shall be deemed to contain the modified group and thus satisfy the written description of all Markush groups used in the appended claims.

[0338] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those skilled in the art in which this disclosure pertains. It should be understood that the present invention is not limited to, but is itself modifiable, the specific methodologies, protocols, and reagents described herein. Technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology are published in The Merck Manual of Diagnosis and Therapy, 20th Edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive According to Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) Published; Immunology by Werner Luttmann, Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WW Norton & Company, 2016 (ISBN 0815345054, 978-0815345053);Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X);Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); 2014 (ISBN 047150338X, 9780471503385) Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737)、WO20 The contents of these publications can be found in issues 18 / 057855A and US10,457,940, and the contents of each of these are incorporated herein by reference in their entirety.

[0339] In any part of the embodiments described herein, the disclosures described herein do not relate to processes for cloning humans, processes for altering the genetic identity of human germline cells, the use of human embryos for industrial or commercial purposes, or processes for altering the genetic identity of animals that may cause disease in animals without any substantial medical benefit to humans or animals, or animals obtained from such processes.

[0340] Other terms are defined herein within the descriptions of various aspects of the present invention.

[0341] All patents and other publications cited throughout this application, including references, issued patents, published patent applications, and concurrently pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methodologies described in such publications, for example, which may be used in conjunction with the technology described herein. These publications only provide their disclosures prior to the filing date of this application. This should not be construed as an admission by the inventors that they are not entitled to precede such disclosures by prior art or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on information available to the applicants and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0342] The descriptions of embodiments of this disclosure are not intended to be exclusive or to limit the disclosure to the exact form disclosed. Specific embodiments of this disclosure and examples thereof are described herein for illustrative purposes, and various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. For example, while the steps or functions of a method are described in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of this disclosure provided herein may be applied to other procedures or methods as needed. Further embodiments may be provided by combining the various embodiments described herein. Aspects of this disclosure may be modified, as needed, to use the compositions, functions and concepts of the above-mentioned references and applications to provide even further embodiments of this disclosure. These and other modifications may be made to this disclosure with respect to the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0343] Specific elements of any of the embodiments described above can be combined with or substituted for elements in other embodiments. Furthermore, while advantages related to certain embodiments of the present disclosure are described in light of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments are required to demonstrate such advantages in order to be included within the scope of the present disclosure.

[0344] The techniques described herein are further illustrated by the following examples, which should not be construed as further limitations.

[0345] Some embodiments of the technology described herein can be defined by any of the following numbered paragraphs. [Examples]

[0346] (Example 1) In one embodiment, an inhibitory RNA that can be used for the treatment of Huntington's disease is described herein. In some embodiments of any of the embodiments, the nucleic acid sequence of the inhibitory RNA includes one of the sequences that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to at least one of the sequences of SEQ ID NOs: 6-17, 40-44, or 50-66, or that maintains the same function as SEQ ID NOs: 6-17, 40-44, or 50-66 (e.g., HTT inhibition).

[0347] Constructs containing artificial miRNAs are described herein. The pEMBL-D(+)-Syn1-hCG intron is a control vector, which has an empty human chorionic gonadotropin (hCG) intron (hCGin) inserted and is driven by a synapsin promoter. Two copies of a control miRNA precursor (random sequence or non-functional mutant) are inserted into hCGin in the vector pEMBL-D(+)-Syn1-hCGin-2x control pre-miR. Two copies of an artificial pre-miR (containing approximately 100-150 bp of adjacent upstream and downstream sequences, perfectly matching the 3'-UTR targeting sequence) are cloned between the hCG introns. The vector pEMBL-D(+)-Syn1-CYP46A1-hCGin-2x artificial pre-miR is a combo construct that can produce both CYP46A1 and artificial miRNA simultaneously. To determine whether the pre-miRNA can be processed into a mature miRNA and combined with an HTT-targeting sequence containing a CAG extension that is fully complementary to the mature miRNA, it is inserted after the luciferase gene. Due to package size limitations, small polyA is used in the construct. Syn1 can be replaced by one or more of the following: a CMV enhancer and / or an ACTB proximal promoter and / or a chimeric ACTB-HBB2 intron, as well as a synthetic nervous system-specific promoter or fragment thereof selected from Tables 10-13 and / or an enhancer, and / or a cis-regulatory element (CRE) selected from Tables 13-15.

[0348] The following sequences are known in the art: pEMBL; synapsin promoter (Syn1); ITR (e.g., derived from AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13); hCG intron; small polyA; CYP46A1; luciferase; HTT targeting sequence; and / or HTT-3'UTR mutant.

[0349] Synapsin-1 (Syn1) is a member of the synapsin gene family. Synapsins encode neuronal phosphoproteins that associate with the cytoplasmic surface of synaptic vesicles. Members of the family are characterized by common protein domains that are involved in the modulation of synapse formation and neurotransmitter release, suggesting potential roles in several neuropsychiatric disorders. Syn1 plays a role in regulating axon formation and synapse formation. The Syn1 protein acts as a substrate for several different protein kinases, and phosphorylation may function in regulating this protein at nerve terminals. Mutations in this gene may be associated with X-linked disorders, primarily neurodegenerative, such as Rett syndrome. Alternatively, spliced ​​transcript variants encoding different isoforms have been identified. In some embodiments of the model, the Syn1 promoter may include the human promoter Syn1 (see, for example, the Syn1 promoter associated with NCBI reference number NG_008437.1 RefSeqGene range 5001~52957;NM_006950.3;NP_008881.2;NM_133499.2;NP_598006.1).

