Compositions for treating neurological disorders

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) 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, the contents of each of which are incorporated herein by reference in their entirety.

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

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

[0004] Abstract One aspect provided herein describes a method for treating a neurological disease or disorder in a subject in need of treatment, the method comprising administering a therapeutically effective amount of at least one of (a) a nucleic acid encoding at least one miRNA and (b) a nucleic acid encoding a CYP46A1 protein to a subject who has or is at risk of developing a neurological disease or disorder.For example, the nucleic acid encoding at least one miRNA is administered alone or in combination with the nucleic acid encoding a CYP46A1 protein.Alternatively, the nucleic acid encoding a CYP46A1 protein is administered alone or in combination with the nucleic acid encoding at least one miRNA.The nucleic acid can be a wild-type or codon-optimized variant.

[0005] Another embodiment described herein provides a composition or combination comprising 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. In one embodiment, described herein is a composition or combination comprising: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (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.

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

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

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

[0009] Another embodiment described herein provides a method for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to the subject. In other embodiments, the method does not comprise administering (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to the subject. In one embodiment, the method described herein is for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated 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 a CYP46A1 protein to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other embodiments, the method does not comprise administering to the subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

[0010] Another embodiment described herein provides a method for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to the subject. In other embodiments, the method does not comprise administering (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to the subject. In one embodiment, the method described herein is for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated 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 to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering to the subject a recombinant viral vector comprising a therapeutically effective amount of (b) an isolated nucleic acid encoding a CYP46A1 protein. In other embodiments, the method does not comprise administering to the subject a recombinant viral vector comprising a therapeutically effective amount of (b) an isolated nucleic acid encoding a 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 repeat spinocerebellar ataxia, Krabbe disease, Batten disease, Refsum disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, delusions, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder. 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 repeat 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 disease-associated alleles thereof.

[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 disease-associated alleles thereof.

[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 at least one miRNA comprises any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260 adjacent to the miRNA scaffold sequence. In some embodiments, the CNS disease is Huntington's disease, and at least one miRNA comprises any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260. In some embodiments, at least one of the miRNAs hybridizes to and inhibits expression of human huntingtin. In some embodiments, the subject comprises a huntingtin gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. In some embodiments, the subject is under 20 years of age.

[0015] In some embodiments, the recombinant viral vector is selected from the group consisting of an AAV vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric viral vector.

[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 comprised in separate recombinant viral vectors. In some embodiments, the isolated nucleic acids of (a) and (b) are comprised in the same recombinant viral vector.

[0017] In some embodiments, (a) and (b) are administered at substantially the same time. In some embodiments, (a) and (b) are administered at different times. In some embodiments, the different times are separated by at least 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 administered before administration of (b). In some embodiments, (b) is administered before administration of (a). In some embodiments, administration of (a), (b), or (a) and (b) is repeated at least once.

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

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

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

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

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

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

[0024] In some embodiments, the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, or AAV12, or a chimera thereof. In some embodiments, the viral vector comprises a capsid protein from an AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, or AAV12, or a chimera thereof. In some embodiments, the capsid protein is an AAV9 capsid protein. In some embodiments, the viral vector is 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 of the aspects, the viral vector comprises a modified viral capsid.

[0026] In some embodiments of any of the aspects, the viral vector comprises a modification to the viral capsid.

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

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

[0029] In some embodiments of any of the aspects, the chemical modification comprises a chemically modified tyrosine residue that has been modified to include a covalently linked monosaccharide or polysaccharide moiety.

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

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

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

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

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

[0035] In some embodiments of any of the aspects, the modified viral capsid is a chimeric or monomelic capsid that further comprises a modification.

[0036] In some embodiments of any of the aspects, the modified viral capsid is of AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, or a mutant modified from, chimeric, mosaic, or rational haploid form thereof.

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

[0038] In some embodiments of any of the aspects, the modified viral capsid may be used for repeated administration. [Brief explanation of the drawings]

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

[0040] [Figure 2] 2 shows the intracranial biodistribution in sagittal sections of CNS-1 (see, e.g., SEQ ID NO: 112), CNS-2 (see, e.g., SEQ ID NO: 113), CNS-3 (see, e.g., SEQ ID NO: 114), CNS-4 (see, e.g., SEQ ID NO: 115), CNS-5 (see, e.g., SEQ ID NO: 122), CNS-6 (see, e.g., SEQ ID NO: 123), CNS-7 (see, e.g., SEQ ID NO: 124), and CNS-8 (see, e.g., SEQ ID NO: 125) delivered by intracerebroventricular (ICV) and intravenous (IV) injection, as well as the transgene GFP under the control of the control promoter hSyn1 (see, e.g., SEQ ID NO: 152). The scale bar is 1 mm.

[0041] [Figure 3A]Figures 3A-3B show images of coronal brain sections. Figure 3A shows the intracranial biodistribution of the transgene GFP under the control of CNS-1 (e.g., see SEQ ID NO: 112), CNS-2 (e.g., see SEQ ID NO: 113), CNS-3 (e.g., see SEQ ID NO: 114), and CNS-4 (e.g., see SEQ ID NO: 115) delivered by ICV in a coronal section. The scale bar is 1 mm. Figure 3B shows the intracranial biodistribution of the transgene GFP under the control of CNS-5 (e.g., see SEQ ID NO: 122), CNS-6 (e.g., see SEQ ID NO: 123), CNS-7 (e.g., see SEQ ID NO: 124), and CNS-8 (e.g., see SEQ ID NO: 125) delivered by ICV, as well as the control promoter hSyn1 (e.g., see SEQ ID NO: 152) in a coronal section. The scale bar is 1 mm. [Figure 3B] Figures 3A-3B show images of coronal brain sections. Figure 3A shows the intracranial biodistribution of the transgene GFP under the control of CNS-1 (e.g., see SEQ ID NO: 112), CNS-2 (e.g., see SEQ ID NO: 113), CNS-3 (e.g., see SEQ ID NO: 114), and CNS-4 (e.g., see SEQ ID NO: 115) delivered by ICV in a coronal section. The scale bar is 1 mm. Figure 3B shows the intracranial biodistribution of the transgene GFP under the control of CNS-5 (e.g., see SEQ ID NO: 122), CNS-6 (e.g., see SEQ ID NO: 123), CNS-7 (e.g., see SEQ ID NO: 124), and CNS-8 (e.g., see SEQ ID NO: 125) delivered by ICV, as well as the control promoter hSyn1 (e.g., see SEQ ID NO: 152) in a coronal section. The scale bar is 1 mm.

[0042] [Figure 4-1]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs: 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO: 152). Data were obtained by quantitative measurement of 10 non-overlapping RGB images of GFP staining intensity by threshold analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification, keeping settings constant across distinct brain regions. Foreground immunostaining was defined by the average of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactive area per field for each region of interest (n = 3). With ICV delivery, expression was highest in the cortical and hippocampal brain regions. CNS1-8 (see, e.g., SEQ ID NOS: 112-115, 122-125) show higher expression in the hippocampus than the hSyn1 control. CNS-1 (see, e.g., SEQ ID NOS: 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1. [Figure 4-2]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs: 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO: 152). Data were obtained by quantitative measurement of 10 non-overlapping RGB images of GFP staining intensity by threshold analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification, keeping settings constant across distinct brain regions. Foreground immunostaining was defined by the average of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactive area per field for each region of interest (n = 3). With ICV delivery, expression was highest in the cortical and hippocampal brain regions. CNS1-8 (see, e.g., SEQ ID NOS: 112-115, 122-125) show higher expression in the hippocampus than the hSyn1 control. CNS-1 (see, e.g., SEQ ID NOS: 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1. [Figure 4-3]Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS1-8 (e.g., see SEQ ID NOs: 112-115, 122-125) or synapsin-1 (e.g., see SEQ ID NO: 152). Data were obtained by quantitative measurement of 10 non-overlapping RGB images of GFP staining intensity by threshold analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were acquired at 40x magnification, keeping settings constant across distinct brain regions. Foreground immunostaining was defined by the average of the highest and lowest signals. Data are expressed as the mean percentage of immunoreactive area per field for each region of interest (n = 3). With ICV delivery, expression was highest in the cortical and hippocampal brain regions. CNS1-8 (see, e.g., SEQ ID NOS: 112-115, 122-125) show higher expression in the hippocampus than the hSyn1 control. CNS-1 (see, e.g., SEQ ID NOS: 112) shows higher expression in the hippocampus, midbrain, and cerebellum compared to ICV-delivered hSyn1.

[0043] [Figure 5A-B]Figures 5A-5B show the tissue expression patterns for the CRE / proximal promoter-engineered faf1 and pitx3 genes from CNS-5, CNS-5_v2, CNS-2, CNS-3, and CNS-4. Figure 5A shows the expression pattern of the faf1 gene in mouse PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. faf1 is expressed in many PNS neurons. Figure 5B shows the expression pattern of the pitx3 gene in PNS neurons from single-cell transcriptomics data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. pixt3 is expressed in sympathetic PNS neurons. Faf1 is expressed in many PNS neurons; therefore, synthetic promoters containing a CRE or proximal promoter engineered from the faf1 gene, such as CNS-5 and CNS-5_v2, are expected to have strong expression in the PNS. pitx3 is expressed in sympathetic PNS neurons; therefore, synthetic promoters containing a CRE engineered from the pitx3 gene, such as CNS-2, CNS-3, or CNS-4, are expected to have expression in PNS sympathetic neurons. Similar analyses of lmx1b and pitx2 revealed no expression in the PNS above the cutoff score for the analysis (trinization score below 0.95; data not shown). Therefore, CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not expected to be active in PNS neurons.

[0044] [Figure 6A] Figure 6A shows the expression pattern of the HTT gene in a sagittal section from an adult mouse brain (taken from the Allen Mouse brain atlas; mouse.brain-map.org). HTT (huntingtin) is highly expressed throughout the brain.

[0045] [Figure 6B] Figure 6B shows the expression pattern of the CYP46A1 gene in coronal sections from an adult mouse brain (obtained from the Allen Mouse brain atlas; available on the World Wide Web at mouse.brain-map.org). CYP46A1 is widely expressed in the brain.

[0046] [Figure 7A] Figure 7A shows the median GFP expression of the synthetic NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, and SP0036, and the control promoter synapsin-1, compared to the control promoter CAG, in neuroblastoma-derived SH-SY5Y cells. NTC indicates non-transfected cells. Data were collected from three biological replicates, each of which represents the average of two technical replicates. Error bars represent standard error.

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

[0048] [Figure 8]Figure 8 shows the levels of HTT transcripts after nucleofection of patient-derived fibroblasts with a plasmid containing miRNA targeting exon 1 of the HTT gene. N=1.

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

[0050] Detailed Description Aspects of the present invention relate to the administration of both an interfering RNA (e.g., an miRNA, such as an artificial miRNA), which, when delivered to a subject, is effective in reducing the expression of a pathogenic gene in the subject, and a nucleic acid encoding a CYP46A1 protein. Thus, the methods and compositions described by the present disclosure are useful in some embodiments for the treatment of neurological diseases or disorders.

[0051] Treatment method The present disclosure provides a method for delivering a nucleic acid and / or a transgene (e.g., an inhibitory RNA such as miRNA, or a nucleic acid encoding CYP46A1) to a subject. The method typically includes administering to a subject an effective amount of at least one of a nucleic acid / inhibitory nucleic acid encoding at least one interfering RNA capable of reducing the expression of a target gene, for example, a pathogenic gene associated with a neurological disease or disorder (e.g., huntingtin (htt) protein), and a nucleic acid encoding CYP46A1. For example, a nucleic acid encoding at least one miRNA is administered alone or in combination with a nucleic acid encoding CYP46A1 protein. Alternatively, a nucleic acid encoding CYP46A1 protein is administered alone or in combination with a nucleic acid encoding at least one miRNA. In some embodiments, one or both of the nucleic acids are provided in a viral vector and / or a viral particle, for example, rAAV.

[0052] Thus, one embodiment described herein provides a method for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to the subject. In other embodiments, the method does not comprise administering (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to the subject. In one embodiment, the method described herein is for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated 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 a CYP46A1 protein to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other embodiments, the method does not comprise administering to the subject a therapeutically effective amount of (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

[0053] Another embodiment described herein provides a method for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering a therapeutically effective amount of (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to the subject. In other embodiments, the method does not comprise administering (b) an isolated nucleic acid encoding a transgene encoding a CYP46A1 protein to the subject. In one embodiment, the method described herein is for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of (a) an isolated 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 to a subject having or at risk of developing a neurological disease or disorder. In some embodiments, the method further comprises administering to the subject a recombinant viral vector comprising a therapeutically effective amount of (b) an isolated nucleic acid encoding a CYP46A1 protein. In other embodiments, the method does not comprise administering to the subject a recombinant viral vector comprising a therapeutically effective amount of (b) an isolated nucleic acid encoding a CYP46A1 protein.

[0054] As used herein, "neurological disease or disorder" can refer to any disease, disorder, or condition that affects or is related to the nervous system, i.e., those that affect the central nervous system (brain and spinal cord) and peripheral nervous system (PNS; e.g., peripheral and cranial nerves), as well as the autonomic nervous system (those portions located in both the central and peripheral nervous systems). More than 600 neurological diseases have been identified in humans. In non-limiting examples, the neurological disease or disorder is septum pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie pupil, Adie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold's syndrome, -Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or cerebellar-spinal degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth syndrome, Batten disease, Becker's myotonia, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Berner-Ross syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury during birth, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysms Aneurysms, brain injury, Brown-Séquard syndrome, spinal-bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, burning pain, cavernoma, cavernous hemangioma, cavernous malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, head injury, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysms, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, hypoxic encephalopathy, cerebral palsy, cerebro-oculofacial-skeletal syndrome (COFS),Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disease, chorea, acanthocytic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic dysregulation, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, agenesis of the corpus callosum, coma, complex regional pain syndrome, congenital facial nerve palsy, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformations, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, giant cytoplasmic sequestration Isophysiology, cytomegalovirus infection, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, dementia, multi-infarct dementia, semantic dementia, subcortical dementia, dementia with Lewy bodies, dentatocerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental coordination disorder, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic dysfunction, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia , progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalopathy (familial infantile), cerebral trigeminal region angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Dusenne and Dejerine-Klumpke palsy, essential tremor, extrapontine myelinolysis, Fabry disease, Fahr's syndrome, syncope, familial Alzheimer's disease, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile seizures, fibromuscular dysplasia , Fisher syndrome, hypotonic infantile syndrome, foot drop, Friedreich's ataxia, frontotemporal dementia, Gaucher disease, systemic gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemihyperglycemic headache, hemifacial spasm, alternating hemiplegia, hereditary neuropathies, hereditary spastic paraplegia, hereditary polyneuropathy,Herpes zoster, varicella zoster oticus, Hirayama syndrome, Holmes-Adie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydranencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelia, corticosteroid excess, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease, infantile spasms, inflammatory myopathy, foramen occipitalis, intestinal lipodystrophy, intracranial cyst, increased intracranial pressure, Isaacs syndrome, Joubert syndrome group, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Klein-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klüber-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral cord syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levine-Critchley syndrome, Lewy bodies Dementia, Lipid storage diseases, Lipoproteinoid proteinosis, Acephaly, Locked-in syndrome, Lou Gehrig's disease, Lupus - neurological sequelae, Lyme disease - neurological complications, Machado-Joseph disease, Megalencephaly, Megalencephaly, Melkerson-Rosenthal syndrome, Meningitis, Meningitis and encephalitis, Menkes disease, Dysensory thigh pain, Metachromatic leukodystrophy, Microcephaly, Migraine, Miller-Fisher syndrome, Minor stroke, Mitochondrial myopathy, Moebius syndrome, Unilateral muscular atrophy, Motor neuron disease, Moyamoya disease, Mucolipidosis, Mucopolysaccharidosis, Multi-infarct dementia, Multi-infarct dementia Focal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, congenital myasthenia, myasthenia gravis, myeloclastic diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, congenital myopathy, thyrotoxic myopathy, myotonia, myotonia congenita, narcolepsy, neuroacanthocytosis, neurodegeneration with cerebral iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological signs of Pompe disease,Neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorders, hereditary neuropathies, neurosarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus ataxia, orthostatic hypotension, overuse injuries, chronic pain, pantothecin Phosphate kinase-related neurodegeneration, paraneoplastic syndromes, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg disease, Pelizaeus-Merzbach disease, Penner-Shocker II syndrome, perineural cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, nerve compression, piriformis syndrome, pituitary tumor, polymyositis, polyglutamine lipid Spinocerebellar ataxia, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, post-measles encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural orthostatic tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive facial hemiatrophy, progressive gait ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, progressive muscular atrophy, pseudo-Torch syndrome syndrome, pseudotoxoplasmosis, pseudotumor cerebri, psychogenic movement disorder, Ramsay-Hunt syndrome I, Ramsay-Hunt syndrome II, Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum's disease, infantile Refsum's disease, repetitive movement disorder, repetitive stress disorder, restless legs syndrome, retroviral-associated myelopathy, Rett's syndrome, Reye's syndrome, rheumatoid encephalitis, Riley-Day syndrome, sacral radicular cyst, chorea (Saint Vitus Dance), salivary gland disease, Sandhoff disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy in infancy (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke,Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, brief, unilateral, neuralgiform (SUNCT) headache, swallowing disorder, spinal and cerebral ataxia, Sydenham chorea, syncope, syphilitic spinal sclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's myotonia, thoracic outlet syndrome, thyrotoxic myopathy, painful tics, Todd's palsy, Tourette's syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer's syndrome, tuberous sclerosis, angioedema, blood vessels of the central and peripheral nervous system The condition may be vascular inflammation, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsaf syndrome, West syndrome, whiplash injury, Whipple disease, Williams syndrome, Wilson disease, Wolman disease, X-linked spinal-bulbar muscular atrophy, neuropathic pain, trauma due to spinal cord or head injury, ophthalmologic diseases and disorders, cerebral infarction, depression, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, delusions, 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, tauopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff disease, Charcot-Marie-Tooth disease, Angelman syndrome, Canavan disease, late-onset infantile neuronal ceroid lipofuscinosis, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodystrophy, hereditary lysosomal storage diseases, e.g., Niemann-Pick disease type C1, and / or neuronal ceroid lipofuscinosis, e.g., Batten disease, progressive supranuclear palsy, corticobasal syndrome, and brain cancer (including astrocytoma and glioblastoma).

