Composition for treating nervous system diseases

Abstract

Aspects of the present disclosure relate to compositions and methods useful for the treatment of nervous system diseases and disorders. In some embodiments, the disclosure provides methods of treating a nervous system disease or disorder comprising administering both a viral vector comprising an interfering nucleic acid (e.g., an artificial miRNA) and a viral vector comprising a CYP46A1 protein. In some embodiments, the disclosure provides methods of treating Huntington's disease comprising administering both a viral vector comprising an interfering nucleic acid (e.g., an artificial miRNA) targeting the Huntington's gene (HTT) and a viral vector comprising a CYP46A1 protein. In some embodiments, the viral vector comprises a modified viral capsid, for example, for preferentially targeting cells in the CNS or PNS.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 303,673, filed January 27, 2022, and U.S. Provisional Application No. 63 / 321,335, filed March 18, 2022, pursuant to 35 USC §119(e), the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The techniques described in this article relate to methods for treating neurological disorders or disorders, such as Huntington's disease. Background Technology

[0003] Huntington's disease (HD) is a devastating, inherited neurodegenerative disorder caused by the amplification of the CAG repeat region in exon 1 of the huntingtin gene. When huntingtin protein (HTT) is expressed systemically, the polyglutamine-amplified protein is particularly toxic to the medial spinous neurons in the striatum and their cortical connections. Patients are plagued by mood symptoms, including depression and anxiety, as well as characteristic motor disturbances and chorea. There is currently no cure for Huntington's disease; treatment options are limited to improving disease symptoms. Summary of the Invention

[0004] One aspect of this document describes a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance at a therapeutically effective amount of at least one of the following: (a) a nucleic acid encoding at least one miRNA; and (b) a nucleic acid encoding the CYP46A1 protein. For example, the nucleic acid encoding at least one miRNA may be administered alone, or in combination with a nucleic acid encoding the CYP46A1 protein. Alternatively, the nucleic acid encoding the CYP46A1 protein may be administered alone, or in combination with a nucleic acid encoding at least one miRNA. The nucleic acid may be wild-type or a codon-optimized variant.

[0005] Another aspect described herein provides compositions or combinations comprising at least one of: (a) an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs; and (b) an isolated nucleic acid encoding a CYP46A1 protein. In one aspect, the compositions or combinations described herein comprise: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs; and (b) a recombinant viral vector comprising an isolated nucleic acid encoding said CYP46A1 protein.

[0006] Another aspect of this document provides compositions or combinations comprising (a) an isolated nucleic acid encoding a transgene, the transgene encoding the isolated nucleic acid, the nucleic acid encoding the CYP46A1 protein. In some aspects, the composition or combination further comprises (b) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In other aspects, the composition or combination does not comprise (b) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In one aspect, the composition or combination described herein comprises: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding the CYP46A1 protein. In some aspects, the composition or combination further comprises: (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other aspects, the composition or combination does not comprise (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

[0007] Another aspect of this document provides compositions or combinations comprising (a) an isolated nucleic acid encoding a transgene, the transgene encoding the isolated nucleic acid, the nucleic acid encoding one or more miRNAs. In some aspects, the composition or combination further comprises (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In other aspects, the composition or combination does not comprise (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein. In one aspect, the composition or combination described herein comprises: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs. In some aspects, the composition or combination further comprises: (b) a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein. In other aspects, the composition or combination does not comprise (b) a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein.

[0008] Another aspect described herein provides a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance at a therapeutically effective amount of at least one of the following: (a) an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs; and (b) an isolated nucleic acid encoding a CYP46A1 protein. In one aspect, this document describes a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance at a therapeutically effective amount of at least one of the following: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs; and (b) a recombinant viral vector comprising an isolated nucleic acid encoding the CYP46A1 protein.

[0009] Another aspect described herein provides a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In some aspects, the method further comprises administering to the subject a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In other aspects, the method does not include administering to the subject (b) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In one aspect, this document describes a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and the second region comprising a transgene encoding a CYP46A1 protein. In some aspects, the method further includes administering to a subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other aspects, the method does not include administering to a subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

[0010] Another aspect described herein provides a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In some aspects, the method further comprises administering to the subject a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In other aspects, the method does not include administering to the subject (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In one aspect, this document describes a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and the second region comprising a transgene encoding one or more miRNAs. In some aspects, the method further includes administering to a subject a therapeutically effective amount of (b) a recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein. In other aspects, the method does not include administering to a subject a therapeutically effective amount of (b) a recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein.

[0011] In some embodiments, the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinal cerebral ataxia, polyglutamine-repetitive spinocerebellar ataxia, Krabbe disease, Batten's 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 or head injury, ophthalmic diseases and disorders, Tay-Sachs disease. Diseases, Lesch-Nyhan's disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurological disorders, psychosis, dementia, delusions, attention deficit disorder, sexual psychological disorders, sleep disorders, pain disorders, eating or weight disorders. In some embodiments, the neurological disease or disorder is a central nervous system (CNS) disease or disorder. In some embodiments, the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine-repetitive spinocerebellar ataxia, amyotrophic lateral sclerosis, and Parkinson's disease.

[0012] In some embodiments, the CNS disease or disorder is Alzheimer's disease, and the 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.

[0013] In some embodiments, the CNS disease or disorder is Parkinson's disease, and the 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.

[0014] In some embodiments, the CNS disease is Huntington's disease, and at least one miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein at least one miRNA comprises a sequence of any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260, said sequence being side-joined with a miRNA backbone sequence. In some embodiments, the CNS disease is Huntington's disease, and at least one miRNA comprises a sequence of any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260. In some embodiments, at least one of the miRNAs hybridizes with and inhibits the expression of the human huntingtin gene. In some embodiments, the subject contains a huntingtin gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. In some embodiments, the subject is less than 20 years old.

[0015] 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.

[0016] In some embodiments, the recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (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.

[0017] In some embodiments, (a) and (b) are applied at substantially the same time. In some embodiments, (a) and (b) are applied at different time points. In some embodiments, the interval between the different time points is at least 1 minute, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year, or longer. In some embodiments, (a) is applied before (b). In some embodiments, (b) is applied before (a). In some embodiments, the application of (a), (b), or (a) and (b) is repeated at least once.

[0018] In some embodiments, the transgene comprises two miRNAs in tandem, each flanked by an intron. In some embodiments, the flanked introns are identical. In some embodiments, the flanked introns originate from the same species. In some embodiments, the flanked introns are hCG introns.

[0019] In some embodiments, the transgene includes a promoter. In some embodiments, the promoter is the synapsin (Syn1) promoter, or the promoters listed in Tables 10-13.

[0020] In some embodiments, the one or more miRNAs are located in the untranslated portion of the transgene. In some embodiments, the untranslated portion is an intron. In some embodiments, the untranslated portion is located between the last codon and the poly-A tail sequence of the nucleic acid sequence encoding the protein, or between the last nucleotide base and the poly-A tail sequence of the promoter sequence.

[0021] In some embodiments, the nucleic acid or viral vector further includes a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

[0022] In some implementations, the ITR variant lacks a functional terminal resolution site (TRS), and optionally, the ITR variant is an ATRS ITR.

[0023] In some embodiments, administration delivers a viral vector or isolated nucleic acid to the central nervous system (CNS) of the subject. In some embodiments, the administration is performed by injection, optionally intravenous injection, or intrastriatal injection.

[0024] In some embodiments, the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof. In some embodiments, the viral vector comprises a capsid protein from AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof. In some embodiments, the capsid protein is the AAV9 capsid protein. In some embodiments, the viral vector is a self-complementary AAV (scAAV). In some embodiments, the viral vector is formulated for delivery to the central nervous system (CNS).

[0025] In some embodiments of any aspect, the viral vector comprises a modified viral capsid.

[0026] In some embodiments of any aspect, the viral vector comprises modifications to the viral capsid.

[0027] In some embodiments of any aspect, the modification is a chemical, non-chemical, or amino acid modification of the viral capsid.

[0028] In some embodiments of any aspect, at least one of the capsid modifications preferentially targets cells in the CNS or PNS.

[0029] In some embodiments of any aspect, the chemical modification includes chemically modified tyrosine residues that are modified to include covalently linked monosaccharide or polysaccharide moieties.

[0030] In some embodiments of any aspect, the chemically modified tyrosine residue comprises a monosaccharide selected from galactose, mannose, N-acetylgalactosamine, GalNac bridge, and mannose-6-phosphate.

[0031] In some embodiments of any aspect, the chemical modification comprises a ligand covalently linked to a primary amino group of the capsid polypeptide via a -CSNH- bond.

[0032] In some embodiments of any aspect, the ligand comprises an arylene or heteroarylene radical covalently bound to the ligand.

[0033] In some embodiments of any aspect, the modified viral capsid is a chimeric capsid or a haploid capsid.

[0034] In some embodiments of any aspect, the modified viral capsid is a haploid capsid.

[0035] In some embodiments of any aspect, the modified viral capsid is further comprising a modified chimeric capsid or a haploid capsid.

[0036] In some embodiments of any aspect, the modified viral capsid is an AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, or a modified form of its mutation, chimera, mosaic, or reasonable haploid.

[0037] In some embodiments of any aspect, the modification alters the antigenic spectrum of the modified viral capsid compared to the unmodified viral capsid.

[0038] In some embodiments of any aspect, the modified viral capsid can be used for repeated administration. Attached Figure Description

[0039] Figure 1 This is a schematic diagram showing the HD plasmid map of pJAL130-CYP46A1 (7314bp), see, for example, SEQ ID NO: 111 and Table 16, which shows the ITR to ITR sequence of the codon-optimized CYP46 sequence from the plasmid (see, for example, SEQ ID NO: 110).

[0040] Figure 2 Intracranial biodistribution of transgenic GFP delivered via intraventricular (ICV) and intravenous (IV) injection, controlled by CNS-1 (see, for example, SEQ ID NO: 112), CNS-2 (see, for example, SEQ ID NO: 113), CNS-3 (see, for example, SEQ ID NO: 114), CNS-4 (see, for example, SEQ ID NO: 115), CNS-5 (see, for example, SEQ ID NO: 122), CNS-6 (see, for example, SEQ ID NO: 123), CNS-7 (see, for example, SEQ ID NO: 124), and CNS-8 (see, for example, SEQ ID NO: 125), as well as the control promoter hSyn1 (see, for example, SEQ ID NO: 152), is shown in sagittal sections. Scale bar is 1 mm.

[0041] Figures 3A-3B An image of a coronal brain slice is shown. Figure 3AIntracranial biodistribution of transgenic GFP delivered via ICV in coronal sections under control of CNS-1 (see, for example, SEQ ID NO: 112), CNS-2 (see, for example, SEQ ID NO: 113), CNS-3 (see, for example, SEQ ID NO: 114), and CNS-4 (see, for example, SEQ ID NO: 115). Scale bar is 1 mm. Figure 3B Intracranial biodistribution of transgenic GFP delivered via ICV in coronal sections controlled by CNS-5 (see, for example, SEQ ID NO: 122), CNS-6 (see, for example, SEQ ID NO: 123), CNS-7 (see, for example, SEQ ID NO: 124), and CNS-8 (see, for example, SEQ ID NO: 125), as well as the control promoter hSyn1 (see, for example, SEQ ID NO: 152). Scale bar is 1 mm.

[0042] Figure 4 The percentage of GFP immunoreactivity in different brain regions following ICV or IV delivery of GFP driven by CNS1-8 (see, for example, SEQ ID NO: 112-SEQ ID NO: 115, SEQ ID NO: 122-SEQ ID NO: 125) or Synapsin-1 (see, for example, SEQ ID NO: 152) is shown. Data were obtained by quantitatively measuring the intensity of GFP staining in 10 non-overlapping RGB images (mean ± SEM) in the cortex, hippocampus, striatum, midbrain, and cerebellum via threshold analysis. Images were taken at a ×40 magnification with discrete brain regions kept at constant settings. Foreground immunostaining was defined by averaging the highest and lowest signals. Data are expressed as the average percentage area of ​​immunoreactivity in each field for each region of interest (n = 3). In the case of ICV delivery, expression was highest in the cortex and hippocampus. CNS1-8 (see, for example, SEQ ID NO: 112-SEQ ID NO: 115, SEQ ID NO: 122-SEQ ID NO: 125) showed higher expression in the hippocampus than the hSyn1 control. In the case of ICV delivery, CNS-1 (see, for example, SEQ ID NO: 112) showed higher expression in the hippocampus, midbrain, and cerebellum compared to hSyn1.

[0043] Figures 5A-5B The tissue expression patterns of the faf1 and pitx3 genes were shown, and CRE / proximal promoters from CNS-5, CNS-5_v2, CNS-2, CNS-3 and CNS-4 were designed accordingly. Figure 5AThe expression pattern of the faf1 gene in mouse PNS neurons is shown in single-cell transcriptome 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 The expression patterns of the pitx3 gene in PNS neurons are shown in single-cell transcriptome 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 promoters containing synthetic proximal promoters or CREs (e.g., CNS-5 and CNS-5_v2) designed from the faf1 gene are expected to have strong expression in PNS. Pixt3 is expressed in sympathetic PNS neurons, therefore promoters containing synthetic CREs (e.g., CNS-2, CNS-3, or CNS-4) designed from the pitx3 gene are expected to have expression in sympathetic PNS neurons. Similar analyses of lmxlb and pitx2 showed no expression in the PNS above the cutoff score of the analysis (trinization score less than 0.95; data not shown), therefore it is expected that CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not active in PNS neurons.

[0044] Figure 6A The expression pattern of the HTT gene in sagittal sections of the adult mouse brain is shown (from the Allen Mouse Brain Atlas; mouse.brain-map.org). HTT (Huntington) is highly expressed throughout the brain.

[0045] Figure 6B The expression pattern of the CYP46A1 gene in coronal sections of the adult mouse brain is shown (from the Allen Mouse Brain Atlas; available at mouse.brain-map.org). CYP46A1 is widely expressed in the brain.

[0046] Figure 7AMedian GFP expression of the synthesized NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036 and the control promoter Synapsin-1 relative to the control promoter CAG is shown in SH-SY5Y cells derived from neuroblastoma. NTC represents untransfected cells. Data were collected from three biological replicates, each of which is the mean of two technical replicates. Error bars represent standard errors.

[0047] Figure 7B Transfection efficiencies in SH-SY5Y cells derived from neuroblastoma are shown when transfected with synthetic NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036 operably linked to GFP, or with the control promoters Synapsin-1 and CAG. NTC represents untransfected cells. Data were collected from three biological replicates, each of which is the average of two technical replicates. Error bars represent standard errors. GFP positivity % indicates the percentage of GFP-positive cells in all cells.

[0048] Figure 8 The levels of HTT transcripts after nuclear infection of patient-derived fibroblasts with a plasmid containing a miRNA targeting exon 1 of the HTT gene are shown. N=1.

[0049] Figure 9 This is a schematic diagram showing the HD plasmid map of dbDNA-CYP46A1 (4,573 bp, see, for example, SEQ ID NO: 194), which shows the ITR-ITR sequence containing the codon-optimized CYP46 sequence (see, for example, SEQ ID NO: 212), the CAG promoter, and the bGH poly(a) signal from the plasmid (see, for example, Table 24). This is a plasmid packaged in rAAVrh.10 for administration to HD patients (see, for example, Example 6). Detailed Implementation

[0050] Various aspects of the present invention relate to the administration of interfering RNA (e.g., miRNA, such as artificial miRNA) and nucleic acid encoding the CYP46A1 protein, said interfering RNA being effective in reducing the expression of a pathogenic gene in a subject when delivered to the subject. Therefore, in some embodiments, the methods and compositions disclosed herein are useful for treating neurological diseases or disorders. Treatment

[0051] This disclosure provides methods for delivering nucleic acids and / or transgenic molecules (e.g., repressive RNAs, such as miRNAs or nucleic acids encoding CYP46A1) to a subject. The methods generally involve administering to a subject an effective amount of at least one nucleic acid encoding at least one interfering RNA / repressive nucleic acid and a nucleic acid encoding CYP46A1, said interfering RNA / repressive nucleic acid being capable of reducing the expression of a target gene (e.g., a pathogenic gene associated with neurological diseases or disorders (e.g., Huntington's (htt) protein)). For example, the nucleic acid encoding at least one miRNA may be administered alone, or in combination with a nucleic acid encoding the CYP46A1 protein. Alternatively, the nucleic acid encoding the CYP46A1 protein may be administered alone, or in combination with a nucleic acid encoding at least one miRNA. In some embodiments, one or both of said nucleic acids are provided in a viral vector and / or in viral particles (e.g., rAAV).

[0052] Therefore, one aspect of this document provides a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene, said transgene encoding the CYP46A1 protein. In some aspects, the method further comprises administering to the subject a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs. In other aspects, the method does not include administering to the subject (b) an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs. In one aspect, this document describes a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and the second region comprising a transgene encoding the CYP46A1 protein. In some aspects, the method further includes administering to a subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other aspects, the method does not include administering to a subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

[0053] Another aspect described herein provides a method for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene, the transgene encoding one or more miRNAs. In some aspects, the method further comprises administering to the subject a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In other aspects, the method does not include administering to the subject (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In one aspect, a method is described herein for treating a neurological disorder or disturbance in a subject in need, the method comprising administering to a subject suffering from or at risk of developing the neurological disorder or disturbance a therapeutically effective amount of (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and the second region comprising a transgene encoding one or more miRNAs. In some aspects, the method further includes administering to a patient a therapeutically effective amount of (b) a recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein. In other aspects, the method does not include administering to a subject a therapeutically effective amount of (b) a recombinant viral vector containing isolated nucleic acid encoding the CYP46A1 protein.

[0054] As used herein, “nervous system disease or disorder” can refer to any disease, disorder, or condition that affects or is related to the nervous system; that is, a disease, disorder, or condition that affects the central nervous system (brain and spinal cord), the peripheral nervous system (PNS; e.g., peripheral nerves and cranial nerves), and the autonomic nervous system (parts of which are located within the central and peripheral nervous systems). More than 600 nervous system diseases have been identified in humans. As non-limiting examples, the neurological disorders or disturbances may include: septum pellucida agenesis, acid lipase disorder, acid maltase deficiency, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's pupil, Adie's syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, cognitive impairment, Aicardi syndrome, Aicardi-Goutieres syndrome, AIDS-related neurological complications, Alexander disease, Alpers' disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangioma, hypoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cysts, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome. Syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth syndrome, Batten's disease, Becker's rigidity, Behçet's disease, Bell's palsy, benign primary blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhardt-Roth syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury during childbirth, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysm, brain injury, Brown-Sequard syndrome. Syndrome, Bulbospinal muscular atrophy, autosomal dominant cerebral arteriosclerosis with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, burning pain, cavernous hemangioma, cavernous hemangiomaAngioma, cavernous malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, cerebrovascular disorders, cerebral hypoxia, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cavernous malformation, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebral oculofacial-bone syndrome (COFS), Charcot-Marie-Tooth disease, cerebellar tonsillar herniation, cholesterol ester storage disease, chorea, neuroacanthosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II. II) Coffin-Lowry syndrome, Colpocephaly, Coma, Complex regional pain syndrome, Congenital bilateral facial paralysis, Congenital myasthenia gravis, Congenital myopathy, Congenital cavernous malformation, Cortical-basal ganglia degeneration, Cranial arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, Cushing's syndrome, Cytomegalovirus infection, Dance-eye-dance-foot syndrome, Dandy-Walker syndrome, Dawson's disease, De Morsier's syndrome, Dejerine-Klumpke paralysis, dementia, multiple infarct dementia, semantic dementia, subcortical dementia, Lewy body dementia, dentate nucleus-cerebellar ataxia, dentate nucleus-red nucleus atrophy, dermatomyositis, developmental motor disorders, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet's syndrome, familial autonomic dysfunction, writing disorders, dyslexia, dysphagia, motor coordination disorders, myoclonic cerebellar coordination disorders, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis of lethargy, encephalocele, encephalopathy (familial infantile), trigeminal neuralgia, epilepsy, epileptic hemiplegia, Erb's paralysis, Erb-Duchenne paralysis and Dejerine-Klumpke paralysis, essential tremor, extrapontine myelinolysis, Fabry disease, Fahr's syndromeSyndrome, syncope, familial Alzheimer's disease, familial autonomic dysfunction, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, infantile relaxation syndrome, foot drop, Friedreich's ataxia, frontotemporal dementia, Gaucher disease, generalized gangliosidoses, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globular leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, traumatic brain injury, headache, continuous Migraine, hemifacial spasm, alternating hemiplegia, hereditary neurosis, hereditary spastic paraplegia, hereditary ataxic polyneuritis-like disease, herpes zoster, otitis herpes zoster, Hirayama syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 related myelopathy, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, normal pressure hydrocephalus, spinal cord injury Water, Cushing's syndrome, somnolence, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, pigmentary disorders, infantile hypotonia, infantile axonal dystrophy, infantile phytate storage disease, infantile Refsum disease, infantile spasms, inflammatory myopathy, occipital cleft with exposed brain malformation, intestinal lipid dystrophy, intracranial cysts, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff's Amnesic syndrome, Krabbe disease, Kugelberg-Welander diseaseDiseases including: Kuru, Lambert-Eaton myasthenia gravis, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment syndrome, lateral medullary syndrome, learning disabilities, Leishman's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levine-Critchley syndrome, Lewy body dementia, lipid storage diseases, lipoprotein deposition diseases, lissencephaly, locked-in syndrome, amyotrophic lateral sclerosis (ALS), lupus-neurological sequelae, Lyme disease-neurological complications, Machado-Joseph disease, macrocephaly, macrocephaly, Melkersson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease. Diseases, including paresthesia of the femoral head, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome, minor stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, moyamoya disease, mucolipid storage disease, mucopolysaccharidosis, multiple infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, congenital myasthenia gravis, myasthenia gravis, and myelinolytic diffuse sclerosis. Sclerosis), infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, thyrotoxic myopathy, myotonia, congenital myotonia, somnolence, neuroacanthosis, neurodegeneration with cerebral iron deposition, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neurogenic myotonia, neuronal ceroid lipofuscin deposition, neuronal migration abnormalities, hereditary neuropathies, neurosarcoidosis, etc. Syphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, visual oculoclonus myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenic acid kinase-dependent neurodegenerative diseases, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, Pena ShokeirII syndrome, peripheral nerve cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorders, phytate storage disease, Pick's disease, nerve pinching, piriformis syndrome, pituitary adenoma, polymyositis, polyglutamine repetitive spinocerebellar ataxia, porencephaly, post-poliomyelitis syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, orthostatic hypotension, orthostatic tachycardia syndrome, post-orthostatic tachycardia syndrome, primary dentate nucleus atrophy. Dentatum Atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerotic gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, progressive muscular atrophy, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic kinesia, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, infantile Refsum disease, repetitive motion disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral nerve root cyst, Saint Vitus chorea, salivary gland disorders, Sandhoff's disease, Schilder's disease, schizophrenia, Seitelberger's diseaseDiseases, epilepsy, semantic dementia, optic septal dysplasia, severe myoclonic epilepsy of infancy (SMEI), shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spastic state, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff person syndrome, striatal nigra degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, unilateral transient neuropathic headache (SUNCT), dysphagia Impairment, spinocerebral ataxia, Sydenham's chorea, syncope, syphilitic myelopathy, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, Tarlov's cyst, Tay-Sachs disease, temporal arteritis, tethered cord syndrome, Thomsen's rigidity, thoracic outlet syndrome, thyrotoxic myopathy, painful spasticity, Todd's paralysis, Tourette syndrome, transient ischemic attack, infectious cavernous encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paralysis, Troyer's syndrome, tuberous sclerosis, vasculatureal neoplasm. Erectile Tumor, vasculitis syndromes of the central and peripheral nervous systems, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, Whiplash, Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linked spinal-bulbar muscular atrophy, neuropathic pain, trauma resulting from spinal cord or head injury, ophthalmic diseases and disorders, cerebral infarction, depression, persistent affective disorders, secondary mood disorders, schizophrenia, drug dependence, neurosis, psychosis, dementia, paranoia, and psychosexual disorders.

[0055] In some embodiments, the CNS disease is selected from the list consisting of: dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathies, Alzheimer's disease, Huntington's disease, Parkinson's disease, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten's disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff's disease, peroneal muscular dystrophy, Angelman's syndrome, Canavan disease, late-stage infantile neuronal ceroid lipofuscin deposition disease, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodermatitis, hereditary lysosomal storage diseases (such as Niemann-Pick disease type C1) and / or neuronal ceroid lipofuscin deposition disease (such as Batten's disease), progressive supranuclear palsy, corticobasal syndrome, and brain cancer (including astrocytoma and glioblastoma).

[0056] As used in this article, "huntington disease" or "HD" refers to a neurodegenerative disease caused by the extension of a trinucleotide repeat in the HTT gene (e.g., CAG, which is translated into polyglutamine or PolyQ bundles) (resulting in the production of pathogenic mutant huntingtin protein (HTT, or mHTT)), characterized by progressively worsening motor, cognitive, and behavioral changes.

[0057] As used herein, “HTT” or “huntington” refers to the gene encoding the huntingtin protein. Normal huntingtin proteins function in nerve cells, and a normal HTT gene typically has approximately 7 to approximately 35 CAG repeats at its 5′ end. In patients with Huntington's disease or at risk of developing Huntington's disease, the HTT gene is typically mutated. In some embodiments, the mutated huntingtin protein accelerates the rate of neuronal cell death in specific regions of the brain. Generally, the severity of HD is related to the size of the trinucleotide repeat expansion in the subject. For example, subjects with a CAG repeat region containing between 36 and 39 repeats (SEQ ID NO: 157) are characterized by “reduced penetrance” HD, while subjects with more than 40 repeats are characterized by “full penetrance” HD. Thus, in some embodiments, subjects with HD or at risk of developing HD have an HTT gene containing between approximately 36 and approximately 39 CAG repeats (e.g., 36, 37, 38, or 39 repeats). In some embodiments, subjects with or at risk of developing HD have an HTT gene containing 40 or more (e.g., 40, 45, 50, 60, 70, 80, 90, 100, 200 or more) CAG repeats (SEQ ID NO: 156). In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats develop HD earlier than subjects with fewer than 100 CAG repeats. In some embodiments, subjects with an HTT gene containing more than 100 CAG repeats may develop HD symptoms before approximately age 20 and are referred to as having juvenile HD (also known as akinetic-rigid HD, or Westphal variant HD). The number of CAG repeats in the HTT gene alleles of a subject can be determined by any suitable method known in the art. For example, nucleic acids (e.g., DNA) can be isolated from a subject's biological sample (e.g., blood), and the number of CAG repeats in the HTT allele can be determined by hybridization-based methods (e.g., PCR or nucleic acid sequencing (e.g., Illumina sequencing, Sanger sequencing, SMRT sequencing, etc.)). The sequence of the HTT gene is known in some species, for example, the human HTT (NCBI Gene ID: 3064) mRNA sequence (NCBI Ref Seq: NM_002111.8, SEQ ID NO: 4) and protein sequence (NCBI Ref Seq: NP_0021012.4, SEQ ID NO: 5). Therefore, in some embodiments related to the treatment of Huntington's disease, one or more repressive nucleic acids (e.g., miRNAs) can hybridize with HTT and / or reduce HTT expression.