[0350] CYP46A1 is a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism, as well as the synthesis of cholesterol, steroids, and other lipids. This endoplasmic reticulum protein is expressed in the brain, where it converts cholesterol to 24S-hydroxycholesterol. Cholesterol cannot cross the blood-brain barrier, but 24S-hydroxycholesterol can be secreted into circulation in the brain and returned to the liver for catabolism. In some embodiments of any aspect, CYP46A1 may include human CYP46A1 (see, for example, NCBI reference number NG_007963.1 RefSeqGene range 4881~47884; NM_006668.2; NP_006659.1). CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease (see, for example, Boussicault et al., CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease, Brain. 2016 Mar, 139(Pt 3):953-70; Kacher et al., CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease, Brain. 2019 Aug 1;142(8):2432-2450; the contents of each of these are incorporated herein by reference in their entirety).

[0351] In some embodiments of any aspect, the miRNA includes a sequence complementary to at least two consecutive bases (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) of the sequence shown in Sequence ID No. 3 or 4 adjacent to the miRNA backbone sequence. In some embodiments of the model, the miRNA includes a sequence complementary to at least two consecutive bases (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) of sequences of untranslated regions (e.g., 5'UTR, 3'UTR), exons, CAG repeats, or CAG jumpers (e.g., CAG5' jumper, CAG3' jumper) adjacent to the miRNA backbone sequence and related to HTT (e.g., NCBI gene ID: 3064; see, e.g., SEQ ID NO: 4).

[0352] Isolated nucleic acids encoding transgenes encoding one or more miRNAs and isolated nucleic acids encoding the CYP46A1 protein, when administered to the same patient, may provide an improved therapeutic effect than either being administered alone. Isolated nucleic acids encoding transgenes encoding one or more miRNAs and isolated nucleic acids encoding the CYP46A1 protein, when administered to the same patient, may provide a synergistic (rather than additive) improved therapeutic effect than either being administered alone. Isolated nucleic acids encoding transgenes encoding one or more miRNAs and isolated nucleic acids encoding the CYP46A1 protein may be administered to a subject sequentially or simultaneously according to any of the methods described herein. rAAV containing the CYP46A1 variant CDS (as shown in SEQ ID NO: 110) is expected to provide a better therapeutic effect in treating neurological disorders, such as Huntington's disease, than, for example, administration of rAAV containing the CYP46A1 non-variant sequence shown in SEQ ID NO: 1. Similarly, rAAVs containing miRNAs (e.g., one or more selected from SEQ ID NOs: 6-17 or 40-44 or 50-66) are expected to provide better therapeutic effects in treating neurological disorders, such as Huntington's disease, when administered with a CYP46A1 variant CDS (as shown in SEQ ID NO: 110) than when administered with a CYP46A1 non-variant sequence (as shown in SEQ ID NO: 1).

[0353] [ka] [ka]

[0354] [ka] [ka] [ka] [ka] [ka] [ka]

[0355] [ka] [ka] [ka]

[0356] [ka]

[0357] [ka] (Example 2)

[0358] The synthetic NS-specific promoter according to the present invention was designed by reviewing scientific literature to identify genes that are highly active in NS cells and their individual promoters.

[0359] During the design of these promoters, we considered the specific shortcomings of known NS-specific promoters. Firstly, known NS-specific promoters that are specific to NS cell types (e.g., synapsin-1, CAMKIIa, and GFAP) are not expressed in the entire cell population (e.g., not in all neurons / astrocytes). This has been shown for GFAP by (Zhang et al., 2019), and A This can be seen from the distribution of Syn-1 in neurons from the Illen brain atlas. Secondly, most known CREs, promoter elements, and promoters are too large to be included in self-complementary AAV vectors (scAAVs) (depending on the size of the transgene, the promoter size may need to be less than 1000 bp, preferably less than 900 bp, more preferably less than 800 bp, and most preferably less than 700 bp). In addition, expression may be required throughout the NS, preferably throughout the CNS or throughout the brain, in specific cell types or combinations of cell types.

[0360] Currently known promoters cannot address these shortcomings, and there is a need in gene therapy to develop short, cell-type NS-specific promoters that have targeted, localized expression as well as expression across the entire NS. For example, the requirement for expression across the entire NS (e.g., the entire brain) is revealed by the expression patterns of the HTT (huntingtin) and CYP46A1 genes in the brain of adult mice, shown in Figures 6A and 6B. Since the HTT (huntingtin) gene is expressed throughout the brain, this could be beneficial for any potential expression product to suppress the defective huntingtin gene and / or to weaken or mitigate the harmful effects of defective huntingtin expressed throughout the brain. Similarly, since the CYP46A1 gene is expressed throughout the brain, this could be beneficial for any potential complementary CYP46A1 expression across the brain.

[0361] Gene expression in all neurons throughout the CNS, as well as in astrocytes and / or oligodendrocytes, may be desirable in the treatment of certain diseases, such as Huntington's disease. Expression in astrocytes and oligodendrocytes may be beneficial because glial cells are involved in Huntington's disease (Shin et al., 2005).

[0362] Accordingly, the present invention demonstrates the design of a tandem NS promoter that is active in multiple NS cell types while addressing some of the drawbacks listed above. For example, the promoter design included a combination of one or more CREs together with the promoter element to broaden cytotropy compared to individual CRE / promoter elements, in order to produce a promoter active in multiple NS cell types and to address the drawback that known promoters are not expressed in the entire cell population. In addition, to address the drawback of known CREs, promoter elements, and promoters that are too large to be included in AAV vectors such as self-complementary AAV vectors (scAAV), some of the CREs and promoter elements of the present invention were shortened using bioinformatics analysis, literature searches, and published genome databases, although these are still expected to be active CREs and promoter elements.