[0056] As used herein, "Huntington's disease" or "HD" refers to a neurodegenerative disease characterized by progressively worsening motor, cognitive, and behavioral changes caused by a trinucleotide repeat expansion (e.g., CAG, which translates to a polyglutamine tract or polyQ tract) in the HTT gene that results in the production of a pathogenic mutant huntingtin protein (HTT, or mHTT).

[0057] As used herein, "HTT" or "huntingtin" refers to the gene encoding the huntingtin protein. Normal huntingtin proteins function in nerve cells, and normal HTT genes typically have about 7 to about 35 CAG repeats at their 5' ends. The HTT gene is often mutated in patients with or at risk of developing Huntington's disease. In some embodiments, mutant huntingtin proteins accelerate the rate of neuronal death in certain regions of the brain. Generally, the severity of HD correlates with the size of the trinucleotide repeat expansion in a subject. For example, subjects with a CAG repeat region containing between 36 and 39 repeats (SEQ ID NO: 157) are characterized as having "low-penetrance" HD, while subjects with more than 40 repeats are characterized as having "full-penetrance" HD. Thus, in some embodiments, a subject having or at risk of having HD has an HTT gene that includes between about 36 and about 39 CAG repeats (e.g., 36, 37, 38, or 39 repeats). In some embodiments, a subject having or at risk of having HD has an HTT gene that includes 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 having an HTT gene that includes more than 100 CAG repeats develop HD earlier than subjects having fewer than 100 CAG repeats. In some embodiments, subjects having an HTT gene that includes more than 100 CAG repeats may develop HD symptoms before about age 20 and are referred to as having early-onset HD (also referred to as akinetic-rigid HD or Westphal variant HD). The number of CAG repeats in the alleles of the HTT gene of a subject can be determined by any suitable modality known in the art.For example, nucleic acid (e.g., DNA) can be isolated from a subject's biological sample (e.g., blood), and the number of CAG repeats of HTT alleles can be determined by hybridization-based methods, such as PCR or nucleic acid sequencing (e.g., Illumina sequencing, Sanger sequencing, SMRT sequencing, etc.). The sequence of HTT gene is known in several species, for example, the mRNA sequence (NCBI Reference Sequence: NM_002111.8, SEQ ID NO: 4) and protein sequence (NCBI Reference Sequence: NP_0021012.4, SEQ ID NO: 5) of human HTT (NCBI Gene ID: 3064).Therefore, in some embodiments for treating Huntington's disease, one or more inhibitory nucleic acids (e.g., miRNA) can hybridize with HTT and / or reduce its 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 difficulties with language, motivation, and self-care. Several genes, including amyloid precursor protein (APP; NCBI gene ID: 351), presenilin 1 (PSEN1; NCBI gene ID 5663), presenilin 2 (PSEN2; NCBI gene ID 5664), ATP-binding cassette subfamily A member 7 (ABCA7; NCBI gene ID 10347), and sortilin-related receptor 1 (SORL1; NCBI gene ID 6653), may contribute to or increase the risk of AD. The sequences of such AD-related genes are known in several species; for example, human mRNA and protein sequences are available in the NCBI database using the provided gene ID numbers. These AD-associated genes and others, as well as their AD-associated 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. Thus, in some embodiments relating to the treatment of Alzheimer's disease, one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with 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 disease caused by a single genetic mutation transmitted 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. Thus, in one embodiment, the subject has a mutation in at least one of PS1, PS2, and APP.

[0060] As used herein, "Parkinson's disease" or "PD" refers to a neurodegenerative disease characterized by progressively worsening shaking and stiffness, and increasing problems with balance, walking, and coordination. Synuclein alpha (SNCA; NCBI Gene ID: 6622), leucine-rich repeat kinase 2 (LRRK2 / PARK8; NCBI Gene ID 120892), glucosylceramidase beta (GBA1; NCBI Gene ID 2629), parkin RBR E3 ubiquitin (PRKN; NCBI Gene ID 5071), PTEN-induced kinase 1 (PINK1; NCBI Gene ID 65018), parkinsonism-associated deglycase (DJ1 / PARK7; NCBI Gene ID 11315), VPS35 retromer complex component (VPS35; NCBI Gene ID 55737), eukaryotic translation initiation factor 4 gamma 1 (EIF4G1; NCBI Gene ID 1981), DnaJ heat shock protein family member C13 (DNAJC13; NCBI Gene ID Several genes may contribute to or increase the risk of PD, including PD-associated PD-associated 2 (PD-1; NCBI Gene ID 23317), coiled-coil helix-coiled-coil helix domain containing 2 (CHCHD2; NCBI Gene ID 51142), and / or ubiquitin C-terminal hydrolase L1 (UCHL1; NCBI Gene ID 7345). The sequences of such PD-associated genes are known in several species; for example, human mRNA and protein sequences are available in the NCBI database using the provided gene ID numbers.These PD-associated genes and others, and their PD-associated alleles (e.g., mutations, SNPs, etc.), are known in the art and are described in, e.g., 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 which is incorporated by reference in its entirety. Thus, in some embodiments for treating Parkinson's disease, one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize to and / or reduce 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 can further include evaluating at least one primary endpoint, at least one secondary endpoint, or a combination thereof after administering a nucleic acid encoding CYP46A1 (wild-type or codon-optimized variant).The time after administration can 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 endpoints include the occurrence of dose-limiting toxicity (DLT), investigational treatment-emergent adverse events (TEAE), serious adverse events (SAE), and combinations thereof. Non-limiting examples of secondary endpoints include anatomical and volumetric measures of brain regions affected by Huntington's disease assessed by MRI, composite Unified Huntington's Disease Rating Scale (cUHDRS) score, mutant huntingtin protein (mHTT) concentrations in blood and / or cerebrospinal fluid (CSF), neurofilament light chain (NfL) levels in blood and / or CSF, 24OH cholesterol concentrations in blood and / or CSF, magnetic resonance spectroscopy (MRS) metabolic profile, positron emission tomography (PET) fluorodeoxyglucose (FDG) striatal profile, and combinations thereof.

[0063] In some embodiments, the anatomical and volumetric measures of brain regions affected by Huntington's disease are determined. The magnitude and variability of the change from baseline, i.e., the measurement of the brain region at the time of rAAV administration of a nucleic acid encoding CYP46A1, as determined by the gradient of progression of atrophy in striatal structures, ventricular dilation, and cortical atrophy, can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5% by volume, greater than or equal to about 1% by volume, greater than or equal to about 2% by volume, greater than or equal to about 3% by volume, greater than or equal to about 4% by volume, greater than or equal to about 5% by volume, greater than or equal to about 6% by volume, greater than or equal to about 7% by volume, or greater than or equal to about 8% by volume. The improvement (e.g., decrease) may be equal to or greater than about 9 vol%, greater than or equal to about 10 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, 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 embodiments, the cUHDRS score is determined and compared to a baseline cUHDRS score, i.e., the cUHDRS score at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline cUHDRS score, the cUHDRS score can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%. The improvement may be greater than or equal to about 10%, greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 25%, or greater than 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 greater.

[0065] In some embodiments, the mHTT concentration in the blood and / or CSF is determined and compared to a corresponding baseline mHTT concentration, i.e., the mHTT concentration in the blood and / or CSF at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline mHTT concentration, the mHTT concentration can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%. The improvement (i.e., decrease) may be equal to, greater than or equal to about 10%, greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 25%, or greater than, for example, 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 greater.

[0066] In some embodiments, the level of NfL in the blood and / or CSF is determined and compared to a corresponding baseline NfL level, i.e., the NfL level in the blood and / or CSF at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline NfL level concentration, the level of NfL can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%. may be improved (i.e., decreased) 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 embodiments, the 24OH cholesterol concentration in the blood and / or CSF is determined and compared to a corresponding baseline 24OH cholesterol concentration, i.e., the 24OH cholesterol concentration in the blood and / or CSF at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline 24OH cholesterol concentration, the 24OH cholesterol concentration can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%. The improvement (i.e., increase) may be greater than or equal to about 10%, greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 25%, or greater than 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 greater.

[0068] In some embodiments, the MRS metabolic profile is determined and compared to a corresponding baseline MRS metabolic profile, i.e., the MRS metabolic profile at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline MRS metabolic profile, the MRS metabolic profile can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, greater than or equal to about 9%, or greater than or equal to about 10%. The improvement may be greater than or equal to about 10%, greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 25%, or greater than 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 greater.

[0069] In some embodiments, the PET FDG profile is determined and compared to a corresponding baseline PET FDG profile, i.e., the PET FDG profile at the time of rAAV administration of a nucleic acid encoding CYP46A1. Compared to the baseline PET FDG profile, the PET FDG profile can be maintained (i.e., stabilized) or can be greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 2%, greater than or equal to about 3%, greater than or equal to about 4%, greater than or equal to about 5%, greater than or equal to about 6%, greater than or equal to about 7%, greater than or equal to about 8%, or greater than or equal to about 9%. The improvement may be equal to, greater than or equal to about 10%, greater than or equal to about 15%, greater than or equal to about 20%, greater than or equal to about 25%, or greater than, e.g., 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 greater.

[0070] In one embodiment, 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 embodiment, the neurological disease or disorder is Parkinson's disease.

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

[0074] An "effective amount" of a substance is an amount sufficient to produce a desired effect. In some embodiments, an effective amount of an isolated nucleic acid is an amount sufficient to transfect (or infect, in the context of rAAV-mediated delivery) a sufficient number of target cells in a target tissue of a subject. In some embodiments, the target tissue is a central nervous system (CNS) tissue (e.g., brain tissue, spinal cord tissue, cerebrospinal fluid (CSF), etc.). In some embodiments, an effective amount of an isolated nucleic acid (e.g., one that can be delivered via rAAV) can be an amount sufficient to have a therapeutic benefit in a subject, for example, to reduce the expression of a pathogenic gene or protein (e.g., HTT), extend the lifespan of a subject, ameliorate one or more symptoms of a disease (e.g., symptoms of Huntington's disease) in a subject, etc. The effective amount depends on various factors, such as the species, age, weight, health, and tissue being targeted of the subject, and therefore may vary between subjects and tissues, as described elsewhere in this disclosure.

[0075] inhibitory RNA In some aspects, the present disclosure provides inhibitory nucleic acids, e.g., miRNAs, that specifically bind to (e.g., hybridize 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, the present disclosure provides inhibitory nucleic acids, e.g., miRNAs, that specifically bind to (e.g., hybridize 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 in exon 1 of human huntingtin mRNA (e.g., SEQ ID NO: 3). As used herein, "consecutive bases" refers to two or more nucleotide bases that are covalently linked (e.g., by one or more phosphodiester bonds) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, at least one miRNA is about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% identical to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleotide bases of a target, e.g., SEQ ID NO: 3 or 4. In some embodiments, the inhibitory RNA is an miRNA that comprises or is encoded by a sequence set forth in any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260.

[0076] In one aspect, described herein is an inhibitory RNA that can be used for the treatment of a neurological disease or disorder. In some embodiments of any of the aspects, the nucleic acid sequence of the inhibitory RNA comprises one of SEQ ID NOS: 6-17, 40-44, 50-66, 158-185, or 217-260, 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 NOS: 6-17, 40-44, 50-66, 158-185, or 217-260 that maintains the same function (e.g., HTT inhibition) as SEQ ID NOS: 3 or 4.

[0077] In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260. In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260 adjacent to a miRNA scaffold sequence.

[0078] In some embodiments, the vectors described herein comprise at least one miRNA, wherein each miRNA comprises a seed sequence complementary to one of SEQ ID NOs: 3, 4, 18-39, or 46-49. In some embodiments, the vectors described herein comprise at least one miRNA, wherein each miRNA comprises a seed sequence complementary to one of SEQ ID NOs: 3, 4, 18-39, or 46-49 adjacent to the miRNA scaffold sequence. In some embodiments, the vectors described herein comprise at least one miRNA, wherein each miRNA comprises a seed sequence substantially complementary to one of SEQ ID NOs: 3, 4, 18-39, or 46-49. In some embodiments, the vectors described herein comprise at least one miRNA, wherein each miRNA comprises a seed sequence substantially complementary to one of SEQ ID NOs: 3, 4, 18-39, or 46-49 adjacent to the miRNA scaffold sequence.

[0079] [Table 1]

[0080] [Table 2]

[0081] [Table 3-1] [Table 3-2]

[0082] In some embodiments, the miRNA comprises SEQ ID NOs: 6 and 11, SEQ ID NOs: 7 and 12, SEQ ID NOs: 8 and 11, SEQ ID NOs: 8 and 16, SEQ ID NOs: 8 and 17, SEQ ID NOs: 9 and 14, or SEQ ID NOs: 10 and 15.

[0083] In some embodiments, the vector comprises a pre-miRNA having the sequence of SEQ ID NO: 40 or 41. These pre-miRNAs comprise a scaffold comprising SEQ ID NO: 8. Alternative first RNA sequences disclosed herein can be substituted for SEQ ID NO: 8 in either SEQ ID NO: 40 and 41.

[0084] In some embodiments, the vector comprises a pri-miRNA having the sequence of SEQ ID NO: 42 or 43. The pri-miRNA of SEQ ID NO: 42 comprises a scaffold comprising SEQ ID NOs: 8 and 16. Alternative RNA sequences disclosed herein can be substituted for SEQ ID NOs: 8 and 16 in SEQ ID NO: 42. The pri-miRNA of SEQ ID NOs: 43 and 44 comprises a scaffold comprising SEQ ID NOs: 8 and 17. Alternative RNA sequences disclosed herein can be substituted for SEQ ID NOs: 8 and 17 in either SEQ ID NOs: 43 and 44.

[0085] [Table 4-1] [Table 4-2]

[0086] In some embodiments, the inhibitory nucleic acid can comprise one or more of SEQ ID NOS: 1-102 and / or 103-249 of International Patent Publication No. WO2017 / 201258. In some embodiments, the inhibitory nucleic acid can comprise one or more of dual combinations selected from SEQ ID NOS: 1-249 of International Patent Publication No. WO2017 / 201258, as provided in Tables 3-5 of International Patent Publication No. WO2017 / 201258. In some embodiments, the vector can comprise one or more of the pri-miRNAs provided in Table 9 or the pri-mRNAs provided in Table 10 of International Patent Publication No. WO2017 / 201258. The contents of International Patent Publication No. WO2017 / 201258 are incorporated herein by reference in their entirety.

[0087] In some embodiments, the inhibitory nucleic acid can comprise one or more of SEQ ID NOS: 914-1013 and / or 1014-1160 of International Patent Publication No. WO2018 / 204803. In some embodiments, the inhibitory nucleic acid can comprise one or more of dual combinations selected from SEQ ID NOS: 914-1160 of International Patent Publication No. WO2018 / 204803 provided in Tables 4-6 of International Patent Publication No. WO2018 / 204803. The contents of International Patent Publication No. WO2018 / 204803 are incorporated herein by reference in their entirety.

[0088] In some embodiments, the inhibitory nucleic acid can comprise one or more of SEQ ID NOS: 916-1015 and / or 1016-1162 of International Patent Publication No. WO 2018 / 204797. In some embodiments, the inhibitory nucleic acid can comprise one or more of SEQ ID NOS: 916-1015, 1016-1162, 1164-1332 and / or 1333-1501 of International Patent Publication No. WO 2018 / 204797. In some embodiments, the inhibitory nucleic acid can comprise one or more of dual combinations selected from SEQ ID NOS: 916-1162 of International Patent Publication No. WO 2018 / 204797 provided in Tables 4-6 of International Patent Publication No. WO 2018 / 204797. In some embodiments, the inhibitory nucleic acid can comprise one or more of dual combinations selected from SEQ ID NOs: 1164-1501 of International Patent Publication No. WO2018 / 204797, as provided in Table 9 of International Patent Publication No. WO2018 / 204797. The contents of International Patent Publication No. WO2018 / 204797 are incorporated herein by reference in their entirety.