[0058] As used herein, "Alzheimer's disease" or "AD" refers to a neurodegenerative disease characterized by progressively worsening memory, disorientation, mood swings, and increasing difficulty with language, motivation, and self-care. Several genes may contribute to or increase the risk of AD, 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 box subfamily A member 7 (ABCA7; NCBI Gene ID 10347), and sortilin-associated receptor 1 (SORL1; NCBI Gene ID 6653). The sequences of these AD-associated genes are known in many species; for example, human mRNA and protein sequences can be obtained from the NCBI database using the provided Gene ID numbers. These AD-related genes and others, as well as their AD-related alleles (e.g., mutations, SNPs, etc.), are known in the art and are further described, for example, in: Sims et al., Nature Neuroscience 2020 23: 311-22; Bellenguez et al., Current Opinion in Neurobiology 2020 61: 40-48; Tabuas-Pereira et al., 2020 Neurogenetics and Psychiatric Genetics 8: 1-16; and Porter et al., “Neurodegeneration and Alzheimer's Disease” 2019 Chapter 15; each of which is incorporated herein by reference in its entirety. Therefore, in some embodiments relating to the treatment of Alzheimer's disease, one or more repressive nucleic acids (e.g., miRNAs) may hybridize with APP, PSEN1, PSEN2, ABCA7, and / or SORL1, and / or reduce the expression of APP, PSEN1, PSEN2, ABCA7, and / or SORL1.

[0059] "Familial Alzheimer's disease" or "FAD" refers to a subset of Alzheimer's diseases caused by a single genetic mutation inherited through an autosomal dominant inheritance pattern. Three genes are known to be associated with or cause FAD: presenilin 1 (PS1) on chromosome 14; presenilin 2 (PS2) on chromosome 1; and amyloid precursor protein (APP) on chromosome 21. Therefore, in one embodiment, the subject has a mutation in at least one of PS1, PS2, and APP.

[0060] As used in this article, "Parkinson's disease" or "PD" refers to a neurodegenerative disease characterized by progressively worsening tremors and rigidity, as well as increasing problems with balance, walking, and coordination. Many genes may contribute to or increase the risk of Parkinson's disease (PD), including synuclein α (SNCA; NCBI Gene ID: 6622), leucine-rich repeat kinase 2 (LRRK2 / PARK8; NCBI Gene ID 120892), glucosamine β (GBA1; NCBI Gene ID 2629), parkin RBR E3 ubiquitin (PRKN; NCBI Gene ID 5071), PTEN-induced kinase 1 (PINK1; NCBI Gene ID 65018), Parkinson's disease-associated desaccharidase (DJ1 / PARK7; NCBI Gene ID 11315), VPS35 retromer complex component (VPS35; NCBI Gene ID 55737), eukaryotic translation initiation factor 4γ1 (EIF4G1; NCBI Gene ID 1981), and DNAJ heat shock protein family member C13 (DNAJC13; NCBI Gene ID 1981). 23317), containing a coil-coil-coil-coil-coil-coil domain 2 (CHCHD2; NCBI Gene ID 51142), and / or ubiquitin C-terminal hydrolase L1 (UCHL1; NCBI Gene ID 7345). Sequences of such PD-related genes are known in many species; for example, human mRNA and protein sequences can be obtained from the NCBI database using the provided Gene ID number.These PD-related genes and others, as well as their PD-related alleles (e.g., mutations, SNPs, etc.), are known in the art and are further described, for example, in the following: 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; Fagan et al., European Journal of Neurology 2017 24:561-e20; Campelo et al., Parkinson's Disease 2017 4318416; and Porter et al., "Neurodegeneration and Alzheimer's "Disease" 2019 Chapter 15; each of these is incorporated herein by reference in its entirety. Thus, in some embodiments relating to the treatment of Parkinson's disease, the one or more inhibitory nucleic acids (e.g., miRNAs) may hybridize with SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, and / or GBA1, and / or reduce the expression of SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, and / or GBA1.

[0061] In one embodiment, the neurological disease or disorder is Huntington's disease.

[0062] When the disease or disorder is Huntington's disease, the method may further include assessing at least one primary outcome measure, at least one secondary outcome measure, or a combination thereof, at a time following administration of a nucleic acid encoding CYP46A1 (wild-type or codon-optimized variant). The time following administration may be, for example, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 52 weeks, 55 weeks, 60 weeks, or longer. Non-limiting examples of primary outcome measures include the incidence of dose-limiting toxicities (DLT), treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and combinations thereof. Non-limiting examples of secondary outcome measurements include anatomical and volumetric measurements of brain regions affected by Huntington's disease as assessed by MRI, Huntington's Disease Unified Comprehensive Rating Scale (cUHDRS) scores, concentrations of mutant huntingtin protein (mHTT) in blood and / or cerebrospinal fluid (CSF), levels of neurofilament light chains (NfL) in blood and / or CSF, concentrations of 24OH cholesterol in blood and / or CSF, magnetic resonance spectroscopy (MRS) metabolic profiles, positron emission tomography (PET) fluorodeoxyglucose (FDG) striatal profiles, and combinations thereof.

[0063] In some implementations, anatomical and volumetric measurements of brain regions affected by Huntington's disease are determined. The variability and magnitude of changes relative to baseline (i.e., measurements of brain regions at the time of rAAV administration of the nucleic acid encoding CYP46A1, determined by the slope of the progression of cortical atrophy, ventricular dilation, and striatal atrophy) can be maintained (i.e., stable) or improved (e.g., reduced) by greater than or equal to about 0.5 vol%, greater than or equal to about 1 vol%, greater than or equal to about 2 vol%, greater than or equal to about 3 vol%, greater than or equal to about 4 vol%, greater than or equal to about 5 vol%, greater than or equal to about 6 vol%, greater than or equal to about 7 vol%, greater than or equal to about 8 vol%, greater than or equal to about 9 vol%, greater than or equal to about 1 vol%, and greater than or equal to about 1 vol%. 0 vol%, greater than or equal to about 15 vol%, greater than or equal to about 20 vol%, greater than or equal to about 25 vol% or more, for example, variations of about 0.5 vol%, about 1 vol%, about 2 vol%, about 3 vol%, about 4 vol%, about 5 vol%, about 6 vol%, about 7 vol%, about 8 vol%, about 9 vol%, about 10 vol%, about 11 vol%, about 12 vol%, about 13 vol%, about 14 vol%, about 15 vol%, about 16 vol%, about 17 vol%, about 18 vol%, about 19 vol%, about 20 vol%, about 25 vol% or more.

[0064] In some implementations, a cUHDRS score is determined and compared with a baseline cUHDRS score (i.e., the cUHDRS score at the time of rAAV administration of the nucleic acid encoding CYP46A1). The cUHDRS score, relative to the baseline cUHDRS score, may be maintained (i.e., stable) or improved by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more.

[0065] In some implementations, the mHTT concentration in blood and / or CSF is determined and compared with the corresponding baseline mHTT concentration (i.e., the mHTT concentration in blood and / or CSF at the time of administration of rAAV encoding the nucleic acid of CYP46A1). Relative to the baseline mHTT concentration, the mHTT concentration may be maintained (i.e., stabilized) or improved (i.e., reduced) by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more.

[0066] In some implementations, the NfL level in blood and / or CSF is determined and compared with the corresponding baseline NfL level (i.e., the NfL level in blood and / or CSF at the time of administration of rAAV encoding the nucleic acid of CYP46A1). The NfL level relative to the baseline NfL level concentration may be maintained (i.e., stable) or improved (i.e., reduced) by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more.

[0067] In some implementations, the concentration of 24OH cholesterol in blood and / or CSF is determined and compared with the corresponding baseline 24OH cholesterol concentration (i.e., the concentration of 24OH cholesterol in blood and / or CSF at the time of administration of rAAV encoding the nucleic acid of CYP46A1). Relative to a baseline 24OH cholesterol concentration, the 24OH cholesterol concentration may be maintained (i.e., stable) or improved (i.e. increased) by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more.

[0068] In some implementations, an MRS metabolic profile is determined and compared with a corresponding baseline MRS metabolic profile (i.e., the MRS metabolic profile at the time of rAAV administration of the nucleic acid encoding CYP46A1). Compared to the baseline MRS metabolic profile, the MRS metabolic profile may be maintained (i.e., stable) or improved by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more.

[0069] In some implementations, a PET FDG profile is determined and compared with a corresponding baseline PET FDG profile (i.e., the PET FDG profile at the time of rAAV administration of the nucleic acid encoding CYP46A1). The PET FDG profile may be maintained (i.e., stable) or improved by more than or equal to about 0.5%, more than or equal to about 1%, more than or equal to about 2%, more than or equal to about 3%, more than or equal to about 4%, more than or equal to about 5%, more than or equal to about 6%, more than or equal to about 7%, more than or equal to about 8%, more than or equal to about 9%, more than or equal to about 10%, more than or equal to about 15%, more than or equal to about 20%, more than or equal to about 25% or more, for example, improved by about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25% or more, relative to the baseline PET FDG profile.

[0070] In one implementation, the neurological disease or disorder is Alzheimer's disease.

[0071] In one embodiment, the neurological disease or disorder is familial Alzheimer's disease.

[0072] In one implementation, the neurological disease or disorder is Parkinson's disease.

[0073] In one embodiment, the neurological disease or disorder is multisystem atrophy.

[0074] 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 infect, in the case of rAAV-mediated delivery) a sufficient number of target cells in a subject’s target tissue. In some embodiments, the target tissue is central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, the effective amount of isolated nucleic acid (e.g., which can be delivered via rAAV) may be an amount sufficient to have a therapeutic benefit in the subject, such as reducing the expression of a pathogenic gene or protein (e.g., HTT), prolonging the lifespan of the subject, or improving symptoms of one or more diseases in the subject (e.g., symptoms of Huntington's disease), etc. The effective amount will depend on various factors, such as species, age, weight, the health of the subject, and the tissue to be targeted, and therefore may vary between subjects and tissues, as described elsewhere in this disclosure. Repressive RNA

[0075] In some aspects, this disclosure provides an inhibitory nucleic acid (e.g., miRNA) that specifically binds to (e.g., hybridizes with) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive bases of a target (e.g., human huntingtin mRNA (e.g., SEQ ID NO: 4)). In some embodiments, this disclosure provides a repressive nucleic acid (e.g., miRNA) that specifically binds to (e.g., hybridizes with) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive bases of exon 1 (e.g., SEQ ID NO: 3) of human huntingtin mRNA. As used herein, “consecutive bases” refers to two or more nucleotide bases covalently bonded (e.g., via one or more phosphodiester bonds, etc.) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, the at least one miRNA is approximately 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% identical to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleotide bases of the target (e.g., SEQ ID NO: 3 or SEQ ID NO: 4). In some embodiments, the repressive RNA is a miRNA comprising any one of the sequences shown in SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260, or a miRNA encoded by any one of the sequences shown in SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260.

[0076] In one respect, this article describes inhibitory RNAs that can be used to treat neurological diseases or disorders. In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of the following: SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185 or SEQ ID NO: 217-SEQ ID NO: 260, or at least one of the sequences of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185 or SEQ ID NO: 217-SEQ ID NO: 260 is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical and retains the same function (e.g., HTT inhibition) as SEQ ID NO: 3 or SEQ ID NO: 4.

[0077] In some embodiments, the vector described herein comprises at least one miRNA, each miRNA comprising the sequence shown in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260, with a miRNA backbone sequence lateralized thereto.

[0078] In some embodiments, the vector described herein comprises at least one miRNA, each miRNA containing a seed sequence complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49. In some embodiments, the vector described herein comprises at least one miRNA, each miRNA containing a seed sequence complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49, flanked by a miRNA backbone sequence. In some embodiments, the vector described herein comprises at least one miRNA, each miRNA containing a seed sequence substantially complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49. In some embodiments, the vector described herein contains at least one miRNA, each miRNA containing a seed sequence substantially complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39 or SEQ ID NO: 46-SEQ ID NO: 49, which is side-joined with a miRNA backbone sequence.

[0079] Table 1: First RNA sequence substantially complementary to SEQ ID NO: 4

[0080] Table 2: Second RNA sequences that are substantially complementary to one or more first RNA sequences provided in Table 1

[0081] Table 3: Target sequences in exon 1 of the human HTT gene targeted by miRNAs, as provided in Tables 1 and 2.

[0082] In some embodiments, the miRNA comprises SEQ ID NO: 6 and SEQ ID NO: 11, SEQ ID NO: 7 and SEQ ID NO: 12; SEQ ID NO: 8 and SEQ ID NO: 11; SEQ ID NO: 8 and SEQ ID NO: 16; SEQ ID NO: 8 and SEQ ID NO: 17; SEQ ID NO: 9 and SEQ ID NO: 14; or SEQ ID NO: 10 and SEQ ID NO: 15.

[0083] In some embodiments, the vector comprises a pre-miRNA having the sequence SEQ ID NO: 40 or SEQ ID NO: 41. These pre-miRNAs include a scaffold containing SEQ ID NO: 8. In either SEQ ID NO: 40 or SEQ ID NO: 41, an alternative first RNA sequence disclosed herein may be used instead of SEQ ID NO: 8.

[0084] In some embodiments, the vector comprises a pri-miRNA having the sequence SEQ ID NO: 42 or SEQ ID NO: 43. The pri-miRNA of SEQ ID NO: 42 comprises a scaffold containing SEQ ID NO: 8 and SEQ ID NO: 16. In SEQ ID NO: 42, alternative RNA sequences disclosed herein may be used instead of SEQ ID NO: 8 and SEQ ID NO: 16. The pri-miRNAs of SEQ ID NO: 43 and SEQ ID NO: 44 comprise a scaffold containing SEQ ID NO: 8 and SEQ ID NO: 17. In either SEQ ID NO: 43 or SEQ ID NO: 44, alternative RNA sequences disclosed herein may be used instead of SEQ ID NO: 8 and SEQ ID NO: 17.

[0085] Table 4: Pre-miRNAs and pri-miRNAs containing miRNAs provided in Tables 1 and 2

[0086] In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 1-SEQ ID NO: 102 and / or SEQ ID NO: 103-SEQ ID NO: 249 of International Patent Publication WO2017 / 201258. In some embodiments, the inhibitory nucleic acid may comprise one or more of the double-stranded combinations selected from SEQ ID NO: 1-SEQ ID NO: 249 of International Patent Publication WO2017 / 201258, which are provided in Tables 3-5 of International Patent Publication WO2017 / 201258. In some embodiments, the vector may comprise one or more of the pri-miRNAs provided in Table 9 or Table 10 of International Patent Publication WO2017 / 201258. The entire contents of International Patent Publication WO2017 / 201258 are incorporated herein by reference.

[0087] In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 914-SEQ ID NO: 1013 and / or SEQ ID NO: 1014-SEQ ID NO: 1160 of International Patent Publication WO2018 / 204803. In some embodiments, the inhibitory nucleic acid may comprise one or more of a combination of duplexes selected from SEQ ID NO: 914-SEQ ID NO: 1160 of International Patent Publication WO2018 / 204803, which are provided in Tables 4-6 of International Patent Publication WO2018 / 204803. The entire contents of International Patent Publication WO2018 / 204803 are incorporated herein by reference.

[0088] In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 916-SEQ ID NO: 1015 and / or SEQ ID NO: 1016-SEQ ID NO: 1162 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 916-SEQ ID NO: 1015, SEQ ID NO: 1016-SEQ ID NO: 1162, SEQ ID NO: 1164-SEQ ID NO: 1332 and / or SEQ ID NO: 1333-SEQ ID NO: 1501 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may comprise one or more of a combination of duplexes selected from SEQ ID NO: 916-SEQ ID NO: 1162 of International Patent Publication WO2018 / 204797, which are provided in Tables 4-6 of International Patent Publication WO2018 / 204797. In some embodiments, the inhibitory nucleic acid may comprise one or more of the duplex combinations selected from SEQ ID NO: 1164-SEQ ID NO: 1501 of International Patent Publication WO2018 / 204797, which are provided in Table 9 of International Patent Publication WO2018 / 204797. The entire contents of International Patent Publication WO2018 / 204797 are incorporated herein by reference.

[0089] In some embodiments, the repressive nucleic acid can target heterozygous SNPs (e.g., sequences complementary to or substantially complementary to heterozygous SNPs in genes encoding gain-of-function mutant huntingtin proteins) within a sample population. In some embodiments, the SNP has an allele frequency of at least 10% in a sample population. In some embodiments, the SNP is present at a genomic locus 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 9,343,943, which is incorporated herein by reference in its entirety. In some embodiments, the target sequence is one of SEQ ID NO: 45-SEQ ID NO: 49. In some embodiments, the repressive nucleic acid sequence comprises one or more of SEQ ID NO: 50-SEQ ID NO: 61. In some embodiments, the repressive nucleic acid sequence comprises at least SEQ ID NO: 50 and SEQ ID NO: 51 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence comprises at least SEQ ID NO: 52 and SEQ ID NO: 53 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence comprises at least SEQ ID NO: 54 and SEQ ID NO: 55 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence comprises at least SEQ ID NO: 56 and SEQ ID NO: 57 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence comprises at least SEQ ID NO: 58 and SEQ ID NO: 59 (e.g., in a double strand). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO: 60 and SEQ ID NO: 61 (e.g., in a duplex).

[0090] Table 5 - Target Sequences

[0091] Table 6: Sensitive and antisense sequences (or first and second RNA sequences) targeting SNPs in the human HTT gene

[0092] In one embodiment, the repressive nucleic acid (e.g., miRNA) is capable of specifically hybridizing to exon 1 of the HTT gene. Exemplary miRNAs that hybridize to exon 1 of the HTT gene are listed in Table 17, namely, SEQ ID NO: 158-SEQ ID NO: 160 and SEQ ID NO: 164-SEQ ID NO: 166.

[0093] In one embodiment, the repressive nucleic acid (e.g., miRNA) is capable of specifically hybridizing to the exon 1 / exon 2 boundary of the HTT gene. Exemplary miRNAs that hybridize to the exon 1 / exon 2 boundary of the HTT gene are listed in Table 17, namely, SEQ ID NO: 161-SEQ ID NO: 163 and SEQ ID NO: 167-SEQ ID NO: 169 (i.e., as indicated by asterisks).

[0094] In one embodiment, the nucleic acid sequence of the repressive RNA includes at least one of SEQ ID NO: 158-SEQ ID NO: 169 or a sequence 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 and retains the same HTT repressive function as at least one of SEQ ID NO: 158-SEQ ID NO: 169.

[0095] In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 158 and SEQ ID NO: 164 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 159 and SEQ ID NO: 165 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 160 and SEQ ID NO: 166 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 161 and SEQ ID NO: 167 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 162 and SEQ ID NO: 168 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 163 and SEQ ID NO: 169 (e.g., in a double strand).

[0096] In one embodiment, the repressive nucleic acid (e.g., miRNA) is capable of specifically hybridizing to intron 1 of the HTT gene. Exemplary miRNAs that hybridize to intron 1 of the HTT gene are listed in Table 18, namely, SEQ ID NO: 170-SEQ ID NO: 174 and SEQ ID NO: 178-SEQ ID NO: 182.

[0097] In one embodiment, the repressive nucleic acid (e.g., miRNA) is capable of specifically hybridizing to the exon 1 / intron 1 boundary of the HTT gene. Exemplary miRNAs that hybridize to the exon 1 / intron 1 boundary of the HTT gene are listed in Table 18, namely, SEQ ID NO: 175-SEQ ID NO: 177 and SEQ ID NO: 183-SEQ ID NO: 185 (i.e., as indicated by asterisks).

[0098] In one embodiment, the nucleic acid sequence of the repressive RNA includes one of SEQ ID NO: 170-SEQ ID NO: 185 or a sequence 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 SEQ ID NO: 170-SEQ ID NO: 185 and retains the same HTT repressive function.

[0099] In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 170 and SEQ ID NO: 178 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 171 and SEQ ID NO: 179 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 172 and SEQ ID NO: 180 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 173 and SEQ ID NO: 181 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 174 and SEQ ID NO: 182 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 175 and SEQ ID NO: 183 (e.g., in a double strand). In some embodiments, the repressive nucleic acid sequence includes at least SEQ ID NO: 176 and SEQ ID NO: 184 (e.g., in a double strand). In some embodiments, the inhibitory nucleic acid sequence includes at least SEQ ID NO: 177 and SEQ ID NO: 185 (e.g., in a double strand).

[0100] In one embodiment, the isolated nucleic acid of this disclosure comprises a sequence encoding a miRNA, said miRNA comprising the sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0101] In some embodiments of any aspect, the nucleic acid sequence of the repressive RNA comprises one of SEQ ID NO: 158-SEQ ID NO: 185 or a sequence 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 SEQ ID NO: 158-SEQ ID NO: 185, the sequence retaining the same function (e.g., HTT inhibition) as SEQ ID NO: 158-SEQ ID NO: 185.

[0102] In one embodiment, the preferred miRNA molecule or its equivalent or analogue or isomiR comprises at least 6 of the 7 nucleotides present in the seed sequences identified in or recognized as SEQ ID NO: 158-SEQ ID NO: 185, and more preferably has a length of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 nucleotides or more.

[0103] This article describes a 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, and wherein each miRNA comprises a sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0104] This article describes a method for treating CNS diseases (e.g., Huntington's disease), the method comprising administering a recombinant AAV to a subject, the 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, and wherein each miRNA comprises a sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0105] This article describes a method for treating CNS diseases (e.g., Huntington's disease) comprising administering to a subject a recombinant AAV and a CYP461 protein or a nucleic acid sequence encoding a CYP46A1 protein, wherein the recombinant AAV comprises a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs, and wherein each miRNA comprises a sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0106] This article describes a method for treating CNS diseases (e.g., Huntington's disease), the method comprising administering to a subject a recombinant AAV and an immunomodulator, the 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, and wherein each miRNA comprises a sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0107] This article describes a method for treating CNS diseases (e.g., Huntington's disease), the method comprising administering to a subject a recombinant AAV, a CYP461 protein or a nucleic acid sequence encoding a CYP46A1 protein, and an immunomodulator, wherein the recombinant AAV comprises a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs, and wherein each miRNA comprises a sequence shown in any one of SEQ ID NO: 158-SEQ ID NO: 185.

[0108] This document describes a recombinant AAV having a genome comprising, substantially consisting of, or consisting of the sequence of SEQ ID NO: 194. In various embodiments, any element described in the sequence of SEQ ID NO: 194 may be replaced by any element described herein.

[0109] For example, in one embodiment, the enhancer element described in the sequence of SEQ ID NO: 194 may be replaced by any enhancer element described herein. In one embodiment, the promoter described in the sequence of SEQ ID NO: 194 may be replaced by any promoter described herein. In one embodiment, the CYP46A1 transgene described in the sequence of SEQ ID NO: 194 may be replaced by any CYP46A1 transgene described herein, for example, a CYP46A1 transgene optimized with different codons. In one embodiment, the poly(A) signal sequence described in the sequence of SEQ ID NO: 194 may be replaced by any poly(A) signal sequence described herein. In one embodiment, the intron element described in the sequence of SEQ ID NO: 194 may be replaced by any intron element described herein. In one embodiment, the prokaryotic telomerase recognition sequence described in the sequence of SEQ ID NO: 194 may be replaced by any prokaryotic telomerase recognition sequence described herein. In one embodiment, any adapter or stuffer sequence described in the sequence of SEQ ID NO: 194 may be replaced by any adapter or stuffer sequence described herein. In one implementation, the ITR described in the sequence of SEQ ID NO: 194 may be replaced by any ITR described herein.

[0110] In one implementation, the sequence of SEQ ID NO: 194 is modified to include any miRNA described herein.

[0111] This document further describes compositions comprising recombinant AAV having a genome comprising, substantially consisting of, or consisting of the sequence of SEQ ID NO: 194.

[0112] This article describes a method for treating CNS diseases or disorders, the method comprising administering a recombinant AAV to a subject, the recombinant AAV having a genome comprising, substantially consisting of, or consisting of the sequence of SEQ ID NO: 194. In one embodiment, an immunomodulatory agent is further administered to the subject.

[0113] This article describes a method for treating Huntington's disease, the method comprising administering to a subject a recombinant AAV having a genome comprising the sequence of SEQ ID NO: 194, substantially consisting of the sequence of SEQ ID NO: 194, or consisting of the sequence of SEQ ID NO: 194.

[0114] Another aspect of this article is a method for treating CNS diseases or disorders (e.g., Huntington's disease), the method comprising administering to a subject a recombinant AAV having a genome comprising the sequence of SEQ ID NO: 194 as modified as described above, substantially consisting of the sequence of SEQ ID NO: 194 as modified as described above, or consisting of the sequence of SEQ ID NO: 194 as modified as described above, for example, the CYP46A1 transgenic sequence of SEQ ID NO: 194 being replaced by another CYP46A1 transgenic sequence as described herein. In one embodiment, an immunomodulator is further administered to the subject.

[0115] In one implementation, an immunomodulator is further administered to the subject.

[0116] In some embodiments, the vector comprises at least one repressive nucleic acid sequence (e.g., miRNA) as described herein, and the vector comprises at least one copy of the at least one repressive nucleic acid sequence. For example, the vector may comprise at least one, at least two, at least three, at least four, at least five or more of the given miRNAs.