[0363] The synthetic NS-specific promoter according to the present invention is operably ligated to the nucleic acid sequences encoding the CYP46A1 transgene and the human influenza hemagglutinin (HA) tag, and experimentally tested in wild-type C57BL6 / J mice. The synthetic NS-specific promoter according to the present invention, operably ligated to the nucleic acid sequences encoding the CYP46A1 transgene and the HA tag, is administered intravenously in a viral vector. The vector copy number is evaluated in brain and spinal cord tissue sections by qPCR analysis of the viral transgene CYP46A1, normalized to an internal genomic DNA copy number control, to confirm equivalent injection doses. Western blotting is performed to evaluate protein expression of the HA-tagged transgene in brain and spinal cord tissue. Finally, immunofluorescence staining is performed on brain and spinal cord tissue sections to evaluate transgene expression within CNS cell types. Similarly, immunofluorescence staining can be performed on PNS tissue sections to evaluate transgene expression within PNS cell types. Specifically, double staining is performed using an HA tag that marks CYP46A1 gene expression, as well as standard markers for neurons, astrocytes, oligodendrocytes, and microglia.

[0364] SP0013 (SEQ ID NO: 74) is predicted to be active in neurons and astrocytes. SP0014 (SEQ ID NO: 75) is predicted to be active in neurons and astrocytes. SP0026 (SEQ ID NO: 76) is predicted to be active in excitatory neurons and astrocytes. SP0027 (SEQ ID NO: 77) is predicted to be active in excitatory neurons and astrocytes. SP0030 (SEQ ID NO: 78) is predicted to be active in neurons and astrocytes. SP0031 (SEQ ID NO: 79) is predicted to be active in neurons and astrocytes. SP0032 (SEQ ID NO: 80) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0033 (SEQ ID NO: 81) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0019 (SEQ ID NO: 82) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0020 (SEQ ID NO: 83) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0021 (SEQ ID NO: 84) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0022 (SEQ ID NO: 85) is predicted to be active in neurons, astrocytes, and oligodendrocytes. SP0028 (SEQ ID NO: 86) is predicted to be active in excitatory neurons, astrocytes, and oligodendrocytes. SP0029 (SEQ ID NO: 87) is predicted to be active in excitatory neurons, astrocytes, and oligodendrocytes. SP0011 (SEQ ID NO: 88) is predicted to be active in neurons and astrocytes. SP0034 (SEQ ID NO: 89) is predicted to be active in neurons and astrocytes. SP0035 (SEQ ID NO: 90) is predicted to be active in neurons and astrocytes. SP0036 (SEQ ID NO: 154) is predicted to be active in neurons and astrocytes.

[0365] Bioinformatics analysis of RNA sequencing data predicts that some of the genes associated with the CRE and / or promoter elements of the present invention (aqp4, cend1, eno2, gfap, s100B, syn1) will be expressed in the dorsal root ganglia and tibial nerve. Therefore, the CRE and / or promoter elements associated with these genes are predicted to be expressed in PNS cells. CRE0001_S100B (SEQ ID NO: 106), CRE0002_S100B (SEQ ID NO: 108), CRE0005_GFAP (SEQ ID NO: 103), CRE0007_GFAP (SEQ ID NO: 104), CRE0009_S100B (SEQ ID NO: 107), CRE0006_GFAP (SEQ ID NO: 99), CRE0008_GFAP (SEQ ID NO: 100), CRE0006_AQP4 (SEQ ID NO: 101), CRE0008_AQP4 (SEQ ID NO: 102), or their functional variants are predicted to be active in PNS cells.

[0366] Bioinformatics analysis of single-cell RNA sequencing data predicts that some of the genes associated with the CRE and / or promoter elements of the present invention (aqp4, cend1, eno2, gfap, s100B, syn1) will be expressed in sensory neurons, PNS sympathetic neurons, and PNS enteric neurons. Therefore, the CRE and / or promoter elements associated with these genes are predicted to be expressed in sensory neurons, PNS sympathetic neurons, and PNS enteric neurons. CRE0001_S100B (SEQ ID NO: 106), CRE0002_S100B (SEQ ID NO: 108), CRE0005_GFAP (SEQ ID NO: 103), CRE0007_GFAP (SEQ ID NO: 104), CRE0009_S100B (SEQ ID NO: 107), CRE0006_GFAP (SEQ ID NO: 99), CRE0008_GFAP (SEQ ID NO: 100), CRE0006_AQP4 (SEQ ID NO: 101), CRE0008_AQP4 (SEQ ID NO: 102), or their functional variants are predicted to be active in sensory neurons, PNS sympathetic neurons, and / or PNS enteric neurons. (Example 3)

[0367] A method for producing a viral vector from Pro10 / HEK293 cells that have been engineered to stably incorporate the CYP46A1 gene is described herein.

[0368] The stable cell line Pro10 / HEK293, described in U.S. Patent No. 9,441,206, is ideal for the scalable generation of AAV vectors. This cell line can be contacted with expression vectors containing the CYP46A1 gene, or a variant thereof, operably ligated to any NS-specific promoter as described herein, for example, in Tables 10-15. Clonal populations having CYP46A1 incorporated into their genomes are selected using methods well known in the art. Pro10 / HEK293 cells stably containing the CYP46A1 gene are transfected with packaging plasmids encoding Rep2 and serotype-specific Cap2:AAV-Rep / Cap, as well as an Ad helper plasmid (XX680: encoding an adenovirus helper sequence).

[0369] Transfection. On the day of transfection, count the cells using a ViCell XR Viability Analyzer (Beckman Coulter) and dilute them for transfection. To mix the transfection cocktail, add the following reagents to a conical tube in this order: plasmid DNA, OPTIMEM® I (Gibco) or OptiPro SFM (Gibco), or other serum-free compatible transfection medium, and then the transfection reagent in a specific ratio to the plasmid DNA. Mix the cocktail by inverting it and incubate at room temperature. Pipette the transfection cocktail into a flask and return it to the shaker / incubator. Perform all optimization studies in a 30 mL culture volume, followed by validation in a larger culture volume. Harvest the cells 48 hours after transfection.