[0089] In some embodiments, the inhibitory nucleic acid can target, for example, include, a sequence complementary to or substantially complementary to a heterozygous SNP in a gene encoding a gain-of-function mutant huntingtin protein. 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 site selected from the group consisting of RS362331, RS4690077, RS363125, RS363075, RS362268, RS362267, RS362307, RS362306, RS362305, RS362304, RS362303, and RS7685686. Such SNPs are described in more detail, for example, in U.S. Patent No. 9,343,943, which is incorporated herein by reference in its entirety. In some embodiments, the target sequence is one of SEQ ID NOs: 45-49. In some embodiments, the inhibitory nucleic acid sequence comprises one or more of SEQ ID NOs: 50-61. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 50 and 51. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 52 and 53. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 54 and 55. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 56 and 57. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 58 and 59. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 60 and 61.

[0090] [Table 5]

[0091] [Table 6]

[0092] In one embodiment, the inhibitory nucleic acid, e.g., miRNA, can specifically hybridize to exon 1 of the HTT gene. Exemplary miRNAs that hybridize to exon 1 of the HTT gene are listed in Table 17, i.e., SEQ ID NOs: 158-160 and 164-166.

[0093] In one embodiment, the inhibitory nucleic acid, e.g., miRNA, can specifically hybridize 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, i.e., SEQ ID NOs: 161-163 and 167-169 (i.e., indicated by an asterisk).

[0094] In one embodiment, the nucleic acid sequence of the inhibitory RNA comprises at least one of SEQ ID NOs: 158-169, 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 at least one of SEQ ID NOs: 158-169 that maintains the same function of inhibiting HTT.

[0095] In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 158 and 164. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 159 and 165. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 160 and 166. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 161 and 167. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 162 and 168. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 163 and 169. [Table 17]

[0096] In one embodiment, the inhibitory nucleic acid, e.g., miRNA, can specifically hybridize to intron 1 of the HTT gene. Exemplary miRNAs that hybridize to intron 1 of the HTT gene are listed in Table 18, i.e., SEQ ID NOs: 170-174 and 178-182.

[0097] In one embodiment, the inhibitory nucleic acid, e.g., miRNA, can specifically hybridize 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 set forth in Table 18, i.e., SEQ ID NOs: 175-177 and 183-185 (i.e., indicated by an asterisk).

[0098] In one embodiment, the nucleic acid sequence of the inhibitory RNA comprises one of SEQ ID NOs: 170-185, 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 NOs: 170-185 that maintains the same function of inhibiting HTT.

[0099] In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 170 and 178. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 171 and 179. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 172 and 180. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 173 and 181. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 174 and 182. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 175 and 183. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 176 and 184. In some embodiments, the inhibitory nucleic acid sequence comprises, e.g., duplex, at least SEQ ID NOs: 177 and 185.

[0100] In one embodiment, an isolated nucleic acid of the present disclosure comprises a sequence encoding a miRNA comprising the sequence set forth in any one of SEQ ID NOs: 158-185.

[0101] In some embodiments of any of the aspects, the nucleic acid sequence of the inhibitory RNA comprises one of SEQ ID NOs: 158-185, 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 NOs: 158-185 that maintains the same function (e.g., HTT inhibition) as SEQ ID NOs: 158-185.

[0102] In one embodiment, a preferred miRNA molecule, or equivalent or mimic or isomiR thereof, comprises at least 6 of the 7 nucleotides present in the seed sequence identified in or as SEQ ID NOs: 158-185, and more preferably has a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides.

[0103] Described herein is a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 158-185.

[0104] Described herein is a method for treating a CNS disease, e.g., Huntington's disease, the method comprising administering to a subject a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, the transgene comprising a nucleic acid sequence encoding one or more miRNAs, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 158-185.

[0105] Described herein is a method for treating a CNS disease, e.g., Huntington's disease, comprising administering to a subject a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 158-185; and a nucleic acid sequence encoding a CYP461 protein or a CYP46A1 protein.

[0106] Described herein is a method for treating a CNS disease, e.g., Huntington's disease, the method comprising administering to a subject a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, the transgene comprising a nucleic acid sequence encoding one or more miRNAs, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 158-185; and an immune modulator.

[0107] Described herein is a method for treating a CNS disease, e.g., Huntington's disease, the method comprising administering to a subject a recombinant AAV comprising a nucleic acid encoding a capsid protein and a transgene, the transgene comprising a nucleic acid sequence encoding one or more miRNAs, each miRNA comprising a sequence set forth in any one of SEQ ID NOs: 158-185; a nucleic acid sequence encoding a CYP461 protein or a CYP46A1 protein; and an immune modulator.

[0108] Described herein is a recombinant AAV having a genome comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 194. In various embodiments, any of the elements set forth in the sequence of SEQ ID NO: 194 can be replaced by any of the elements described herein.

[0109] For example, in one embodiment, the enhancer element set forth in sequence SEQ ID NO: 194 can be replaced with any of the enhancer elements described herein. In one embodiment, the promoter set forth in sequence SEQ ID NO: 194 can be replaced with any of the promoters described herein. In one embodiment, the CYP46A1 transgene set forth in sequence SEQ ID NO: 194 can be replaced with any of the CYP46A1 transgenes described herein, e.g., a different codon-optimized CYP46A1 transgene. In one embodiment, the poly(A) signal sequence set forth in sequence SEQ ID NO: 194 can be replaced with any of the poly(A) signal sequences described herein. In one embodiment, the intron element set forth in sequence SEQ ID NO: 194 can be replaced with any of the intron elements described herein. In one embodiment, the protelomerase recognition sequence set forth in sequence SEQ ID NO: 194 can be replaced with any of the protelomerase recognition sequences described herein. In one embodiment, any of the linker or stuffer sequences set forth in sequence SEQ ID NO: 194 can be replaced with any of the linker or stuffer sequences described herein. In one embodiment, the ITR set forth in sequence SEQ ID NO: 194 can be replaced by any of the ITRs described herein.

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

[0111] Further described herein is a composition comprising a recombinant AAV having a genome comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:194.

[0112] Described herein are methods for treating a CNS disease or disorder, the method comprising administering to a subject a recombinant AAV having a genome comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 194. In one embodiment, the subject is further administered an immune modulator.

[0113] Described herein is a method for treating Huntington's disease, the method comprising administering to a subject a recombinant AAV having a genome comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 194.

[0114] Another aspect herein is a method for treating a CNS disease or disorder, e.g., Huntington's disease, the method comprising administering to a subject a recombinant AAV having a genome comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 194 amended as described herein above, e.g., the CYP46A1 transgene sequence set forth in SEQ ID NO: 194 replaced by another CYP46A1 transgene sequence described herein. In one embodiment, the subject is further administered an immune modulator.

[0115] In one embodiment, the subject is further administered an immune modulator.

[0116] In some embodiments, a vector comprising at least one inhibitory nucleic acid sequence, e.g., an miRNA, described herein, comprises at least one copy of at least one inhibitory nucleic acid sequence. For example, a vector can comprise at least 1, at least 2, at least 3, at least 3, at least 4, at least 5, or more copies of a given miRNA. [Table 18]

[0117] In some embodiments, inhibitory nucleic acid, for example, miRNA, can specifically hybridize or target polymorphism, mutation or SNP in one of the genes disclosed herein.Methods for selecting inhibitory nucleic acid sequences that target polymorphisms, for example, SNPs, in HTT gene are known in the art.For example, such methods are disclosed in U.S. Patent No. 8,679,750 and U.S. Patent No. 7,947,658, each of which is incorporated herein by reference in its entirety.In some embodiments, inhibitory nucleic acid can comprise one or more sequences, for example, SEQ ID NO: 1-342 in U.S. Patent No. 8,679,750 or U.S. Patent No. 7,947,658.

[0118] In some embodiments, the inhibitory nucleic acid can include one or more of SEQ ID NOs: 62-66.

[0119] [Table 7]

[0120] Further suitable sequences are known in the art, e.g., U.S. Pat. No. 7,951,934, Miniarikova et al. Molecular Therapy - Nucleic Acids 2015 5:e297; and Kordasiweicz et al. Neuron 2012 74:1031-1044, each of which is incorporated herein by reference in its entirety.

[0121] In some embodiments of any of the aspects, the inhibitory RNA (e.g., miRNA) binds to and / or targets the 5' untranslated region (UTR) of the target. In some embodiments of any of the aspects, the inhibitory RNA (e.g., miRNA) binds to and / or targets one or more exons of the target. In some embodiments of any of the aspects, the inhibitory RNA (e.g., miRNA) binds to and / or targets the 5' UTR, exon 1, CAG repeat, CAG5'-jumper, or CAG3'jumper of HTT.

[0122] In some embodiments, the inhibitory RNA and / or vector does not comprise any of the sequences set forth in SEQ ID NOs: 67-73. In some embodiments, the inhibitory RNA and / or vector does not comprise any sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any of SEQ ID NOs: 67-73. .

[0123] In some embodiments, the inhibitory RNA and / or vector does not comprise any of the sequences set forth in SEQ ID NOs: 67-73 or 135-151. In some embodiments, the inhibitory RNA and / or vector does not comprise any sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any of SEQ ID NOs: 67-73 or 135-151.

[0124] In some embodiments, the inhibitory RNA and / or vector comprises the sequence of any of SEQ ID NOs: 67-73. In some embodiments, the inhibitory RNA and / or vector comprises a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any of SEQ ID NOs: 67-73.

[0125] In some embodiments, the inhibitory RNA and / or vector comprises the sequence of any of SEQ ID NOs: 67-73 or 135-151. In some embodiments, the inhibitory RNA and / or vector comprises a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any of SEQ ID NOs: 67-73 or 135-151. See, e.g., International Patent Application No. WO2021 / 127455, the contents of which are incorporated herein by reference in their entirety.

[0126] [Table 8-1] [Table 8-2]

[0127] Suitable sequences for use in inhibitory nucleic acid (for example, miRNA) targeting AD and / or PD-related targets are known in the art, see, for example, International Patent Publication No. WO2011 / 133890, WO2012 / 036433, WO2013 / 007874; US Patent Application Publication No. US2016 / 0264965; US Patent Nos. 7,829,694, 8,415,319, 10,125,363, 10,011,835.The contents of the aforementioned references are incorporated herein by reference in their entirety.

[0128] In some embodiments of any of the aspects, the agent for treating neurological diseases or disorders is or comprises an inhibitory nucleic acid. In some embodiments of any of the aspects, the inhibitor of the expression of a given gene can be an inhibitory nucleic acid. As used herein, "inhibitory nucleic acid" refers to a nucleic acid molecule that can inhibit the expression of a target, such as double-stranded RNA (dsRNA), inhibitory RNA (iRNA), etc.

[0129] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can comprise an RNA strand (antisense strand) having a region 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of a targeted mRNA transcript. The use of these iRNAs allows for targeted degradation of the mRNA transcript, resulting in a decrease in target expression and / or activity.

[0130] As used herein, the term "iRNA" refers to an agent that contains RNA (or a modified nucleic acid, as described herein below) and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, the iRNAs described herein result in inhibition of target expression and / or activity. In some embodiments of any of the aspects, contacting a cell with an inhibitor (e.g., an iRNA) results in a reduction in target mRNA levels in the cell 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%, or 100%, up to 100%, of the target mRNA levels found in cells without the iRNA present. In some embodiments of any of the aspects, administering an inhibitor (e.g., an iRNA) to a subject can result in a reduction in target mRNA levels in the subject of at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, and up to 100%, of the target mRNA levels found in the subject in the absence of the iRNA.

[0131] In some embodiments of any of the aspects, the iRNA may be a dsRNA. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize and form a duplex structure under conditions for which the dsRNA is used. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally perfectly complementary, to the target sequence. The target sequence may be derived from the sequence of an mRNA formed during expression of the target; for example, it may span one or more intron boundaries. The other strand (the sense strand) comprises a region that is complementary to the antisense strand such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length, inclusive; more generally, between 18 and 25 base pairs in length, inclusive; even more generally, between 19 and 24 base pairs in length, inclusive; and most generally, between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length, inclusive, more commonly between 18 and 25 base pairs in length, even more commonly between 19 and 24 base pairs in length, inclusive, and most commonly between 19 and 21 base pairs in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive; in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As those skilled in the art will recognize, the targeted region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). dsRNA with duplexes as short as 9 base pairs can mediate RNAi-directed RNA cleavage in some circumstances. In most cases, the target is at least 15 nucleotides in length, preferably 15 to 30 nucleotides in length. Exemplary embodiments of types of inhibitory nucleic acids may include, for example, siRNA, shRNA, miRNA, and / or amiRNA, which are well known in the art.

[0132] In some embodiments of any of the aspects, the inhibitory RNA is an miRNA. MicroRNAs (miRNAs) are small RNAs of 17 to 25 nucleotides that function as regulators of gene expression in eukaryotes. "MicroRNAs" or "miRNAs" are small non-coding RNA molecules that can mediate transcriptional or post-translational gene silencing. Typically, miRNAs are transcribed as hairpin or stem-loop (e.g., self-complementary single-stranded backbones) duplex structures, referred to as primary miRNAs (pri-miRNAs), which are enzymatically processed into pre-miRNAs (e.g., by Drosha, DGCR8, Pasha, etc.). The duplex structure comprises a) a first RNA sequence with a region of complementarity that is substantially complementary, and generally perfectly complementary, to the target sequence, and b) a second RNA sequence region that is 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 may be derived from the sequence of an mRNA formed during expression of the target, for example, it may span one or more intron boundaries. Generally, the duplex structure is between 15 and 30 base pairs in length, inclusive, more generally between 18 and 25 base pairs in length, inclusive, even more generally between 19 and 24 base pairs in length, inclusive, and most generally between 19 and 21 base pairs in length, inclusive.

[0133] miRNAs are initially expressed in the nucleus as part of a long primary transcript called the primary miRNA (pri-miRNA). The length of a pri-miRNA can vary. In some embodiments, a pri-miRNA ranges in length 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, a pri-miRNA is longer than 200 base pairs in length (e.g., 2500, 5000, 7000, 9000, or more base pairs in length).

[0134] Inside the nucleus, pri-miRNAs are partially digested by the enzyme Drosha to form hairpin precursor miRNAs (pre-miRNAs) of 65-120 nucleotides in length, which are then transported to the cytoplasm for further processing by Dicer into shorter, active mature miRNAs. In animals, these short RNAs contain a 5'-proximal "seed" region (2-8 nucleotides) that is thought to be the primary determinant of pairing specificity for the 3'-untranslated region (3'-UTR) of the target mRNA. Pre-miRNAs, also characterized by a hairpin or stem-loop duplex structure, can vary in length. In some embodiments, pre-miRNAs range in size from about 40 base pairs to about 500 base pairs in length. In some embodiments, pre-miRNAs range in size from about 50-100 base pairs in length. In some embodiments, the pre-miRNA ranges in size 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-miRNAs are transported to the cytoplasm and enzymatically processed by Dicer to initially produce incomplete miRNAs / miRNAs. * This generates double-stranded and then single-stranded mature miRNA molecules, which are then loaded into the RNA-induced silencing complex (RISC). Typically, mature miRNA molecules range in size from about 19 to about 30 base pairs in length. In some embodiments, mature miRNA molecules are about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or 30 base pairs in length. In some embodiments, the isolated nucleic acids of the present disclosure comprise sequences encoding pri-miRNAs, pre-miRNAs, or mature miRNAs comprising the sequences set forth in any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260.

[0136] In the context of the present invention, miRNA molecules or their equivalents, mimics, or isomiRs may be synthetic, natural, recombinant, or mature miRNAs or human miRNAs or parts of mature miRNAs or human miRNAs, or may be derived from human miRNAs as further defined in the section providing the general definition. Human miRNA molecules are miRNA molecules found in human cells, tissues, organs, or body fluids (i.e., endogenous human miRNA molecules). Human miRNA molecules may also be derived from endogenous human miRNA molecules by nucleotide substitution, deletion, and / or addition. miRNA molecules, or their equivalents or mimics, may be single-stranded or double-stranded RNA molecules. Preferably, the miRNA molecule, or equivalent or mimic thereof, is 6 to 30 nucleotides in length, preferably 12 to 30 nucleotides in length, preferably 15 to 28 nucleotides in length, more preferably the molecule is 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 in length or longer.