[0117] In some embodiments, the repressive nucleic acid (e.g., miRNA) may specifically hybridize to, or target a polymorphism, mutation, or SNP in one of the genes disclosed herein. Methods for selecting a repressive nucleic acid sequence that targets a polymorphism (e.g., SNP) in the HTT gene are known in the art. Such methods are disclosed, for example, in U.S. Patents 8,679,750 and 7,947,658, each of which is incorporated herein by reference in its entirety. In some embodiments, the repressive nucleic acid may comprise, for example, one or more sequences from SEQ ID NO: 1-SEQ ID NO: 342 of U.S. Patents 8,679,750 or 7,947,658.

[0118] In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 62-SEQ ID NO: 66.

[0119] Table 7. In some embodiments, the uppercase letters contain 2'-O-(2-methoxy)ethyl modification.

[0120] Further suitable sequences are known in the art, for example in U.S. Patent 7,951,934; Miniarikava et al., Molecular Therapy-Nucleic Acids 20155:e297; and Kordasiweicz et al., Neuron 201274:1031-1044; all of which are incorporated herein by reference in their entirety.

[0121] In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5′ untranslated region (UTR) of a target. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets one or more exons of a target. In some embodiments of any aspect, a repressive RNA (e.g., miRNA) binds to and / or targets the 5′ UTR of the HTT, exon 1, CAG repeat, CAG 5′-jumper, or CAG 3′-jumper.

[0122] In some embodiments, the repressive RNA and / or vector does not contain a sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the repressive RNA and / or vector does not contain a sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with any one of SEQ ID NO: 67-SEQ ID NO: 73.

[0123] In some embodiments, the repressive RNA and / or vector does not contain any of the sequences in SEQ ID NO: 67-SEQ ID NO: 73 or SEQ ID NO: 135-SEQ ID NO: 151. In some embodiments, the repressive RNA and / or vector does not contain any sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with any of SEQ ID NO: 67-SEQ ID NO: 73 or SEQ ID NO: 135-SEQ ID NO: 151.

[0124] In some embodiments, the repressive RNA and / or vector does indeed contain a sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the repressive RNA and / or vector does indeed contain a sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with any one of SEQ ID NO: 67-SEQ ID NO: 73.

[0125] In some embodiments, the repressive RNA and / or vector does indeed comprise a sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73 or SEQ ID NO: 135-SEQ ID NO: 151. In some embodiments, the repressive RNA and / or vector does indeed comprise a sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% sequence identity with any one of SEQ ID NO: 67-SEQ ID NO: 73 or SEQ ID NO: 135-SEQ ID NO: 151. See, for example, International Patent Application WO 2021 / 127455, the contents of which are incorporated herein by reference in their entirety.

[0126] Table 8

[0127] Suitable sequences for use in repressive nucleic acids (e.g., miRNAs) targeting AD and / or PD-related targets are known in the art, for example, see International Patent Publications WO2011 / 133890, WO2012 / 036433, WO2013 / 007874; US Patent Publication US2016 / 0264965; US Patent Nos. 7,829,694, 8,415,319, 10,125,363, 10,011,835. The contents of the foregoing references are incorporated herein by reference in their entirety.

[0128] In some embodiments of any aspect, the agent for treating neurological disorders or disturbances is or contains a repressive nucleic acid. In some embodiments of any aspect, an inhibitor of the expression of a given gene may be a repressive nucleic acid. As used herein, "repressive nucleic acid" refers to a nucleic acid molecule capable of inhibiting the expression of a target, such as double-stranded RNA (dsRNA), repressive RNA (iRNA), etc.

[0129] Double-stranded RNA molecules (dsRNAs) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The repressive nucleic acids described herein may contain an RNA strand (antisense strand) having a region of less than 30 nucleotides in length (i.e., 15–30 nucleotides in length, typically 19–24 nucleotides), said region being substantially complementary to at least a portion of the targeted mRNA transcript. Using these iRNAs enables the degradation of the targeted mRNA transcript, resulting in reduced expression and / or activity of the target.

[0130] As used herein, the term "iRNA" refers to an agent containing RNA (or a modified nucleic acid as described below) that mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of any aspect, iRNA as described herein causes inhibition of the expression and / or activity of a target. In some embodiments of any aspect, contacting cells with an inhibitor (e.g., iRNA) results in a reduction of the target mRNA level in the cells by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in cells where iRNA is not present. In some embodiments of any aspect, administration of an inhibitor (e.g., iRNA) to a subject may reduce the level of the target mRNA in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the level of the target mRNA found in subjects without iRNA.

[0131] In some embodiments of any aspect, the iRNA may be dsRNA. The dsRNA comprises two RNA strands sufficiently complementary to hybridize and form a duplex structure under the conditions under which the dsRNA will be used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary to, and typically perfectly complementary to, the target sequence. The target sequence may be derived from the sequence of mRNA formed during the expression of the target, for example, it may cross the boundaries of one or more introns. The other strand (the sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when they bind under suitable conditions. Generally, the length of the duplex structure is between 15 and 30 base pairs, more typically between 18 and 25 base pairs, even more typically between 19 and 24 base pairs, and most typically between 19 and 21 base pairs. Similarly, the length of the region complementary to the target sequence is between 15 and 30 base pairs, more typically between 18 and 25 base pairs, even more typically between 19 and 24 base pairs, and most typically between 19 and 21 base pairs. In some embodiments of any aspect, the length of the dsRNA is between 15 and 20 nucleotides, and in other embodiments, the length of the dsRNA is between 25 and 30 nucleotides. As those skilled in the art will recognize, the targeted region of the RNA to be cleaved will often be part of a larger RNA molecule (typically an mRNA molecule). In relevant cases, a “part” of the mRNA target is a contiguous sequence of the mRNA target that is long enough to serve as a substrate for targeted RNAi cleavage (i.e., cleavage via the RISC pathway). In some cases, dsDNA with a short double helix of as little as 9 base pairs can mediate RNAi-directed RNA cleavage. Most typically, the target length will be at least 15 nucleotides, preferably 15-30 nucleotides. Exemplary embodiments of repressive nucleic acid types may include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art.

[0132] In some embodiments of any aspect, the inhibitor is a miRNA. MicroRNA (miRNA) is a small RNA of 17-25 nucleotides that functions as a regulator of gene expression in eukaryotic cells. “MicroRNA” or “miRNA” is a small non-coding RNA molecule capable of mediating transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed into hairpin or stem-loop (e.g., a self-complementary single-stranded backbone) duplex structures, referred to as primary miRNAs (pri-miRNAs), which are enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into pre-miRNAs. The duplex structure comprises: a) a first RNA sequence, which is a complementary region substantially complementary to, and generally perfectly complementary to, the target sequence; and b) a second RNA sequence region complementary to the first RNA sequence strand such that the two sequences hybridize and form a duplex structure when combined under suitable conditions. The target sequence can be derived from the sequence of mRNA formed during the expression of the target, for example, it can cross the boundaries of one or more introns. Generally, the length of a double-stranded structure is between 15 and 30 base pairs, more commonly between 18 and 25 base pairs, even more commonly between 19 and 24 base pairs, and most commonly between 19 and 21 base pairs.

[0133] miRNAs are initially expressed in the cell nucleus as part of a long primary transcript called primary miRNA (pri-miRNA). The length of pri-miRNA can vary. In some embodiments, the length of pri-miRNA ranges from about 100 to about 5000 base pairs (e.g., about 100, about 200, about 500, about 1000, about 1200, about 1500, about 1800, or about 2000 base pairs). In some embodiments, the length of pri-miRNA is greater than 200 base pairs (e.g., 2500, 5000, 7000, 9000, or more base pairs).

[0134] Within the cell nucleus, pri-miRNA is partially digested by the Drosha enzyme to form hairpin precursor miRNA (pre-miRNA) of 65-120 nucleotides in length. This pre-miRNA is exported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs, which are the active molecules. In animals, these short RNAs contain a 5′ proximal “seed” region (nucleotides 2 to 8), which appears to be the primary determinant of the pairing specificity between the miRNA and the 3′ untranslated region (3′-UTR) of the target mRNA. The length of the pre-miRNA (also characterized by a hairpin or stem-loop double-stranded structure) can also vary. In some embodiments, the pre-miRNA size ranges from approximately 40 base pairs to approximately 500 base pairs. In other embodiments, the pre-miRNA size ranges from approximately 50 to 100 base pairs. In some implementations, the pre-miRNA size ranges from about 50 to about 90 base pairs in length (e.g., about 50, about 52, about 54, about 56, about 58, about 60, about 62, about 64, about 66, about 68, about 70, about 72, about 74, about 76, about 78, about 80, about 82, about 84, about 86, about 88, or about 90 base pairs in length).

[0135] Generally, pre-miRNA is exported to the cytoplasm and enzymatically processed by Dicer to first produce an incomplete miRNA / miRNA* duplex, followed by a single-stranded mature miRNA molecule, which is then loaded into the RNA-induced silencing complex (RISC). Typically, the size of the mature miRNA molecule ranges from approximately 19 to approximately 30 base pairs in length. In some embodiments, the mature miRNA molecule length is approximately 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs. In some embodiments, the isolated nucleic acid of this disclosure comprises a sequence encoding pri-miRNA, pre-miRNA, or mature miRNA, comprising any one of the sequences shown in SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260.

[0136] In the context of this invention, a miRNA molecule or its equivalent or mimetic, or isomiR, can be synthetic, natural, recombinant, mature, or a portion of a mature miRNA, or a human miRNA, or derived from a human miRNA, as further defined in the section specifically for general definitions. A human miRNA molecule is a miRNA molecule found in human cells, tissues, organs, or body fluids (i.e., endogenous human miRNA molecules). A human miRNA molecule can also be a human miRNA molecule derived from an endogenous human miRNA molecule by substitution, deletion, and / or addition of nucleotides. A miRNA molecule or its equivalent or mimetic can be a single-stranded or double-stranded RNA molecule. Preferably, the miRNA molecule or its equivalent or analogue has a length of 6 to 30 nucleotides, more preferably 12 to 30 nucleotides, more preferably 15 to 28 nucleotides, and even more preferably, the molecule has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

[0137] In a preferred embodiment, the miRNA molecule or its equivalent, mimetic, or isomiR comprises at least six of the seven nucleotides present in the seed sequence of the miRNA molecule or its equivalent, mimetic, or isomiR. Preferably, in this embodiment, the miRNA molecule or its equivalent, mimetic, or isomiR is 6 to 30 nucleotides in length, and more preferably comprises at least six of the seven nucleotides present in the seed sequence of the miRNA molecule or its equivalent. Even more preferably, the miRNA molecule or its equivalent or analogue or isomiR has a length of 15 to 28 nucleotides, and even more preferably, it 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 more.

[0138] Therefore, preferred miRNA molecules or their equivalents or mimics or isomiRs contain those identified as SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260, or in SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260. At least 6 of the 7 nucleotides in the seed sequence identified in NO: 260, and more preferably of 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 more.

[0139] Delivery vectors for miRNAs include, but are not limited to, liposomes, polymer nanoparticles, viral systems, conjugates of lipid or receptor-binding molecules, exosomes, and phages; see, for example, Baumann and Winkler, miRNA-based therapies: Strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents, Future Med Chem. 2014, 6(17): 1967-1984; U.S. Patents 8,900,627, 9,421,173, and 9,555,060; WO 2019 / 177550; the contents of which are incorporated herein by reference in their entirety.

[0140] The microRNA sequence includes a "seed" region, which is the sequence in the region from position 2 to position 8 of the mature microRNA, and whose sequence has perfect Watson-Crick complementarity with the miRNA target sequence of the nucleic acid. In one implementation, the viral genome can be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0141] The term substantial complementarity means that complete complementarity between the first and second RNA sequences is not required, or that complete complementarity between the first RNA sequence and the reference or target sequence (e.g., SEQ ID NO: 3 or SEQ ID NO: 4) is not required. In one embodiment, substantial complementarity between the RNA sequence and the target consists of: no mismatches, having one mismatched nucleotide, or having two mismatched nucleotides. It is understood that a one-mismatched nucleotide refers to a nucleotide that does not pair with a target base along the entire length of the RNA sequence that pairs with the target base. No mismatches means that all nucleotides pair with the target base, and having two mismatches means that two nucleotides do not pair with the target base.

[0142] miRNAs and / or transgenes containing one or more miRNAs may be provided in or contain scaffold sequences. As used herein, a "scaffold" refers to a portion of the miRNA coding sequence outside the mature double-stranded structure. For example, a scaffold may contain loop and / or stem regions. Thus, scaffolds are useful in the generation, encoding, and / or expression of the miRNAs described herein. The scaffolds used in the compositions and methods described herein may be sequences of endogenous and / or naturally occurring miRNA scaffolds, sequences obtained from endogenous and / or naturally occurring miRNA scaffolds, and / or sequences derived from endogenous and / or naturally occurring miRNA scaffolds, such as human miRNAs. In some embodiments, the scaffold sequences used in the compositions and methods described herein may be sequences of endogenous and / or naturally occurring miRNA scaffolds, sequences obtained from endogenous and / or naturally occurring miRNA scaffolds, and / or sequences derived from endogenous and / or naturally occurring miRNA scaffolds of miRNAs overexpressed in one or more NS and / or CNS diseases.

[0143] In one embodiment, the scaffold sequence used in the compositions and methods described herein may be a sequence of an endogenous and / or naturally occurring miRNA scaffold, a sequence obtained from an endogenous and / or naturally occurring miRNA scaffold, and / or a sequence derived from an endogenous and / or naturally occurring miRNA scaffold overexpressed in Huntington's disease. Exemplary scaffolds overexpressed in Huntington's disease are described in Table 24 herein.

[0144] In one embodiment, the scaffold sequence used in the compositions and methods described herein may be a sequence of an endogenous and / or naturally occurring miRNA scaffold, a sequence obtained from an endogenous and / or naturally occurring miRNA scaffold, and / or a sequence derived from an endogenous and / or naturally occurring miRNA scaffold overexpressed in Huntington's disease.

[0145] In one embodiment, the miRNA used in the vector described herein is selected from... Table 24. Nucleic acid

[0146] In some aspects, this disclosure provides isolated nucleic acids that are useful for reducing (e.g., inhibiting) the expression of pathogenic genes (e.g., HTT) or encoding CYP46A1. A “nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, the proteins and nucleic acids of this disclosure are isolated. As used herein, the term “isolated” means artificially produced. As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) produced by clonal recombination; (iii) purified, such as by cutting and gel separation; or (iv) synthesized by, for example, chemical synthesis. Isolated nucleic acids are nucleic acids that can be readily manipulated using recombinant DNA techniques well known in the art. Thus, nucleotide sequences contained in vectors in which the 5′ and 3′ restriction sites are known or whose polymerase chain reaction (PCR) primer sequences have been disclosed are considered isolated, but nucleic acid sequences present in their natural state in their natural host are not. Isolated nucleic acids may be substantially purified, but are not required to be. For example, nucleic acids isolated within a cloning or expression vector are not pure because they may contain only a very small percentage of the substance found in the cell in which they reside. However, such nucleic acids are isolated, as the term is used herein, because they can be readily manipulated using standard techniques known to those skilled in the art. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or that has been artificially produced (e.g., through chemical synthesis, through recombinant DNA technology, etc.).

[0147] Those skilled in the art will also recognize that conserved amino acid substitutions can be made to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, this disclosure includes sequence alterations that result in conserved amino acid substitutions. As used herein, a conserved amino acid substitution is an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution has been made. Variants can be prepared according to methods of altering polypeptide sequences known to those skilled in the art, such as those found in references that have documented such methods, for example, *Molecular Cloning: A Laboratory Manual*, eds. J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or *Current Protocols in Molecular Biology*, eds. F. Mausubel et al., John Wiley & Sons, Inc., New York. Conserved substitution of amino acids includes substitutions between amino acids within the following group: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, conserved amino acid substitutions can be performed on the amino acid sequences of the proteins and peptides disclosed in this paper.

[0148] The isolated nucleic acid of the present invention may be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described herein comprises a region (e.g., a first region) 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 within a capsid protein and administered to and / or delivered to a subject to selected target cells. A “recombinant AAV (rAAV) vector” typically consists of at least a transgene and its regulatory sequence, and 5′ and 3′ AAV inverted terminal repeats (ITRs). As disclosed elsewhere herein, the transgene may comprise one or more regions encoding one or more repressive RNAs (e.g., miRNAs) containing nucleic acids targeting the subject’s endogenous mRNA. As described elsewhere herein, the transgene may also comprise regions encoding, for example, proteins and / or expression control sequences (e.g., poly-A tails).

[0149] Generally, the ITR sequence is approximately 145 bp in length. Preferably, the sequence encoding the ITR is used in the molecule in its entirety, although some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope of the art. (See, for example, the following articles: Sambrook et al., “Molecular Cloning. A Laboratory Manual,” 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70: 520-532 (1996)). An example of such a molecule used in this invention is a transgenic “cis-acting” plasmid in which the selected transgenic sequence and associated regulatory element are flanked by 5′ and 3′ AAV ITR sequences. The AAV ITR sequence can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., an rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region encoding an AAV2 ITR (e.g., a first region).

[0150] In some embodiments, the isolated nucleic acid further includes a region containing a second AAV ITR (e.g., a second region, a third region, a fourth region, etc.). In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, and variants thereof. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal cleavage site (TRS). The term "lacking a terminal cleavage site" can refer to an AAV ITR containing a mutation that negates the function of the terminal cleavage site (TRS) of the ITR (e.g., a sense mutation, such as a nonsynonymous mutation, or a missense mutation), or to a truncated AAV ITR (e.g., an ATRSITR) lacking a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors containing ITRs lacking functional TRS generate self-complementary rAAV vectors, as described, for example, by McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0151] In addition to the major elements identified above for the recombinant AAV vector, the vector also contains conventional control elements that are operatively linked to transgenic elements in a manner that allows them to be transcribed, translated, and / or expressed in cells transfected with the vector or infected with a virus produced in this invention. As used herein, "operatively linked" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that control the gene of interest by acting trans or at a distance. Expression control sequences include: suitable transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals (e.g., splicing and polyadenylation (polyA) signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product when desired. Some expression control sequences (including native, constitutive, inducible, and / or tissue-specific promoters) are known in the art and can be used.

[0152] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be operatively linked when they are covalently linked in such a manner that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. Two DNA sequences are said to be operatively linked if the nucleic acid sequence is expected to be translated into a functional protein, if the induction of a promoter in the 5′ regulatory sequence causes the coding sequence to be transcribed, and if the nature of the link between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operatively linked to a nucleic acid sequence if it can influence the transcription of the DNA sequence such that the resulting transcript may be translated into the desired protein or polypeptide. Similarly, two or more coding regions are operatively linked when the result of transcription from a common promoter is the expression of two or more proteins already translated within the frame. In some embodiments, operatively linked coding sequences produce fusion proteins. In some embodiments, operatively linked coding sequences produce functional RNA (e.g., miRNA).

[0153] In some aspects, this disclosure provides isolated nucleic acids comprising transgenes, wherein the transgenes comprise nucleic acid sequences encoding one or more microRNAs (e.g., miRNAs).

[0154] It should be understood that, in some embodiments, the isolated nucleic acid or vector (e.g., an rAAV vector) contains nucleic acid sequences encoding more than one (e.g., multiple, such as 2, 3, 4, 5, 10 or more) miRNAs. In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds to) the same target gene (e.g., an isolated nucleic acid encoding three distinct miRNAs, wherein each miRNA targets the HTT gene). In some embodiments, each of the more than one miRNA targets (e.g., hybridizes or specifically binds to) a different target gene.

[0155] In some respects, this disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) encoding one or more artificial miRNAs. As used herein, “artificial miRNA” or “amiRNA” refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone, which is a precursor miRNA capable of producing a functional mature miRNA), wherein the miRNA and miRNA* (e.g., the transit strand of a miRNA duplex) sequences have been replaced with corresponding amiRNA / amiRNA* sequences that guide efficient RNA silencing of the targeted gene, as described, for example, by Eamens et al. (2014), Methods Mol. Biol. 1062: 211-224. For example, in some embodiments, the artificial miRNA comprises a miR-155pri-miRNA backbone into which a sequence encoding a mature HTT-specific miRNA (e.g., any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260) is inserted to replace the endogenous miR-155 mature miRNA coding sequence. In some embodiments, the miRNA (e.g., artificial miRNA) described herein comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a miR-64 backbone sequence, or a miR-122 backbone sequence.

[0156] The transgenic region (e.g., second, third, fourth, etc.) can be located at any suitable location on the isolated nucleic acid. This region can be located at any untranslated portion of the nucleic acid, including, for example, introns, 5′ or 3→ untranslated regions, etc.

[0157] In some cases, it may be desirable to locate the region (e.g., second region, third region, fourth region, etc.) upstream of the first codon of the nucleic acid sequence encoding the protein (e.g., protein-coding sequence). For example, the region may be located between the first codon and 2000 nucleotides upstream of the first codon in the protein-coding sequence. The region may be located between the first codon and 1000 nucleotides upstream of the first codon in the protein-coding sequence. The region may be located between the first codon and 500 nucleotides upstream of the first codon in the protein-coding sequence. The region may be located between the first codon and 250 nucleotides upstream of the first codon in the protein-coding sequence. The region may be located between the first codon and 150 nucleotides upstream of the first codon in the protein-coding sequence. In some cases (e.g., when the transgene lacks a protein-coding sequence), it may be desirable to locate the region (e.g., second region, third region, fourth region, etc.) upstream of the poly-A tail of the transgene. For example, the region may be located between the first base and 2000 nucleotides upstream of the first base of the poly-A tail. The region can be located between the first base of the poly-A tail and 1000 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 500 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 250 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 150 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 100 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 50 nucleotides upstream of that first base. The region can be located between the first base of the poly-A tail and 20 nucleotides upstream of that first base. In some embodiments, the region is located between the last nucleotide base of the promoter sequence and the first nucleotide base of the poly-A tail sequence.

[0158] In some cases, the region can be located downstream of the last base of the poly-A tail of the transgene. The region can be between the last base of the poly-A tail and 2000 nucleotides downstream of that last base. The region can be between the last base of the poly-A tail and 1000 nucleotides downstream of that last base. The region can be between the last base of the poly-A tail and 500 nucleotides downstream of that last base. The region can be between the last base of the poly-A tail and 250 nucleotides downstream of that last base. The region can be between the last base of the poly-A tail and 150 nucleotides downstream of that last base.

[0159] It should be understood that when a transgene encodes more than one miRNA, each miRNA can be located at any suitable location within the transgene. For example, the nucleic acid encoding the first miRNA can be located within an intron of the transgene, and the nucleic acid sequence encoding the second miRNA can be located in another untranslated region (e.g., between the last codon of the protein-coding sequence and the first base of the poly-A tail of the transgene).

[0160] promoter

[0161] In some embodiments, the transgene further comprises a nucleic acid sequence encoding one or more expression control sequences (e.g., promoters, etc.). Expression control sequences include: appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals (e.g., splicing and polyadenylation (polyA) signals); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance protein stability; and sequences that enhance the secretion of the encoded product when desired. A large number of expression control sequences (including native, constitutive, inducible, and / or tissue-specific promoters) are known in the art and can be used.

[0162] A promoter is a DNA sequence that is recognized and required by the cell's synthetic apparatus or introduced synthetic apparatus to initiate the specific transcription of a gene. The phrases "operably positioned," "under the control of," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid to control the initiation of RNA polymerase and gene expression.

[0163] For nucleic acids encoding proteins, a polyadenylated sequence is typically inserted after the transgene sequence and before the 3′AAV ITR sequence. The rAAV constructs useful in this disclosure may also contain introns, 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-40T intron sequence. Another vector element that can be used is the internal ribosome entry site (IRES). IRES sequences are used to generate more than one polypeptide from a single gene transcript. IRES sequences will be used to generate proteins containing more than one polypeptide chain. The selection of these and other common vector elements is routine, and many such sequences are available [see, for example, Sambrook et al., and the references cited therein, e.g., pp. 3.18-3.26 and 16.17-16.27; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989]. In some embodiments, the foot-and-mouth disease virus 2A sequence is contained in a polyprotein; this is a small peptide (about 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; pp. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; pp. 8124-8127; Furler, S et al., GeneTherapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., GeneTherapy, 1999; 6: 198-208; de Felipe, P et al., Human GeneTherapy, 2000; 11: 1921-1931; and Klump, H et al., GeneTherapy, 2001; 8: 811-817).

[0164] Examples of constitutive promoters include, but are not limited to, the retroviral Rausch's sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is the U6 promoter. In some embodiments, the promoter is the human synapsin (hSYN) promoter. In some embodiments, the promoter is the GFA2 promoter.

[0165] In one embodiment, the promoter is a CAG promoter. In one embodiment, the CAG promoter has the sequence of SEQ ID NO: 193. In some embodiments, the CAG promoter comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence identical to any one of SEQ ID NO: 193, or consists of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence identical to any one of SEQ ID NO: 193. The CAG promoter is capable of driving, for example, the expression of transgenes operatively linked thereto in target neurons and astrocytes.

[0166] The underlined text (e.g., 453bp-819bp of SEQ ID NO: 193) represents the CMV enhancer.

[0167] Bold, italic text (e.g., 821bp-1094bp of SEQ ID NO: 193) indicates the proximal promoter of ACTB.

[0168] Double-underlined text (e.g., 1191bp-2065bp of SEQ ID NO: 193) represents ACTB introns.

[0169] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors (e.g., temperature), or specific physiological states (e.g., acute phase, specific differentiation state of the cell, or in cells undergoing replication only). Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described, and those skilled in the art can readily select from them. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-induced sheep metallothionein (MT) promoter, the dexamethasone (Dex)-induced mouse mammary tumor virus (MMTV) promoter, and the T7 polymerase promoter system (WO... 98 / 10088); Ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93: 3346-3351 (1996)), Tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992)), Tetracycline induction system (Gossen et al., Science, 268: 1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2: 512-518 (1998)), RU486 induction system (Wang et al., Nat. Biotech., 15: 239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)), and the rapamycin induction system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that may be useful in this case are those that are regulated by specific physiological states (e.g., temperature, acute phase, specific differentiation state of the cell, or only in replicating cells).

[0170] In another embodiment, the natural promoter of the transgene will be used. A natural promoter may be preferred when it is desired that the expression of the transgene mimic natural expression. A natural promoter may be used when the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. In a further embodiment, other natural expression control elements (e.g., enhancer elements, polyadenylation sites, or Kozak concordant sequences) may also be used to mimic natural expression.