[0370] rAAV generation using a Wave bioreactor system. Seeds are seeded into wave bags two days before transfection. Two days after seeding into wave bags, a count of cell cultures is obtained, and the cell cultures are then grown / diluted before transfection. The cell cultures are then transfected into the wave bioreactor. The cell cultures are harvested from the wave bioreactor bags at least 48 hours after transfection.

[0371] Titer. AAV titer was calculated after DNase digestion using qPCR against a standard curve (AAV ITR specific) and primers specific to the CYP46A1 gene. Recovery of suspension cells from shaker flasks and 60 Wave bioreactor bags. 48 hours after transfection, the cell cultures were collected into 500 mL polypropylene conical tubes (Corning) by either injection from a shaker flask or pumping from a Wave bioreactor bag. The cell cultures were then centrifuged at 655 × g for 10 minutes using a Sorvall RC3C plus centrifuge and an H6000A rotor. The supernatant was discarded, and the cells were resuspended in 1 × PBS, transferred to a 50 mL conical tube, and centrifuged at 655 × g for 10 minutes. At this point, the pellet could be stored at NLT -60°C or purification could be continued.

[0372] Titration of rAAV from cell lysates using qPCR. Take 10 mL of cell culture and centrifuge at 655 × g for 10 minutes using a Sorvall RC3C plus centrifuge and H6000A rotor. Decant the supernatant from the cell pellet. Then resuspend the cell pellet in 5 mL of DNase buffer (5 mM CaCl2, 5 mM MgCl2, 50 mM Tris-HCl pH 8.0) and sonicate to efficiently lyse the cells. Then take 300 μL and place it in a 1.5 mL microfuge tube. Then add 140 units of DNase I to each sample and incubate at 37 °C for 1 hour. To determine the effectiveness of DNase digestion, add 4–5 mg of CYP46A1 plasmid to untransfected cell lysates with and without DNase. Add 50 μL of EDTA / sarcosyl solution (6.3% sarcosyl, 62.5 mM EDTA, pH 8.0) to each tube and incubate at 70°C for 20 minutes. Then, add 50 μL of proteinase K (10 mg / mL) and incubate at 55°C for at least 2 hours. Inactivate the proteinase K by boiling the samples for 15 minutes. Take aliquots from each sample and analyze by qPCR. To efficiently determine how much rAAV vector is generated per cell, perform two qPCR reactions. One qPCR reaction consists of a set of primers designed to bind to homologous sequences in the backbone of plasmids XX680, pXR2, and CYP46A1. The second qPCR reaction consists of a set of primers that bind to and amplify a region within the CYP46A1 gene. qPCR is performed using Roche Sybr green reagent and a Light cycler 480. The sample is denatured at 95°C for 10 minutes, followed by 45 cycles (10 seconds at 90°C, 10 seconds at 62°C, and 10 seconds at 72°C) and a melting curve (one cycle consists of 30 seconds at 99°C and 1 minute at 65°C).

[0373] Purification of rAAV from crude lysate. Each cell pellet is adjusted to a final volume of 10 mL. The pellet is sonicated for 4 minutes in a short vortex burst of 1 second on, 1 second off, at a yield of 30%. After sonication, 550 U of DNase is added and incubated at 37°C for 45 minutes. The pellet is then centrifuged at 9400 × g using a Sorvall RCSB centrifuge and HS-4 rotor to pelletize the cell debris, and the clarified lysate is transferred to a Type 70Ti centrifuge tube (Beckman 361625). With regard to the recovery and lysis of suspension HEK293 cells for rAAV isolation, those skilled in the art may perform a clarification step using mechanical methods such as microfluidization or chemical methods such as detergents, followed by deep filtration or tangential flow filtration (TFF).

[0374] AAV vector purification. The clarified AAV lysate is as recognized and described in the following manuscripts, which are incorporated herein by reference in their entirety, by a person skilled in the art (Allay et al., Davidoff et al., Kaludov et al., Zolotukhin et al., Zolotukhin et al., etc.). The solution is purified by column chromatography. (Example 4)

[0375] The selection of NS-specific promoters according to the present invention was tested in neuroblastoma-derived SH-SY5Y cells. material and method

[0376] Cell maintenance and transfection. SH-SY5Y cells were cultured in HAM F12 medium containing 1 mM L-glutamine (Gibco 11765-054), 15% heat-inactivated FBS (ThermoFisher 10500064), 1% non-essential amino acids (Merck M1745-100ML), and 1% penicillin / streptomycin (ThermoFisher 15140122). Cells were passaged twice weekly at a ratio of 1:3 to 1:4 to maintain a healthy cell density between 70 and 80%. Cells were kept below 20 passages. For transfection, cells were transfected. 5 Individual cells were seeded in 48-well adhesive plates. 24 hours after seeding, 300 ng of plasmid was transfected into the cells using Lipofectamine 3000 reagent (ThermoFisher L3000008).

[0377] Plasmids transfected into the SHSY5Y cell line include SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0033, SP0011, SP0034, SP0035, or SP0036 operably linked to GFP.