[0137] In a preferred embodiment, the miRNA molecule, or its equivalent, mimic, or isomiR, comprises at least 6 of the 7 nucleotides present in the seed sequence of said miRNA molecule, or its equivalent, mimic, or isomiR. Preferably, in this embodiment, the miRNA molecule, or its equivalent, mimic, or isomiR, is 6 to 30 nucleotides in length, more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence of said miRNA molecule or equivalent. Even more preferably, the miRNA molecule, or its equivalent, mimic, or isomiR, is 15 to 28 nucleotides in length, more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence, and even more preferably, the miRNA molecule is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0138] Thus, preferred miRNA molecules, or equivalents or mimetics or isomiRs thereof, comprise at least 6 of the 7 nucleotides present in the seed sequence identified in or as SEQ ID NOs: 6-17, 40-44, 50-66, 158-185 or 217-260, and more preferably have a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides.

[0139] Delivery vehicles for miRNA include, but are not limited to, liposomes, polymeric nanoparticles, viral systems, lipid or receptor-binding molecule conjugation, exosomes, and bacteriophages; see, e.g., Baumann and Winkler, miRNA-based therapies: Strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents, Future Med Chem. 2014, 6(17): 1967-1984; U.S. Patent No. 8,900,627; U.S. Patent No. 9,421,173; U.S. Patent No. 9,555,060; WO2019 / 177550, the contents of each of which are incorporated herein by reference in their entirety.

[0140] The microRNA sequence comprises the "seed" region, i.e., the sequence of the region from positions 2 to 8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence of the nucleic acid. In one embodiment, the viral genome may be engineered to include, alter, or remove at least one miRNA binding site, sequence, or seed region.

[0141] The term "substantial complementarity" does not mean that the first and second RNA sequences are completely complementary, or that the first RNA sequence and the reference or target sequence (for example, SEQ ID NO: 3 or 4) are completely complementary.In one embodiment, the substantial complementarity between an RNA sequence and a target consists of having no mismatch, having one mismatched nucleotide, or having two mismatched nucleotides.It is understood that one mismatched nucleotide means that one nucleotide does not base pair with the target throughout the entire length of the RNA sequence that base pairs with the target.No mismatch means that all nucleotides are base paired with the target, and having two mismatches means that two nucleotides are not base paired with the target.

[0142] The miRNA and / or transgene containing one or more miRNAs may be provided in or include a scaffold sequence. As used herein, "scaffold" refers to the portion of the miRNA coding sequence that is outside the mature duplex structure. For example, the scaffold may include a loop and / or stem region. Thus, the scaffold is useful for generating, encoding, and / or expressing the miRNAs described herein. The scaffold used in the compositions and methods described herein may be, obtained from, and / or derived from an endogenous and / or naturally occurring miRNA scaffold, for example, the sequence of a human miRNA. In some embodiments, the scaffold sequence used in the compositions and methods described herein may be, obtained from, and / or derived from the sequence of an endogenous and / or naturally occurring miRNA scaffold of a miRNA that is overexpressed in one or more NS and / or CNS diseases.

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

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

[0145] In one embodiment, the miRNA used in the vectors described herein is selected from Table 24. [Table 24-1] [Table 24-2] [Table 24-3]

[0146] nucleic acid In some aspects, the present disclosure provides isolated nucleic acids 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 the present disclosure are isolated. As used herein, the term "isolated" means artificially produced. As used herein with respect to nucleic acids, the term "isolated" means (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, such as by cleavage and gel separation, or (iv) synthesized, e.g., by chemical synthesis. An isolated nucleic acid is one that can be readily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector for which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences are disclosed is considered isolated, whereas a nucleic acid sequence native to its natural host is not. Isolated nucleic acid may be substantially purified, but need not be.For example, the nucleic acid isolated in a cloning or expression vector is not pure, in that it may only comprise a small percentage of the material in the cell in which it exists.However, as this term is used herein, such nucleic acid is isolated because it can be easily manipulated by standard techniques known to those skilled in the art.When used herein with respect to protein or peptide, the term "isolated" refers to a protein or peptide that is isolated from its natural environment or artificially produced (for example, by chemical synthesis, by recombinant DNA technology, etc.).

[0147] Those skilled in the art will also recognize that conservative amino acid substitutions may be made to provide functionally equivalent variants or homologs of capsid proteins. In some aspects, the present disclosure encompasses sequence changes that result in conservative amino acid substitutions. As used herein, a conservative amino acid substitution refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, methods found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made within the following groups of amino acids: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus, conservative amino acid substitutions can be made to the amino acid sequences of the proteins and polypeptides disclosed herein.

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

[0149] Generally, the ITR sequence is about 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some degree of minor modification of these sequences is acceptable. The ability to modify these ITR sequences is within the skill of the art (see, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol., 70:520-532 (1996)). An example of such a molecule used in the present invention is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including the mammalian AAV types identified by the present invention. In some embodiments, the isolated nucleic acid (e.g., rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

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

[0151] In addition to the key elements identified above for recombinant AAV vectors, the vector also contains conventional control elements operably linked to the transgene elements in a manner that allows its transcription, translation, and / or expression in cells transfected with the vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Several expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0152] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of 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. Thus, a promoter region will be operably linked to a nucleic acid sequence if it is capable of effecting transcription of its DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins that are translated in frame. In some embodiments, the operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequences result in a functional RNA (e.g., miRNA).

[0153] In some aspects, the present disclosure provides an isolated nucleic acid comprising a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more microRNAs (e.g., miRNAs).

[0154] It should be appreciated that an isolated nucleic acid or vector (e.g., an rAAV vector) may, in some embodiments, comprise a nucleic acid sequence encoding two or more (e.g., a plurality, e.g., 2, 3, 4, 5, 10, or more) miRNAs. In some embodiments, each of the two or more miRNAs targets (e.g., hybridizes to or specifically binds to) the same target gene (e.g., an isolated nucleic acid encoding three unique miRNAs, where each miRNA targets the HTT gene). In some embodiments, each of the two or more miRNAs targets (e.g., hybridizes to or specifically binds to) a different target gene.

[0155] In some aspects, the present disclosure provides isolated nucleic acids and vectors (e.g., rAAV vectors) encoding one or more artificial miRNAs. As used herein, "artificial miRNA" or "amiRNA" refers to miRNAs and miRNAs, e.g., as described in Eamens et al. (2014), Methods Mol. Biol. 1062:211-224. * The corresponding amiRNA / amiRNA (e.g., passenger strand of an miRNA duplex) sequence directs highly efficient RNA silencing of targeted genes. * The term "miR-155" refers to an endogenous pri-miRNA or pre-miRNA (e.g., a miRNA backbone, which is a precursor miRNA capable of generating a functional mature miRNA) in which a sequence encoding a mature HTT-specific miRNA (e.g., any one of SEQ ID NOS: 6-17, 40-44, 50-66, 158-185, or 217-260) has been inserted in place of the endogenous miR-155 mature miRNA coding sequence. In some embodiments, miRNAs (e.g., artificial miRNAs) described by the present disclosure include a miR-155 backbone sequence, a miR-30 backbone sequence, a miR-64 backbone sequence, or a miR-122 backbone sequence.

[0156] The region containing the transgene (e.g., the second region, third region, fourth region, etc.) may be located in any suitable location in the isolated nucleic acid. The region may be located in any untranslated portion of the nucleic acid, including, for example, an intron, a 5' or 3' untranslated region, etc.

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

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

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

[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 start sequences, stop sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0162] "Promoter" refers to a DNA sequence recognized by or introduced into the synthetic machinery of a cell necessary to initiate the specific transcription of a gene. The phrases "operably positioned," "under control," or "under transcriptional control" mean that the promoter is in the correct location and orientation with respect to the nucleic acid to control the initiation of RNA polymerase and expression of the gene.

[0163] For nucleic acids encoding proteins, a polyadenylation sequence is generally inserted after the transgene sequence and before the 3' AAV ITR sequence. The rAAV constructs useful in this disclosure may also contain an intron, which is preferably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. Another vector element that can be used is an internal ribosome entry site (IRES). IRES sequences are used to generate two or more polypeptides from a single gene transcript. IRES sequences will be used to generate proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional, and many such sequences are available (see, e.g., Sambrook et al., and the references cited therein, e.g., pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). In some embodiments, the foot-and-mouth disease virus 2A sequence is included in the polyprotein, which is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459).The cleavage activity of 2A sequences has previously been demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retrovirus) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; pp. 8124-8127; Furler, S et al., Gene Therapy, 2001; pp. 864-873; and Halpin, C et al., The Plant Journal, 1999; pp. 453-459; de Felipe, P et al., Gene Therapy, 1999; pp. 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; pp. 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001;8:811-817).

[0164] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter [Invitrogen]. In some embodiments, the promoter is an enhanced chicken β-actin promoter. In some embodiments, the promoter is a U6 promoter. In some embodiments, the promoter is a human synapsin (hSYN) promoter. In some embodiments, the promoter is a 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 or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 193. The CAG promoter can drive expression, for example, the expression of a transgene to which it is operably linked, in target neurons and astrocytes. [ka] [ka]

[0166] The underlined text (eg, bp 453 to 819 of SEQ ID NO: 193) indicates the CMV enhancer.

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

[0168] Double underlined text (eg, bp 1191-2065 of SEQ ID NO: 193) indicates the ACTB intron.

[0169] Inducible promoters allow the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as the presence of an acute phase, a specific differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from various commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by an exogenously supplied promoter include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088), the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268: 1766-1769 (1995), and Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1995)). (1998)), the RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997) ) and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

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

[0171] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: 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 beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3: 1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7: 1503-14 (1996); the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24: 185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), the CD2 promoter (Hansal et al., J. Immunol., 161: 1063-8 (1996)), among others that will be apparent to those skilled in the art. (1998)); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal promoters such as the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). NS-specific promoters contemplated for use in the present methods and compositions also include those described in Patent Applications GB2013940.8, filed September 4, 2020, and GB2005732.9, filed April 20, 2020, which are incorporated herein by reference in their entireties. 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%, at least 98% identity to a promoter of Table 10.In some embodiments, the NS-specific promoter is a 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 to a promoter in Table 10 and retains the NS-specific promoter activity of a promoter in Table 10.

[0172] CNS-specific promoters contemplated for use in the present methods and compositions also include those described in International Patent Application No. 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 promoter is a promoter in Tables 11-13, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter in Tables 11-13. In some embodiments, the CNS-specific promoter is a promoter in Tables 11-13, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a promoter in Tables 11-13, and retaining the CNS-specific promoter activity of a promoter 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 of Tables 13-15, or a CRE having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a CRE of Tables 13-15. In some embodiments, the CRE is a CRE of Tables 13-15, or a CRE having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to a CRE of Tables 13-15 that retains the activity of a CRE of Tables 13-15.

[0174] In some embodiments, the CRE may include one or more CREs known in the art. For example, in one embodiment, the one or more CREs may be selected from SEQ ID NOs: 19-24, 27, 28, 37, and 38 of Patent Application No. GB2013940.8, filed September 4, 2020. For example, in one embodiment, the one or more CREs may be selected from SEQ ID NOs: 1-8 of WO2019 / 199867A1, SEQ ID NOs: 1-7 of WO2020 / 076614A1, and SEQ ID NOs: 25-51, 177-178, and 188 of WO2020 / 097121. The foregoing references are incorporated herein by reference in their entireties.

[0175] [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7]

[0176] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

Table 11-5

Table 11-6

[0177]

Table 12

[0178]

Table 13-1

Table 13-2

[0179]

Table 14-1

Table 14-2

[0180]

Table 15-1

Table 15-2

Table 15-3

[0181] In a further aspect of the invention, there is provided a synthetic central nervous system (CNS)-specific promoter comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 187, 188 or 189, or a functional variant thereof. In some embodiments, the synthetic CNS-specific promoter comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 187, 188 or 189.

[0182] Thus, the present invention provides a variety of synthetic CNS-specific promoters and functional variants thereof. It is generally preferred that a promoter according to the present invention that is a variant of any one of SEQ ID NOs: 187, 188, or 189 retains 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 NOs: 187, 188, or 189).

[0183] In some embodiments of the invention, the CNS-specific promoter has a length of 1200 or fewer nucleotides, 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, 700 or fewer nucleotides.

[0184] In another aspect of the present invention, a synthetic CNS-specific promoter is provided, comprising a cis-regulatory element (CRE) according to any one of SEQ ID NOs: 191 or 192, or a functional variant thereof. In some embodiments, the CRE may be operably linked 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 NOs: 191 or 192 comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 191 or 192.

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

[0186] In another embodiment, the synthetic CNS-specific promoter comprises or consists of CRE00011 (SEQ ID NO: 192) or a functional variant thereof operably linked to CRE151 (SEQ ID NO: 190) or a functional variant thereof.

[0187] Element CRE151 is a promoter element that functions in combination with one or more CNS-specific CREs to provide CNS-specific transcription from the promoter in which it is contained. Its sequence and functional variants are discussed further below.

[0188] Elements CRE00010 and CRE00011 are CNS-specific CREs. They function in combination with promoter elements to modulate, typically enhance, CNS-specific transcripts from the promoters in which they are contained. Their sequences and functional variants thereof are discussed further below.

[0189] In embodiments in which CRE00010 is operably linked to CRE151, CRE00010 may be contiguous with (i.e., located directly adjacent to) the adjacent CRE151 or may be separated by a spacer or other sequence.

[0190] In embodiments in which CRE00011 is operably linked to CRE151, CRE00011 may be contiguous with (i.e., located directly adjacent to) the adjacent CRE151 or may be separated by a spacer or other sequence.

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

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

[0193] In embodiments in which CRE00010 is operably linked to CRE527, CRE00010 may be contiguous with (i.e., located directly adjacent to) the adjacent CRE527 or may be separated by a spacer or other sequence.

[0194] In one embodiment, the synthetic CNS-specific promoter comprises or consists of CRE00010 (SEQ ID NO: 191) or a functional variant thereof operably 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 there is provided a minimal promoter comprising or consisting of the sequence set forth in SEQ ID NO: 211 or a functional variant thereof. Suitably, the functional variant of the minimal promoter comprises a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 211.

[0196] In another aspect of the present invention there is provided a synthetic promoter comprising a 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 a sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 211.

[0197] In a further aspect of the present invention there is provided a CRE comprising or consisting of a sequence as set forth in any one of SEQ ID NO: 191 or SEQ ID NO: 192, or a functional variant thereof.

[0198] The CRE according to the present invention may be combined with an additional CRE to form a cis-regulatory module (CRM). Preferably, the additional CRE may be a CRE according to SEQ ID NO: 191 or 192, or a functional variant thereof, or may be another CRE. Preferably, the additional CRE is CNS-specific.

[0199] Preferably, in another aspect, a CRM is provided comprising a CRE of the present invention. Preferably, 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 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, a functional variant of a CRE according to any one of SEQ ID NOs: 191 and / or 192 comprises or consists of a sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 191 or 192.

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

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

[0202] The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in one or more of various parts of the CNS. The CNS is mainly composed of the brain and spinal cord. The retina, optic nerve, olfactory nerve, and olfactory epithelium may be considered parts of the CNS alongside the brain and spinal cord. This is because they are directly connected to brain tissue without intervening nerve fibers. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in the brain and spinal cord. Preferably, the CRE, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in the brain but not in the spinal cord or any other part of the CNS. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in the spinal cord but not in the brain. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in the brain.

[0203] Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in one or more of various regions in the brain. Non-limiting examples of brain regions include the frontal lobe, parietal lobe, occipital lobe, temporal lobe (including the hippocampus and amygdala), cerebellum, midbrain, pons, medulla, and diencephalon (including the thalamus and hypothalamus). Non-limiting examples of spinal cord regions include the cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae, and caudal vertebrae. 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 not in other parts of the CNS, such as the spinal cord. 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, six, seven, eight, or nine of the brain regions listed above. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in a majority of brain regions, i.e., at least five, at least six, at least seven, at least eight, or all nine of the nine brain regions listed above. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in four to six of the brain regions listed above. In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in two to four of the brain regions listed above, 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 may be active in the above-mentioned regions of the brain and spinal cord.In some embodiments, the CRE, CRM, minimal promoter or synthetic CNS-specific promoter of the present invention is active in the spinal cord but not in other parts of the CNS, such as 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 spinal cord regions listed above.In some embodiments, the CRE, CRM, minimal promoter or synthetic CNS-specific promoter of the present invention is active in most of the regions in the spinal cord, i.e., in at least three, at least four, or all five of the five spinal cord regions listed above.