[0171] 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 include the β-actin promoter; the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); and the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene. Ther., 7:1503-14 (1996)); osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); osteosialin promoter (Chen et al., J. BoneMiner. Res., 11:654-64 (1996)); CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter; neuronal promoters, such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. A cad. Sci. USA, 88: 5611-5 (1991)), and neuron-specific vgf gene promoters (Piccioli et al., Neuron, 15: 373-84 (1995)), as well as other promoters obvious to those skilled in the art. NS-specific promoters for use in this method and composition also include those described in patent application GB2013940.8, filed September 4, 2020, and patent application 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 a promoter of Table 10.In some embodiments, the NS-specific promoter is the promoter in Table 10, or a promoter that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoter in Table 10 and retains the NS-specific promoter activity of the promoter in Table 10.

[0172] CNS-specific promoters for use in this method and composition also include those described in International Patent Application PCT / GB2021 / 050939, filed April 19, 2021, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the CNS-specific promoters are those listed in Tables 11-13, or promoters having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters listed in Tables 11-13. In some embodiments, the CNS-specific promoters are those listed in Tables 11-13, or promoters having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with the promoters listed in Tables 11-13 and retaining the CNS-specific promoter activity of the promoters listed in Tables 11-13.

[0173] 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 from Tables 13-15, or a CRE 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 and retaining the activity of the CREs from Tables 13-15.

[0174] In some embodiments, the CRE may comprise one or more CREs known in the art. For example, in one embodiment, the one or more CREs may be selected from SEQ ID NO: 19-SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 37, and SEQ ID NO: 38 of patent application GB2013940.8 filed on September 4, 2020. For example, in one embodiment, the one or more CREs may be selected from: SEQ ID NO: 1-SEQ ID NO: 8 of WO 2019 / 199867A1, SEQ ID NO: 1-SEQ ID NO: 7 of WO 2020 / 076614A1, and SEQ ID NO: 25-SEQ ID NO: 51, SEQ ID NO: 177-SEQ ID NO: 178, and SEQ ID NO: 188 of WO2020 / 097121. The foregoing references are incorporated herein by reference in their entirety.

[0175] Table 10 - NS-specific promoters

[0176] Table 11 - CNS-specific promoters

[0177] The promoters included in Tables 12-11 are minimal / proximal promoters.

[0178] Table 13 - Overview of synthesized CNS-specific promoters

[0179] Table 14 - Exemplary CREs

[0180] Table 15. Cis-regulatory elements (CREs) included in the promoters of Table 11.

[0181] In a further aspect of the invention, a synthetic central nervous system (CNS)-specific promoter is provided, the promoter comprising a sequence according to any one of SEQ ID NO: 187, SEQ ID NO: 188 or SEQ ID NO: 189 or a functional variant thereof, or consisting of a sequence according to any one of SEQ ID NO: 187, SEQ ID NO: 188 or SEQ ID NO: 189 or a functional variant thereof. In some embodiments, the synthesized CNS-specific promoter comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identical to any one of SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189, or consists of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identical to any one of SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189.

[0182] Therefore, the present invention provides various synthetic CNS-specific promoters and their functional variants. Generally preferred are the promoters according to the invention being variants of any one of SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189, which retain at least 25%, at least 50%, at least 75%, at least 80%, at least 85%, 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 at least 100% of the activity of the reference CNS-specific promoter (any one of SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189).

[0183] In some embodiments of the present invention, the CNS-specific promoter is 1200 or fewer nucleotides in length, preferably 1175 or fewer, 1150 or fewer, 1125 or fewer, 1100 or fewer, 1075 or fewer, 1050 or fewer, 1025 or fewer, 1000 or fewer, 975 or fewer, 950 or fewer, 925 or fewer, 900 or fewer, 875 or fewer, 850 or fewer, 825 or fewer, 800 or fewer, 775 or fewer, 750 or fewer, 740 or fewer, 730 or fewer, 720 or fewer, 710 or fewer, or 700 or fewer nucleotides.

[0184] In another aspect of the invention, a synthesized CNS-specific promoter is provided, the promoter comprising a cis-regulatory element (CRE) or a functional variant thereof according to any one of SEQ ID NO: 191 or SEQ ID NO: 192. In some embodiments, the CRE may be operatively connected to a promoter element. In some embodiments, the promoter element may be a minimal promoter or a proximal promoter. Preferably, the proximal promoter is a CNS-specific proximal promoter. In some embodiments, the minimal promoter is a CNS-specific minimal promoter. In some embodiments, the functional variant of the CRE according to any one of SEQ ID NO: 191 or SEQ ID NO: 192 comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 191 or SEQ ID NO: 192, or consists of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 191 or SEQ ID NO: 192.

[0185] In some embodiments, the synthesized CNS-specific promoter comprises or consists of CRE00010 (SEQ ID NO: 191) or a functional variant thereof, wherein the CRE00010 or a functional variant thereof is operatively linked to CRE151 (SEQ ID NO: 190) or a functional variant thereof.

[0186] In other embodiments, the synthesized CNS-specific promoter comprises or consists of CRE00011 (SEQ ID NO: 192) or a functional variant thereof, wherein CRE00011 or a functional variant thereof is operatively linked to CRE151 (SEQ ID NO: 190) or a functional variant thereof.

[0187] The element CRE151 is a promoter element. It functions by binding to one or more CNS-specific CREs to provide CNS-specific transcription from the promoters that contain them. Its sequence and functional variants will be discussed further below.

[0188] The elements CRE00010 and CRE00011 are CNS-specific CREs. They function by binding to promoter elements to regulate (and typically enhance) CNS-specific transcription by promoters containing them. Their sequences and functional variants will be discussed further below.

[0189] In an embodiment where CRE00010 is operatively connected to CRE151, CRE00010 may be continuous with adjacent CRE151 (i.e., directly adjacent) or separated by a gap or other sequence.

[0190] In an implementation where CRE00011 is operatively connected to CRE151, CRE00011 may be continuous with adjacent CRE151 (i.e., directly adjacent) or separated by a gap or other sequence.

[0191] In another embodiment, the synthesized CNS-specific promoter comprises or consists of CRE00010 (SEQ ID NO: 191) or a functional variant thereof, wherein the CRE00010 or a functional variant thereof is operatively linked to CRE527 (SEQ ID NO: 211) or a functional variant thereof.

[0192] The element CRE527 is a promoter element, suitably a minimal promoter element. In some embodiments, the element CRE527 is a CNS-specific promoter element.

[0193] In an implementation where CRE00010 is operatively connected to CRE527, CRE00010 may be continuous with adjacent CRE527 (i.e., directly adjacent) or separated by a gap or other sequence.

[0194] In one embodiment, the synthesized CNS-specific promoter comprises or consists of CRE00010 (SEQ ID NO: 191) or a functional variant thereof, wherein the CRE00010 or a functional variant thereof is operatively linked to CRE527 (SEQ ID NO: 211) or CRE151 (SEQ ID NO: 190) or a functional variant thereof.

[0195] In a further aspect of the invention, a minimal promoter is provided, which comprises, or consists of, the sequence according to SEQ ID NO: 211 or a functional variant thereof. Suitably, the functional variant of the minimal promoter comprises a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211.

[0196] In another aspect of the invention, a synthetic promoter is provided, the promoter comprising the minimal promoter according to SEQ ID NO: 211 or a functional variant thereof. Suitably, the functional variant of the minimal promoter according to SEQ ID NO: 211 comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 211.

[0197] In a further aspect of the invention, a CRE is provided, the CRE comprising a sequence according to any one of SEQ ID NO: 191 or SEQ ID NO: 192 or a functional variant thereof, or consisting of a sequence according to any one of SEQ ID NO: 191 or SEQ ID NO: 192 or a functional variant thereof.

[0198] The CRE according to the invention can be combined with additional CREs to form a cis-regulatory module (CRM). Suitablely, the additional CRE can be a CRE according to SEQ ID NO: 191 or SEQ ID NO: 192 or a functional variant thereof, or it can be other CREs. Suitablely, the additional CRE is CNS-specific.

[0199] Suitably, in another aspect, a CRM incorporating the CRE of the present invention is provided. Suitably, in some embodiments, the CRM comprises a CRE according to SEQ ID NO: 191 and / or SEQ ID NO: 192, or a functional variant thereof. In one embodiment, the CRM comprises a CRE according to SEQ ID NO: 191, or a functional variant thereof. In another embodiment, the CRM comprises a CRE according to SEQ ID NO: 192, or a functional variant thereof. In yet another embodiment, the CRM comprises a CRE according to SEQ ID NO: 191, or a functional variant thereof, and a CRE according to SEQ ID NO: 192, or a functional variant thereof. In some embodiments, the functional variant of the CRE according to any one of SEQ ID NO: 191 and / or SEQ ID NO: 192 comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 191 or SEQ ID NO: 192, or consists of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same sequence as SEQ ID NO: 191 or SEQ ID NO: 192.

[0200] In one aspect of the invention, a synthetic central nervous system (CNS) specific promoter comprising the CRM of the present invention is provided.

[0201] The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in one or more specific regions of the CNS, preferably in specific regions of the brain, or in specific brain cell types or cell types or combinations thereof.

[0202] The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in one or more parts of the CNS, which is primarily composed of the brain and spinal cord. The retina, optic nerve, olfactory nerve, and olfactory epithelium are sometimes considered as parts of the CNS alongside the brain and spinal cord because they connect directly to brain tissue without requiring intermediate nerve fibers. Suitably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in both the brain and spinal cord. Suitably, the CRE, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in the brain but inactive in the spinal cord or any other part of the CNS. Suitably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in the spinal cord but inactive in the brain. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in the brain.

[0203] Suitablely, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in one or more regions of the brain. Non-limiting examples of brain regions include: frontal lobe, parietal lobe, occipital lobe, temporal lobe (including hippocampus and amygdala), cerebellum, midbrain, pons, medulla, and diencephalon (including thalamus and hypothalamus). Non-limiting examples of spinal cord regions include: cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacrum, and coccyx. In some embodiments, it may be desirable for the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention to exhibit broad activity in the brain. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in all parts of the brain or CNS (pan-CNS), preferably in all regions of the brain. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in the brain but inactive in other parts of the CNS (e.g., spinal cord). In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the aforementioned brain regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in most brain regions, i.e., at least 5, at least 6, at least 7, at least 8, or all 9 of the aforementioned 9 brain regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in 4 to 6 of the aforementioned brain regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in 2 to 4 of the aforementioned brain regions, such as the midbrain, temporal lobe, and diencephalon. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in both the aforementioned brain regions and spinal cord regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in the spinal cord but inactive in other parts of the CNS (e.g., the brain). In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in one, two, three, four, or five of the aforementioned spinal cord regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in most regions of the spinal cord, i.e., at least three, at least four, or all five of the five aforementioned spinal cord regions.

[0204] In some embodiments, it is desirable that the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention exhibit major activity in one region of the CNS (suitably in one region of the brain). Suitably, it is desirable that the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention exhibit activity in one region of the brain, but not activity or only minimal activity in the rest of the brain or CNS. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active only in one region of the CNS of the brain (e.g., the midbrain). In some preferred embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is specifically active in the midbrain (midbrain-specific). In one preferred embodiment, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is specifically active in the midbrain (midbrain-specific), but not activity or only minimal activity in other regions of the brain. The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in various cells of the CNS. The main cell types in the brain are neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells. Other cell types may be present, especially under inflammatory conditions. In some embodiments, it may be desirable for the promoter to be active in many different cell types. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in almost all cells of the CNS (e.g., neurons, astrocytes, oligodendrocytes, microglia, ependymal cells). In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in at least four CNS cell types (e.g., neurons, astrocytes, microglia, and oligodendrocytes) from the CNS cell types listed above. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in at least three CNS cell types (e.g., neurons, astrocytes, and oligodendrocytes) from the CNS cell types listed above. In some embodiments, it is desirable for the promoter to be active in a limited number of CNS cell types, or in no more than one CNS cell type. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in no more than four, three, two, or one CNS cell types from the CNS cell types listed above.In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in no more than two of the CNS cell types listed above (e.g., neurons and oligodendrocytes). In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in only one of the CNS cell types listed above (e.g., neurons). In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in a specific subtype of CNS cell (e.g., dopaminergic neurons). In some particularly preferred embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in dopaminergic neurons. In some preferred embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in dopaminergic neurons but inactive in other CNS cell types or other CNS cell subtypes. In some preferred embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in GABAergic or glutamatergic neurons. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in specific types or subtypes of CNS cells and in specific regions of the brain. The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may or may not be active in tissues other than the CNS. Non-limiting examples of tissues other than the CNS are: heart, liver, kidney, skeletal muscle, and spleen. Suitably, in some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is inactive or minimally active in tissues or cells other than the CNS. Suitably, in ICV delivery, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in no more than one, two, three, or four tissues other than the aforementioned CNS. Appropriately, during IV delivery, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention are active in no more than one, two, three, or four tissues other than the aforementioned CNS.

[0205] Suitably, in some embodiments, it is desirable that the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in the CNS, but also active in other tissues besides the CNS. Suitably, in ICV delivery, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in at least one, two, three, four, or five tissues besides the aforementioned CNS. Suitably, in IV delivery, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in at least one, two, three, four, or five tissues besides the aforementioned CNS.

[0206] In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention can be active in both the CNS and the peripheral nervous system (PNS). If the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in both the CNS and PNS, then the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be referred to as nervous system-specific (NS-specific). The PNS refers to the portion of the nervous system other than the brain and spinal cord. Non-limiting examples of the peripheral nervous system include cranial nerves, brachial plexus, thoracic and abdominal nerves, lumbar plexus, sacral plexus, and neuromuscular junctions. In some embodiments, it is desirable for the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention to exhibit broad activity in the PNS. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in one, two, three, four, five, or six of the aforementioned PNS regions. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in most regions of the PNS, i.e., active in at least 4, at least 5, or all 6 regions of the aforementioned 6 regions of the PNS.

[0207] CNS-specific promoters can be expressed in other non-CNS cells. However, they are expressed at higher levels in CNS cells, such as neurons in the brain and spinal cord, as well as neuronal support cells or non-neuronal cells located in the brain and spinal cord. For example, in cells located in the CNS (including neurons and non-neuronal cells in the brain and spinal cord), CNS-specific promoters express genes at a rate of at least 25%, or at least 35%, or at least 45%, or at least 55%, or at least 65%, or at least 75%, or at least 80%, or at least 90%, or at least 95%, or any integer between 25% and 95%, compared to cells located outside the CNS.

[0208] In a further aspect of the invention, an expression cassette is provided comprising a synthetically produced CNS-specific promoter according to any aspect of the invention, the promoter being operatively linked to a sequence encoding an expression product (suitably a gene, such as a transgene). In one embodiment, an expression cassette is provided comprising any one of CNS-10, CNS-11, or CNS-13, or a functional variant thereof, operatively linked to a sequence encoding an expression product (suitably a gene, such as a transgene).

[0209] In a further aspect of the invention, an expression cassette is provided comprising a CNS-10 or a functional variant thereof operatively linked to a sequence encoding an expression product (suitably a gene, such as a transgene).

[0210] In another aspect of the invention, an expression cassette is provided, the expression cassette comprising a CNS-11 or a functional variant thereof operatively linked to a sequence encoding an expression product (suitably a gene, such as a transgene).

[0211] In another aspect of the invention, an expression cassette is provided, the expression cassette comprising a CNS-13 or a functional variant thereof operatively linked to a sequence encoding an expression product (suitably a gene, such as a transgene).

[0212] In some embodiments, the transgene is selected from any one of the following groups: NPC1, EAAT2, NPY, CYP46A1, GLB1, APOE (or APOE2), HEX, CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, SUMF1, DCTN1, PRPH, SOD1, NEFH, GBA, IDUA, NAGLU, GUSB, ARSA, MANB, AADC, GDNF, NTN, ASP, MECP2, PTCHD1, GJB1, UBE3A, HEXA, FXN, MOG, and SLC6A3. Suitably, in some embodiments, the expression product is a therapeutic expression product. In some embodiments, the expression product is a reporter gene, such as, but not limited to, GFP, RFP, YFP, or luciferase. In some embodiments, when the transgene is GDNF, the expression product is a GDNF protein. In some particularly preferred embodiments, the transgene is a codon-optimized GDNF. In some embodiments, the transgene is a CpG-depleted GDNF. More preferably, in some embodiments, the transgene is a CpG-depleted and codon-optimized GDNF.

[0213] In a further aspect, a vector is provided comprising a CNS-specific promoter or expression cassette synthesized according to the invention. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a gene therapy vector, suitably an AAV vector, an adenovirus vector, a retroviral vector, or a lentiviral vector. In a preferred embodiment, the vector is an AAV vector.

[0214] In a further aspect, according to the present invention, a virion (viral particle) is provided, said virion comprising a viral vector.

[0215] In a further aspect, a pharmaceutical composition is provided comprising a CNS-specific promoter, expression cassette, vector, or virion synthesized according to the invention.

[0216] In a further aspect, synthetic CNS-specific promoters, expression cassettes, vectors, virions, or pharmaceutical compositions according to the invention are provided for use in the treatment (i.e., prevention or treatment of a medical condition or disease). Suitably, the condition or disease is related to abnormal gene expression, optionally related to abnormal gene expression in the CNS. Suitably, the use is for gene therapy, preferably for treating diseases involving abnormal gene expression. In some embodiments, the disease is a CNS-related disease. CNS-related diseases include, but are not limited to, dopamine transporter deficiency syndrome (DTDS), Alzheimer's disease, epilepsy, Parkinson's disease, multiple sclerosis, motor neuron disease, and Huntington's disease. In a preferred embodiment, the disease affects dopaminergic neurons in the central nervous system.

[0217] Appropriately, the gene therapy involves expressing a therapeutic expression product in the CNS.

[0218] In a further aspect, cells are provided that comprise synthetic CNS-specific promoters, expression cassettes, vectors, or virions as described herein. In some embodiments, the cells are eukaryotic cells, optionally mammalian cells, and optionally human cells. In some embodiments, the cells are CNS cells, optionally human CNS cells. Suitably, in some embodiments, the CNS cells are neurons, astrocytes, oligodendrocytes, microglia, or ependymal cells. In a preferred embodiment, the CNS cells are neurons, and even more preferably dopaminergic neurons. The synthetic CNS promoter or expression cassette may be in a vector or may be in the genome of the cell.

[0219] In a further aspect, a synthetic CNS-specific promoter, expression cassette, vector, or virion according to the invention is provided for use in manufacturing pharmaceutical compositions for treating the medical conditions or diseases discussed herein. In some embodiments, the disease is a CNS-related disease.

[0220] In a further aspect, a method for expressing a therapeutic transgene in cells (preferably CNS cells) is provided, wherein the method comprises introducing a synthetically produced CNS-specific expression cassette, vector, or virion as described herein into the cells. In some preferred embodiments, the therapeutic transgene is GDNF.

[0221] In a further aspect, a method for treating a subject in need (preferred) is provided, the method comprising: administering to the subject in need an expression cassette, vector, virion, or pharmaceutical composition as described herein, the expression cassette, vector, virion, or pharmaceutical composition comprising a sequence encoding a therapeutic product, the sequence being operatively linked to a promoter according to the invention; and expressing a therapeutic amount of the therapeutic product in the subject's CNS.

[0222] In one implementation, the therapeutic product is GDNF protein.

[0223] In some embodiments, the expression cassette, vector, virion, or pharmaceutical composition described herein is administered directly to the CNS of a subject.

[0224] In some embodiments, the method further includes introducing the expression cassette, vector, virion, or pharmaceutical composition of the present invention into the CNS of a subject, said expression cassette, vector, virion, or pharmaceutical composition containing a gene encoding a therapeutic product. In some embodiments, the vector is a viral gene therapy vector, preferably an AAV vector.

[0225] In a further aspect, a method for generating an expression product is provided, the method comprising: introducing the synthetic CNS-specific expression cassette of the present invention into CNS cells; and expressing genes present in the synthetic CNS-specific expression cassette.

[0226] The method suitably includes maintaining the CNS cells to express genes under appropriate conditions. In culture, this may include incubating the cells or tissue containing the cells under appropriate culture conditions. Expression can, of course, be in vitro, ex vivo, or in vivo, for example, in one or more cells in the subject's CNS.

[0227] Suitable of the method, the method includes the step of introducing a synthetic CNS-specific expression cassette into CNS cells. Various methods for transfecting CNS cells are well known in the art. A preferred method for transfecting CNS cells is to transduce the cells using a viral vector (e.g., an AAV vector) containing a synthetic CNS-specific expression cassette.

[0228] CRE and its functional variants

[0229] This paper discloses CREs that can be used to construct CNS-specific promoters. Appropriately, the CREs are CNS-specific. These CREs are typically derived from genomic promoter and enhancer sequences, but they are used in this paper in situations entirely different from their native genomic environments. Typically, the CREs constitute small portions of larger genomic regulatory domains that control the expression of genes they are typically associated with. Unexpectedly, these CREs (many of which are very small) can be isolated from their normal environments and retain CNS-specific regulatory activity. This is surprising because removing regulatory sequences from the complex and “three-dimensional” natural environment of the genome usually results in a significant loss of activity, so there is no reason to expect a given CRE to retain the activity level observed after removal from its natural environment. Even more surprising is the retention of CNS-specific activity when the CREs are in AAV vectors. This is especially true because AAV vectors contain inverted terminal repeats (ITRs) and have a different DNA structure compared to the genome, and it is known that both ITRs and DNA structure affect CRE activity. It should be noted that the CRE sequences of this invention can be altered without resulting in a significant loss of activity. Functional variants of CRE can be prepared by modifying the sequence of CRE, provided that modifications that significantly impair CRE activity are avoided. Given the information provided in this disclosure, modifying CRE to provide functional variants is straightforward. Furthermore, this disclosure provides a method for easily evaluating the functionality of any given CRE variant (see, for example, Example 3).

[0230] The specific size of the CRE according to the invention is advantageous because it allows the CRE (more specifically, the promoters containing them) to be provided in the vector while occupying a minimal amount of vector payload. This is especially important when the CRE is used in a vector with limited capacity (e.g., an AAV-based vector).

[0231] The CRE of this invention comprises specific CNS-specific TFBSs. It is generally expected that these CNS-specific TFBSs will retain functionality in functional variants of the CRE. Those skilled in the art are well aware that TFBS sequences can vary while retaining functionality. Therefore, the sequences of the TFBSs are typically described by a common sequence, in which some degree of variation is usually present. A position weight matrix (PWM) can be used to illustrate further information about the variations occurring in the TFBS; the position weight matrix represents the frequency of a given nucleotide typically found at a given position in the common sequence. Details of the TF common sequence and the associated position weight matrix can be found, for example, in the Jaspar or Transfac databases http: / / jaspar.genereg.net / and http: / / gene-regulation.com / pub / databases.html. This information allows those skilled in the art to modify the sequences in any given TFBS of the CRE in a manner that preserves (and in some cases even enhances) the CRE's functionality. Therefore, those skilled in the art have sufficient guidance on how any TFBS of a given TF can be modified (while maintaining the ability to bind the desired TF); for example, the Jaspar system will score a hypothetical TFBS based on its similarity to a given PWM. Furthermore, all PWM scans of CREs in the JASPAR database can be performed to identify / analyze all TFBSs. Those skilled in the art can certainly find other guidance in the literature, and routine experiments can be used to confirm the binding of TFs to the putative TFBSs in any variant CRE. It is evident that significant sequence modifications can be made to the CRE (and even to the TFBSs within the CRE) while preserving function. The CRE of this invention can be used in conjunction with a variety of suitable minimal promoters or CNS-specific proximal promoters. Functional variants of the CRE include sequences that differ from the reference CRE element but substantially retain the activity of a CNS-specific CRE. Those skilled in the art will recognize that the sequence of the CRE can be altered while retaining its ability to recruit suitable CNS-specific transcription factors (TFs) and thereby enhance expression. Functional variants of the CRE compared to the reference CRE can include substitutions, deletions, and / or insertions, as long as they do not render the CRE substantially nonfunctional.

[0232] In some embodiments, a functional variant of the CRE can be considered as a CRE that substantially retains its activity when it replaces a reference CRE in a promoter. For example, a CNS-specific promoter containing a functional variant of a given CRE preferably retains at least 80% of its activity, more preferably at least 90%, more preferably at least 95%, and even more preferably 100% of its activity (compared to a reference promoter containing an unmodified CRE). Suitably, the functional variant of the CRE retains a significant level of sequence identity with the reference CRE. Suitably, the functional variant contains at least 70% of the sequence identical to the reference CRE, more preferably at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence identical to the reference CRE.

[0233] Promoter elements and their functional variants

[0234] One or more promoter elements disclosed herein may be minimal promoters or proximal promoters. Suitably, in some embodiments, the minimal promoter or proximal promoter may be a non-tissue-specific (or ubiquitous) minimal promoter or proximal promoter. In another embodiment, the minimal promoter or proximal promoter may be a CNS-specific minimal promoter or a CNS-specific proximal promoter. While the CRE of the present invention can be used in combination with a range of suitable minimal promoters or CNS-specific proximal promoters, some promoter elements synergize with one or more CREs to significantly contribute to the activity of CNS-specific promoters.

[0235] Functional variants of promoter elements include sequences different from those of the reference promoter element, but essentially retain their activity as promoter elements. Those skilled in the art will understand that the sequence of a promoter element can be altered while retaining its ability to recruit RNA polymerase II and, where applicable, bind CNS-specific TFs to enhance expression. Functional variants of the promoter element compared to the reference promoter element may include substitutions, deletions, and / or insertions, provided that such substitutions, deletions, and / or insertions do not render the promoter element nonfunctional.

[0236] In some implementations, functional variants of CRE151 can be considered as promoter elements that substantially retain their activity when a reference promoter element is replaced in the promoter. Suitable assays for determining CNS-specific promoter activity are disclosed herein, for example in Example 3.

[0237] Suitablely, the functional variants of the promoter element retain a significant level of sequence identity with the reference promoter element. Suitablely, the functional element of CRE151 comprises at least 70% of the sequence identical to SEQ ID NO: 190, more preferably at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 190. As discussed above, the functional variants of CRE151 substantially retain the ability of the reference promoter element as a promoter element. For example, when a functional variant of CRE151 is substituted into a promoter containing the reference promoter element, the modified promoter retains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and more preferably 100% of its activity. Appropriately, functional variants of CRE151 include sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 190.

[0238] In some implementations, functional variants of CRE527 can be considered as promoter elements that substantially retain their activity when a reference promoter element is replaced in the promoter. Suitable assays for determining CNS-specific promoter activity are disclosed herein, such as those in Example 1.