[0378] Flow cytometry. 48 hours after transfection, SH-SY5Y cells were washed with PBS before dissociation with 0.05% trypsin. Cells were collected and resuspended in 90% PBS, 10% FBS solution. GFP expression of cells was measured using Attune Nxt. Flow cytometry was performed using an Acoustic Focusing Cytometer. Cell viability dye 7-AAD (ThermoFisher 00-6993-50) was mixed with a control cell population to identify and eliminate dead cells. GFP expression was measured in a population of living single cells using a 488 nm blue laser with a 510 / 10 nm band-pass filter. Untransfected cells were used to gate for GFP-negative and GFP-positive cells. The number of GFP-positive single cells and the median GFP fluorescence of all GFP-positive cells were calculated using Attune Nxt software. result

[0379] The results of this experiment are shown in Figures 7A and 7B. Neuroblastoma-derived SH-SY5Y cells transfected with expression cassettes containing SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, or SP0036 operably linked to GFP were evaluated by flow cytometry for median GFP expression and percentage of GFP-positive cells. Expression cassettes containing known promoters of synapsin-1 and CAG operably linked to GFP were used as controls. All tested promoters had comparable transfection efficiency and median GFP expression to the neuron-specific control promoter of synapsin-1 (see Figures 7A and 7B). The control promoter CAG showed 2–3 times higher transfection efficiency (Figure 7B) and approximately 2.5 times higher median GFP expression compared to the control promoter synapsin-1 and the tested synthetic NS-specific promoter (Figure 7A).

[0380] The synthetic NS-specific promoter SP0028 (SEQ ID NO: 86) has a similar design to the synthetic NS-specific promoter SP0019 (SEQ ID NO: 82), which contains both identical elements. Therefore, SP0028 (SEQ ID NO: 86) can be expected to perform similarly to SP0019 (SEQ ID NO: 82).

[0381] The synthetic NS-specific promoter SP0029 (SEQ ID NO: 87) has a similar design to the synthetic NS-specific promoter SP0021 (SEQ ID NO: 84), which contains both identical elements. Therefore, SP0029 (SEQ ID NO: 87) can be expected to perform similarly to SP0021 (SEQ ID NO: 84).

[0382] The synthetic NS-specific promoter SP0026 (SEQ ID NO: 76) has a similar design to the synthetic NS-specific promoter SP0013 (SEQ ID NO: 74), which contains both of the same elements. Therefore, SP0026 (SEQ ID NO: 76) can be expected to perform similarly to SP0013 (SEQ ID NO: 74).

[0383] The synthetic NS-specific promoter SP0027 (SEQ ID NO: 77) has a similar design to the synthetic NS-specific promoter SP0014 (SEQ ID NO: 75), which contains both of the same elements. Therefore, SP0027 (SEQ ID NO: 77) can be expected to perform similarly to SP0014 (SEQ ID NO: 75).

[0384] The synthetic NS-specific promoter SP0033 (SEQ ID NO: 81) has a similar design to SP0021 (SEQ ID NO: 84), which contains both identical and similar elements. Therefore, SP0033 (SEQ ID NO: 81) is a shorter version of SP0021 (SEQ ID NO: 84) and can therefore be expected to perform similarly. (Example 5)

[0385] Modified vectors containing CYP46A1 or GFP and covalent mannosylation of the vector are compared to the parent's unmodified rAAV. Delivery of CYP46A1 by rAAV drives abundant secretion of CYP46A1 from transduced neurons, which can be visually detected by immunohistochemical testing and quantified by ELISA of tissue extracts. For example, after injection of modified AAV-CYP46A1 into the thalamus by conversion-enhancement delivery as described in U.S. Patent Application No. 13 / 146,640 or catheter delivery in monkeys, the degree of CYP46A1 immunopositive staining is evaluated in the ipsilateral frontal cortex. Expression of CYP46A1 delivered with the modified vector was significantly enhanced compared to the unmodified vector, extending significantly from the prefrontal association cortex (cortical regions 9 and 10) to the frontal lobe oculomotor cortex (region 8), premotor cortex (region 6), primary somatosensory cortex (regions 3, 1, and 2) to the primary motor cortex (region 4), including expression in the cingulate cortex (regions 23, 24, and 32) and Broca's area (regions 44 and 45). In addition to very strong staining of individual neuronal cell bodies and cellular processes, CYP46A1 staining was observed across multiple layers of the frontal cortex with the highest intensity gradient in superficial layers III and IV compared to the same dose of the unmodified vector.

[0386] Delivery of the modified vector containing GFP, compared to the parent vector, will also be tested in the monkey model described in U.S. Patent Application No. 13 / 146,640. The relative amounts of the modified vector in the anterior thalamic nucleus (AN), dorsomedial nucleus (MD), ventroanteroposterior nucleus (VA), ventrolateral nucleus (VL), and ventroposterior nucleus (VP) are significantly higher than those of the unmodified vector. In addition, the modified vector is distributed more broadly and efficiently throughout the cortex compared to the unmodified vector. The percentage of positive cells is significantly higher in each region and area compared to the parent vector. More efficient transduction in the superficial layers 1–6 is also expected. Delivery to all multiple lobes of the cerebral cortex or cortical regions 1–4, 6, and 8–10 can be achieved. [Table A]

[0387] Surgical delivery. Modified or unmodified rAAV vectors containing GFP, under the control of a cytomegalovirus promoter, were injected into the right thalamus of six adult rhesus monkeys using a convection-enhanced delivery (CED) protocol. All experiments were conducted in accordance with National Institutes of Health guidelines and protocols approved by the University of California San Francisco's Institutional Animal Care and Use Committee.

[0388] Immunostaining with antibodies against CYP46A1 (1:500, AF-212-NA, R&D Systems) and GFP (1:500, AB3080, Chemicon) is performed on a Zamboni-fixed 40 μm coronal section covering the entire frontal cortex and extending posteriorly to the thalamic level. Localization of CYP46A1 and GFP immunopositive neurons is analyzed relative to the rhesus monkey brain in stereotactic coordinates to identify specific areas of immunostaining in the cortex and thalamus.