[0204] In some embodiments, it may be desirable for the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention to exhibit predominant activity in one region of the CNS, preferably in one region of the brain. Preferably, it may be desirable for the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention to exhibit activity in one region of the brain, but exhibit no 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 of the CNS regions of the brain listed above, for example, 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 a 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 exhibits no or only minimal activity in other regions of the brain. The CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may be active in various cells of the CNS. The predominant cell types in the brain are neurons, astrocytes, oligodendrocytes, microglia, and ependymal cells. Other cell types may be present, particularly in inflammatory conditions. In some embodiments, it may be desirable for a 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 substantially all cells of the CNS (e.g., neurons, astrocytes, oligodendrocytes, microglia, and 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 from the CNS cell types listed above, such as neurons, astrocytes, microglia, and oligodendrocytes.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 from the CNS cell types listed above, such as neurons, astrocytes, and oligodendrocytes. In some embodiments, it may be desirable for a 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 CNS cell types from the CNS cell types listed above, such as 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 CNS cell type from the CNS cell types listed above, such as 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 cells, such as 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 not 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 a specific type or subtype of CNS cells, and in a specific region 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 outside the CNS. Non-limiting examples of tissues outside the CNS include the heart, liver, kidney, skeletal muscle, and spleen. Preferably, in some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is not active or only minimally active in tissues or cells outside the CNS. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in 1, 2, 3, or 4 or less of the tissues outside the CNS listed above when delivered via ICV. Preferably, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention is active in 1, 2, 3, or 4 or less of the tissues outside the CNS listed above when delivered via IV.

[0205] Preferably, in some embodiments, it may be desirable for the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention to be active in the CNS but also active in other tissues outside the CNS. Preferably, 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 of the tissues outside the CNS listed above upon ICV delivery. Preferably, 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 of the tissues outside the CNS listed above upon IV delivery.

[0206] In some embodiments, the CRE, CRM, minimal promoter, or synthetic CNS-specific promoter of the present invention may 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 the PNS, 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 parts of the nervous system other than the brain and spinal cord. Non-limiting examples of the peripheral nervous system include the cranial nerves, brachial plexus, thoracic and abdominal nerves, lumbar and sacral plexuses, and the neuromuscular junction. 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 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 regions of the PNS listed above. In some embodiments, the CRE, CRM, minimal promoter or synthetic CNS-specific promoter of the present invention is active in a majority of regions in the PNS, i.e., at least four, at least five, or all six of the six regions of the PNS listed above.

[0207] A CNS-specific promoter may be expressed in other non-CNS cells. However, it has a higher degree of expression in CNS cells, such as neurons in the brain and spinal cord, and non-neuronal cells or neuron-supporting cells located in the brain and spinal cord. For example, a CNS-specific promoter expresses a gene 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%, higher in cells located in the CNS, including neurons and non-neuronal cells located in the brain and spinal cord, compared to cells located outside the CNS.

[0208] In a further aspect of the invention, there is provided an expression cassette comprising a synthetic CNS-specific promoter according to any of the aspects of the invention operably linked to a sequence encoding an expression product, preferably a gene, e.g., a transgene. In one embodiment, there is provided an expression cassette comprising any one of CNS-10, CNS-11 or CNS-13, or a functional variant thereof, operably linked to a sequence encoding an expression product, preferably a gene, e.g., a transgene.

[0209] In a further aspect of the present invention, there is provided an expression cassette comprising CNS-10 or a functional variant thereof operably linked to a sequence encoding an expression product, preferably a gene, e.g., a transgene.

[0210] In another aspect of the present invention, there is provided an expression cassette comprising CNS-11 or a functional variant thereof operably linked to a sequence encoding an expression product, preferably a gene, e.g., a transgene.

[0211] In another aspect of the present invention, there is provided an expression cassette comprising CNS-13 or a functional variant thereof operably linked to a sequence encoding an expression product, preferably a gene, e.g., a transgene.

[0212] In some embodiments, the transgene is any one of the genes selected from the group consisting of 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, for example, but not limited to, GFP, RFP, YFP, or luciferase. In some embodiments, when the transgene is GDNF, the expression product is GDNF protein. In some particularly preferred embodiments, the transgene is codon-optimized GDNF. In some embodiments, the transgene is CpG-deleted GDNF. More preferably, in some embodiments, the transgene is CpG-deleted and codon-optimized GDNF.

[0213] In a further aspect, a vector is provided comprising the synthetic CNS-specific promoter or expression cassette according to the present 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, preferably an AAV vector, an adenoviral vector, a retroviral vector, or a lentiviral vector. In a preferred embodiment, the vector is an AAV vector.

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

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

[0216] In a further aspect, a synthetic CNS-specific promoter, expression cassette, vector, virion, or pharmaceutical composition according to the present invention is provided for use in therapy, i.e., in the prevention or treatment of a medical condition or disease. Preferably, the condition or disease is associated with abnormal gene expression, optionally in the CNS. Preferably, the use is for gene therapy, preferably for use in the treatment of 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 results in dopaminergic neurons in the CNS.

[0217] Preferably, the gene therapy involves expression of a therapeutic expression product in the CNS.

[0218] In a further aspect, a cell is provided comprising the synthetic CNS-specific promoter, expression cassette, vector, or virion described herein. In some embodiments, the cell is a eukaryotic cell, optionally a mammalian cell, optionally a human cell. In some embodiments, the cell is a CNS cell, optionally a human CNS cell. Suitably, in some embodiments, the CNS cell is a neuron, an astrocyte, an oligodendrocyte, a microglial cell, or an ependymal cell. In a preferred embodiment, the CNS cell is a neuron, even more preferably a dopaminergic neuron. The synthetic CNS promoter or expression cassette can be present in a vector or in the genome of the cell.

[0219] In a further aspect, there is provided a synthetic CNS-specific promoter, expression cassette, vector, or virion according to the invention for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease discussed herein. In some embodiments, the disease is a CNS-related disease.

[0220] In a further aspect, there is provided a method of expressing a therapeutic transgene in a cell, preferably a CNS cell, the method comprising introducing into the cell a synthetic CNS-specific expression cassette, vector, or virion described herein. In some preferred embodiments, the therapeutic transgene is GDNF.

[0221] In a further aspect, there is provided a method of treating a subject, preferably a human, in need of treatment, the method comprising administering to a subject in need of treatment an expression cassette, vector, virion or pharmaceutical composition as described herein comprising a sequence encoding a therapeutic product operably linked to a promoter according to the invention; and expressing a therapeutic amount of the therapeutic product in the CNS of said subject.

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

[0223] In some embodiments, an 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 comprises introducing into the CNS of the subject an expression cassette, vector, virion, or pharmaceutical composition of the present invention comprising 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 producing an expression product is provided, the method comprising introducing a synthetic CNS-specific expression cassette of the invention into a CNS cell; and expressing a gene present in the synthetic CNS-specific expression cassette.

[0226] The method preferably includes maintaining the CNS cells under conditions suitable for expression of the gene. In culture, this may include incubating the cells or tissue containing the cells under suitable culture conditions. Expression may, of course, be in vitro, ex vivo, or in vivo, for example, in one or more cells in the CNS of a subject.

[0227] Preferably, the method comprises introducing a synthetic CNS-specific expression cassette into a CNS cell. A wide range of methods for transfecting CNS cells are known in the art. A preferred method for transfecting CNS cells is to transduce the cells with a viral vector, e.g., an AAV vector, containing the synthetic CNS-specific expression cassette.

[0228] CRE and its functional variants

[0229] Disclosed herein are CREs that can be used in the construction of CNS-specific promoters. Preferably, the CREs are CNS-specific. These CREs are generally derived from genomic promoter and enhancer sequences, but they are used herein in a context completely different from their native genomic environment. Generally, CREs represent small portions of very large genomic regulatory domains, which control the expression of the genes they normally associate with. Surprisingly, we have found that these CREs, many of which are very small, can be isolated from their normal environment and retain CNS-specific regulatory activity. This is surprising because removal of regulatory sequences from the complex, "three-dimensional" natural context in the genome often results in a significant loss of activity, and therefore there is no reason to expect a given CRE to retain its observed activity level when removed from its natural environment. This is even more surprising if the CRE retains its CNS-specific activity in an AAV vector. This is particularly surprising because AAV vectors contain inverted terminal repeats (ITRs) and have a different DNA structure compared to the genome, and both the ITR and DNA structures are known to affect the activity of CREs. It should be noted that the sequence of the CRE of the present invention can be changed without causing substantial loss of activity.Functional variants of CRE can be prepared by modifying the sequence of CRE, provided that modifications that are significantly harmful to the activity of CRE are avoided.In view of the information provided in this disclosure, the modification of CRE to provide functional variants is straightforward.In addition, this disclosure provides a methodology for simply evaluating the functionality of any given CRE variant (see, for example, Example 3).

[0230] The size of a particular CRE according to the present invention is advantageous because it allows the CRE, more particularly the promoter containing it, to be provided in the vector while occupying a minimal amount of payload of the vector, which is particularly important when the CRE is used in a vector with limited capacity, such as an AAV-based vector.

[0231] The CRE of the present invention contains certain CNS-specific TFBSs. Generally, it is desirable that these CNS-specific TFBSs retain functionality in functional variants of CREs. Those skilled in the art are well aware that even if the TFBS sequence is changed, functionality can still be maintained. With this in mind, the sequences for TFBSs are typically represented by consensus sequences, which typically have some degree of variation. Further information about the variations occurring in TFBSs can be displayed using a position weight matrix (PWM), which represents the frequency with which a given nucleotide typically finds the consensus sequence at a given position. Details of TF consensus sequences and associated position weight matrices 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 sequence in any given TFBS of a CRE in a manner that retains, or in some cases even increases, CRE functionality. With this in mind, those skilled in the art have ample guidance on how to modify the TFBS for any given TF while maintaining its ability to bind to the desired TF; for example, the Jaspar system scores putative TFBSs based on their similarity to a given PWM. Furthermore, CREs can be scanned against all PWMs from the JASPAR database to identify / analyze all TFBSs. Those skilled in the art can, of course, find additional guidance in the literature and use routine experimentation to confirm TF binding to putative TFBSs in any variant CRE. It should be clear that significant sequence modifications can be made in CREs while retaining function, even within the TFBSs in the CRE. The CREs of the present invention can be used in combination with a wide range of suitable minimal promoters or CNS-specific proximal promoters. Functional variants of CREs include sequences that differ from the reference CRE element but substantially retain activity as a CNS-specific CRE.It will be appreciated by those skilled in the art that it is possible to vary the sequence of a CRE while retaining its ability to recruit suitable CNS-specific transcription factors (TFs) and thereby enhance expression. Functional variants of a CRE can include substitutions, deletions, and / or insertions compared to a reference CRE, provided that they do not render the CRE substantially non-functional.

[0232] In some embodiments, a functional variant of a CRE can be considered a CRE that substantially retains its activity when substituted for a reference CRE in a promoter. For example, a CNS-specific promoter comprising 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 comprising an unmodified CRE). Preferably, a functional variant of a CRE retains a significant level of sequence identity to the reference CRE. Preferably, a functional variant comprises a sequence that is at least 70% identical to the reference CRE, more preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference CRE.

[0233] Promoter elements and their functional variants

[0234] One or more promoter elements disclosed herein may be a minimal promoter or a proximal promoter. Preferably, 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. The CREs of the present invention can be used in combination with a range of suitable minimal promoters or CNS-specific proximal promoters, but some promoter elements act synergistically with one or more CREs to significantly contribute to the activity of the CNS-specific promoter.

[0235] Functional variants of promoter elements include sequences that differ from reference promoter elements but substantially retain their activity as promoter elements.Those skilled in the art will understand that promoter element sequences can be altered while retaining the ability to recruit RNA polymerase II and, where appropriate, bind to CNS-specific TFs to enhance expression.Functional variants of promoter elements can include substitutions, deletions, and / or insertions compared to reference promoter elements, provided that the substitutions, deletions, and / or insertions do not render the promoter element non-functional.

[0236] In some embodiments, a functional variant of CRE151 can be considered a promoter element that substantially retains its activity when substituted for a reference promoter element in a promoter. Suitable assays for assaying CNS-specific promoter activity are disclosed herein, for example, in Example 3.

[0237] Preferably, a functional variant of a promoter element retains a significant level of sequence identity to the reference promoter element. Preferably, the functional element of CRE151 comprises a sequence that is at least 70% identical to SEQ ID NO: 190, more preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 190. As discussed above, a functional variant of CRE151 substantially retains the ability of the reference promoter element to act as a promoter element. For example, when a functional variant of CRE151 is substituted for a promoter comprising a 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 even more preferably 100% of its activity. Suitably, a functional variant of CRE151 comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:190.

[0238] In some embodiments, a functional variant of CRE527 can be considered a promoter element that substantially retains its activity when substituted for a reference promoter element in a promoter. Suitable assays for assaying CNS-specific promoter activity are disclosed herein, for example, in Example 1.

[0239] Preferably, functional variants of promoter elements retain a significant level of sequence identity to the reference promoter element. Preferably, the functional element of CRE527 comprises a sequence that is at least 70% identical to SEQ ID NO:211, more preferably at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:211. As discussed above, functional variants of CRE527 substantially retain the ability of the reference promoter element to act as a promoter element. For example, when a functional variant of CRE527 is substituted for a promoter comprising 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 even more preferably 100% of its activity. Suitably, a functional variant of CRE527 comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:211.

[0240] Retention of activity can be assessed by comparing expression of a suitable reporter under the control of a reference promoter with an otherwise identical promoter containing the replaced promoter element under equivalent conditions. Suitable assays for assessing CNS-specific promoter activity are disclosed herein, e.g., in Example 3.

[0241] Promoter elements used in the present invention can be natural (ie, obtained or derived from a naturally occurring gene promoter) or synthetic (ie, not occurring in nature).

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

[0243] A functional variant of a reference synthetic CNS-specific promoter is a promoter that contains a sequence that differs from the reference synthetic CNS-specific promoter but substantially retains CNS-specific promoter activity. Those skilled in the art will recognize that it is possible to alter the sequence of a synthetic CNS-specific promoter while retaining its ability to recruit suitable CNS-specific transcription factors (TFs) and recruit RNA polymerase II to provide CNS-specific expression of an operably linked sequence (e.g., an open reading frame). A functional variant of a synthetic CNS-specific promoter can contain substitutions, deletions, and / or insertions compared to the reference promoter (e.g., CNS-10, CNS-11, or CNS-13), provided that such substitutions, deletions, and / or insertions do not render the synthetic CNS-specific promoter substantially non-functional.

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

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

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

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

[0248] Functional variants of a synthetic CNS-specific promoter often retain a significant level of sequence similarity to the reference synthetic CNS-specific promoter.

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

[0250] Suitably, in one embodiment, a functional variant of any one of CNS-10, CNS-11 or CNS-13 is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 187, 188 or 189.

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

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

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

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

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

[0256] A functional variant of a given synthetic CNS-specific promoter can include one or more additional CREs relative to those present in the reference synthetic CNS-specific promoter. The additional CREs can be CREs disclosed herein, or they can be other CREs.

[0257] Functional variants of a given synthetic CNS-specific promoter can contain one or more additional regulatory elements compared to a reference synthetic CNS-specific promoter. For example, they can include inducible elements, intronic elements, boundary control elements, insulators, locus control regions, response elements, binding sites, terminal repeat segments, response sites, stabilizing elements, destabilizing elements, splicing elements, and the like, provided that they do not render the promoter substantially non-functional. Functional variants can also include 5'UTR sequences.

[0258] Functional variants of a given synthetic CNS-specific promoter can include additional spacers between adjacent CREs and / or between the CRE and the promoter element.

[0259] Preferred synthetic CNS-specific promoters of the invention exhibit CNS-specific promoter activity that is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, or 400% of the activity of the synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE promoter in CNS cells. In some embodiments, synthetic CNS-specific promoters of the invention exhibit CNS-specific promoter activity that is at least 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, or 200% of the activity of the human TH promoter. In some embodiments, the synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at a level that is at least 100% of the activity of the synapsin-1, Camk2a, GFAP, MBP, IBA1 or NSE promoter 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 promoter.

[0260] In some embodiments, the synthetic CNS-specific promoters of the present invention are suitable for promoting CNS-specific transgene expression at a level that is at least 100% of the activity of the human TH promoter, preferably 150% or 200% of the activity of the human TH promoter. While higher levels of promoter activity are often preferred, this is not always the case, and therefore, in some cases, more moderate levels of expression may be preferred. In some cases, more moderate levels of expression may be preferred, for example, to prevent protein toxicity or accumulation. The activity of a given synthetic CNS-specific promoter of the present invention relative 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, when the two promoters are provided in otherwise equivalent expression constructs and under equivalent conditions.

[0261] In some embodiments, a synthetic CNS-specific promoter of the invention can increase expression of a gene (e.g., a therapeutic gene or gene of interest) in a subject's CNS or CNS cells 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 compared to a known CNS-specific promoter, preferably synapsin-1, Camk2a, GFAP, MBP, IBA1, or NSE. In another embodiment, a synthetic CNS-specific promoter of the invention can increase expression of a gene (e.g., a therapeutic gene or gene of interest) in a subject's neurons, preferably dopaminergic neurons, by at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 200%, at least 300%, or more compared to the human TH promoter. In a preferred embodiment, the synthetic CNS-specific promoter of the present invention is capable of increasing expression of a gene, preferably a transgene, in neurons, optionally dopaminergic neurons.