[0239] Suitablely, the functional variants of the promoter element retain a significant level of sequence identity with the reference promoter element. Suitablely, the functional element of CRE527 comprises at least 70% of the sequence identical to SEQ ID NO: 211, more preferably at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 211. As discussed above, the functional variants of CRE527 substantially retain the ability of the reference promoter element as a promoter element. For example, when a functional variant of CRE527 is substituted into a promoter containing the reference promoter element, the modified promoter retains at least 80% of its activity, more preferably at least 90% of its activity, more preferably at least 95% of its activity, and more preferably 100% of its activity. Appropriately, functional variants of CRE527 include sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 211.

[0240] The retention of activity can be assessed by comparing the expression of an appropriate reporter gene under the control of a reference promoter with that of another identical promoter containing a substituted promoter element under equivalent conditions. Suitable assays for assessing CNS-specific promoter activity are disclosed herein, for example, in Example 3.

[0241] The promoter elements used in this invention may be natural (i.e. obtained or derived from naturally occurring gene promoters) or synthetic (i.e., not naturally occurring).

[0242] Synthesized CNS-specific promoters and their functional variants

[0243] Functional variants of the reference synthetic CNS-specific promoter are promoters that contain a sequence different from that of the reference synthetic CNS-specific promoter but substantially retain CNS-specific promoter activity. Those skilled in the art will understand that the sequence of a synthetic CNS-specific promoter can be altered while retaining its ability to recruit suitable CNS-specific transcription factors (TFs) and RNA polymerase II to provide CNS-specific expression of operably linked sequences (e.g., open reading frames). Functional variants of the synthetic CNS-specific promoter may include substitutions, deletions, and / or insertions compared to the reference promoter, provided that such substitutions, deletions, and / or insertions do not render the synthetic CNS-specific promoter substantially non-functional compared to the reference promoter (e.g., CNS-10, CNS-11, or CNS-13).

[0244] Therefore, in some embodiments, functional variants of the promoter according to the invention can be considered as variants that substantially retain the CNS-specific promoter activity of the reference promoter. For example, functional variants of the promoter according to the invention preferably retain at least 70% of the activity of the reference promoter, more preferably at least 80%, more preferably 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%, and more preferably at 100% of the activity.

[0245] In some embodiments, functional variants of CNS-10 can be considered as variants that substantially retain the CNS-specific promoter activity of the reference promoter. For example, functional variants of CNS-10 preferably retain at least 70% of the reference promoter activity, more preferably at least 80% of its activity, even more preferably 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% of its activity, and even more preferably at least 100% of its activity.

[0246] In some embodiments, functional variants of CNS-11 can be considered as variants that substantially retain the CNS-specific promoter activity of the reference promoter. For example, functional variants of CNS-11 preferably retain at least 70% of the reference promoter activity, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 100%.

[0247] In some embodiments, functional variants of CNS-13 can be considered as variants that substantially retain the CNS-specific promoter activity of the reference promoter. For example, functional variants of CNS-13 preferably retain at least 70% of the reference promoter activity, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and even more preferably at least 100%.

[0248] Functional variants of synthetic CNS-specific promoters typically retain a significant level of sequence similarity to reference synthetic CNS-specific promoters.

[0249] Therefore, in some embodiments, the functional variants of the CNS-specific promoter synthesized according to the present invention can be considered as variants that substantially retain the CNS-specific promoter activity of the reference promoter. For example, the functional variants of the CNS-specific promoter synthesized according to the present invention preferably retain at least 70% of the activity of the reference promoter, more preferably at least 80%, more preferably 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%, and more preferably at 100% of the activity.

[0250] Suitablely, in one embodiment, a functional variant of any one of CNS-10, CNS-11, or CNS-13 is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 187, SEQ ID NO: 188, or SEQ ID NO: 189.

[0251] In some embodiments, the functional variant of CNS-10 comprises at least 70% of the sequence identical to that of a reference synthetic CNS-specific promoter, more preferably at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of a reference synthetic CNS-specific promoter. Suitably, the functional variant of CNS-10 comprises at least 70% of the sequence identical to that of SEQ ID NO: 187, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 187.

[0252] In some embodiments, the functional variant of CNS-11 comprises at least 70% of the sequence identical to that of a reference synthetic CNS-specific promoter, more preferably at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of a reference synthetic CNS-specific promoter. Suitably, the functional variant of CNS-11 comprises at least 70% of the sequence identical to that of SEQ ID NO: 188, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that of SEQ ID NO: 188.

[0253] In some embodiments, functional variants of CNS-13 contain at least 70% of the same sequence as a reference synthetic CNS-specific promoter, more preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence as a reference synthetic CNS-specific promoter. Suitably, functional variants of CNS-11 contain at least 70% of the same sequence as SEQ ID NO: 189, more preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence as SEQ ID NO: 189.

[0254] The activity of functional variants can be assessed by comparing the expression of an appropriate reporter gene under the control of a reference synthetic CNS-specific promoter with that of a putative functional variant under equivalent conditions. Suitable assays for assessing CNS-specific promoter activity are disclosed herein, for example, in Example 3.

[0255] A given functional variant of a synthetic CNS-specific promoter may include one or more functional variants of CRE and / or functional variants of promoter elements present in a reference synthetic CNS-specific promoter. A functional variant of CNS-10 may include a functional variant of CRE00010 and / or a functional variant of CRE151. A functional variant of CNS-11 may include a functional variant of CRE00011 and / or a functional variant of CRE151. A functional variant of CNS-13 may include a functional variant of CRE00010 and / or a functional variant of CRE527.

[0256] A given synthetic CNS-specific promoter functional variant may contain one or more additional CREs in addition to those present in the reference synthetic CNS-specific promoters. These additional CREs may be the CREs disclosed herein or other CREs.

[0257] Compared to a reference synthetic CNS-specific promoter, a functional variant of a given synthetic CNS-specific promoter may contain one or more additional regulatory elements. For example, they may include inducible elements, intronic elements, boundary control elements, insulators, locus control regions, responsive elements, binding sites, terminal repeats, stabilizing elements, destabilizing elements, and splicing elements, provided that they do not render the promoter substantially nonfunctional. Functional variants may also include a 5′ UTR sequence.

[0258] A given synthetic CNS-specific promoter functional variant may contain additional spacer regions between adjacent CREs and / or between CREs and promoter elements.

[0259] The preferred synthetic CNS-specific promoters of this invention exhibit CNS-specific promoter activity, which is at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 300%, or at least 400% of the activity of Synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE promoters in CNS cells. In some embodiments, the synthesized CNS-specific promoters of the present invention exhibit CNS-specific promoter activity of at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, or at least 200% of the activity of human TH promoters. In some embodiments, the synthesized CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at levels of at least 100% of the activity of Synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE promoters in CNS cells, preferably 150%, 200%, 300%, or 500% of the activity of any one of the Synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE promoters.

[0260] In some embodiments, the synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at a level of at least 100%, preferably 150% or 200% of the activity of the human TH promoter. In many cases, a higher level of promoter activity is preferred, but this is not always the case; therefore, in some cases, a more moderate expression level may be preferred. In some cases, a more moderate expression level may be preferred, for example, to prevent toxicity or protein accumulation. When two promoters are provided under equivalent conditions and in otherwise equivalent expression constructs, the activity of a given synthetic CNS-specific promoter of the present invention compared to a reference promoter (e.g., a control promoter) can be assessed by comparing the CNS-specific expression of a reporter gene under the control of the synthetic CNS-specific promoter with the expression of the same reporter gene under the control of a reference promoter.

[0261] In some embodiments, the synthetic CNS-specific promoters of the present invention, relative to known CNS-specific promoters (suitably Synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE), are capable of increasing the expression of genes (e.g., therapeutic genes or target genes) in the CNS or CNS cells of a subject by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, at least 500%, at least 1000%, or more. In another embodiment, the synthetic CNS-specific promoters of the present invention are capable of increasing the expression of genes (e.g., therapeutic genes or genes of interest) in the neurons (suitably dopaminergic neurons) of a subject by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, or more, relative to human TH promoters. In a preferred embodiment, the synthetic CNS-specific promoters of the present invention are capable of increasing the expression of genes (preferably transgenic) in neurons (optionally dopaminergic neurons).

[0262] The preferred synthetic CNS-specific promoters of the present invention exhibit activity in non-CNS cells (e.g., HEK293 cells) at 50% or less of the activity compared to CMV-IE, preferably 25% or less, more preferably 10% or less, and in some cases 5% or less, or 1% or less, compared to CMV-IE. In some embodiments, the promoter is CNS-specific if it is more active in the CNS than in non-CNS cells or tissues. The promoters of the present invention can be active in all types of CNS cells (neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells). In some embodiments, the synthetic CNS-specific promoters of the present invention are more active in neurons than in other CNS cells (e.g., microglia, astrocytes, ependymal cells, and oligodendrocytes). In a preferred embodiment, the synthesized CNS-specific promoter is more active in dopaminergic neurons than in other cells of the CNS (e.g., microglia, astrocytes, ependymal cells, and oligodendrocytes).

[0263] Those skilled in the art will understand that the activity of the synthetic CNS-specific promoters of the present invention may vary depending on the route of administration to the subject. Suitably, in some embodiments, when delivered intraventricularly (ICV), the synthetic CNS-specific promoters are more active in dopaminergic neurons compared to other cells in the CNS. Alternatively, in some embodiments, when delivered intravenously (IV), the synthetic CNS-specific promoters are more active in dopaminergic neurons compared to other cells in the CNS. Suitably, in one embodiment, when administered via ICV or IV, a CNS-specific promoter comprising a sequence according to any one of SEQ ID NO: 187 or a functional variant thereof, or consisting of a sequence according to any one of SEQ ID NO: 187 or a functional variant thereof, is active in dopaminergic neurons. In one embodiment, when administered via ICV or IV, a CNS-specific promoter comprising a sequence according to any one of SEQ ID NO: 188 or a functional variant thereof, or consisting of a sequence according to any one of SEQ ID NO: 188 or a functional variant thereof, is active in dopaminergic neurons. Alternatively, in one embodiment, when administered via ICV delivery, a CNS-specific promoter comprising a sequence according to any one of SEQ ID NO: 189 or a functional variant thereof, or consisting of a sequence according to any one of SEQ ID NO: 189 or a functional variant thereof, is activated in dopaminergic neurons.

[0264] The synthetic CNS-specific promoters of the present invention can be active in specific regions, specific CNS cells, or CNS cell subtypes, or both, of the CNS. In some embodiments, the synthetic CNS-specific promoters of the present invention can be active in specific CNS cell types (e.g., neurons) across all regions of the CNS. In other embodiments, the synthetic CNS-specific promoters of the present invention can be active in specific CNS cell types (e.g., neurons) within no more than one region of the CNS (e.g., the midbrain). In some embodiments, the synthetic CNS-specific promoters of the present invention can be active in all CNS cells across all regions of the CNS. In some embodiments, the synthetic CNS-specific promoters of the present invention can be active in no more than one region of the CNS (e.g., the midbrain). Suitably, in some embodiments, the synthetic CNS-specific promoters of the present invention can be active only in dopaminergic neurons of the midbrain.

[0265] CNS-10 and CNS-13 are expected to be particularly active in dopaminergic neurons. CNS-11 is expected to be particularly active in the medial entorhinal cortex. This has been confirmed in preliminary in vivo experiments, where neuronal tropism was primarily observed with CNS-10, CNS-11, and CNS-13. Specifically, CNS-10 and CNS-13 showed expression in mixed neurons and minimal astrocytes.

[0266] Various aspects of this disclosure relate to isolated nucleic acids containing more than one promoter (e.g., two, three, four, five or more promoters). For example, in the context of a construct having a transgene containing a first region encoding a protein and a second region encoding a repressive RNA (e.g., miRNA), it may be desirable to drive the expression of the protein-coding region using a first promoter sequence (e.g., a first promoter sequence operatively linked to the protein-coding region) and to drive the expression of the repressive RNA-coding region using a second promoter sequence (e.g., a second promoter sequence operatively linked to the repressive RNA-coding region). Generally, the first and second promoter sequences can be the same promoter sequence or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter driving the expression of the protein-coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include U6 and HI promoter sequences. In some embodiments, the second promoter sequence (e.g., the promoter sequence driving the expression of the repressive RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of the polII promoter sequence include the T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the polIII promoter sequence drives the expression of a repressive RNA (e.g., miRNA) coding region. In some embodiments, the polII promoter sequence drives the expression of a protein-coding region.

[0267] In some embodiments, the nucleic acid comprises a transgene encoding a protein. The protein may be a therapeutic protein (e.g., a peptide, protein, or polypeptide useful for treating or preventing a disease state in a mammalian subject) or a reporter protein. In some embodiments, the 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 with SEQ ID NO: 2. In some embodiments, the therapeutic protein is useful for treating or preventing Huntington's disease, such as polyglutamine-binding peptide 1 (QBP1), PTD-QBP1, ED11, C4 intracellular antibody, VL12.3 intracellular antibody, MW7 intracellular antibody, Happ1 antibody, Happ3 antibody, mEM48 intracellular antibody, specific monoclonal antibodies (e.g., 1C2), and peptide P42 and its variants, as described in Marelli et al. (2016) Orphanet Journal of Rare Disease 11:24; doi: 10.1186 / s 13023-016-0405-3. In some embodiments, the therapeutic protein is a wild-type Huntington's protein (e.g., a Huntington's protein having a PolyQ repeat region containing fewer than 36 repeat sequences). CYP46A1

[0268] Cholesterol 24-hydroxylase is a neuronal enzyme encoded by the CYP46A1 gene. It converts cholesterol into 24-hydroxycholesterol and plays a crucial role in the outflow of cholesterol from the brain (Dietschy, JM et al., 2004). Cholesterol in the brain is primarily produced in situ but cannot be degraded, and the intact blood-brain barrier restricts direct transport of cholesterol from the brain (Dietschy, JM et al., 2004). 24-hydroxycholesterol can cross the cell membrane and the blood-brain barrier and reach the liver, where it is degraded.

[0269] CYP46A1 is neuroprotective in cellular models of HD (see, for example, WO2012 / 049314). Furthermore, CYP46A1 mRNA is reduced in the striatum (a more vulnerable brain structure in the disease) of the R6 / 2 transgenic HD mouse model.

[0270] In the early stages of Alzheimer's disease (AD), the concentration of 24-hydroxycholesterol (24-hydroxycholesterol) in the CSF and peripheral circulation is high. In the later stages of AD, the concentration of 24-hydroxycholesterol may decrease, which may reflect neuronal loss (Kolsch, H. et al., 2004). CYP46A1 is expressed around the amyloid core of neuroinflammatory plaques in the brains of AD patients (Brown, J. et al., 3rd ed., 2004).

[0271] Agonism of cholesterol 24-hydroxylase encoded by CYP46A1 has provided significant reductions in neuropathology and improvements in cognitive deficits in mouse models of CNS diseases. For example, in a Huntington's disease model, co-expression of CYP46A1 and ExpHtt promoted a strong and significant reduction (58% vs. 27.5%) in ExpHtt aggregate formation (WO2012 / 049314). (See also International Patent Publication WO2009 / 034127; which is incorporated herein by reference in its entirety). The methods described herein relate to the binding of CYP46A1 agonism to the administration of miRNAs targeting specific other targets. For example, the methods may involve the administration of viral vectors to treat neurological diseases or disorders, wherein the vectors express CYP46A1 in cells of the central nervous system.

[0272] In some embodiments, this document describes a viral vector for treating neurological disorders or disturbances, the vector comprising a nucleic acid encoding cholesterol 24-hydroxylase. In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence 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 more sequence identity with 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 more sequence identity with 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.

[0273] Further descriptions of CY46A1 and its therapeutic uses (e.g., for Alzheimer's disease, ALS, and ataxia) are set forth in the art (e.g., in WO 2012 / 049314, WO 2009 / 034127, WO 2018 / 138371, and WO2020 / 089154). The sequences, methods, and compositions described therein may be used in the methods and compositions described herein. All of the foregoing references are incorporated herein by reference in their entirety. The term "gene" refers to a polynucleotide containing at least one open reading frame that, upon transcription or translation, encodes a specific polypeptide or protein.

[0274] The term "coding sequence" or "sequence encoding a specific protein" refers to a nucleic acid sequence that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo. The boundaries of a coding sequence are defined by a 5′ (amino)-terminal start codon and a 3′ (carboxyl)-terminal translation stop codon. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

[0275] The cDNA sequence of CYP46A1 is disclosed in Genbank accession number NM_006668 (SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2. This invention utilizes nucleic acid constructs comprising sequence SEQ ID NO: 1 or variants thereof to treat neurological diseases or disorders. The variants include naturally occurring variants, for example, due to allelic variants between individuals (e.g., polymorphism), alternative splicing forms, etc. The term variant also includes CYP46A1 gene sequences from other sources or organisms. Variant sequences are preferably substantially homologous to SEQ ID NO: 1 and / or SEQ ID NO: 2, i.e., nucleotide sequences exhibiting at least about 75%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% nucleotide sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid construct comprises a sequence having at least 95% sequence identity with SEQ ID NO: 1 and retaining the activity (e.g., the ability to convert cholesterol to 24-hydroxycholesterol) of SEQ ID NO: 1 or SEQ ID NO: 2. Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the sequence (or its complementary strand) as defined above under stringent hybridization conditions. Typical stringent hybridization conditions include temperatures above 30°C, preferably above 35°C, more preferably above 42°C, and / or salinities below about 500 mM, preferably below 200 mM. Hybridization conditions can be adjusted by a person skilled in the art by modifying the temperature, salinity, and / or the concentration of other reagents (e.g., SDS, SSC, etc.).

[0276] In one embodiment, the CYP46A1 is a codon-optimized CYP46A1. Exemplary codon-optimized CYP46A1 intended for use herein are provided as SEQ ID NO: 109 and SEQ ID NO: 110. In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 109. 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 more sequence identity with SEQ ID NO: 109. In some embodiments, the viral vector comprises a nucleic acid sequence of SEQ ID NO: 110. In some embodiments, the viral vector comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity with the sequence of SEQ ID NO: 110.

[0277] In one embodiment, the CYP46A1 nucleic acid sequence is codon-optimized, i.e., a codon-optimized variant. In another embodiment, the wild-type CYP46A1 nucleic acid sequence (e.g., SEQ ID NO: 1) is codon-optimized, i.e., a codon-optimized variant. Exemplary methods of codon optimization known in the art are described below. In one embodiment, more than one codon optimization method is utilized. For example, the CYP46A1 nucleic acid sequence is modified to have reduced CpG motifs and increased GC content.

[0278] For example, at least one or more CpG islands (cytosine bases followed immediately by guanine bases (CpG), where cytosine tends to be methylated) typically appearing at or near the transcription start site in the CYP46A1 nucleic acid sequence are deleted and / or substituted. In one embodiment, a reduction or absence of CpG islands may enhance in vivo expression or reduce the innate immune response to the transgene. In one embodiment, at least one CpG motif is deleted and / or substituted, for example, at least one, two, three, four, five, or more CpG motifs are deleted and / or substituted. In one embodiment, the wild-type CYP46A1 nucleic acid sequence is optimized to contain 0% CpG. In one embodiment, the wild-type CYP46A1 nucleic acid sequence is optimized to contain no more than 10%, or no more than 20%, or no more than 30%, or no more than 40%, or no more than 50%, or no more than 60%, or no more than 70%, or no more than 80%, or no more than 90% of CpG present in the wild-type CYP46A1 nucleic acid sequence. Exemplary modifications to the wild-type CYP46A1 nucleic acid sequence (SEQ ID NO: 1) that cause the removal of CpG islands are described in Table 23 below.

[0279] Alternatively, codon optimization can be achieved by editing the nucleic acid to remove alternative reading frames. In one embodiment, the deletion of alternative reading frames can enhance in vivo expression or reduce the innate immune response to the transgene. In one embodiment, at least one alternative reading frame (e.g., at least two, at least three, or more alternative reading frames) is deleted. Exemplary modifications to the wild-type CYP46A1 nucleic acid sequence (SEQ ID NO: 1) that cause the removal of alternative reading frames are described in Table 23 below.

[0280] In various embodiments, the CYP46A1 nucleic acid sequence is codon-optimized by increasing the total GC content (i.e., the percentage of guanine and cytosine in the entire coding sequence); removing hidden splice donor or acceptor sites; and / or adding or removing ribosome entry and / or initiation sites (e.g., Kozak sequences).

[0281] In one embodiment, the wild-type CYP46A1 nucleic acid (e.g., SEQ ID NO: 1) is codon-optimized by including at least one modification provided in Table 23 below. The “new elements” described in Table 23 are intended to be exemplary and not limiting. For example, T222 as described in Table 23 can be replaced with “A”. However, T22 can also be replaced with “G” or “C”, and each modification can result in the removal of an alternative reading frame. In one embodiment, the wild-type CYP46A1 nucleic acid (e.g., SEQ ID NO: 1) is codon-optimized by including at least two or more modifications provided in Table 23 below.

[0282] In one embodiment, the wild-type CYP46A1 nucleic acid (e.g., SEQ ID NO: 1) is codon optimized by modifying the untranslated region of the wild-type CYP46A1 nucleic acid.

[0283] In one embodiment, the CYP46A1 protein is encoded by a codon-optimized CYP46A1 nucleic acid sequence, for example, for any one or more of the following: (1) enhancing in vivo expression, (2) reducing CpG islands, or (3) reducing innate immune responses.

[0284] In some implementations, the codon-optimized CYP46A1 variant exhibits enhanced long-term expression relative to the wild-type CYP46A1, for example, over a period of at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0285] In some implementations, the codon-optimized CYP46A1 variant exhibits a reduced immune response relative to the wild-type CYP46A1.

[0286] In some respects, rAAV encoding CYP46A1 and / or CYP46A1 variants is produced using closed linear DNA molecules lacking bacterial backbone sequences.

[0287] In one aspect, this document provides a composition comprising isolated nucleic acid containing a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 110. In another aspect, this document provides a composition comprising a recombinant viral vector containing isolated nucleic acid containing a sequence that is at least 80% identical (e.g., 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, compared to SEQ ID NO: 1, contains 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 of SEQ ID NO: 110. The aforementioned composition can be used, for example, to treat neurological diseases or disorders as described herein in the absence of administered miRNA.

[0288] SEQ ID NO: 1 CYP46A1 mRNA

[0289] SEQ ID NO: 2 CYP46A1 amino acid sequence carrier

[0290] Without being bound by any particular theory, allele-specific silencing of pathogenic genes (e.g., mutant huntingington (HTT)) can provide improved safety in subjects due to the preservation of wild-type expression and function in cells, compared to non-allele-specific silencing (e.g., silencing of wild-type and mutant HTT alleles). For example, aspects of the present invention relate to the inventors' knowledge and understanding that isolated nucleic acids and vectors that introduce one or more repressive RNA (e.g., miRNA) sequences that target the HTT gene in a non-allele-specific manner, while simultaneously driving the expression of hardened wild-type HTT genes (wild-type HTT genes not targeted by miRNAs), can achieve concurrent mutant HTT knockdown (e.g., in CNS tissues) and increased expression of wild-type HTT. Generally, the nucleic acids encoding endogenous wild-type and mutant HTT mRNAs are sequenced sufficiently different from the nucleic acids encoding the transgenic "hardened" wild-type HTT mRNAs such that the "hardened" wild-type HTT transgenic mRNA is not targeted by said one or more repressive RNAs (e.g., miRNAs). For example, this can be achieved by introducing one or more silent mutations into the HTT transgenic sequence, such that it encodes the same protein as the endogenous wild-type HTT gene but with a different nucleic acid sequence. In this case, the exogenous mRNA can be referred to as "hardened". Alternatively, the repressive RNA (e.g., miRNA) can target the 5′ and / or 3′ untranslated regions of the endogenous wild-type HTT mRNA. These 5′ and / or 3′ regions can then be removed or replaced in the transgenic mRNA, so that the transgenic mRNA is not targeted by one or more repressive RNAs.

[0291] Reporter sequences that can be provided in transgenes (e.g., nucleic acid sequences encoding reporter proteins) include, but are not limited to, DNA sequences encoding β-lactamases, β-galactosidase (LacZ), alkaline phosphatases, thymidine kinases, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferases, and others well known in the art. When associated with regulatory elements driving their expression, the reporter sequence provides a signal detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectroscopic methods, fluorescence-activated cell sorting assays, and immunological assays (including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). For example, when the marker sequence is the LacZ gene, the presence of a vector carrying the signal is detected by measuring β-galactosidase activity. When the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be measured visually by color or light generation in a photometer. For example, such reporters can be used to verify the tissue-specific targeting ability of nucleic acids and the tissue-specific promoter regulatory activity. Recombinant adeno-associated virus (rAAV).

[0292] In some embodiments, the vector is adeno-associated virus (AAV) or recombinant AAV. In some aspects, this disclosure provides isolated AAV. As used herein with respect to AAV, the term "isolated" refers to AAV that has been artificially produced or obtained. Isolated AAV can be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV". Recombinant AAV (rAAV) preferably has tissue-specific targeting capability, such that the transgene and / or nuclease of the rAAV will be specifically delivered to one or more predetermined tissues. The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Therefore, rAAVs with capsids suitable for the targeted tissues can be selected.

[0293] AAV vectors are preferably used as self-complementary double-stranded AAV vectors (scAAV) to overcome one of the limiting steps in AAV transduction (i.e., the conversion from single-stranded to double-stranded AAV), although the use of single-stranded AAV vectors (ssAAV) is also covered herein. In some embodiments of the invention, the AAV vector is chimeric, meaning it contains components from at least two AAV serotypes, such as the ITR of AAV2 and the capsid protein of AAV5. AAV9 is known to transduce CNS cells and tissues particularly efficiently, and therefore AAV9 and its derivatives are of particular interest for targeting CNS cells and tissues. AAV2g9 is known to transduce CNS cells and tissues particularly efficiently, and therefore AAV2g9 and its derivatives are of particular interest for targeting CNS cells and tissues. AAVrh10 is known to transduce CNS cells and tissues particularly efficiently, and therefore AAVrh10 and its derivatives are of particular interest for targeting CNS cells and tissues. AAVrh10 is particularly preferred because systemic or intravenous delivery of AAVrh10 has been found to provide high transgenic expression in the central nervous system, as described in (Tanguy et al., 2015), which is incorporated herein by reference. AAVDJ8 is known to transduce CNS cells and tissues particularly effectively, and therefore AAVDJ8 and its derivatives are of particular interest for targeting CNS cells and tissues. AAVDJ8 is preferred because it has been shown to effectively target multiple regions of the brain and effectively target astrocytes, as described in (Hammond et al., 2017), which is incorporated herein by reference. AAV1, AAV2, AAV4, AAV5, and AAV8 are also known to target CNS cells and tissues, and therefore these AAV serotypes and their derivatives are also of particular interest for targeting CNS cells and tissues. AAV-PHP.eB is also of particular interest because it is known to effectively cross the blood-brain barrier (Mathiesen et al., 2020).