[0389] CYP46A1 protein ELISA. Tissue punches derived from 3 mm coronal blocks of fresh-frozen tissue were collected from several cortical and thalamic regions. Methods and materials, as well as striatal regions of monkeys injected with modified vectors. Expressed surgical delivery was quantified by ELISA assays using human CYP46A1 clDNA or GFP cDNA with commercially available ELISA kits (Emax ELISA, Promega, Wis.). (Example 6)

[0390] Next, to determine whether altering the capsid modification would allow for re-administration, the modified vector containing CYP46A1 from Example 5 was redesigned to consist of the same capsid as in Example 5, containing the same payload (i.e., CYP46A1) but with a different chemical modification. Adult rhesus monkeys were administered the first modified vector containing CYP46A1 from Example 2, and 14 days after administration, they were administered either a second dose of the same vector or the redesigned modified capsid. CYP46A1 expression was evaluated using the ELISA assay described above in Example 5. It was found that re-administration of the same vector resulted in significantly reduced expression, which may be due to neutralizing antibodies that developed against the vector after the first administration. Notably, the expression of the redesigned vector was high and broad, indicating that the changes in the capsid modification enabled the expression of the redesigned vector. In certain embodiments, for example, the following items are provided: (Item 1) A method for treating a neurological disorder or impairment in a subject requiring treatment, wherein the method is a therapeutically effective amount (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs, and (b) Isolated nucleic acid encoding the CYP46A1 protein A method comprising administering to a subject having or at risk of developing the aforementioned neurological disease or disorder. (Item 2) A method for treating a neurological disorder or impairment in a subject requiring treatment, wherein the method is a therapeutically effective amount (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat sequence (ITR) or a variant thereof, and (ii) a second region comprising a transgene encoding one or more miRNAs, and (b) Recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein A method comprising administering to a subject having or at risk of developing the aforementioned neurological disease or disorder. (Item 3) The method according to any one of items 1 to 2, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma resulting from spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan syndrome, epilepsy, cerebral infarction, depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, delusional disorder, attention deficit disorder, psychosexial disorder, sleep disorders, painful disorders, eating or weight disorders. (Item 4) The aforementioned neurological disease or disorder is a central nervous system (CNS) disease or disorder, items 1-3 One of the methods described above. (Item 5) The method according to any one of items 1 to 4, wherein the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, amyotrophic lateral sclerosis, and Parkinson's disease. (Item 6) The method according to any one of items 1 to 5, wherein the CNS disease or disorder is Alzheimer's disease, and at least one miRNA comprises a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and their disease-associated alleles. (Item 7) The method according to any one of items 1 to 5, wherein the CNS disease or disorder is Parkinson's disease, and at least one miRNA comprises a seed sequence complementary to SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and their disease-associated alleles. (Item 8) The method according to any one of items 1 to 5, wherein the CNS disease is Huntington's disease, and at least one miRNA contains a seed sequence complementary to SEQ ID NO: 4, or at least one miRNA contains one of the sequences of SEQ ID NOs: 6-17, 40-44, or 50-66 adjacent to the miRNA backbone sequence. (Item 9) The method according to any one of items 1 to 8, wherein the CNS disease is Huntington's disease, and at least one miRNA contains one of the sequences of sequence numbers 6-17, 40-44, or 50-66. (Item 10) The method according to any one of items 8-9, wherein at least one of the miRNAs hybridizes with human huntingtin and inhibits its expression. (Item 11) The method according to any one of items 8 to 10, wherein the subject comprises a huntingtin gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. (Item 12) The method described in any of items 8 to 11, wherein the subject is under 20 years of age. (Item 13) The method according to any one of items 1 to 12, wherein the recombinant viral vector is selected from the group consisting of AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors. (Item 14) The method according to any one of items 2 to 13, wherein the recombinant viral vector containing (a) is the same as the recombinant viral vector containing (b). (Item 15) The method according to any one of items 1 to 13, wherein the isolated nucleic acids of (a) and (b) are contained in separate recombinant viral vectors. (Item 16) The method according to any one of items 1 to 14, wherein the isolated nucleic acids of (a) and (b) are contained in the same recombinant viral vector. (Item 17) (a) and (b) are administered at substantially the same time, according to one of items 1 to 16. Method of description. (Item 18) The method according to any one of items 1-13 and 15, wherein (a) and (b) are administered at different time points. (Item 19) The method described in item 18, wherein the aforementioned different time points are separated by at least one minute, at least one hour, at least one day, at least one week, at least one month, at least one year, or longer. (Item 20) The method described in any of items 18-19, wherein (a) is administered before the administration of (b). (Item 21) (b) is administered before the administration of (a) as described in any of items 18-19. (Item 22) The method according to any one of items 1 to 21, wherein the administration of (a), (b), or (a) and (b) is repeated at least once. (Item 23) The method according to any of items 1 to 22, wherein the transgene contains two miRNAs adjacent to an intron in tandem. (Item 24) The method described in item 23, wherein the adjacent introns are identical. (Item 25) The method described in item 23, wherein the adjacent introns are of the same species origin. (Item 26) The method according to item 23, wherein the adjacent intron is an hCG intron. (Item 27) The method according to any one of items 1 to 26, wherein the introduced gene includes a promoter. (Item 28) The method according to item 27, wherein the promoter is a synapsin (Syn1) promoter or one of the promoters in Tables 10-13. (Item 29) The method according to any one of items 1 to 28, wherein the one or more miRNAs are located in the untranslated portion of the transgene. (Item 30) The method described in item 29, wherein the aforementioned untranslated portion is an intron. (Item 31) The method according to item 30, wherein the untranslated portion is between the last codon of the protein-coding nucleic acid sequence and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence. (Item 32) The method according to any one of items 1 to 31, further comprising a third region containing a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. (Item 33) The method according to any one of items 1 to 33, wherein the ITR variant lacks a functional