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

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

[0264] The synthetic CNS-specific promoters of the present invention may be active in specific regions of the CNS, specific CNS cells or CNS cell subtypes, or both. In some embodiments, the synthetic CNS-specific promoters of the present invention may be active in specific CNS cell types, such as neurons, within all regions of the CNS. In other embodiments, the synthetic CNS-specific promoters of the present invention may be active in specific CNS cell types, such as neurons, within one or fewer regions of the CNS, such as the midbrain. In some embodiments, the synthetic CNS-specific promoters of the present invention may be active in all CNS cells in all regions of the CNS. In some embodiments, the synthetic CNS-specific promoters of the present invention may be active in one or fewer regions of the CNS, such as the midbrain. Suitably, in some embodiments, the synthetic CNS-specific promoters of the present invention may be active only in dopaminergic neurons in 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 is demonstrated in preliminary in vivo experiments, where a predominantly neuronal tropism was observed for CNS-10, CNS-11, and CNS-13. Notably, CNS-10 and CNS-13 showed a mixture of neuronal and minimal astroglial expression.

[0266] Aspects of the present disclosure relate to isolated nucleic acids comprising two or more promoters (e.g., two, three, four, five, or more promoters). For example, in the context of a construct having a transgene comprising a first region encoding a protein and a second region encoding an inhibitory RNA (e.g., miRNA), it may be desirable to drive expression of the protein-coding region using a first promoter sequence (e.g., a first promoter sequence operably linked to the protein-coding region) and to drive expression of the inhibitory RNA-coding region with a second promoter sequence (e.g., a second promoter sequence operably linked to the inhibitory RNA-coding region). Generally, the first promoter sequence and the second promoter sequence can be the same promoter sequence or different promoter sequences. In some embodiments, the first promoter sequence (e.g., the promoter driving expression of the protein-coding region) is an RNA polymerase III (pol III) promoter sequence. Non-limiting examples of pol III promoter sequences include U6 and HI promoter sequences. In some embodiments, the second promoter sequence (e.g., the promoter sequence driving the expression of an inhibitory RNA) is an RNA polymerase II (pol II) promoter sequence. Non-limiting examples of pol II promoter sequences include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the pol III promoter sequence drives the expression of an inhibitory RNA (e.g., miRNA) coding region. In some embodiments, the pol II 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 can 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 comprising SEQ ID NO:2. In some embodiments, the protein encodes a protein having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO:2. In some embodiments, the therapeutic protein is useful for treating or preventing Huntington's disease, e.g., polyglutamine-binding peptide 1 (QBP1), PTD-QBP1, ED11, C4 intrabody, VL12.3 intrabody, MW7 intrabody, Happ1 antibody, Happ3 antibody, mEM48 intrabody, certain monoclonal antibodies (e.g., 1C2), and peptide P42, and variants thereof, as described in Marelli et al. (2016) Orphanet Journal of Rare Disease 11:24; doi: 10.1186 / s l3023- 016-0405-3. In some embodiments, the therapeutic protein is wild-type huntingtin protein (e.g., a huntingtin protein having a PolyQ repeat region containing fewer than 36 repeats).

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

[0269] CYP46A1 is neuroprotective in cellular models of HD (see, e.g., WO2012 / 049314), and there is a reduction in CYP46A1 mRNA in the striatum, a brain structure more vulnerable to disease in the R6 / 2 transgenic HD mouse model.

[0270] During the early stages of AD, 24-hydroxycholesterol concentrations are high in the CSF and peripheral circulation. In the later stages of AD, 24-hydroxycholesterol concentrations may decrease, reflecting neuronal loss (Kolsch, H. et al., 2004). CYP46A1 is expressed around the amyloid core of senile plaques in the brains of AD patients (Brown, J., 3rd et al., 2004).

[0271] Agonism of cholesterol 24-hydroxylase encoded by CYP46A1 significantly reduces neuropathology and improves cognitive impairment in mouse models of CNS disease.For example, co-expression of CYP46A1 with ExpHtt in Huntington's disease models strongly and significantly reduces the formation of ExpHtt aggregates (58% vs. 27.5%) (WO2012 / 049314) (see also International Patent Publication No. WO2009 / 034127, which is incorporated herein by reference in its entirety).The methods described herein relate to the agonism of CYP46A1 in combination with the administration of miRNA that targets certain other targets.For example, the methods may relate to the administration of a viral vector for the treatment of neurological diseases or disorders, wherein the vector expresses CYP46A1 in cells of the central nervous system.

[0272]

[0009] In some embodiments, described herein is a viral vector for treating a neurological disease or disorder, 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 of SEQ ID NO:2. In some embodiments, the viral vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO:2. In some embodiments, the viral vector comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more sequence identity to SEQ ID NO:1. In some embodiments, the viral vector comprises the sequence of SEQ ID NO:1. In some embodiments, the viral vector may be an adeno-associated virus (AAV) vector.

[0273] Further description of CY46A1 and its therapeutic uses (e.g., for Alzheimer's disease, ALS, and ataxia) can be found in the art, for example, in WO2012 / 049314, WO2009 / 034127, WO2018 / 138371, and WO2020 / 089154. The sequences, methods, and compositions described therein can be utilized in the methods and compositions described herein. The aforementioned references are incorporated herein by reference in their entirety. The term "gene" refers to a polynucleotide containing at least one open reading frame that can encode a specific polypeptide or protein after being transcribed or translated.

[0274] The terms "coding sequence" or "sequence encoding a particular protein" refer to a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. 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 for CYP46A1 is disclosed in Genbank Accession No. NM_006668 (SEQ ID NO: 1). The amino acid sequence is set forth in SEQ ID NO: 2. The present invention provides for the use of nucleic acid constructs comprising the sequence of SEQ ID NO: 1 or variants thereof for the treatment of neurological diseases or disorders. Variants include, for example, naturally occurring variants resulting from allelic variation (e.g., polymorphisms) between individuals, alternative splice forms, and the like. The term variant also includes CYP46A1 gene sequences from other sources or organisms. Variants are preferably substantially homologous to SEQ ID NO: 1 and / or 2, i.e., typically exhibit at least about 75%, preferably at least about 85%, more preferably at least about 90%, and more preferably at least about 95% nucleotide sequence identity with SEQ ID NO: 1 or 2. In some embodiments, nucleic acid constructs comprise a sequence having at least 95% sequence identity to SEQ ID NO: 1 and retaining the activity of SEQ ID NO: 1 or 2 (e.g., the ability to convert cholesterol to 24-hydroxycholesterol). Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the above-defined sequences (or their complementary strands) under stringent hybridization conditions. Typical stringent hybridization conditions include a temperature above 30°C, preferably above 35°C, more preferably above 42°C, and / or a salt content of less than about 500mM, preferably less than 200mM. Hybridization conditions can be adjusted by those skilled in the art by modifying the temperature, salt content, and / or the concentration of other reagents such as SDS and SSC.

[0276] In one embodiment, the CYP46A1 is codon-optimized CYP46A1. Exemplary codon-optimized CYP46A1 contemplated for use herein are provided in SEQ ID NOs: 109 and 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 to 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 to 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 one 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 for codon optimization are known in the art and are described herein below. In one embodiment, more methods for codon optimization are utilized. For example, the CYP46A1 nucleic acid sequence is modified to reduce CpG motifs and increase GC content.

[0278] For example, at least one or more CpG islands (guanine bases (CpG) immediately following a cytosine base, where cytosines in such positions tend to be methylated), which typically occur at or near the transcription start site of the CYP46A1 nucleic acid sequence, are deleted and / or substituted. In one embodiment, deleting or reducing the number of CpG islands can enhance in vivo expression or reduce the innate immune response of the transgene. In one embodiment, at least one CpG motif is deleted and / or substituted, for example, at least 1, 2, 3, 4, 5, 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 10% or less of the CpGs, or 20% or less of the CpGs, or 30% or less of the CpGs, or 40% or less of the CpGs, or 50% or less of the CpGs, or 60% or less of the CpGs, or 70% or less of the CpGs, or 80% or less of the CpGs, or 90% or less of the CpGs present in the wild-type CYP46A1 nucleic acid sequence. Exemplary modifications to the wild-type CYP46A1 nucleic acid sequence (SEQ ID NO: 1) that result in the removal of CpG islands are listed 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 natural immune response of the transgene. In one embodiment, at least one alternative reading frame is deleted, for example, at least two, at least three, or more alternative reading frames are deleted. Exemplary modifications to the wild-type CYP46A1 nucleic acid sequence (SEQ ID NO: 1) that result in the removal of alternative reading frames are listed in Table 23 below.

[0280] In various embodiments, the CYP46A1 nucleic acid sequence is codon-optimized through increasing the overall GC content, i.e., the percentage of guanine and cytosine throughout the coding sequence; removing cryptic splice donor or acceptor sites; and / or adding or removing ribosome entry and / or start sites, e.g., Kozak sequences.

[0281] In one embodiment, a 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" listed in Table 23 are meant to be exemplary and not limiting. For example, T222 can be replaced with "A" as listed in Table 23. However, T22 can also be replaced with "G" or "C," each modification of which may result in the elimination of an alternative reading frame. In one embodiment, a 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. [Table 23]

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

[0283] In one embodiment, the CYP46A1 protein is encoded by a CYP46A1 nucleic acid sequence that has been codon-optimized, for example, for one or more of: (1) enhanced in vivo expression, (2) reduced CpG islands, or (3) reduced innate immune response.

[0284] In some embodiments, the codon-optimized CYP46A1 variant exhibits enhanced long-term expression compared to wild-type CYP46A1, such as for 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 embodiments, the codon-optimized CYP46A1 variant exhibits a reduced immune response compared to wild-type CYP46A1.

[0286] In some embodiments, rAAV encoding CYP46A1 and / or CYP46A1 variants is generated by using a closed linear DNA molecule lacking bacterial backbone sequences.

[0287] In one aspect, provided herein is a composition comprising an isolated nucleic acid comprising a sequence 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 one aspect, provided herein is a composition comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence 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 a CYP46A1 protein comprises 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 compared to SEQ ID NO: 1. The foregoing compositions can be used, for example, in the absence of administered miRNA to treat a neurological disease or disorder described herein.

[0288] [ka] [ka] [ka]

[0289] [ka] [ka]

[0290] vector Without wishing to be bound by any particular theory, allele-specific silencing of a pathogenic gene, such as mutant huntingtin (HTT), may provide an improved safety profile in subjects compared to non-allele-specific silencing (e.g., silencing both wild-type and mutant HTT alleles), since wild-type expression and function are preserved in cells. For example, embodiments of the present invention relate to the inventors' recognition and understanding that isolated nucleic acids and vectors incorporating one or more inhibitory RNA (e.g., miRNA) sequences that target the HTT gene in a non-allele-specific manner, while driving the expression of a hardened wild-type HTT gene (a wild-type HTT gene that is not targeted by miRNA), can achieve concomitant mutant HTT knockdown, for example, in CNS tissues with increased expression of wild-type HTT. Generally, the sequences of the nucleic acids encoding endogenous wild-type and mutant HTT mRNAs and the nucleic acid of the transgene encoding the "hardened" wild-type HTT mRNA are sufficiently different so that the "hardened" wild-type HTT transgene mRNA is not targeted by one or more inhibitory RNAs (e.g., miRNAs). This can be achieved, for example, by introducing one or more silent mutations into the HTT transgene sequence, resulting in a protein encoding 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, inhibitory RNAs (e.g., miRNAs) can target the 5' and / or 3' untranslated regions of the endogenous wild-type HTT mRNA. These 5' and / or 3' regions can then be removed or replaced in the transgene mRNA so that the transgene mRNA is not targeted by one or more inhibitory RNAs.

[0291] Reporter sequences (e.g., nucleic acid sequences encoding reporter proteins) that can be provided in a transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with regulatory elements that drive their expression, reporter sequences provide signals that can be detected by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorescent assays or other spectroscopic assays, fluorescent-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is a LacZ gene, the presence of a signal-carrying vector is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-carrying vector may be visually measured by color or light production in a luminescence spectrometer. Such reporters can be useful, for example, to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of nucleic acids, such as recombinant adeno-associated viruses (rAAVs).

[0292] In some embodiments, the vector is an adeno-associated virus (AAV) or a recombinant AAV. In some aspects, the present disclosure provides an isolated AAV. When used herein with respect to AAV, the term "isolated" refers to an artificially produced or obtained AAV. An isolated AAV may be produced using recombinant methods. Such an AAV is referred to herein as a "recombinant AAV." Recombinant AAV (rAAV) preferably has tissue-specific targeting capabilities, so that the nuclease and / or transgene of the rAAV is specifically delivered to one or more predetermined tissues. The AAV capsid is an important factor in determining these tissue-specific targeting capabilities. Therefore, a rAAV having a capsid appropriate for the tissue to be targeted can be selected.

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

[0294] Methods for obtaining recombinant AAVs with desired capsid proteins are well known in the art (see, e.g., US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing host cells containing a nucleic acid sequence encoding the AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to enable packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the AAV cap gene. AAVs contain three capsid proteins, designated virion proteins 1 to 3 (VP1, VP2, and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, 72 kDa, and 62 kDa, respectively. In some embodiments, during translation, capsid protein forms a spherical 60-mer protein shell around the viral genome.In some embodiments, the function of capsid protein is to protect the viral genome, deliver the genome, and interact with the host.In some aspects, capsid protein delivers the viral genome to the host in a tissue-specific manner.

[0295] In some embodiments, the recombinant AAV (rAAV) capsid protein is of an AAV serotype selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV 2G9, AAV 2.5G9, AAV9, AAVDJ8, AAV-PHP.eB, and AAV10. In some embodiments, the AAV capsid protein is of a serotype derived from a non-human primate, such as the AAVrh10 serotype. In some embodiments, the AAV capsid protein is of 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 from serotype AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 2G9, AAV 2.5G9, AAV rh8, AAV rh10, AAV rh74, AAV10, or AAV11, or a chimera thereof. In certain embodiments, the rAAV comprises a chemically modified capsid disclosed in WO2017 / 212019, for example, a mannose ligand is chemically coupled to AAV2. The rAAV with the chemically modified capsid disclosed in WO2017 / 212019 is incorporated herein by reference in its entirety. In further embodiments, the AAV capsid proteins and viral capsids of the present invention may be polyploid (also referred to as rational singular), in that they may contain different combinations of VP1, VP2, and VP3 AAV serotypes from AAV serotypes in a single AAV capsid, as described in PCT / US18 / 22725, PCT / US2018 / 044632, or US 10,550,405, which are incorporated by reference.

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

[0297] In one embodiment, the AAV is AAVrhlO or a derivative thereof.

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

[0299] The components cultured in the host cell to package the rAAV vector into an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components using methods known to those of skill in the art. Most preferably, such a stable host cell contains the required components under the control of an inducible promoter. However, the required components may also be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein in the discussion of regulatory elements suitable for use with transgenes. In yet another alternative, the selected stable host cell may contain selected 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 can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by those skilled in the art. In some embodiments, the present disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a protein (e.g., a wild-type huntingtin protein, optionally a "hardened" wild-type huntingtin protein). In some embodiments, the present disclosure relates to a composition comprising the host cell described above. In some embodiments, the composition comprising the host cell further comprises a cryopreservation agent.

[0300] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to generate the rAAV of the present disclosure may be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure are known to those skilled in 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 selection of a suitable method is not a limitation on the present disclosure. See, for example, K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0301] In some embodiments, recombinant AAV may be produced using a triple transfection method (described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include the pHLP19 vector described in U.S. Pat. No. 6,001,650 and the pRep6cap6 vector described in U.S. Pat. No. 6,156,303, both of which are incorporated herein by reference in their entireties. Accessory function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (i.e., "accessory functions") on which AAV replication depends. Accessory functions include those functions required for AAV replication, including, but not limited to, moieties involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0302] In some embodiments, the triple transfection method uses a closed linear DNA molecule lacking bacterial backbone sequences, for example, as described in PCT / US2021 / 013689, published as WO / 2021 / 146591, which is incorporated by reference herein in its entirety.

[0303] In some aspects, the present disclosure provides a transfected host cell. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are generally known in the art. 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. Using such techniques, one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into a suitable host cell.

[0304] "Host cell" refers to any cell that harbors or is capable of harboring a substance of interest. Host cells are often mammalian cells. Host cells may be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNA involved in the generation of recombinant AAV. The term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" may refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in overall genetic or DNA complement to the original parent due to natural, accidental, or deliberate mutations.

[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. In many cases, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during the storage or transfer of such clonal populations. Thus, the cells derived from the cell line referred to may not be exactly identical to the ancestral cell or culture, and the cell line referred to includes such variants.

[0306] As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA.

[0307] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which, when associated with the appropriate control elements, is capable of replication and can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles as well as viral vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Certain vectors are also capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0308] A cloning vector is one that can replicate autonomously or integrate into the genome of a host cell, and is further characterized by one or more endonuclease restriction sites, at which the vector can be cut in a determinable manner and the desired DNA sequence can be ligated so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within a host cell, such as a host bacterium, or can occur only once per host before the host reproduces by mitosis. In the case of a phage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0309] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in identifying cells transformed or transfected with the vector, or cells that have not been transformed or transfected. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity is detectable 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 autonomous replication and expression of structural gene products present in the DNA segments to which they are operably linked.