[0294] Methods for obtaining recombinant AAV with the desired capsid protein are well known in the art (see, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing host cells containing a nucleic acid sequence encoding the AAV capsid protein; a functional rep gene; a recombinant AAV vector consisting of an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper functions to allow the recombinant AAV vector to be packaged within the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the AAV cap gene. AAV comprises three capsid proteins, viral particle proteins 1 through 3 (named VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, VP1, VP2, and VP3 have molecular weights of approximately 87 kDa, approximately 72 kDa, and approximately 62 kDa, respectively. In some embodiments, during translation, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some implementations, the capsid protein functions to protect the viral genome, deliver the genome, and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0295] In some embodiments, the recombinant AAV (rAAV) capsid protein has an AAV serotype selected from the group consisting of: AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV2G9, AAV2.5G9, AAV9, AAVDJ8, AAV-PHP.eB, and AAV10. In some embodiments, the AAV capsid protein has a serotype derived from a non-human primate, such as the AAVrh10 serotype. In some embodiments, the AAV capsid protein has the AAV9 serotype. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, or AAV13 capsid protein or a chimera thereof. In some embodiments, the rAAV comprises a capsid protein or a chimera thereof from serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV2.5G9, AAVrh8, AAVrh10, AAVrh74, AAV10, or AAV11. In certain embodiments, the rAAV comprises a chemically modified capsid as disclosed in WO2017 / 212019, for example, a mannose ligand chemically coupled to AAV2. The rAAVs with chemically modified capsids disclosed in WO2017 / 212019 are incorporated herein by reference in their entirety. As a further embodiment, the AAV capsid protein and viral capsid of the present invention can be polyploid (also known as rational haploid) because they can contain different combinations of VP1, VP2 and VP3 AAV serotypes in a single AAV capsid, as described in PCT / US18 / 22725, PCT / US2018 / 044632 or US10,550,405, which are incorporated herein by reference.

[0296] In one implementation, AAV is AAV9 or a derivative thereof.

[0297] In one implementation, AAV is AAVrh10 or a derivative thereof.

[0298] In another implementation, AAV is AAV-PHP.eB or a derivative thereof.

[0299] The components to be cultured in host cells to package the rAAV vector in an AAV capsid can be provided to the host cells in a trans configuration. Alternatively, any one or more desired components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper function) can be provided by stable host cells engineered to contain one or more of the desired components using methods known to those skilled in the art. Most preferably, such stable host cells will contain the desired components under the control of an inducible promoter. However, the desired components may be under the control of a constitutive promoter. In the discussion of regulatory elements suitable for use with transgenes, examples of suitable inducible and constitutive promoters are provided herein. In yet another alternative, selected stable host cells may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells derived from 293 cells (which contain E1 helper function under the control of a constitutive promoter) but containing rep and / or cap proteins under the control of an inducible promoter can be generated. Other stable host cells can be generated by those skilled in the art. In some embodiments, this disclosure relates to host cells containing nucleic acids comprising a coding sequence for a protein (e.g., wild-type huntingtin, optionally "hardened" wild-type huntingtin). In some embodiments, this disclosure relates to compositions comprising the aforementioned host cells. In some embodiments, the compositions comprising the aforementioned host cells further comprise a cryoprotectant.

[0300] The recombinant AAV vector, rep sequence, cap sequence, and helper function required for the production of the rAAV of this disclosure can be delivered to the packaging host cell using any suitable gene element (vector). Selected gene elements can be delivered by any suitable method, including those described herein. 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. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the choice of suitable method is not a limitation of this disclosure. See, for example, K. Fisher et al., J. Viroli., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0301] In some embodiments, a triple transfection method (described in detail in U.S. Patent No. 6,001,650) can be used to produce recombinant AAV. Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (containing a transgene), an AAV helper vector, and an accessory function vector to be packaged into AAV particles. The AAV helper vector encodes “AAV helper function” sequences (i.e., rep and cap) that function in trans for productive AAV replication and capsid formation. Preferably, the AAV helper vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with this disclosure include pHLP19 (described in U.S. Patent No. 6,001,650) and the pRep6cap6 vector (described in U.S. Patent No. 6,156,303), the entire contents of which are incorporated herein by reference. The accessory function vector encodes the nucleotide sequence of non-AAV-derived viral and / or cellular functions (i.e., "accessory functions") upon which AAV replication depends. Accessory functions include those required for AAV replication, including but not limited to those involved in: activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus.

[0302] In some respects, triple transfection methods use closed linear DNA molecules that lack a bacterial backbone sequence, for example, as described in PCT / US2021 / 013689 (published as WO / 2021 / 146591), which is incorporated herein by reference in its entirety.

[0303] In some respects, 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 foreign DNA is introduced into the cell membrane. Many transfection techniques are well known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology; Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more foreign nucleic acids (e.g., nucleotide integrators and other nucleic acid molecules) into a suitable host cell.

[0304] "Host cell" refers to any cell that contains or is capable of containing the substance of interest. Typically, host cells are mammalian cells. Host cells can serve as recipients of AAV helper constructs, AAV small gene plasmids, accessory functional vectors, or other transfer DNA associated with the production of recombinant AAV. The term includes the progeny of the transfected original cell. Therefore, as used herein, "host cell" can refer to a cell that has been transfected with a foreign DNA sequence. It is understood that, due to natural, accidental, or intentional mutations, the progeny of a single parent cell may not necessarily be morphologically identical to the original parent, or in terms of genome or total DNA complementarity.

[0305] As used herein, the term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that the karyotype can undergo spontaneous or induced changes during the storage or transfer of such clonal populations. Therefore, cells derived from the referred cell line may not be exactly identical to the progenitor cell or culture, and the referred cell line includes such variants.

[0306] As used herein, the term “recombinant cell” refers to a cell in which a foreign DNA segment (e.g., a DNA segment that leads to the transcription of a bioactive polypeptide or the production of a bioactive nucleic acid (e.g., RNA)) is introduced.

[0307] As used herein, the term "vector" includes any genetic element, such as plasmids, bacteriophages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, etc., that is capable of replication when associated with appropriate control elements and that is capable of transferring gene sequences between cells. Therefore, the term includes cloning tools and expression tools, 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 attached. Another type of vector is a viral vector, in which an additional DNA segment is attached to a viral genome. Specific vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors). Furthermore, specific vectors are capable of directing the expression of the gene operatively attached to them. Such vectors are referred to herein as "expression vectors." Generally, expression vectors useful in recombinant DNA technologies are typically in the form of plasmids. In this application, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which provide equivalent functionality.

[0308] A cloning vector is a vector capable of autonomously replicating or integrating into the genome of a host cell. A further characteristic is the presence of one or more endonuclease restriction sites at which the vector can be cleaved in a deterministic manner, and where a desired DNA sequence can be ligated, thereby enabling the new recombinant vector to retain its ability to replicate within the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the copy number of the plasmid increases within the host cell (e.g., host bacteria), or replication of the desired sequence can occur only once per host before the host reproduces through mitosis. In the case of bacteriophages, replication can occur actively during lysis or passively during lysogenicity.

[0309] Expression vectors are vectors into which a desired DNA sequence can be inserted by restriction and ligation, thereby operatively binding to a regulatory sequence and expressing it as an RNA transcript. The vector may further contain one or more marker sequences suitable for recognizing cells that have been or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds; genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase); and genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomously replicating and expressing structural gene products present in the DNA segments operatively linked to them.

[0310] This invention further provides vectors comprising the synthesized CNS-specific promoters or expression cassettes of this invention. In some embodiments of this invention, the vector is a plasmid. Such plasmids may contain a variety of other functional nucleic acid sequences, such as one or more optional markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments of this invention, the vector is a viral vector.

[0311] In some embodiments of the invention, the vector is an expression vector for expression in eukaryotic cells. Examples of eukaryotic expression vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; pSVK3, pBPV, pMSG, and pSVL, available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP, available from Clontech. Many other vectors are well known and commercially available. For mammalian cell adenovirus vectors, pSV and pCMV family vectors are particularly well known, non-limiting examples. Many well-known yeast expression vectors exist, including, but not limited to, yeast integration plasmids (YIp) and yeast replication plasmids (YRp). For plants, Agrobacterium Ti plasmids are exemplary expression vectors, and suitable expression vectors are also provided by plant viruses, such as tobacco mosaic virus (TMV), potato virus X, and cowpea mosaic virus.

[0312] In some preferred embodiments, the vector is a gene therapy vector. Various gene therapy vectors are known in the art, and AAV vectors, adenovirus vectors, retroviral vectors, and lentiviral vectors may be mentioned. When the vector is a gene therapy vector, it preferably contains a nucleic acid sequence operatively linked to a synthetic CNS-specific promoter of the present invention, the nucleic acid sequence encoding a therapeutic product (suitably a therapeutic protein). The therapeutic protein may be a secretible protein. Non-limiting examples of secretible proteins have been discussed above, and exemplary secretible therapeutic proteins include coagulation factors such as factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, toxic proteins, etc.

[0313] In some embodiments, a useful vector is considered to be one in which the nucleic acid segment to be transcribed is located under the transcriptional control of a promoter. Two DNA sequences are said to be operatively conjugated if the coding sequence is to be translated into a functional protein, if the induction of a promoter in the 5′ regulatory sequence induces transcription of the coding sequence, and if the nature of the connection between the two DNA sequences does not (1) introduce a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, a promoter region is operatively conjugated to a coding sequence if it enables transcription of the DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.

[0314] A “promoter” is a DNA sequence that is recognized by and required by the cell’s synthetic apparatus or an introduced synthetic apparatus to initiate specific transcription of a gene. When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, various transcriptional control sequences (e.g., promoter / enhancer sequences) can be used to guide its expression. A promoter can be a natural promoter, i.e., a promoter of a gene in its endogenous environment, which provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter is unregulated, allowing for continuous transcription of its associated gene. Various conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule.

[0315] As used herein, the term "synthetic promoter" refers to a promoter that does not exist in nature. In its present context, it typically includes the CRE of the present invention operatively linked to a promoter element (e.g., a minimal (or core) promoter or a CNS-specific proximal promoter). One or more CREs of the present invention are used to enhance CNS-specific transcription of genes operatively linked to a synthetic promoter. A portion of the synthetic promoter may be naturally occurring (e.g., a minimal promoter or one or more CREs within a promoter), but the synthetic promoter as a complete entity is not naturally occurring.

[0316] The precise nature of the regulatory sequences required for gene expression can vary between species or cell types, but generally may include 5′ non-transcriptional and 5′ non-translational sequences, respectively, involved in transcription initiation and translation, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5′ non-transcriptional regulatory sequences will include promoter regions containing promoter sequences for transcriptional control of operatively conjugated genes. As desired, the regulatory sequences may also include enhancer sequences or upstream activator sequences. The vectors of the present invention may optionally contain 5′ leader or signal sequences. The selection and design of suitable vectors are within the competence and discretion of those skilled in the art.

[0317] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into them. The heterologous DNA (RNA) is placed under the operative control of transcriptional elements to allow expression of the heterologous DNA in the host cell.

[0318] The phrases “operably positioned,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term “expression vector or construct” means any type of genetic construct containing nucleic acids in which some or all of the nucleic acid coding sequences can be transcribed. In some embodiments, expression involves the transcription of nucleic acids, for example, to generate a biologically active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.

[0319] The foregoing methods for packaging the recombinant vector into a desired AAV capsid to produce the rAAV of this disclosure are not intended to be limiting, and other suitable methods will be apparent to those skilled in the art.

[0320] In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequences provided herein (including those provided in the sequence listing) may be replaced by any other nucleotide suitable for pairing with adenosine nucleotide bases (e.g., via Watson-Crick base pairs). For example, in some embodiments, any one or more thymidine (T) nucleotides in the sequences provided herein (including those provided in the sequence listing) may be suitably replaced with uridine (U) nucleotides, or vice versa.

[0321] In some embodiments of any aspect, nucleic acids (e.g., miRNAs) are chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and / or modified by methods well-established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5′-end modifications (phosphorylation, conjugation, reverse linkage, etc.), 3→-end modifications (conjugation, DNA nucleotides, reverse linkage, etc.); (b) base modifications, such as substitution with a stable base, destabilization of a base, or base pairing with an expanded chaperone library, base removal (base-free nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2→ or 4′ position) or sugar substitution; and (d) backbone modifications, including modification or substitution of phosphodiester bonds. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids comprising a modified backbone or lacking natural internucleotide linkages. Nucleic acids having a modified backbone particularly include nucleic acids without phosphorus atoms in their backbone. For the purposes of this application, and as sometimes mentioned in the art, modified nucleic acids lacking phosphorus atoms in their internucleotide backbone may also be considered oligonucleotides. In some embodiments of any aspect, the modified nucleic acid will have phosphorus atoms in its internucleotide backbone.

[0322] For example, modified nucleic acid backbones may include thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates (including 3′-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3′-aminoaminophosphates and aminoalkylaminophosphates), borophosphates, thiocarbonylaminophosphates, thionoalkylphosphonates, and thionoalkylphosphotriesters with normal 3′-5′ linkages, their 2′-5′ analogues, and those with opposite polarities, wherein adjacent nucleoside unit pairs are linked in a 3′-5′ to 5′-3′ or 2′-5 to 5′-2′ configuration. Various salts, mixed salts, and free acid forms are also included. Modified nucleic acid backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleosides, mixed heteroatoms and alkyl or cycloalkyl nucleosides, or one or more short-chain heteroatoms or heterocycles. These backbones include those with the following characteristics: morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformyl and thioformacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; other backbones with mixed N, O, S, and CH2 components; and backbones with... Oligonucleotides with heteroatomic skeletons, and especially --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [referred to as methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the native phosphodiester skeleton is represented as --O--P--O--CH2--].

[0323] In other nucleic acid mimics, both the sugar and nucleoside linkers (i.e., the backbone) of the nucleotide units are replaced with new groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomer (showing an RNA mimic with excellent hybridization properties) is called peptide nucleic acid (PNA). In PNA compounds, the RNA sugar backbone is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bound to the nitrogen atom of the amide portion of the backbone.

[0324] Nucleic acids can also be modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety containing an additional bridging link between the 2′ and 4′ carbons. This structure effectively “locks” the ribose in a 3′-inner conformation. Adding 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).

[0325] 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-ynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and ynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. Exemplary suitable modifications include O[(CH2)] n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are 1 to approximately 10. In some embodiments in any aspect, the nucleic acid at the 2′ position comprises one of the following: C1 to C 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl groups, O-alkylaryl or O-aryl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups that improve the pharmacokinetic properties of nucleic acids, or groups that improve the pharmacodynamic properties of nucleic acids, and other substituents with similar properties. In some embodiments in any aspect, the modification includes 2′-methoxyethoxy (2′-O--CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2′-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in the examples below; and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e. 2′-O--CH2--O--CH2--N(CH2)2, which is also described in the examples below.

[0326] Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), and 2′-fluorine (2′-F). Similar modifications can also be made at other positions on nucleic acids, particularly at the 3′ position of the 3′ terminal nucleotide or the 5′ position of the 5′ terminal nucleotide on 2′-5′ linked dsRNAs. Nucleic acids can also have sugar mimics, such as a cyclobutyl moiety, to replace the furanopentose.

[0327] Nucleic acids may also include modifications or substitutions of nucleobases (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may include other synthetic and natural nucleobases, including but not limited to 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiourea pyrimidine, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyluracil and cytosine, 6-azolyl uracil, and 6-aminoazolyl uracil. Azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenine and guanine; 5-halogenated (especially 5-bromo), 5-trifluoromethyl, and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazoguanine and 7-deazoadenine; and 3-deazoguanine and 3-deazoadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids characteristic of this invention. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine). 5-methylcytosine substitution has been shown to improve the stability of nucleic acid duplexes by 0.6 °C–1.2 °C (Sanghvi, YS, Crooke, ST, and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276–278), and is an exemplary base substitution, even more so when combined with 2′-O-methoxyethyl sugar modifications. In some embodiments of any aspect, the modified nucleobases may include d5SICS and dNAM, which are non-limiting examples of non-natural nucleobases that may be used alone or together as base pairs (see, for example, Leconte et al., J. Am. Chem. Soc. 2008, 130, 7, 2336-2343; Malyshev et al., PNAS. 2012, 109(30)12005-12010).In some embodiments of any aspect, the oligonucleotide tag (e.g., Oligopaint) contains any modified nucleobase known in the art, i.e., any nucleobase derived from unmodified and / or natural nucleobases.

[0328] The preparation of the modified nucleic acids, backbones, and nucleobases described above is well known in the art.

[0329] Another modification of the nucleic acid characterized in this invention involves chemically linking the nucleic acid to one or more ligands, portions or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties or cellular uptake of the nucleic acid. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556); bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060); thioethers, such as beryl-S-triphenylmethylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3: 2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538); and aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO). J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54); phospholipids, such as di-hexadecyl-racemic glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphonate / salt (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14: 969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron). Lett., 1995, 36: 3651-3654); palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229-237); or octadecylamine or hexano-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923-937).

[0330] In some embodiments of any aspect, the vector is pEMBL. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-only intron. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x control pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-CYP46A1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-luc-HTT-3′UTR / mutant. In some embodiments in any aspect, the vector comprises at least one of the following: at least one (e.g., two) ITRs; a Synl promoter; at least one (e.g., two) hCG introns; at least one (e.g., two) copies of a premiR (e.g., a control pre-miR, an artificial pre-miR; SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260); small polyA; CYP46A1; luciferase; an HTT targeting sequence; and / or an HTT-3′UTR / mutant. In some embodiments, the vector comprises neuron-specific synthetic promoters selected from Tables 10-13 and / or CREs selected from Tables 13-15. In a particular aspect of the embodiment, the miRNA targets the wild-type HTT allele. In other aspects of the embodiment, the miRNA targets the mutant HTT allele. In yet another implementation, the miRNA targets both wild-type and mutant HTT alleles. In yet another implementation, the miRNA targets any HTT mRNA.

[0331] In some implementations, one or more of the recombinantly expressed genes can be integrated into the cell's genome.

[0332] Nucleic acid molecules encoding the enzyme of the claimed invention can be introduced into one or more cells using methods and techniques standard in the art. For example, nucleic acid molecules can be introduced using standard protocols, such as transformation by chemical transformation and electroporation, transduction, particle bombardment, etc. Expression of nucleic acid molecules encoding the enzyme of the claimed invention can also be achieved by integrating said nucleic acid molecules into the genome.

[0333] In some embodiments, the promoter is a synapsin (Syn1) promoter (see, for example, SEQ ID NO: 152). In one aspect, the promoter comprises a nucleic acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 152. In one aspect, the compositions provided herein comprise a recombinant viral vector comprising a promoter that comprises a nucleic acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 152.

[0334] Synapsin-1 (SEQ ID NO: 152)

[0335] In one aspect, this document provides compositions comprising isolated nucleic acids containing a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 111. In another aspect, this document provides compositions comprising recombinant viral vectors containing isolated nucleic acids containing a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 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, an enhancer, and / or a cis-regulatory element (CRE; see, for example, 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 introns. In some embodiments, the introns comprise ACTB introns / chimeric ACTB-HBB2 introns. See, for example, SEQ ID NO: 111, Table 16. The foregoing compositions can be used, for example, in the absence of administered miRNA, to treat the neurological diseases or disorders described herein.

[0336] SEQ ID NO: 111, pJAL130-CYP46A1 (4036bp), ITR sequence of codon-optimized CYP46 sequence to ITR (see, for example, SEQ ID NO: 110; see, for example) Figure 1 ).

[0337] Bold text (e.g., nucleotides (nt) 1-130 of SEQ ID NO: 111) indicates the left ITR.

[0338] Italicized text (e.g., nt 182-436 of SEQ ID NO: 111) indicates an enhancer.

[0339] Bold italic text (e.g., nt 550-804 of SEQ ID NO: 111) indicates a promoter.

[0340] Double-underlined text (e.g., nt 824-1892 of SEQ ID NO: 111) indicates an intron.

[0341] Bold double-underlined text (e.g., nt 1966-3465 of SEQ ID NO: 111) represents the codon-optimized CYP46A1 coding sequence (CDS) (see, for example, SEQ ID NO: 110).

[0342] Italic double-underlined text (e.g., nt 3629-3853 of SEQ ID NO: 111) represents polyA.

[0343] Bold italic double-underlined text (e.g., nt3907-4036 of SEQ ID NO: 111) indicates the right ITR.

[0344] Table 16

[0345] In one aspect, this document provides compositions comprising isolated nucleic acids containing at least 80% identical sequences to SEQ ID NO: 194, for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical sequences. In another aspect, this document provides compositions comprising a recombinant viral vector containing isolated nucleic acids containing at least 80% identical sequences to SEQ ID NO: 194 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical sequences). 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, an enhancer, and / or a cis-regulatory element (CRE; see, for example, 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 promoter is a CAG promoter. In some embodiments, the vector further comprises introns. In some embodiments, the introns comprise ACTB introns / chimeric ACTB-HBB2 introns. The foregoing compositions can be used, for example, in the absence of administered miRNA, to treat neurological diseases or disorders as described herein.

[0346] SEQ ID NO: 194, dbDNA-CYP46A1 (4,573 bp), ITR-ITR sequence of codon-optimized CYP46 sequence (see, for example, SEQ ID NO: 212; see, for example) Figure 9 SEQ ID NO: 212 is the sequence of SEQ ID NO: 110 with three additional base pairs at the C-terminus, which are translated to a stop codon.

[0347] Base pairs 1-28 of SEQ ID NO: 194 describe the TelN prokaryotic telomerase recognition sequence; that is, SEQ ID NO: 195.

[0348] Base pairs 29-37 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 196.

[0349] The base pairs 38-249 of SEQ ID NO: 194 describe the 5′ filling sequence; that is, SEQ ID NO: 197.

[0350] Base pairs 250-271 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 198.

[0351] The base pairs 272-401 of SEQ ID NO: 194 describe the L-ITR sequence; that is, SEQ ID NO: 199.

[0352] The base pairs 402-452 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 200.

[0353] The base pairs 453-2073 of SEQ ID NO: 194 describe the CAG promoter sequence; that is, SEQ ID NO: 193.

[0354] The base pairs 2074-2114 of SEQ ID NO: 194 describe the HBB2 intron sequence; that is, SEQ ID NO: 201.

[0355] The base pairs 2115-2236 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ TD NO: 202.

[0356] Base pairs 2237-2379 of SEQ ID NO: 194 describe the CYP46A1 sequence; that is, SEQ ID NO: 212 (SEQ ID NO: 212 is the sequence of SEQ ID NO: 110 with three additional base pairs at the C-terminus, which are translation stop codons).

[0357] The base pairs 2380-3899 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 203.

[0358] The base pairs 3900-4124 of SEQ ID NO: 194 describe the bGH poly(A) signal sequence; that is, SEQ ID NO: 204.

[0359] The base pairs 4125-4177 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 205.

[0360] The base pairs 4178-4307 of SEQ ID NO: 194 describe the R-ITR sequence; that is, SEQ ID NO: 206.

[0361] The base pairs 4308-4321 of SEQ ID NO: 194 describe the linker sequence; that is, SEQ ID NO: 207.

[0362] The base pairs 4322-4533 of SEQ ID NO: 194 describe the 3′ filling sequence; that is, SEQ ID NO: 208.

[0363] The base pairs 4534-4545 of SEQ ID NO: 194 describe the linker sequence; that is, ID NO: 209.

[0364] Base pairs 4546-4573 of SEQ ID NO: 194 describe the TelN prokaryotic telomerase recognition sequence; that is, SEQ ID NO: 210. Synthetic CNS-specific expression cassette

[0365] The present invention also provides a synthetic CNS-specific expression cassette comprising a synthetic CNS-specific promoter of the present invention, the promoter being operatively linked to a sequence encoding an expression product, the expression product being suitably a gene (e.g., a transgene).

[0366] The gene typically encodes a desired gene expression product, such as a polypeptide (protein) or RNA. The gene can be a full-length cDNA or genomic DNA sequence, or any mutant or subunit or fragment thereof having at least some of the desired biological activity.

[0367] When a gene encodes a protein, the protein can be virtually any type of protein. By way of non-limiting examples, the protein can be an enzyme, an antibody (e.g., a monoclonal antibody), an antibody fragment, a viral protein (e.g., REP-CAP, REV, VSV-G, or RD114), a therapeutic protein (e.g., FVIII), or a toxic protein (e.g., Caspase 3, Caspase 8, or Caspase 9).

[0368] Various expression products suitable for treating the above-mentioned conditions have been described in the art. Suitably, the nucleic acid encoding the expression product of CRE operatively linked to the promoter or minimal / proximal promoter according to the invention can be one of the genes selected from the group consisting of: NPC1, EAAT2, NPY, CYP46A1, GLB 1, APOE (e.g., ApoE2, ApoE3 or ApoE4), HEX, CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, SUMF1, DCTN1, PRPH, SOD1, NEFH, GBA, IDUA, NAGLU, GUSB, ARSA, MANB, AADC, GDNF, NTN, ASP, MECP2, PTCHD1, GJB1, UBE3A, HEXA, MOG or SLC6A3. Alternatively or concurrently, the expression product of CRE, operably linked to the promoter or minimal / proximal promoter according to the invention, can be a miRNA / CRISPR Cas9 targeting the disease allele.

[0369] In some embodiments, useful expression products include dystrophin (including microdystrophin), β-1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthase I, α-1 antitrypsin, ornithine transcarbamoylase, argininosuccinate synthase, argininosuccinate lyase, arginase, fumarate acetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, bile pigmentogen deaminase, cystathionine β-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylase, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR), and dopaminergic transporter (hDAT).