terminal isolation site (TRS), and optionally the ITR variant is an ATRS ITR. (Item 34) The method according to any one of items 1 to 33, wherein the administration results in the delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the target. (Item 35) The aforementioned administration is by injection, and if necessary, by intravenous or intrastriatal injection, items 1-34. One of the methods described above. (Item 36) The method according to any one of items 2 to 35, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimera thereof. (Item 37) The method according to any one of items 2 to 36, wherein the viral vector comprises a capsid protein derived from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimeric thereof. (Item 38) The method according to item 37, wherein the capsid protein is AAV9 capsid protein. (Item 39) The method according to any of items 2 to 38, wherein the viral vector is a self-complementary AAV (scAAV). (Item 40) The method according to any one of items 2 to 39, wherein the viral vector is formulated for delivery to the central nervous system (CNS). (Item 41) (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs, and (b) Isolated nucleic acid encoding the CYP46A1 protein A composition or combination containing the following: (Item 42) (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat sequence (ITR) or a variant thereof, and (ii) a second region comprising a transgene encoding one or more miRNAs, and (b) Recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein A composition or combination containing the following: (Item 43) A composition or combination described in any of items 41 to 42 for use in a method for treating a neurological disorder or impairment in a subject requiring treatment, wherein the method comprises administering a therapeutically effective amount of the composition or combination to a subject having or at risk of developing the neurological disorder or impairment. (Item 44) The composition or combination described in item 43, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal ataxia, Krabbe disease, polyglutamine repeat spinocerebellar ataxia, Batten disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma resulting from spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan syndrome, epilepsy, cerebral infarction, depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, psychosexial disorder, sleep disorders, painful disorders, eating or weight disorders. (Item 45) The composition or combination described in item 44, wherein the neurological disease or disorder is a central nervous system (CNS) disease or disorder. (Item 46) The composition or combination according to item 45, wherein the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, amyotrophic lateral sclerosis, and Parkinson's disease. (Item 47) A composition or combination according to any one of items 41 to 46, wherein at least one miRNA comprises a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and their disease-associated alleles. (Item 48) A composition or combination described in any of items 41 to 46, wherein at least one miRNA comprises a seed sequence complementary to SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and their disease-associated alleles. (Item 49) A composition or combination according to any one of items 41 to 46, wherein at least one miRNA contains a seed sequence complementary to SEQ ID NO: 4, or at least one miRNA contains one of the sequences from SEQ ID NOs: 6-17, 40-44, or 50-66 adjacent to the miRNA backbone sequence. (Item 50) A composition or combination according to any of items 41-46, wherein at least one miRNA contains one of the sequences of sequence numbers 6-17, 40-44, or 50-66. (Item 51) A composition or combination according to any one of items 49 to 50, wherein at least one of the miRNAs hybridizes with human huntingtin and inhibits its expression. (Item 52) A composition or combination according to any one of items 49 to 51, wherein the subject comprises a huntingtin gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. (Item 53) The composition or combination described in any of items 49 to 52, wherein the subject is under 20 years of age. (Item 54) The composition or combination described in any of items 42 to 53, wherein the recombinant viral vector is selected from the group consisting of AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors. (Item 55) A composition or combination according to any one of items 42 to 54, wherein the recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (b). (Item 56) The compositions or combinations described in any of items 41 to 54, wherein the isolated nucleic acids of (a) and (b) are contained in separate recombinant viral vectors. (Item 57) A composition or combination according to any of items 41 to 55, wherein the isolated nucleic acids of (a) and (b) are contained in the same recombinant viral vector. (Item 58) A composition or combination according to any of items 41 to 57, wherein (a) and (b) are administered at substantially the same time. (Item 59) (a) and (b) are administered at different time points, the composition or combination described in any of items 41-54 and 56. (Item 60) The composition or combination described in item 59, wherein the aforementioned different time points are separated by at least one minute, at least one hour, at least one day, at least one week, at least one month, at least one year, or longer. (Item 61) (a) is administered before the administration of (b), the composition or combination described in any of items 59 to 60. (Item 62) (b) is administered prior to the administration of (a), and is one of the compositions or combinations described in any of items 59-60. (Item 63) A composition or combination according to any of items 59 to 60, wherein the administration of (a), (b), or (a) and (b) is repeated at least once. (Item 64) The composition or combination according to any one of items 41 to 65, wherein the transgene comprises two miRNAs adjacent to an intron in tandem. (Item 65) The composition or combination described in item 64, wherein the adjacent introns are identical. (Item 66) The composition or combination described in item 64, wherein the adjacent introns are of the same species. (Item 67) The composition or combination according to item 64, wherein the adjacent intron is an hCG intron. (Item 68) The aforementioned introduced gene comprises a promoter, and is one of the compositions or combinations described in any of items 41 to 67. (Item 69) The composition or combination described in item 68, wherein the promoter is a synapsin (Syn1) promoter or one of the promoters in Tables 10-13. (Item 70) The composition or combination according to any one of items 41 to 69, wherein one or more miRNAs are located in the untranslated portion of the transgene. (Item 71) The composition or combination described in item 70, wherein the untranslated portion is an intron. (Item 72) The composition or combination described in item 70, wherein the untranslated portion is between the last codon of the protein-coding nucleic acid sequence and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence. (Item 73) A composition or combination according to any one of items 41 to 72, further comprising a third region containing a second adeno-associated virus (AAV) inverted terminal repeat sequence (ITR) or a variant thereof. (Item 74) The composition or combination according to any one of items 41 to 73, wherein the ITR variant lacks a functional end-isolation site (TRS), and optionally