[0310] The present invention further provides a vector comprising the synthetic CNS-specific promoter or expression cassette of the present invention. In some embodiments of the present invention, the vector is a plasmid. Such a plasmid may contain various other functional nucleic acid sequences, such as one or more selection markers, one or more origins of replication, multiple cloning sites, etc. In some embodiments of the present invention, the vector is a viral vector.

[0311] In some embodiments of the present 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 cells, the adenovirus vectors pSV and pCMV series vectors are particularly well known, non-limiting examples. There are many well-known yeast expression vectors, including, but not limited to, yeast integrating plasmids (YIp) and yeast replicating plasmids (YRp). For plants, the Ti plasmid of Agrobacterium is an exemplary expression vector, and plant viruses also provide suitable expression vectors, for example, 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, including AAV vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors. When the vector is a gene therapy vector, the vector preferably comprises a nucleic acid sequence operably linked to the synthetic CNS-specific promoter of the present invention that encodes a therapeutic product, preferably a therapeutic protein. The therapeutic protein may be a secretable protein. Non-limiting examples of secretable proteins are discussed above, and exemplary secretable therapeutic proteins include clotting factors, such as factor VIII or factor IX, insulin, erythropoietin, lipoprotein lipase, antibodies or nanobodies, growth factors, cytokines, chemokines, plasma factors, toxic proteins, and the like.

[0313] In some embodiments, useful vectors are intended to be those vectors in which the nucleic acid segment to be transcribed is placed under the transcriptional control of a promoter.When it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably linked if the induction of the promoter in the 5'regulatory sequence results in the transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frameshift mutations, (2) does not interfere with the promoter region's ability to direct the transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into protein.Therefore, a promoter region will be operably linked to a coding sequence if the promoter region can cause the transcription of that DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide.

[0314] A "promoter" refers to a DNA sequence recognized by or introduced into the synthetic machinery of a cell, necessary to initiate the specific transcription of a gene. When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, various transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., a promoter of a gene in its endogenous context, which provides normal regulation of the gene's expression. In some embodiments, the promoter can be constitutive, i.e., the promoter is not regulated and allows continuous transcription of its associated gene. Various conditional promoters can also be used, for example, promoters controlled by the presence or absence of a molecule.

[0315] The term "synthetic promoter" as used herein relates to a promoter that does not occur in nature. In this context, it typically comprises a CRE of the present invention operably 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 act to enhance the CNS-specific transcription of a gene operably linked to the synthetic promoter. Although portions of a synthetic promoter may occur in nature (e.g., a minimal promoter or one or more CREs in a promoter), the synthetic promoter as a complete entity does not occur in nature.

[0316] The exact nature of the regulatory sequences necessary for gene expression may vary between species or cell types, but generally may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also optionally include enhancer sequences or upstream activator sequences. Vectors of the present invention may also optionally include 5' leader or signal sequences. The selection and design of appropriate vectors is within the ability and discretion of one 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, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing heterologous DNA (RNA) into the cells. This heterologous DNA (RNA) is placed under the operable control of transcriptional elements that allow the expression of the heterologous DNA in the host cell.

[0318] The phrases "operably positioned," "under control," or "under transcriptional control" mean that a promoter is in the correct position and orientation relative to a nucleic acid to control the initiation of RNA polymerase and expression of a gene. The term "expression vector or construct" refers to any type of genetic construct containing a nucleic acid capable of transcribing part or all of a nucleic acid coding sequence. In some embodiments, expression includes transcription of a nucleic acid, for example, to produce a biologically active polypeptide product or functional RNA (e.g., guide RNA) from the transcribed gene.

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

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

[0321] In some embodiments of any of the aspects, nucleic acids (e.g., miRNAs) are chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference. Modifications include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.), (b) base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that base-pair with a wide range of partners, base removal (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) backbone modifications, including modification or replacement of phosphodiester linkages. Specific examples of nucleic acid compounds useful in the embodiments described herein include, but are not limited to, nucleic acids containing modified backbones or not containing natural internucleoside linkages. Nucleic acids with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification and as sometimes referred to in the art, modified nucleic acids that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides. In some embodiments of any of the aspects, the modified nucleic acid has a phosphorus atom in its internucleoside backbone.

[0322] Modified nucleic acid backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl phosphonates and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included. Modified nucleic acid backbones that do not contain phosphorus atoms therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others with mixed N, O, S, and CH2 constituent moieties, and heteroatoms. Included are oligonucleosides having atomic backbones, and in particular those having --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [known as the methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [where the native phosphodiester backbone is represented as --O--P--O--CH2--].

[0323] In other nucleic acid mimics, both the sugar and internucleoside linkages, i.e., the backbone, of nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0324] Nucleic acid can also be modified to include one or more locked nucleic acids (LNA).Locked nucleic acids are nucleotides with modified ribose moieties, in which the ribose moiety includes an extra bridge connecting the 2' and 4' carbons.This structure effectively "locks" the ribose in the 3'-endo structure conformation.The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol. Canc. Ther. 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0325] Modified nucleic acids can also contain one or more substituted sugar moieties. The nucleic acids described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted, C1-C10 alkyl, or C2-C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, the nucleic acid comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, or a group for improving the pharmacodynamic properties of a nucleic acid, and other substituents with similar properties. In some embodiments of any of the aspects, the modification includes 2'-methoxyethoxy (2'-O--CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples herein 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, as also described in the Examples herein below.

[0326] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in nucleic acids, particularly the 3' position of the sugar in the 3'-terminal nucleotide or in 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. Nucleic acids can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0327] Nucleic acids can also contain modifications or substitutions of nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. Other synthetic and natural nucleobases may be included, including pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification. In some embodiments of any of the aspects, modified nucleobases can include d5SICS and dNAM, which are non-limiting examples of unnatural nucleobases that can be used separately or together as base pairs (see, e.g., 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 of the aspects, the oligonucleotide tag (e.g., Oligopaint) comprises any modified nucleobase known in the art, i.e., any nucleobase that is modified from an unmodified and / or naturally occurring nucleobase.

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

[0329] Another modification of nucleic acids featured in the present invention involves chemically linking to the nucleic acid one or more ligands, moieties, 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, cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterols (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 20:533-538), 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-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or lipid moiety such as octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0330] In some embodiments of any of the aspects, the vector is pEMBL. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1-hCG intron only. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1-hCGin-2x regulatory pre-miR. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1-hCGin-2x artificial pre-miR. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1-CYP46A1-hCGin-2x artificial pre-miR. In some embodiments of any of the aspects, the vector is pEMBL-D(+)Syn1-luc-HTT-3'UTR / mutant. In some embodiments of any of the aspects, the vector comprises at least one of the following: at least one (e.g., two) ITRs; a Syn1 promoter; at least one (e.g., two) hCG intron; at least one (e.g., two) copies of a premiR (e.g., a control premiR; an artificial premiR; SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260); a small polyA; CYP46A1; luciferase; an HTT targeting sequence; and / or an HTT-3'UTR / mutant. In some embodiments, the vector comprises a neuron-specific synthetic promoter selected from Tables 10-13 and / or a CRE selected from Tables 13-15. In certain aspects of the embodiments, the miRNA targets a wild-type HTT allele. In other aspects of the embodiments, the miRNA targets a mutant HTT allele. In yet other embodiments, the miRNA targets both the wild-type and mutant HTT alleles. In yet another embodiment, the miRNA targets any HTT mRNA.

[0331] In some embodiments, one or more of the recombinantly expressed genes may be integrated into the genome of the cell.

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

[0333] In some embodiments, the promoter is a synapsin (Syn1) promoter (see, e.g., SEQ ID NO: 152). In one aspect, the promoter comprises a nucleic acid sequence 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, provided herein is a composition comprising a recombinant viral vector comprising a promoter comprising a nucleic acid sequence 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] [ka]

[0335] In one aspect, provided herein is a composition comprising an isolated nucleic acid comprising a sequence 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 one aspect, provided herein is a composition comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence 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, e.g., Tables 10-13) or a fragment thereof, an enhancer, and / or a cis-regulatory element (CRE; see, e.g., Tables 13-15). In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the promoter is an ACTB proximal promoter. In some embodiments, the vector further comprises an intron. In some embodiments, the intron comprises an ACTB intron / chimeric ACTB-HBB2 intron. See, for example, SEQ ID NO: 111, Table 16. The aforementioned compositions can be used in the absence of administered miRNA, for example, to treat the neurological diseases or disorders described herein.

[0336] Codon-optimized CYP46 sequence SEQ ID NO: 111, pJAL130-CYP46A1, 4036 bp ITR-ITR sequence (see, e.g., SEQ ID NO: 110; see, e.g., Figure 1).

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

[0338] Italicized text (eg, nt 182-436 of SEQ ID NO: 111) indicates enhancers.

[0339] Bold italicized text (eg, nt 550-804 of SEQ ID NO: 111) indicates the promoter.

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

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

[0342] Italicized, double-underlined text (eg, nt 3629-3853 of SEQ ID NO: 111) indicates polyA.

[0343] Bold, italicized, double-underlined text (eg, nt 3907-4036 of SEQ ID NO: 111) indicates the right ITR. [ka] [ka]

[0344] [Table 16-1] [Table 16-2]

[0345] In one aspect, provided herein is a composition comprising an isolated nucleic acid comprising a sequence 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: 194. In one aspect, provided herein is a composition comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence 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: 194. In some embodiments, the vector (e.g., rAAV) comprises a promoter (e.g., a synthetic nervous system-specific promoter; see, e.g., Tables 10-13) or a fragment thereof, an enhancer, and / or a cis-regulatory element (CRE; see, e.g., Tables 13-15). In some embodiments, the enhancer is a CMV enhancer. In some embodiments, the promoter is an ACTB proximal promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the vector further comprises an intron. In some embodiments, the intron comprises an ACTB intron / chimeric ACTB-HBB2 intron. The aforementioned compositions can be used in the absence of administered miRNA, for example, to treat a neurological disease or disorder described herein.

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

[0347] Base pairs 1 to 28 of SEQ ID NO:194 set forth the TelN protelomerase recognition sequence, i.e., SEQ ID NO:195.

[0348] Base pairs 29 to 37 of SEQ ID NO:194 represent a linker sequence, ie, SEQ ID NO:196.

[0349] Base pairs 38 to 249 of SEQ ID NO:194 describe the 5' stuffer sequence, i.e., SEQ ID NO:197.

[0350] Base pairs 250 to 271 of SEQ ID NO:194 represent a linker sequence, ie, SEQ ID NO:198.

[0351] Base pairs 272 to 401 of SEQ ID NO:194 describe the L-ITR sequence, ie, SEQ ID NO:199.

[0352] Base pairs 402 to 452 of SEQ ID NO:194 represent a linker sequence, ie, SEQ ID NO:200.

[0353] Base pairs 453 to 2073 of SEQ ID NO: 194 describe the CAG promoter sequence, i.e., SEQ ID NO: 193.

[0354] Base pairs 2074 to 2114 of SEQ ID NO: 194 describe the HBB2 intron sequence, ie, SEQ ID NO: 201.

[0355] Base pairs 2115 to 2236 of SEQ ID NO: 194 represent a linker sequence, i.e., SEQ ID NO: 202.

[0356] Base pairs 2237-2379 of SEQ ID NO: 194 set forth the CYP46A1 sequence, i.e., SEQ ID NO: 212 (SEQ ID NO: 212 is the sequence of SEQ ID NO: 110 with an additional 3 base pairs at the C-terminus that translates to a stop codon).

[0357] Base pairs 2380 to 3899 of SEQ ID NO: 194 represent a linker sequence, i.e., SEQ ID NO: 203.

[0358] Base pairs 3900 to 4124 of SEQ ID NO:194 describe the bGH poly(A) signal sequence, ie, SEQ ID NO:204.

[0359] Base pairs 4125 to 4177 of SEQ ID NO:194 represent a linker sequence, i.e., SEQ ID NO:205.

[0360] Base pairs 4178 to 4307 of SEQ ID NO:194 describe the R-ITR sequence, i.e., SEQ ID NO:206.

[0361] Base pairs 4308 to 4321 of SEQ ID NO:194 represent a linker sequence, i.e., SEQ ID NO:207.

[0362] Base pairs 4322 to 4533 of SEQ ID NO:194 describe the 3' stuffer sequence, i.e., SEQ ID NO:208.

[0363] Base pairs 4534 to 4545 of SEQ ID NO: 194 represent a linker sequence, i.e., SEQ ID NO: 209.

[0364] Base pairs 4546 to 4573 of SEQ ID NO: 194 describe the TelN protelomerase recognition sequence, i.e., SEQ ID NO: 210. [Table 25]

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

[0366] A gene typically encodes a desired gene expression product, e.g., a polypeptide (protein) or RNA. A gene may be a full-length cDNA or genomic DNA sequence, or any fragment, subunit, or mutant thereof that has at least some desired biological activity.

[0367] If the gene encodes a protein, it can be essentially any kind of protein. As 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, 8, or 9).

[0368] Various expression products suitable for treating the above-mentioned conditions have been described in the art. Preferably, the nucleic acid encoding the expression product operably linked to the CRE, minimal / proximal promoter or promoter according to the present invention can be one of the genes selected from the group consisting of NPC1, EAAT2, NPY, CYP46A1, GLB1, 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. Additionally or alternatively, the expression product operably linked to a CRE, minimal / proximal promoter or promoter according to the present invention may be a miRNA / CRISPR Cas9 directed to a disease allele.

[0369] In some embodiments, useful expression products include dystrophin (including micro-dystrophin), beta 1,4-n-acetylgalactosamine galactosyltransferase (GALGT2), carbamoyl synthetase I, alpha-1 antitrypsin, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, glucose-6-phosphatase, porphobilinogen deaminase, cystatin beta hydroxylase, and the like. These include β-synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, cystic fibrosis transmembrane conductance regulator (CFTR), and dopaminergic transporter (hDAT).

[0370] Still other useful expression products include enzymes useful in enzyme replacement therapy, which are useful in a variety of conditions resulting from a deficiency in enzyme activity. For example, enzymes containing mannose-6-phosphate may be used in treatments for lysosomal storage diseases (e.g., suitable genes include those encoding β-glucuronidase (GUSB)).

[0371] In some embodiments, exemplary polypeptide expression products include neuroprotective polypeptides and anti-angiogenic polypeptides. Suitable polypeptides 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-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelium-derived factor (PEDF), Wnt polypeptides, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibodies, soluble VEGFR, factor VIII (FVIII), factor IX (FIX), and members of the hedgehog family (such as sonic hedgehog, Indian hedgehog, and desert hedgehog).

[0372] In some embodiments, useful therapeutic expression products include, but are 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 growth factor I and II (IGF-I and IGF-II), TGFα, activin, inhibin, or bone morphogenetic protein. hormones and growth and differentiation factors, including any one of the transforming growth factor alpha superfamily, including any of bone marrow protein (BMP) BMP1-15, any one of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurturin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, 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 (IL) IL-1 through 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 alpha and beta, interferons (alpha, beta, and gamma), stem cell factor, and flk-2 / flt3 ligand. Gene products produced by the immune system are also useful in the present 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 accelerating factor (DAF), CR1, CF2, and CD59.

[0374] In some embodiments, useful expression products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. 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 present invention also encompasses the use of gene products, for example, members of the steroid hormone receptor superfamily, including glucocorticoid and estrogen receptors, vitamin D receptors, and other nuclear receptors. Additionally, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MyoD, and myogenin, ETS box-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, STATs, GATA box-binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0375] In some embodiments, useful expression products include non-naturally occurring polypeptides, for example, chimeric or hybrid polypeptides having a non-naturally occurring amino acid sequence containing insertions, deletions, or amino acid substitutions.

[0376] Further suitable expression products include microRNA (miRNA), interfering RNA, antisense RNA, ribozymes and aptamers.

[0377] In some embodiments of the present invention, the synthetic CNS-specific expression cassette comprises a gene useful for gene editing, such as a gene encoding a site-specific nuclease, such as a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats system (CRISPR-Cas). Preferably, the site-specific nuclease is adapted to edit the desired target genomic locus by making a cut (typically a site-specific double-strand cut) and then repairing it via non-homologous end joining (NHEJ) or homology-dependent repair (HDR), resulting in the desired edit. Editing can be partial or complete repair of a dysfunctional gene or knockdown or knockout of a functional gene. Alternatively, editing can be via base editing or prime editing using a suitable system known in the art.

[0378] Preferably, the synthetic CNS-specific expression cassette comprises a sequence providing or encoding one or more, preferably all, of a ribosome binding site, a start codon, a stop codon, and a transcription termination sequence. Preferably, the expression cassette comprises a nucleic acid encoding a post-transcriptional regulatory element. Preferably, the expression cassette comprises a nucleic acid encoding a polyA element.