[0370] Other useful expression products include enzymes that can be used in enzyme replacement therapy and can be used for various conditions caused by enzyme activity defects. For example, enzymes containing mannose-6-phosphate can be used to treat lysosomal storage diseases (e.g., suitable genes include genes encoding β-glucuronidase (GUSB)).

[0371] In some embodiments, exemplary peptide expression products include neuroprotective peptides and anti-angiogenic peptides. Suitable peptides include, but are not limited to, glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-β), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-6 (NT-6), epidermal growth factor (EGF), pigment epithelial-derived factor (PEDF), Wnt peptide, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibody, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and hedgehog family members (e.g., sonic hedgehog factor, Indian hedgehog factor, and desert hedgehog factor).

[0372] In some embodiments, useful therapeutic expression products include hormones and growth and differentiation factors, including but not limited to insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor I and insulin-like growth factor II (IGF-I and IGF-II), and any of the transforming growth factor α superfamily (including TGFa, activin, inhibin, or any bone morphogenetic protein (BMP)). 1-15) Any one of the heregluin / neuromodulatory protein / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor NT-3 and neurotrophic factor NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorins / collapsins family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), eprins, noggin, sound hedgehog factor and tyrosine hydroxylase.

[0373] In some embodiments, useful expression products include proteins that regulate the immune system, including but not limited to cytokines and lymphokines such as thrombopoietin (TPO), interleukins (ILs) IL-1 to IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemoattractant proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor α and tumor necrosis factor β, interferons (α, β, and γ), stem cell factors, flk-2 ligand / flt3 ligand. Gene products generated by the immune system can also be used in this invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T-cell receptors, chimeric T-cell receptors, single-chain T-cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins, such as complement regulatory protein, membrane cofactor protein (MCP), decay accelerator factor (DAF), CR1, CF2, and CD59.

[0374] In some embodiments, useful expression products include any one of receptors for growth factors, cytokines, lymphokines, regulatory proteins, immune system proteins, and hormones. Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and / or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. The invention also covers the use of gene products, such as members of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MyoD and myopoietin, ETS-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-binding proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding proteins, STAT, GATA box-binding proteins (e.g., GATA-3), and the forkhead family of winged helical proteins.

[0375] In some embodiments, useful expression products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions.

[0376] Further suitable expression products include microRNAs (miRNAs), interfering RNAs, antisense RNAs, ribozymes, and aptamers.

[0377] In some embodiments of the invention, the synthesized CNS-specific expression cassette contains genes suitable for gene editing, such as genes encoding site-specific nucleases, such as broad-spectrum nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly spaced short palindromic repeat systems (CRISPR-Cas). Suitably, the site-specific nuclease is adapted to edit the desired target genomic site by performing a cut (typically a site-specific double-strand break), followed by repair via non-homologous end joining (NHEJ) or homology-dependent repair (HDR), thereby producing the desired edit. The edit can be a partial or complete repair of a dysfunctional gene, or a knockdown or knockout of a functional gene. Alternatively, the edit can be performed using suitable systems known in the art via base editing or prime editing.

[0378] Suitably, the synthesized CNS-specific expression cassette comprises sequences providing or encoding one or more (and preferably all) of the following: a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Suitably, the expression cassette comprises nucleic acids encoding post-transcriptional regulatory elements. Suitably, the expression cassette comprises nucleic acids encoding polyA elements. Modified outer shell

[0379] In one embodiment, the capsid described herein is further modified to increase tropism for the CNS. The compositions provided herein comprise a modified viral capsid containing a payload, wherein the payload comprises a nucleic acid sequence side-joined by an inverted terminal repeat (ITR) targeting central nervous system disorders and a regulatory sequence, and wherein the modification is chemical, non-chemical, or amino acid modification. In some embodiments, the nucleic acid sequence of the payload comprises (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) an isolated nucleic acid encoding a CYP46A1 protein. In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

[0380] This document further provides a composition 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 payload comprises a nucleic acid sequence side-attached by an inverted terminal repeat (ITR) targeting central nervous system disorders and a regulatory sequence, wherein the first modified viral capsid and at least the second modified viral capsid are identical, and the first and second payloads are different, and wherein the modification is a chemical, non-chemical, or amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

[0381] This document further provides a composition comprising (a) a first modified capsid containing a first payload, and (b) at least a second modified capsid containing a second payload, wherein the payload comprises a nucleic acid sequence side-attached by an inverted terminal repeat (ITR) targeting central nervous system disorders and a regulatory sequence, wherein the first modified capsid and at least the second modified capsid are different, and the first and second payloads may be the same or different, and wherein the modification is a chemical, non-chemical, or amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload comprises isolated nucleic acid encoding a transgene of one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload comprises isolated nucleic acid encoding a CYP46A1 protein.

[0382] Another aspect described herein provides compositions or combinations comprising at least one of: (a) an isolated nucleic acid encoding a transgene, said transgene encoding one or more miRNAs; and (b) an isolated nucleic acid encoding a CYP46A1 protein. In one aspect, the compositions or combinations described herein comprise: (a) a recombinant viral vector containing isolated nucleic acids, said nucleic acids comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs; and (b) a recombinant viral vector containing isolated nucleic acids encoding said CYP46A1 protein.

[0383] Another aspect described herein provides compositions or combinations comprising: (a) an isolated nucleic acid encoding a transgene, said transgene encoding an isolated nucleic acid that encodes a CYP46A1 protein. In some aspects, said compositions or combinations further comprise: (b) an isolated nucleic acid encoding a transgene that encodes one or more miRNAs. In other aspects, said compositions or combinations do not comprise: (b) an isolated nucleic acid encoding a transgene that encodes one or more miRNAs. In one aspect, said compositions or combinations are described herein comprising: (a) a recombinant viral vector containing an isolated nucleic acid comprising: (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and the second region comprising a transgene encoding a CYP46A1 protein. In some aspects, said compositions or combinations further comprise: (b) a recombinant viral vector containing an isolated nucleic acid encoding one or more miRNAs. In other aspects, said compositions or combinations do not comprise: (b) a recombinant viral vector containing an isolated nucleic acid encoding one or more miRNAs.

[0384] Another aspect described herein provides a composition or combination comprising (a) an isolated nucleic acid encoding a transgene, the transgene encoding the isolated nucleic acid, the isolated nucleic acid encoding one or more miRNAs. In some aspects, the composition or combination further comprises (b) an isolated nucleic acid encoding a transgene, the transgene encoding a CYP46A1 protein. In other aspects, the composition or combination does not comprise (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein. In one aspect, a composition or combination is described herein comprising: (a) a recombinant viral vector containing an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs. In some aspects, the composition or combination further comprises: (b) a recombinant viral vector containing an isolated nucleic acid encoding a CYP46A1 protein. In other aspects, the composition or combination does not comprise (b) a recombinant viral vector containing an isolated nucleic acid encoding a CYP46A1 protein.

[0385] In certain embodiments, the modified viral capsid includes modifications that preferentially target the CNS or PNS. For example, the modified viral capsid has an increased tendency toward the CNS and / or a decreased tendency toward at least a second site (e.g., the liver). Preferential targeting of the CNS does not preclude targeting of other sites, but rather indicates that it targets the CNS more strongly than other sites.

[0386] In one embodiment, the modified viral capsid includes modifications that cause it to target either the CNS or the PNS. For example, modifications to a capsid that typically targets non-CNS sites (e.g., the liver) can redirect the capsid to now target both CNS and non-CNS sites. In such embodiments, CNS targeting is not required to be preferred.

[0387] In one embodiment, the modification of the capsid is an amino acid modification, such as the deletion, insertion, or substitution of amino acids. In one embodiment, the amino acid modification increases the affinity for the CNS or PNS. In one embodiment, the amino acid modification targets the modified capsid to the CNS or PNS.

[0388] In one embodiment, the modified viral capsid has a nucleic acid sequence that is 90% identical to SEQ ID NO: 1-SEQ ID NO: 4 of U.S. Patent Application No. 16 / 511,913, or consists of a nucleic acid sequence that is 90% identical to SEQ ID NO: 1-SEQ ID NO: 4 of U.S. Patent Application No. 16 / 511,913, or substantially consists of a nucleic acid sequence that is 90% identical to SEQ ID NO: 1-SEQ ID NO: 4 of U.S. Patent Application No. 16 / 511,913, the contents of which are incorporated herein by reference in their entirety. This U.S. patent application describes a chimeric AAV capsid sequence that exhibits a dominant tendency towards oligodendrocytes and can be used to create AAV vectors for transducing oligodendrocytes in the CNS of a subject.

[0389] In one embodiment, the modified viral capsid is an AAV capsid protein comprising one or more amino acid substitutions, wherein the substitutions introduce novel glycan binding sites into the AAV capsid protein. In some embodiments, the amino acid substitutions are at amino acids 266, 463-475, and 499-502 in AAV2, or at 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, the contents of which are incorporated herein by reference in their entirety.

[0390] In one embodiment, the modified viral capsid is an AAV capsid protein comprising, consisting of, or substantially consisting of, AAV2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT / US2020 / 029493, the contents of which are incorporated herein by reference in their entirety), AAV2.5 capsid protein comprising one or more amino acid substitutions that introduce novel glycan binding sites. Such amino acid substitutions can enable the capsid to target neurons and glial cells (e.g., astrocytes). In embodiments of the capsid protein, capsid, viral vector, and method described in International Patent Application No. PCT / US2020 / 029493, the one or more amino acid substitutions include A267S, SQAGASDIRDQSR464-476SX1AGX2SX3X4X5X6QX7R (SEQ ID NO: 153 and SEQ ID NO: 154, respectively) (where X... 1-7 It can be any amino acid), and EYSW 500-503 (SEQ ID NO: 155)EX8X9W, where X 8-9 It can be any amino acid. In embodiments of the capsid protein, capsid, viral vector, and method 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 protein, capsid, viral vector, and method 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, wherein the novel 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; the contents of which are incorporated herein by reference in their entirety.

[0391] In one embodiment, the modified viral capsid is an AAV capsid protein particle comprising a surface-bound peptide, wherein 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, as described, for example, in U.S. Patent Application No. 16 / 956,306; the contents of which are incorporated herein by reference in their entirety. Such an AAV capsid allows for the delivery (e.g., of a payload) across the blood-brain barrier.

[0392] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of said capsid protein contains modifications (e.g., replacing tyrosine residues with non-tyrosine residues and / or replacing threonine residues with non-threonine residues) at positions corresponding to: one or more, or each of Y705, Y731, and T492 of wild-type AAV1 capsid protein (e.g., SEQ 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, or each of Y436, Y693, and Y719 of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); or one or more, or each of Y705, Y731, and T492 of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191). This AAV capsid targets neurons and astrocytes.

[0393] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein) wherein the capsid protein contains a Y to F (tyrosine to phenylalanine) modification or a T to V (threonine to valine) modification at a position in the VP3 region of the capsid corresponding to: one or more, or each of Y705F, Y731F, and T492V of wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); one or more, or each of Y436F, Y693F, and Y719F of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); or one or more, or each of Y705F, Y731F, and T492V of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191). This AAV capsid targets neurons and astrocytes.

[0394] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of the capsid protein contains modifications (e.g., replacing tyrosine residues with non-tyrosine residues and / or replacing threonine residues with non-threonine residues) at positions corresponding to: one or each of Y705, Y731, and T492 of wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); one or each of Y436, Y693, and Y719 of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); or one or each of Y705, Y731, and T492 of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191). This AAV capsid targets neurons and astrocytes.

[0395] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein) containing a Y to F (tyrosine to phenylalanine) modification or a T to V (threonine to valine) modification at a position in the VP3 region of the capsid protein corresponding to: one or more of Y705F, Y731F, and T492V of wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); one or more of Y436F, Y693F, and Y719F of 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 Y705F, Y731F, and T492V of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. Patent Application No. 16 / 565,191). This AAV capsid targets neurons and astrocytes.

[0396] In one embodiment, amino acid modification allows the modified capsid to evade neutralizing antibodies, such as those generated against a viral vector (e.g., a viral vector of the same serotype). In another embodiment, amino acid modification allows the modified capsid to be used for repeated administration; for example, the modification would make the capsid have a therapeutic effect upon re-administration.

[0397] In one embodiment, the modified viral capsid is a chimeric capsid. As used herein, a “chimeric” capsid protein refers to an AAV capsid protein (e.g., any one or more of VP1, VP2, or VP3) that has been modified by substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues relative to the wild type, and by insertion or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues relative to the wild type. In some embodiments, complete or partial domains, functional regions, epitopes, etc., from one AAV serotype can replace corresponding wild-type domains, functional regions, epitopes, etc., from different AAV serotypes in any combination to produce the chimeric capsid protein of the present invention. The generation of chimeric capsid proteins can be carried out according to methods well known in the art, and numerous chimeric capsid proteins that can be included in the capsids of the present invention are described in the literature and herein.

[0398] In one embodiment, the modified viral capsid is a haploid capsid. As used herein, the term "haploid AAV" should refer to the AAV described in International Application WO2018 / 170310 or U.S. Application US2018 / 037149, which are incorporated herein by reference in their entirety. In some embodiments, the virion population is a haploid AAV population, in which virion particles can be constructed in which at least one viral protein from the group consisting of AAV capsid proteins, VP1, VP2, and VP3 differs 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 protein is of the same type (e.g., all AAV2 VP1). In one instance, 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, and only VP3 is non-chimeric. For example, the viral particle consists only of: VP1 / VP2 from chimeric AAV2 / 8 (N-terminus of AAV2 and C-terminus of AAV8) paired with VP3 from AAV2 only; or only chimeric VP1 / VP2 28m-2P3 (N-terminus of AAV8 and C-terminus of AAV2, without VP3 start codon mutation) paired with VP3 from AAV2 only. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins comes from a completely different serotype. For example, only chimeric VP1 / VP2 28m-2P3 pairs with VP3 from AAV3 only. In another example, no chimeric protein is present.

[0399] 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 may, for example, affect the tissue tropism or cellular tropism of the modified capsid, as well as others.

[0400] Modifications can directly alter the properties of the capsid (including biochemical properties, such as receptor binding), thereby changing the behavior of the capsid itself, or can allow for further modifications (such as ligand attachment, which in turn modifies the behavior of the capsid in a desired manner).

[0401] In one embodiment, chemical modification of cysteine ​​residues (which may be naturally occurring or introduced through genetic modification of the capsid polypeptide coding sequence) allows for covalent attachment of ligands by forming disulfide bonds (see, for example, WO 2005 / 106046, the contents of which are incorporated herein by reference).

[0402] Various ligands have been considered, including, but not limited to, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by target cells (see, for example, WO 2000 / 002654, which is incorporated herein by reference).

[0403] WO2015 / 062516 (the contents of which are also incorporated herein by reference) describes the insertion of an amino acid containing an azide group through genetic modification of the capsid gene, followed by chemical coupling of a ligand via the azide group.

[0404] Horowitz et al., Bioconjugate Chem., 22: 529-532 (2011) described the tendency to modify AAV capsids by saccharification or chemical coupling of sugar moieties. As described herein, this method, and similar methods, have been considered for capsid modification.

[0405] In other embodiments, coating the viral capsid with a polymer such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl)methacrylamide (pHPMA) is particularly considered. This modification can, for example, reduce specific and nonspecific interactions with non-target tissues.

[0406] In other embodiments, carbodiimide coupling is particularly considered. See, for example, Joo et al., ACS Nano 5, entitled “Enhanced Real-time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates” (2011).

[0407] In other embodiments, the viral capsid may be modified, for example, as described in WO 2017 / 212019, and also see US National Phase 16 / 308,740, the contents of which are incorporated herein by reference. The methods described therein couple the viral capsid to the ligand via bonds containing -CSNH- and aromatic moieties. While genetically modified viral capsids can be further modified using this method, the modifications described therein do not require genetic modification of the viral capsid. The ligands described therein include, for example, targeting agents, spatial shielding agents for avoiding neutralizing antibody interactions, labeling agents, or magnetic agents. The 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 can mimic cell adhesion proteins and bind to integrins), vitamins, and small molecules.

[0408] In one embodiment, the chemical modification of the present invention is the modification described in International Patent Application PCT / EP2017 / 064089, the contents of which are incorporated herein by reference in their entirety.

[0409] In one embodiment, the chemical modification of the present invention is the modification described in International Patent Application PCT / EP2020 / 069554, the contents of which are incorporated herein by reference in their entirety.

[0410] In one embodiment, the capsid has at least one chemically modified tyrosine residue therein, wherein the chemically modified tyrosine residue is of formula (I):

[0411] in:

[0412] -X1 is selected from the following group:

[0413] -Ar is an optionally substituted aryl or heteroaryl moiety.

[0414] In one embodiment, the capsid has at least one chemically modified tyrosine residue of formula (Ia):

[0415] in:

[0416] -X i And Ar, as defined above,

[0417] - The spacer region is a group used to attach the "Ar" group to the functional moiety "M", which preferably contains up to 1000 carbon atoms and is preferably in the form of a chemical chain optionally containing heteroatoms and / or cyclic moieties.

[0418] -n is 0 or 1; and

[0419] -M is a functional part that includes steric hindrance agents, labeling agents, cell type-specific ligands, or drug components.

[0420] In one embodiment, Xi is of formula (a) and / or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl and anthracene.

[0421] In one embodiment, the capsid has at least one chemically modified tyrosine, which is of formula (Ic):

[0422] in:

[0423] -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-.

[0424] -X2 is located at the para, meta, or ortho position of the phenyl group, preferably at the para position.

[0425] - The interval, n, and M are as defined above.

[0426] In one implementation, when present, the "spacer region" is selected from the group consisting of: saturated or unsaturated, straight-chain or branched C2-C. 40 Hydrocarbon chains, optionally substituted polyethylene glycol, polypropylene glycol, pHPMA (a polymer of N-(2-hydroxypropyl)methacrylamide), polylactic-co-glycolic acid copolymer (PLGA), polymers of alkyl diamines, and combinations thereof, and / or

[0427] “M” includes or is composed of cell-type targeting ligands, which are preferably selected from monosaccharides or polysaccharides, hormones (including steroid hormones), peptides (e.g., RGD peptides (e.g., Arg-Gly-Asp, a cell adhesion motif that mimics cell adhesion proteins and binds to integrins), muscle-targeting peptides (MTPs) or Angiopep-2), proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies (including heavy chain antibodies) and fragments thereof (e.g., antigen-binding fragments (Fab), Fab' (which is an antigen-binding fragment further containing a free thiol group) and VHH), single-chain fragment variable (ScFv), spiegelmers, peptide aptamers, vitamins, and drugs (e.g., cannabinoid receptor 1 (CB1) and / or cannabinoid receptor 2 (CB2) ligands).

[0428] In one implementation, the "spacer region" (when present) is selected from the group consisting of: C2-C of straight or branched chains. 20Polymers of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyl diamines, and combinations thereof, wherein the polymer having 2 to 20 monomers and / or “M” contains or is composed of cell type-specific ligands derived from: proteins selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor 13FGF; monosaccharides or polysaccharides containing one or more galactose, mannose, N-acetylgalactosamine residues, GalNac bridges, or mannose-6-phosphate; MTPs selected from SEQ ID NO: 1 to SEQ ID NO: 7; and vitamins (e.g., folic acid).

[0429] In one embodiment, the capsid further comprises at least one additional chemically modified amino acid residue, which is different from a tyrosine residue, preferably having an amino group chemically modified with a group of formula (V):

[0430] in:

[0431] -N* is the nitrogen atom of the amino group of an amino acid residue (e.g., a lysine or arginine residue), and

[0432] Ar, the spacer, n, and M have the same definitions as Ar, the spacer, n, and M in formula (II) of claim 2.

[0433] In one embodiment, the capsid is incubated with a chemical reagent bearing a reactive group selected from aryldiazo and 4-phenyl-1,2,4-triazol-3,5-dione (PTAD) moieties under conditions that facilitate the reaction of the reactive group with tyrosine residues present in the capsid, thereby forming a covalent bond.

[0434] In one embodiment, the capsid is incubated with a chemical reagent of formula VId to obtain at least one chemically modified tyrosine residue in the capsid of formula Ic. Pharmaceutical Composition

[0435] The expression cassettes, vectors, or virions of the present invention can be formulated into pharmaceutical compositions together with pharmaceutically acceptable excipients (i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc.). The pharmaceutical compositions can be provided in the form of kits.

[0436] Therefore, a further aspect of the present invention provides pharmaceutical compositions comprising expression cassettes, vectors, or viruses as described herein.

[0437] In all aspects, the pharmaceutical composition comprises a phosphate buffer. The phosphate buffer comprises about 1 mM to about 50 mM of phosphate, for example, at concentrations of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. The phosphate is prepared from a combination of diphosphates (e.g., Na₂HPO₄, K₂HPO₄) and monophosphates (e.g., NaH₂PO₄, KH₂PO₄) with a diphosphate:monophosphate molar ratio of about 1:10 to about 10:1. For example, in various exemplary embodiments, the 10 mM phosphate includes 9.5 mM diphosphate and 0.5 mM monophosphate, 9 mM diphosphate and 1 mM monophosphate, 8.5 mM diphosphate and 1.5 mM monophosphate, or 8 mM diphosphate and 2 mM monophosphate. The pH of the phosphate buffer is from about 6.5 to about 7.5, for example, pH 6.5, 6.6, 6.7, 6.8, 6.9, 6.95, 7.0, 7.05, 7.1, 7.2, 7.3, 7.4, or 7.5. Phosphate buffer may also contain NaCl at concentrations of about 50 mM to about 200 mM, such as about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 135 mM, about 136 mM, about 137 mM, about 138 mM, about 139 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about 200 mM. The phosphate buffer may also contain KCl at a concentration of about 0.5 mM to about 10 mM, for example, at concentrations of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 2 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM. The phosphate buffer may also contain CaCl2 at a concentration of about 0.20 mM to about 10 mM, for example, at concentrations of about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.81 mM, about 0.82 mM, about 0.83 mM, about 0.84 mM, about 0.85 mM, about 0.86 mM, about 0.87 mM, about 0.88 mM, about 0.89 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM.The phosphate buffer may also contain MgCl2 at a concentration of about 0.10 mM to about 1 mM, for example, at concentrations of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, 0.41 mM, 0.42 mM, 0.43 mM, 0.44 mM, 0.45 mM, 0.46 mM, 0.47 mM, 0.48 mM, 0.49 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM. The phosphate buffer may also contain poloxamer 188 (e.g., Pluronic) at a concentration of about 0.0001 wt% to about 0.005 wt%. TM F-68 nonionic surfactant, for example, at concentrations of about 0.0001 wt%, about 0.0002 wt%, about 0.0003 wt%, about 0.0004 wt%, about 0.0005 wt%, about 0.0006 wt%, about 0.0007 wt%, about 0.0008 wt%, about 0.0009 wt%, about 0.001 wt%, about 0.0015 wt%, about 0.002 wt%, about 0.0025 wt%, about 0.003 wt%, about 0.0035 wt%, about 0.004 wt%, about 0.0045 wt%, or about 0.005 wt%. The phosphate buffer may also contain sorbitol at a concentration of about 0.005 wt% to about 10 wt%, for example, at concentrations of about 0.005 wt%, about 0.075 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt%. The rAAV containing a nucleic acid (e.g., AAVrh.10 containing a CAG promoter and the nucleic acid containing at least 80%, for example at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 194; AAVrh10.CAG.hCYP46A1) can have a titer of approximately 0.1 × 10⁻⁶ in phosphate buffer. 9 vg / μl to approximately 5×10 10 vg / μl, for example, a titer of approximately 0.1 × 10⁻⁶. 9 vg / μl, approximately 0.2×10 9 vg / μl, approximately 0.3×10 9vg / μl, approximately 0.4×10 9 vg / μl, approximately 0.5×10 9 vg / μl, approximately 0.6×10 9 vg / μl, approximately 0.7×10 9 vg / μl, approximately 0.8×10 9 vg / μl, approximately 0.9×10 9 vg / μl, approximately 1×10 9 vg / μl, approximately 2×10 9 vg / μl, approximately 3×10 9 vg / μl, approximately 4×10 9 vg / μl, approximately 5×10 9 vg / μl, approximately 6×10 9 vg / μl, approximately 7×10 9 vg / μl, approximately 8×10 9 vg / μl, approximately 9×10 9 vg / μl, approximately 1×10 10 vg / μl, approximately 1×10 10 vg / μl, approximately 1×10 10 vg / μl, approximately 4×10 10 vg / μl or approximately 5×10 10 vg / μl.

[0438] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 0.4 × 10⁻⁶ ppm. 9 vg / μlAAVrh10.CAG.hCYP46A1.

[0439] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 0.4 × 10⁻⁶ ppm. 9 vg / μl AAVrh10.CAG.hCYP46A1.

[0440] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 1.1 × 10⁻⁶ ppm. 9vg / μlAAVrh10.CAG.hCYP46A1.

[0441] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 1.1 × 10⁻⁶ ppm. 9 vg / μl AAVrh10.CAG.hCYP46A1.

[0442] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 3 × 10 9 vg / μlAAVrhl0.CAG.hCYP46A1.

[0443] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 3 × 10 9 vg / μl AAVrh10.CAG.hCYP46A1.

[0444] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 7 × 10⁻⁶ ppm. 9 vg / μlAAVrh10.CAG.hCYP46A1.

[0445] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 7 × 10⁻⁶ ppm. 9 vg / μl AAVrh10.CAG.hCYP46A1.

[0446] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 8.2 × 10⁻⁶ ppm. 9 vg / μlAAVrh10.CAG.hCYP46A1.

[0447] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 8.2 × 10⁻⁶ ppm. 9 vg / μl AAVrh10.CAG.hCYP46A1.

[0448] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 3 × 10 10 vg / μlAAVrh10.CAG.hCYP46A1.

[0449] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM Na2HPO4 and 1 mM KH2PO4), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl2, 0.49 mM MgCl2, 0.001 wt% poloxamer 188 (pH 7.2), and 3 × 10 10 vg / μl AAVrh10.CAG.hCYP46A1.

[0450] In an alternative embodiment, the pharmaceutical composition is equivalent to any of the exemplary compositions described above, but with a phosphate concentration of about 9.53 mM, comprising about 8.06 mM Na2HPO4 and about 1.47 mM KH2PO4.

[0451] In an alternative embodiment, the pharmaceutical composition is equivalent to any of the exemplary compositions described above, but with a sorbitol concentration of about 0.005 wt% to about 10 wt%. application

[0452] The rAAV disclosed herein can be delivered to a subject in a composition according to any suitable method known in the art. For example, rAAV preferably suspended in a physiologically compatible carrier (i.e., in the composition) can be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (such as a macaque). In some embodiments, the host animal does not include humans.