the ITR variant is an ATRS ITR. (Item 75) A composition or combination according to any one of items 41 to 74, wherein the administration results in the delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the target. (Item 76) The aforementioned administration is by injection, intravenous injection or intrastriatal injection as necessary, and is one of the compositions or combinations described in any of items 41 to 75. (Item 77) A composition or combination according to any one of items 42 to 76, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a chimera thereof. (Item 78) The composition according to any one of items 42 to 77, wherein the viral vector comprises a capsid protein derived from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimeric thereof. (Item 79) The composition or combination described in item 78, wherein the capsid protein is the AAV9 capsid protein. (Item 80) The composition or combination described in any of items 42 to 79, wherein the viral vector is a self-complementary AAV (scAAV). (Item 81) A composition or combination according to any one of items 42 to 80, wherein the viral vector is formulated for delivery to the central nervous system (CNS). (Item 82) A composition comprising an isolated nucleic acid encoding the CYP46A1 protein, wherein the nucleic acid comprises a sequence that is at least 80% identical to SEQ ID NO: 110, or at least 80% identical to SEQ ID NO: 111, or at least 80% identical to SEQ ID NO: 153. (Item 83) A composition comprising a recombinant viral vector containing an isolated nucleic acid encoding the CYP46A1 protein, wherein the nucleic acid contains a sequence that is at least 80% identical to SEQ ID NO: 110, or at least 80% identical to SEQ ID NO: 111, or at least 80% identical to SEQ ID NO: 153. (Item 84) A method for treating a neurological disorder or impairment in a subject requiring treatment, the method comprising administering a therapeutically effective amount of the composition described in item 82 or 83 to a subject having or at risk of developing the neurological disorder or impairment. (Item 85) The method according to item 84, wherein the neurological disorder or disability is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma resulting from spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan syndrome, epilepsy, cerebral infarction, depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, paranoia, attention deficit disorder, psychosexial disorder, sleep disorders, painful disorders, eating or weight disorders. (Item 86) The aforementioned neurological disease or disorder is a central nervous system (CNS) disease or disorder, item 84~ The method described in any of the 85 methods. (Item 87) The method according to any one of items 84 to 86, wherein the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, amyotrophic lateral sclerosis, and Parkinson's disease. (Item 88) The composition or method according to any one of items 83 to 87, wherein the recombinant viral vector is selected from the group consisting of AAV vectors, adenovirus vectors, lentivirus vectors, retrovirus vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors. (Item 89) The method according to any one of items 84 to 88, wherein the administration is repeated at least once. (Item 90) The method according to any one of items 84 to 89, wherein the administration results in the delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the target. (Item 91) The administration is by injection, intravenous injection or intrastriatal injection as necessary, as described in any of items 84 to 90. (Item 92) The composition or method according to any one of items 83 to 91, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimera thereof. (Item 93) A composition or method according to any one of items 83 to 92, wherein the viral vector comprises a capsid protein derived from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a chimeric thereof. (Item 94) The composition or method according to item 93, wherein the capsid protein is AAV9 capsid protein. (Item 95) The composition or method according to any one of items 83 to 94, wherein the viral vector is a self-complementary AAV (scAAV). (Item 96) A composition or method according to any one of items 83 to 95, wherein the viral vector is formulated for delivery to the central nervous system (CNS). (Item 97) The composition or method according to any one of items 82 to 96, wherein the nucleic acid contains a sequence that is at least 90% identical to sequence number 110. (Item 98) The composition or method according to any one of items 82 to 96, wherein the nucleic acid contains at least 95% of the same sequence as sequence number 110. (Item 99) The composition or method according to any one of items 82 to 96, wherein the nucleic acid contains the same sequence as sequence number 110. (Item 100) The composition or method according to any one of items 2-40, 42-81, or 83-99, wherein the viral vector comprises a modified viral capsid. (Item 101) The composition or method according to any one of items 2-40, 42-81, or 83-99, wherein the viral vector includes modifications to the viral capsid. (Item 102) The composition or method according to item 100 or 101, wherein the modification is a chemical modification, non-chemical modification, or amino acid modification of the viral capsid. (Item 103) The composition or method according to item 100 or 101, wherein at least one of the capsid modifications preferentially targets cells in the CNS or PNS. (Item 104) The composition or method according to item 100 or 101, wherein the chemical modification comprises a chemically modified tyrosine residue that is modified to include a covalently linked monosaccharide or polysaccharide moiety. (Item 105) The composition or method according to item 104, wherein the chemically modified tyrosine residue comprises a monosaccharide selected from galactose, mannose, N-acetylgalactosamine, cross-linked GalNac, and mannose-6-phosphate. (Item 106) The composition or method according to item 100 or 101, wherein the chemical modification includes a ligand covalently linked to a primary amino group of a capsid polypeptide via a -CSNH- linkage. (Item 107) The composition or method according to item 106, wherein the ligand comprises an arylene or heteroarylene radical covalently bonded to the ligand. (Item 108) The composition or method according to any one of items 100 to 107, wherein the modified viral capsid is a chimeric capsid or a singular capsid. (Item 109) The composition or method according to any one of items 100 to 107, wherein the modified viral capsid is a singular capsid. (Item 110) The composition or method according to any one of items 100 to 107, wherein the modified viral capsid is a chimeric capsid or singular capsid further comprising the modification. (Item 111) The composition or method according to any one of items 100 to 110, wherein the modified viral capsid is derived from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or their mutant variants, chimeras, mosaics, or rational singular forms. (Item 112) A composition or method according to any one of items 100 to 111, wherein the modification alters the antigenic profile of the modified viral capsid compared to an unmodified viral capsid. (Item 113) The composition or method according to any one of items 100 to 112, wherein the modified viral capsid can be used for repeated administration.

Claims

[Claim 1] The invention described in the specification.

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