[0453] Delivery of rAAV to mammalian subjects can be performed, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream can be done by injection into a vein, artery, or any other vascular conduit. In some embodiments, the rAAV is administered into the bloodstream via isolated limb perfusion (a technique well-known in the surgical field), a method primarily allowing the technician to isolate the limb from the systemic circulation before administering the rAAV virion. Variations of the isolated limb perfusion technique (described in U.S. Patent No. 6,177,403) can also be employed by the technician to administer the virion into the vascular system of the isolated limb to potentially enhance transduction to muscle cells or tissues. Furthermore, in certain cases, it may be desirable to deliver the virion to the subject's CNS. "CNS" refers to all cells and tissues of the brain and spinal cord in vertebrates. Therefore, the term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), intercellular spaces, bone, cartilage, etc. Recombinant AAV can be delivered directly to the CNS or brain using needles, catheters, or related devices, employing neurosurgical techniques known in the art (e.g., stereotactic injection, 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) into, for example, ventricular regions and the striatum (e.g., the putamen and / or caudate nucleus of the dorsal striatum), spinal cord and neuromuscular junction, or cerebellar lobules. In some embodiments, rAAV as described in this disclosure is administered via intravenous injection. In some embodiments, rAAV is administered via intracerebral injection. In some embodiments, rAAV is administered via intrathecal injection. In some embodiments, rAAV is administered via intrathecal injection. In some embodiments, rAAV is administered via intrastriatal injection. In some embodiments, rAAV is delivered via intracranial injection. In some embodiments, rAAV is delivered via injection through the cerebellomedullary cistern. In some embodiments, rAAV is delivered via injection through the lateral ventricle.

[0454] In some embodiments, the rAAV is administered by multiple injections along a single trajectory. For example, a cannula can be positioned through the brain at a first location within a target structure (e.g., the putamen) and a first injection can be made at the first location. The cannula is then extended or retracted along the same single trajectory to a second location within the target structure and a second injection can be made at the second location. Further injections can be made at other locations within the target structure by extending or retracting the cannula along the same single trajectory. Brain structures can be targeted by positioning the cannula through the brain at a trajectory-guided MRI location. The trajectory can be anterior (e.g., through the frontal bone) or posterior (e.g., through the occipital bone). In other embodiments, multiple trajectories are used to target a single tissue or structure.

[0455] Various aspects of this disclosure relate to compositions comprising recombinant AAV, said recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, said transgene comprising a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises a sequence shown in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185, or SEQ ID NO: 217-SEQ ID NO: 260. 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, AAVrh10, AAV11, AAV12, or AAV13 ITR. In some embodiments, the composition further comprises a pharmaceutically acceptable vector. The compositions disclosed herein may comprise a single rAAV or a combination of rAAVs with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the compositions comprise one, two, three, four, five, six, seven, eight, nine, ten or more different rAAVs, each having one or more different transgenes.

[0456] Those skilled in the art can readily select a suitable carrier based on the indication targeted by rAAV. For example, a suitable carrier includes saline, which can be prepared with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of this disclosure.

[0457] Optionally, in addition to rAAV and the carrier, the compositions disclosed herein may also contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0458] Administering rAAV in sufficient quantities to transfect cells of the desired tissue and provide adequate levels of gene transfer and expression without excessive adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intravenous delivery to the liver), oral administration, inhalation (including intranasal and intratracheal delivery), intraocular administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, and other parenteral administration routes. Routes of administration may be combined if desired. In some embodiments, all or at least one nucleic acid sequence disclosed herein is delivered via a nonviral DNA construct containing at least one DD-ITR. For example, one or more of the nucleic acids described herein may be delivered using a nonviral DNA construct as described in WO 2019 / 246554. WO 2019 / 246554 is incorporated herein by reference in its entirety.

[0459] The dose of rAAV virus required to achieve a specific "therapeutic effect" (e.g., in units of genome copies per kilogram of body weight (GC / kg)) will vary based on several factors, including but not limited to: the route of rAAV virus 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. Based on the above factors, as well as other factors well known in the art, those skilled in the art can readily determine the dose range of rAAV virus for treating patients with specific diseases or disorders.

[0460] An effective amount of rAAV is an amount sufficient to target and infect an animal and the desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health status of the subject, and the tissue to be targeted, and therefore can vary from animal to tissue. For example, the effective amount of rAAV is typically found in an amount containing about 10 9 One to 10 16 The range is approximately 1 mL to 100 mL of solution containing one genome copy. In some cases, approximately 10 11 One to 10 13 The dosage between rAAV genome copies is appropriate. In a particular embodiment, 10 12One or 10 13 One rAAV genome copy is effective in targeting CNS tissues. In some cases, stable transgenic animals are produced by multiple doses of rAAV.

[0461] In other cases, the administered dose depends on the rAAV titer and the volume of tissue to be injected. For example, the titer of the rAAV (or rAAV composition) may be about 0.1 × 10⁻⁶. 9 vg / μL to approximately 1.5×10 9 vg / μL, for example, a titer of approximately 0.1 × 10⁻⁶. 9 vg / μL, approximately 0.2×10 9 vg / μL, approximately 0.3×10 9 vg / μL, approximately 0.4×10 9 vg / μL, approximately 0.5×10 9 vg / μL, approximately 0.6×10 9 vg / μL, approximately 0.7×10 9 vg / μL, approximately 0.8×10 9 vg / μL, approximately 0.9×10 9 vg / μL, approximately 1×10 9 vg / μL, approximately 1.1×10 9 vg / μL, approximately 1.2×10 9 vg / μL, approximately 1.3×10 9 vg / μL, approximately 1.4×10 9 vg / μL or approximately 2.5×10 9vg / μL. The amount of titer to be injected can independently depend on the volume of at least one target tissue or structure determined by MRI. For example, the volume of the tissue to be injected can be from about 1% to about 90% of the total volume of the tissue or structure, such as about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% of the tissue or structure volume. Similarly, the volume of the tissue or structure to be injected can be at least about 5%, at least about 10%, at least about 15%, 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%, or more of the total volume of the tissue or structure. When targeting (i.e., injecting) more than one tissue or structure, the volumes of the tissues or structures to be injected can be the same or different. For example, in some embodiments, the titer is injected in separate volumes into the left and right putamen, for example, the separate volumes being equivalent to, for example, about 25% of the total volume of each putamen. In other embodiments, the titer is injected in separate volumes into the left and right tail nuclei, for example, the separate volumes being equivalent to about 25% of the total volume of each tail nucleus. In other embodiments, the left and right capsids, as well as the left and right tail nuclei, are all titrated, for example, based on about 25% of the total volume of each capsid and tail nucleus.

[0462] In some embodiments, the rAAV dose is administered to the subject no more than once per calendar day (e.g., during a 24-hour period). In some embodiments, the rAAV dose is administered to the subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, the rAAV dose is administered to the subject no more than once per calendar week (e.g., during 7 calendar days). In some embodiments, the rAAV dose is administered to the subject no more than once every two weeks (e.g., once between two calendar weeks). In some embodiments, the rAAV dose is administered to the subject no more than once per calendar month (e.g., once every 30 calendar days). In some embodiments, the rAAV dose is administered to the subject no more than once every six calendar months. In some embodiments, the rAAV dose is administered to the subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0463] In some embodiments, the rAAV composition is formulated to reduce the aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., -10). 13In cases of GC / mL or higher. Methods for reducing rAAV aggregation are well known in the art, including, for example, the addition of surfactants, pH adjustment, salt concentration adjustment, etc. (see, for example, Wright FR et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0464] Pharmaceutically acceptable excipient and carrier solutions are well known to those skilled in the art, as are suitable dosing and treatment regimens for the use of the specific compositions described herein in a variety of treatment regimens.

[0465] Typically, these dosage forms may contain at least about 0.1% or more of the active compound, although the percentage of the active ingredient can, of course, vary and can conveniently range from about 1% or 2% to about 70% or 80% or more of the total dosage form by weight or volume. Naturally, the amount of the active compound in each therapeutically useful composition can be prepared in such a manner that a suitable do...

Claims

1. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject suffering from or at risk of developing the neurological disease or disorder a therapeutically effective amount of at least one of: (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) An isolated nucleic acid encoding a CYP46A1 protein.

2. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject suffering from or at risk of developing the neurological disease or disorder a therapeutically effective amount of at least one of: (a) a recombinant viral vector comprising an isolated nucleic acid, the nucleic acid comprising (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 the CYP46A1 protein.

3. The method according to any one of claims 1 to 2, wherein: The nervous system disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinocerebellar ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger disease, neuropathic pain, trauma caused by spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusions, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder.

4. The method according to any one of claims 1 to 3, wherein: The nervous system disease or disorder is a central nervous system (CNS) disease or disorder.

5. The method according to any one of claims 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.

6. The method according to any one of claims 1 to 5, wherein: The CNS disease or disorder is Alzheimer's disease, and the at least one miRNA comprises a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and disease-associated alleles thereof.

7. The method according to any one of claims 1 to 5, wherein: The CNS disease or disorder is Parkinson's disease, and the at least one miRNA comprises a seed sequence complementary to SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and disease-associated alleles thereof.

8. The method according to any one of claims 1 to 5, wherein: The CNS disease is Huntington's disease and at least one miRNA comprises a seed sequence complementary to SEQ ID NO:4, or wherein at least one miRNA comprises a sequence of any one of SEQ ID NO:6-SEQ ID NO:17, SEQ ID NO:40-SEQ ID NO:44, SEQ ID NO:50-SEQ ID NO:66, SEQ ID NO:158-SEQ ID NO:185 or SEQ ID NO:217-SEQ ID NO:260, flanked by a miRNA backbone sequence.

9. The method according to any one of claims 1 to 8, wherein: The CNS disease is Huntington's disease, and the at least one miRNA comprises the sequence of any one of SEQ ID NO:6-SEQ ID NO:17, SEQ ID NO:40-SEQ ID NO:44, SEQ ID NO:50-SEQ ID NO:66, SEQ ID NO:158-SEQ ID NO:185, or SEQ ID NO:217-SEQ ID NO:

260.

10. The method according to any one of claims 8 to 9, wherein: At least one of the miRNAs hybridizes with the human Huntington's gene and inhibits the expression of the human Huntington's gene.

11. The method according to any one of claims 8 to 10, wherein: The subject comprises a Huntington gene having greater than 36 CAG repeats, greater than 40 repeats, or greater than 100 repeats.

12. The method according to any one of claims 8 to 11, wherein: The subject is less than 20 years old.

13. The method according to any one of claims 1 to 12, wherein: The recombinant viral vector is selected from the group consisting of: AAV vector, adenoviral vector, lentiviral vector, retroviral vector, herpes virus vector, alphavirus vector, poxvirus vector, baculovirus vector and chimeric virus vector.

14. The method according to any one of claims 2 to 13, wherein: The recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (b).

15. The method according to any one of claims 1 to 13, wherein: The isolated nucleic acids of (a) and (b) are contained in separate recombinant viral vectors.

16. The method of any one of claims 1 to 14, wherein: The isolated nucleic acids of (a) and (b) are contained in the same recombinant viral vector.

17. The method according to any one of claims 1 to 16, wherein: (a) and (b) are administered at substantially the same time.

18. The method of any one of claims 1 to 13 and 15, wherein: (a) and (b) were administered at different time points.

19. The method of claim 18, wherein: The different time points are separated by at least 1 minute, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year, or longer.

20. The method according to any one of claims 18 to 19, wherein: (a) is applied before the application of (b).

21. The method of any one of claims 18-19, wherein: (b) is applied before the application of (a).

22. The method of any one of claims 1 to 21, wherein: The administration of (a), (b), or (a) and (b) is repeated at least once.

23. The method of any one of claims 1 to 22, wherein: The transgene comprises two miRNAs in tandem flanked by introns.

24. The method of claim 23, wherein: The flanking introns are identical.

25. The method of claim 23, wherein: The flanking introns are from the same species.

26. The method of claim 23, wherein: The flanking intron is the hCG intron.

27. The method of any one of claims 1 to 26, wherein: The transgene comprises a promoter.

28. The method of claim 27, wherein: The promoter is synapsin (Syn1) promoter or the promoters in Table 10 to Table 13.

29. The method of any one of claims 1 to 28, wherein: The one or more miRNAs are located in the untranslated portion of the transgene.

30. The method of claim 29, wherein: The untranslated portions are introns.

31. The method of claim 30, wherein: The non-translated portion is between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence, or between the last nucleotide base of the promoter sequence and the poly-A tail sequence.

32. The method of any one of claims 1-31, further comprising a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

33. The method of any one of claims 1 to 32, wherein: The ITR variant lacks a functional terminal resolution site (TRS), optionally wherein the ITR variant is an ATRS ITR.

34. The method of any one of claims 1 to 33, wherein: The administration results in delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the subject.

35. The method of any one of claims 1 to 34, wherein: The administration is by injection, optionally intravenous or intrastriatal.

36. The method of any one of claims 2 to 35, wherein: The viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof.

37. The method of any one of claims 2-36, wherein the viral vector comprises a capsid protein from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof.

38. The method of claim 37, wherein: The capsid protein is AAV9 capsid protein.

39. The method of any one of claims 2 to 38, wherein: The viral vector is a self-complementary AAV (scAAV).

40. The method of any one of claims 2 to 39, wherein: The viral vector is formulated for delivery to the central nervous system (CNS).

41. A composition or combination comprising at least one of the following: (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) An isolated nucleic acid encoding a CYP46A1 protein.

42. A composition or combination comprising at least one of the following: (a) a recombinant viral vector comprising an isolated nucleic acid, the nucleic acid comprising (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.

43. A composition or combination as described in any one of claims 41-42, for use in a method for treating a nervous system disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition or combination to a subject suffering from, or at risk of developing, the nervous system disease or disorder.

44. The composition or combination of claim 43, wherein: The nervous system disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinocerebral ataxia, Krabbe disease, polyglutamine repeat spinocerebellar ataxia, Batten's 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 caused by spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusions, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder.

45. The composition or combination of claim 44, wherein: The nervous system disease or disorder is a central nervous system (CNS) disease or disorder.

46. ​​The composition or combination of claim 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.

47. A composition or combination as described in any one of claims 41-46, wherein The at least one miRNA comprises a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and disease-associated alleles thereof.

48. A composition or combination as described in any one of claims 41-46, wherein, The at least one miRNA comprises a seed sequence complementary to SNCA, LRRK2 / PARK8, PRKN, PINK1, DJ1 / PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and disease-associated alleles thereof.

49. A composition or combination as described in any one of claims 41-46, wherein, The at least one miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein the at least one miRNA comprises a sequence of any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, SEQ ID NO: 50-SEQ ID NO: 66, SEQ ID NO: 158-SEQ ID NO: 185 or SEQ ID NO: 217-SEQ ID NO: 260, wherein the sequence is flanked by a miRNA backbone sequence.

50. A composition or combination as described in any one of claims 41-46, wherein, The at least one miRNA comprises the sequence of any one of SEQ ID NO:6-SEQ ID NO:17, SEQ ID NO:40-SEQ ID NO:44, SEQ ID NO:50-SEQ ID NO:66, SEQ ID NO:158-SEQ ID NO:185, or SEQ ID NO:217-SEQ ID NO:

260.

51. A composition or combination as described in any one of claims 49-50, wherein, At least one of the miRNAs hybridizes with the human Huntington's gene and inhibits the expression of the human Huntington's gene.

52. A composition or combination as described in any one of claims 49-51, wherein, The subject comprises a Huntington gene having greater than 36 CAG repeats, greater than 40 repeats, or greater than 100 repeats.

53. A composition or combination as described in any one of claims 49-52, wherein, The subject is less than 20 years old.

54. A composition or combination as described in any one of claims 42-53, wherein, The recombinant viral vector is selected from the group consisting of: AAV vector, adenoviral vector, lentiviral vector, retroviral vector, herpes virus vector, alphavirus vector, poxvirus vector, baculovirus vector and chimeric virus vector.

55. A composition or combination as described in any one of claims 42-54, wherein, The recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (b).

56. A composition or combination as described in any one of claims 41-54, wherein, The isolated nucleic acids of (a) and (b) are contained in separate recombinant viral vectors.

57. A composition or combination as described in any one of claims 41-55, wherein, The isolated nucleic acids of (a) and (b) are contained in the same recombinant viral vector.

58. A composition or combination as described in any one of claims 41-57, wherein, (a) and (b) are administered at substantially the same time.

59. A composition or combination as described in any one of claims 41-54 and 56, wherein, (a) and (b) were administered at different time points.

60. The composition or combination of claim 59, wherein: The different time points are separated by at least 1 min, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year or longer.

61. A composition or combination as described in any one of claims 59-60, wherein, (a) is applied before the application of (b).

62. A composition or combination as described in any one of claims 59-60, wherein, (b) is applied before the application of (a).

63. A composition or combination as described in any one of claims 59-60, wherein, The administration of (a), (b), or (a) and (b) is repeated at least once.

64. A composition or combination as described in any one of claims 41-63, wherein, The transgene comprises two miRNAs in tandem flanked by introns.

65. The composition or combination of claim 64, wherein: The flanking introns are identical.

66. The composition or combination of claim 64, wherein: The flanking introns are from the same species.

67. The composition or combination of claim 64, wherein: The flanking intron is the hCG intron.

68. A composition or combination as described in any one of claims 41-67, wherein, The transgene comprises a promoter.

69. The composition or combination of claim 68, wherein: The promoter is synapsin (Syn1) promoter or the promoters in Table 10 to Table 13.

70. A composition or combination as described in any one of claims 41-69, wherein, The one or more miRNAs are located in the untranslated portion of the transgene.

71. The composition or combination of claim 70, wherein: The untranslated portions are introns.

72. The composition or combination of claim 70, wherein: The non-translated portion is between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence, or between the last nucleotide base of the promoter sequence and the poly-A tail sequence.

73. The composition or combination of any one of claims 41-72, further comprising a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof.

74. A composition or combination as described in any one of claims 41-73, wherein, The ITR variant lacks a functional terminal resolution site (TRS), optionally wherein the ITR variant is an ATRS ITR.

75. A composition or combination as described in any one of claims 41-74, wherein, The administration results in delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the subject.

76. A composition or combination as described in any one of claims 41-75, wherein, The administration is by injection, optionally intravenous or intrastriatal.

77. A composition or combination as described in any one of claims 42-76, wherein, The viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, or a chimera thereof.

78. The composition of any one of claims 42-77, wherein the viral vector comprises a capsid protein from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof.

79. The composition or combination of claim 78, wherein: The capsid protein is AAV9 capsid protein.

80. A composition or combination as described in any one of claims 42-79, wherein, The viral vector is a self-complementary AAV (scAAV).

81. A composition or combination as described in any one of claims 42-80, wherein, The viral vector is formulated for delivery to the central nervous system (CNS).

82. A composition comprising an isolated nucleic acid encoding a CYP46A1 protein, said nucleic acid comprising a sequence at least 80% identical to SEQ ID NO:

110.

83. A composition comprising a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein, wherein the nucleic acid comprises a sequence at least 80% identical to SEQ ID NO:

110.

84. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject suffering from or at risk of developing the neurological disease or disorder a therapeutically effective amount of the composition of claim 82 or 83.

85. The method of claim 84, wherein: The nervous system disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinocerebellar ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger disease, neuropathic pain, trauma caused by spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusions, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder.

86. The method of any one of claims 84-85, wherein The nervous system disease or disorder is a central nervous system (CNS) disease or disorder.

87. The method of any one of claims 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.

88. The composition or method of any one of claims 83-87, wherein The recombinant viral vector is selected from the group consisting of: AAV vector, adenoviral vector, lentiviral vector, retroviral vector, herpes virus vector, alphavirus vector, poxvirus vector, baculovirus vector and chimeric virus vector.

89. The method of any one of claims 84 to 88, wherein The administration is repeated at least once.

90. The method of any one of claims 84-89, wherein The administration results in delivery of the viral vector or isolated nucleic acid to the central nervous system (CNS) of the subject.

91. The method of any one of claims 84 to 90, wherein: The administration is performed by injection, optionally intravenous injection or intrastriatal injection.

92. The composition or method of any one of claims 83-91, wherein The viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof.

93. The composition or method of any of claims 83-92, wherein the viral vector comprises a capsid protein from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, or AAV12, or a chimera thereof.

94. The composition or method of claim 93, wherein: The capsid protein is AAV9 capsid protein.

95. The composition or method of any one of claims 83-94, wherein The viral vector is a self-complementary AAV (scAAV).

96. The composition or method of any one of claims 83-95, wherein The viral vector is formulated for delivery to the central nervous system (CNS).

97. The composition or method of any one of claims 82-96, wherein The nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO:

110.

98. The composition or method of any one of claims 82-96, wherein The nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO:

110.

99. The composition or method of any one of claims 82-96, wherein The nucleic acid comprises a sequence identical to SEQ ID NO:

110.

100. The composition or method of any one of claims 2-40, 42-81, 83-99, wherein The viral vector comprises a modified viral capsid.

101. The composition or method of any one of claims 2-40, 42-81, 83-99, wherein The viral vector comprises a modification of the viral capsid.

102. The composition or method of claim 100 or 101, wherein: The modification is a chemical, non-chemical or amino acid modification of the viral capsid.

103. The composition or method of claim 100 or 101, wherein At least one of the capsid modifications is preferentially targeted to cells in the CNS or PNS.

104. The composition or method of claim 100 or 101, wherein The chemical modification comprises a chemically modified tyrosine residue modified to comprise a covalently attached monosaccharide or polysaccharide moiety.

105. The composition or method of claim 104, wherein: The chemically modified tyrosine residue comprises a monosaccharide selected from galactose, mannose, N-acetylgalactosamine, a GalNac bridge and mannose-6-phosphate.

106. The composition or method of claim 100 or 101, wherein The chemical modification comprises a ligand covalently attached to a primary amino group of the capsid polypeptide via a -CSNH- bond.

107. The composition or method of claim 106, wherein: The ligand comprises an arylene or heteroarylene radical covalently bonded to the ligand.

108. The composition or method of any one of claims 100-107, wherein The modified viral capsid is a chimeric capsid or a haploid capsid.

109. The composition or method of any one of claims 100-107, wherein The modified viral capsid is a haploid capsid.

110. The composition or method of any one of claims 100-107, wherein The modified viral capsid further comprises a modified chimeric capsid or haploid capsid.

111. The composition or method of any one of claims 100-110, wherein The modified viral capsid is AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, or a mutant modified form, chimera, mosaic or reasonable haploid thereof.

112. The composition or method of any one of claims 100-111, wherein The modifications alter the antigenic profile of the modified viral capsid compared to the unmodified viral capsid.

113. The composition or method of any one of claims 100-112, wherein: The modified viral capsids can be used for repeated administration.

114. A synthetic CNS-specific promoter, comprising or consisting of a sequence according to any one of SEQ ID NO: 187 to SEQ ID NO: 189 or a functional variant thereof.

115. The synthetic CNS-specific promoter of claim 114, wherein The functional variant is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 187-SEQ ID NO:

189.

116. A CRE comprising or consisting of a sequence according to any one of SEQ ID NO: 191 or SEQ ID NO: 192 or a functional variant thereof.

117. The CRE of claim 116, comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NO: 191 or SEQ ID NO:

192.

118. A CRM comprising the CRE of claim 116 or 117.

119. A minimal promoter comprising or consisting of a sequence according to SEQ ID NO: 211 or a functional variant thereof.

120. A CNS-specific promoter comprising the CRE of claim 116 or 117, the CRM of claim 118, or the minimal promoter of claim 119.

121. An expression cassette comprising the synthetic CNS-specific promoter of any one of claims 114, 115 or 120, operably linked to a sequence encoding an expression product.

122. A vector comprising the synthetic CNS-specific promoter of any one of claims 114, 115 or 120 or the expression cassette of claim 121.

123. The vector of claim 122, wherein The vector is a viral vector.

124. The vector of claim 123, wherein The viral vector is an AAV vector.

125. A virosome comprising the vector of claim 123 or 124.

126. A pharmaceutical composition comprising the synthetic CNS-specific promoter of any one of claims 114, 115 or 120, the expression cassette of claim 121, the vector of any one of claims 122-124, or the virosome of claim 125.

127. The synthetic CNS-specific promoter of any one of claims 114, 115 or 120, the expression cassette of claim 121, the vector of any one of claims 122-124, the virosome of claim 125 or the pharmaceutical composition of claim 126 for use in therapy.

128. A synthetic CNS-specific promoter, expression cassette, vector, virion or pharmaceutical composition as claimed in claim 127 for use in gene therapy, suitably wherein the gene therapy involves expression of a therapeutic expression product in the CNS.

129. The synthetic CNS-specific promoter, expression cassette, vector, virosome or pharmaceutical composition of claim 127 or 128 for use in gene therapy of CNS-related diseases.

130. A cell comprising the synthetic CNS-specific promoter of any one of claims 114, 115 or 120, the expression cassette of claim 121, the vector of any one of claims 122-124, or the virosome of claim 125.

131. The cell of claim 130, wherein The cell is a CNS cell, optionally a human CNS cell, preferably a neuron, more preferably a dopaminergic neuron.

132. The synthetic CNS-specific promoter of any one of claims 114, 115 or 120, the expression cassette of claim 121, the vector of any one of claims 122-124 or the virosome of claim 125 for use in the manufacture of a pharmaceutical composition for treating a medical condition or disease, optionally wherein the disease is a CNS-related disease.

133. A method for producing an expression product, the method comprising: introducing the synthetic CNS-specific expression cassette of claim 121 into a cell, optionally a CNS cell; And, the gene present in the synthetic CNS-specific expression cassette is expressed.

134. A method of expressing a therapeutic transgene in a cell, optionally a CNS cell, wherein: The method comprises introducing into the cell an expression cassette of claim 121, a vector of any one of claims 122-124, or a virosome of claim 125.

135. A method of treating a subject in need thereof, wherein: The method comprises administering to the subject in need thereof an expression cassette as claimed in claim 120, a vector as claimed in any one of claims 122-124, or a virosome as claimed in claim 125, or a pharmaceutical composition as claimed in claim 127 or 128, wherein the expression cassette, vector, virosome or pharmaceutical composition comprises a sequence encoding an operably linked therapeutic product.

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