[0379] Modified capsid In one embodiment, the capsid described herein is further modified to increase tropism for the CNS. Provided herein is a composition comprising a modified viral capsid comprising a payload, wherein the payload comprises a regulatory sequence and a nucleic acid sequence flanked by inverted terminal repeats (ITRs) that target a central nervous system disorder, and the modification is a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the payload 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] Further provided herein is a composition comprising (a) a first modified viral capsid comprising a first payload, and (b) at least a second modified viral capsid comprising a second payload, wherein the payload comprises a regulatory sequence and a nucleic acid sequence flanked by inverted terminal repeats (ITRs) targeting a central nervous system disorder, wherein the first and at least a second modified viral capsid are the same, and the first and second payloads are different, and the modification is a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

[0381] Further provided herein is a composition comprising (a) a first modified capsid comprising a first payload, and (b) at least a second modified capsid comprising a second payload, wherein the payload comprises a regulatory sequence and a nucleic acid sequence flanked by inverted terminal repeats (ITRs) targeting a central nervous system disorder, wherein the first and at least the second modified capsids are different, and the first and second payloads can be the same or different, and the modification is a chemical modification, a non-chemical modification, or an amino acid modification. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the first or second payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

[0382] Another embodiment described herein provides a composition or combination comprising 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. In one embodiment, a composition or combination is described herein comprising: (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (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.

[0383] Another embodiment described herein provides a composition or combination comprising (a) an isolated nucleic acid encoding a transgene encoding an isolated nucleic acid encoding a CYP46A1 protein. In some embodiments, the composition or combination further comprises (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In other embodiments, the composition or combination does not comprise (b) an isolated nucleic acid encoding a transgene encoding one or more miRNAs. In one embodiment, a composition or combination is described herein comprising (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and (ii) a second region comprising a transgene encoding a CYP46A1 protein. In some embodiments, the composition or combination further comprises (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs. In other embodiments, the composition or combination does not comprise (b) a recombinant viral vector comprising an isolated nucleic acid encoding one or more miRNAs.

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

[0385] In certain embodiments, modified viral capsid comprises modifications that cause its preferential targeting to CNS or PNS.For example, modified viral capsid has increased tropism to CNS, and / or reduced tropism to at least a second location, for example, liver.Preferential targeting to CNS does not exclude targeting to other sites, but rather indicates that it is more highly targeted to CNS compared with other sites.

[0386] In one embodiment, the modified viral capsid comprises a modification that results in its targeting of the CNS or PNS. For example, modifications to a capsid that typically targets a non-CNS site (e.g., the liver) can redirect the capsid to immediately target both CNS and non-CNS sites. In such an embodiment, CNS targeting does not need to be preferential.

[0387] In one embodiment, the capsid modification is an amino acid modification, such as an amino acid deletion, insertion, or substitution. In one embodiment, the amino acid modification increases tropism to 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 comprises, consists of, or consists essentially of a nucleic acid sequence whose contents are 90% identical to SEQ ID NOS: 1-4 of U.S. Patent Application No. 16 / 511,913, the entire contents of which are incorporated herein by reference. This U.S. patent application describes chimeric AAV capsid sequences that exhibit a dominant tropism for oligodendrocytes and can be used to generate AAV vectors that transduce oligodendrocytes in the CNS of a subject.

[0389] In one embodiment, the modified viral capsid is an AAV capsid protein containing one or more amino acid substitutions, wherein the substitutions introduce new glycan binding sites into the AAV capsid protein. In some embodiments, the amino acid substitutions are at amino acid 266, amino acids 463-475, and amino acids 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 comprises, consists of, or essentially consists of an AAV 2.5 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) containing one or more amino acid substitutions that introduce new glycan binding sites. Such amino acid substitutions can target the capsid to neurons and glial cells, such as astrocytes. In embodiments of the capsid proteins, capsids, viral vectors, and methods described in International Patent Application No. PCT / US2020 / 029493, the one or more amino acid substitutions include A267S, SQAGASDIRDQSR464-476SX1AGX2SX3X4X5X6QX7R (SEQ ID NOs: 153 and 154, respectively) (wherein X1-7 can be any amino acid), and EYSW500-503 (SEQ ID NO: 155)EX8X9W (wherein X8-9 can be any amino acid). In embodiments of the capsid protein, capsid, viral vector, and method described herein, X1 is V or a conservative substitution thereof; X2 is P or a conservative substitution thereof; X3 is N or a conservative substitution thereof; X4 is M or a conservative substitution thereof; X5 is A or a conservative substitution thereof; X6 is V or a conservative substitution thereof; X7 is G or a conservative substitution thereof; X8 is F or a conservative substitution thereof; and / or X9 is A or a conservative 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 new glycan binding site is a galactose binding site. Such AAV capsid proteins are further described, for example, in International Patent Application No. PCT / US2020 / 029493, 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, such as those described in U.S. Patent Application No. 16 / 956,306, 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, the contents of which are incorporated herein by reference in their entirety. Such AAV capsids cross the blood-brain barrier, enabling, for example, delivery of a payload.

[0392] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of the capsid protein has a modification (e.g., replacement of a tyrosine residue with a non-tyrosine residue and / or replacement of a threonine residue with a non-threonine residue) to a 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). The capsid protein may contain one or more of the following residues: Y705, Y731, and T492 of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16 / 565,191); one or more of the following residues: 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 of the following residues: Y705, Y731, and T492 of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 ...). Such AAV capsids target neurons and astrocytes.

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

[0394] In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., an AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of the capsid protein has modifications (e.g., replacement of a tyrosine residue with a non-tyrosine residue, and / or replacement of a threonine residue with a non-threonine residue) at Y705, Y731, and Y732 of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191). One or more of Y436, Y693, and Y719, or each of them, of wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 in U.S. Patent Application No. 16 / 565,191); or one or more of Y705, Y731, and T492, or each of them, of wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 in U.S. Patent Application No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0395] In one embodiment, the modified viral capsid replaces a Y to F (tyrosine to phenylalanine) or a T to V (threonine to valine) modification in the VP3 region of the capsid protein with one or more of: Y705F, Y731F, and T492V of the wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16 / 565,191); The AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein) contains one or more of Y436F, Y693F, and Y719F, or each of them, in SEQ ID NO:2 of U.S. Patent Application No. 16 / 565,191; or one or more of Y705F, Y731F, and T492V, or each of them, in a position corresponding to a wild-type AAV6 capsid protein (e.g., SEQ ID NO:3 of U.S. Patent Application No. 16 / 565,191). Such AAV capsids target neurons and astrocytes.

[0396] In one embodiment, the amino acid modification allows the modified capsid to avoid, for example, neutralizing antibodies raised against, for example, a viral vector of the same serotype, hi one embodiment, the amino acid modification allows the modified capsid to be used for repeated administration, e.g., the modification allows the capsid to have a therapeutic effect upon re-administration.

[0397] In one embodiment, the modified viral capsid is a chimeric capsid. A "chimeric" capsid protein, as used herein, refers to an AAV capsid protein (e.g., any one or more of VP1, VP2, or VP3) that has been modified by the substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein compared to the wild-type, as well as the insertion and / or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence compared to the wild-type. In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype can be substituted for corresponding wild-type domains, functional regions, epitopes, etc. from a different AAV serotype in any combination to generate a chimeric capsid protein of the present invention. The production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a number of chimeric capsid proteins that can be included in the capsids of the invention have been described in the literature and herein.

[0398] In one embodiment, the modified viral capsid is a monoploid capsid. As used herein, the term "monoploid AAV" refers to the AAV described in International Application No. WO2018 / 170310 or US Application No. US2018 / 037149, the entire contents of which are incorporated herein by reference. In some embodiments, the population of virions is a monoploid AAV population capable of assembling virion particles, wherein at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins 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 example, at least one viral protein 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 not chimeric. For example, only viral particles composed of VP1 / VP2 from chimeric AAV2 / 8 (N-terminus of AAV2 and C-terminus of AAV8) paired with only VP3 from AAV2, or only chimeric VP1 / VP2 28m-2P3 (N-terminus from AAV8 without VP3 start codon mutation and C-terminus from AAV2) paired with only VP3 from AAV2. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are not chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1 / VP2 28m-2P3 paired with VP3 from AAV3. In another example, no chimeric protein exists.

[0399] In some embodiments of the technology described herein, the modified viral capsid comprises one or more modifications, such as chemical, non-chemical, or amino acid modifications to the capsid, which can, for example, alter the tissue-type or cell-type tropism of the modified capsid, among other things.

[0400] The modifications can directly alter the properties of the capsid, including biochemical properties such as receptor binding, so that the modifications themselves alter the behavior of the capsid, or can allow for further modifications, such as the attachment of ligands, that in turn alter 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 by genetic modification of the capsid polypeptide coding sequence, allows for covalent attachment of a ligand via disulfide bond formation (see, e.g., WO2005 / 106046, the contents of which are incorporated herein by reference).

[0402] A variety of ligands are contemplated, including, but not limited to, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by the target cell (see, e.g., WO2000 / 002654, which is incorporated herein by reference).

[0403] WO2015 / 062516, the contents of which are also incorporated herein by reference, describes genetic modification of capsid genes to insert amino acids containing azide groups, followed by chemical conjugation of ligands via the azide groups.

[0404] Altering AAV capsid tropism by glycosylation, or chemical conjugation of sugar moieties, is described by Horowitz et al., Bioconjugate Chem. 22: 529-532 (2011). This and similar approaches are contemplated for the capsid modifications described herein.

[0405] In other embodiments, coating of viral capsids with polymers such as polyethylene glycol (PEG) or poly-(N-hydroxypropyl) methacrylamide (pHPMA) is specifically contemplated. Such modifications can, for example, reduce specific and nonspecific interactions with non-target tissues.

[0406] In other embodiments, carbodiimide coupling is specifically contemplated. See, e.g., 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, viral capsids can be modified, for example, as described in WO 2017 / 212019 (see also U.S. National Phase Publication No. USSN 16 / 308,740, the contents of which are each incorporated herein by reference). The method described therein couples the viral capsid to a ligand via a bond containing -CSNH- and an aromatic moiety. Genetically modified viral capsids can be further modified using this method, but the modifications described therein do not require genetic modification of the viral capsid. The ligands described therein include, for example, targeting agents, steric shielding agents to avoid 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 mimics cell adhesion proteins and can bind to integrins), vitamins, and small molecules.

[0408] In one embodiment, the chemical modification of the present invention is a modification described in International Patent Application No. 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 a modification described in International Patent Application No. 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 in the capsid, wherein said chemically modified tyrosine residue has a structure represented by formula (I): [ka] It is of

[0411] [In the formula,

[0412] - X1 [ka] is selected from the group consisting of

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

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

[0415] [In the formula,

[0416] Xi and Ar are as defined herein above,

[0417] - spacer is a group for linking the "Ar" group to the functional moiety "M", which is in the form of a chemical chain containing up to 1000 carbon atoms and preferably containing, optionally, heteroatoms and / or cyclic moieties;

[0418] - n is 0 or 1,

[0419] - M is a functional moiety including a steric agent, labeling agent, cell type-specific ligand or drug moiety].

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

[0421] In one embodiment, the capsid has formula (Ic): [ka] and at least one chemically modified tyrosine having

[0422] [In the formula,

[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 in the para, meta or ortho position of the phenyl group, preferably in the para position;

[0425] - the spacer, n and M are as defined herein above].

[0426] In one embodiment, the "spacer", when present, is selected from the group consisting of an optionally substituted saturated or unsaturated, linear or branched C2-C40 hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-hydroxypropyl) methacrylamide), polylactic-co-glycolic acid (PLGA), polymers of alkyldiamines, and combinations thereof; and / or

[0427] "M" comprises or consists of a cell-type targeting ligand, preferably selected from mono- or polysaccharides, hormones including steroid hormones, peptides such as RGD peptides (e.g., Arg-Gly-Asp, a cell adhesion motif that mimics cell adhesion proteins and can bind to integrins), muscle-targeting peptide (MTP) or Angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies and fragments thereof including heavy chain antibodies, such as antigen-binding fragments (Fab), Fab' (which are antigen-binding fragments that further comprise a free sulfhydryl group), and VHHs, single-chain variable fragments (ScFv), spiegelmers, peptide aptamers, vitamins, and drugs, e.g., cannabinoid receptor 1 (CB1) and / or cannabinoid receptor 2 (CB2) ligands.

[0428] In one embodiment, the "spacer" (if present) is selected from the group consisting of linear or branched C2-C20 alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyldiamines, and combinations thereof, said polymers having 2 to 20 monomers, and / or "M" comprises or consists of a cell-type specific ligand derived from a protein selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor 13 (FGF), a mono- or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNac or mannose-6-phosphate, an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a vitamin, e.g., folic acid.

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

[0430] [In the formula,

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

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

[0433] In one embodiment, the capsid is incubated with a chemical reagent having a reactive group selected from an aryldiazonium and a 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moiety under conditions conducive to reacting said reactive group with a tyrosine residue present in the capsid to form a covalent bond.

[0434] In one embodiment, the capsid is incubated with a chemical reagent of formula VId to form a compound of formula Ic [ka] At least one chemically modified tyrosine residue in the capsid of the antibody is obtained.

[0435] Pharmaceutical Composition The expression cassettes, vectors or virions of the invention may be formulated into pharmaceutical compositions using a pharmaceutically acceptable excipient, i.e., one or more pharmaceutically acceptable carrier substances and / or additives, such as buffers, carriers, excipients, stabilizers, etc. The pharmaceutical composition may be provided in the form of a kit.

[0436] Thus, a further aspect of the present invention provides a pharmaceutical composition comprising an expression cassette, vector or virion as described herein.

[0437] In various embodiments, the pharmaceutical composition comprises a phosphate buffer solution containing about 1 mM to about 50 mM phosphate, e.g., 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 phosphate. The phosphate solution is prepared from a combination of a dibasic phosphate (e.g., NaHPO, KHPO) and a monobasic phosphate (e.g., NaHPO, KHPO) in a molar ratio of about 1:10 to about 10:1 dibasic phosphate:monobasic phosphate. For example, in various exemplary embodiments, 10 mM phosphate comprises 9.5 mM dibasic phosphate and 0.5 mM monobasic phosphate, 9 mM dibasic phosphate and 1 mM monobasic phosphate, 8.5 mM dibasic phosphate and 1.5 mM monobasic phosphate, or 8 mM dibasic phosphate and 2 mM monobasic phosphate. The pH of the phosphate buffer is about 6.5 to about 7.5, e.g., 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. The phosphate buffer may also contain NaCl at a concentration of about 50 mM to about 200 mM, for example, 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, 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, e.g., 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 MgCl at a concentration of about 0.10 mM to about 1 mM, e.g., 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™ F-68 nonionic surfactant) at a concentration of about 0.0001% to about 0.005% by weight, e.g., about 0.0001%, about 0.0002%, about 0.0003%, about 0.0004%, about 0.0005%, about 0.0006%, about 0.0007%, about 0.0008%, about 0.0009%, about 0.001%, about 0.0015%, about 0.002%, about 0.0025%, about 0.003%, about 0.0035%, about 0.004%, about 0.0045%, or about 0.005% by weight. The phosphate buffer may also contain sorbitol at a concentration of about 0.005% to about 10% by weight, e.g., about 0.005%, about 0.075%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight.The rAAV comprising the nucleic acid (e.g., AAVrh.10 comprising a CAG promoter and a nucleic acid comprising a sequence 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: 194; AAVrh10.CAG.hCYP46A1) is about 0.1 x 10. 9 vg / μl ~ approx. 5×10 10 Titer in phosphate buffer in vg / μl, e.g., about 0.1×10 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 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 It may have a titer of vg / μl.

[0438] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 0.4×10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0439] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 0.4×10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0440] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 1.1 x 10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0441] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 1.1 x 10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0442] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 3×10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0443] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 3×10 9vg / μl of AAVrh10.CAG.hCYP46A1.

[0444] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 7×10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0445] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 7×10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0446] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 8.2 x 10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0447] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 8.2 x 10 9 vg / μl of AAVrh10.CAG.hCYP46A1.

[0448] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 3×10 10vg / μl of AAVrh10.CAG.hCYP46A1.

[0449] In some embodiments, the pharmaceutical composition comprises 10 mM phosphate (9 mM NaHPO and 1 mM KHPO), 137 mM NaCl, 2.7 mM KCl, 0.88 mM CaCl, 0.49 mM MgCl, 0.001% by weight poloxamer 188, pH 7.2, and 3×10 10 vg / μl of AAVrh10.CAG.hCYP46A1.

[0450] In an alternative embodiment, the pharmaceutical composition has a phosphate concentration of about 9.53 mM, comprising about 8.06 mM NaHPO and about 1.47 mM KHPO, but is equivalent to any of the above exemplified compositions.

[0451] In an alternative embodiment, the pharmaceutical composition has a concentration of sorbitol of about 0.005% to about 10% by weight, but equivalent to any of the above exemplified compositions.

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

[0453] Delivery of rAAV to a mammalian subject may be, for example, by intramuscular injection or by administration into the mammalian subject's bloodstream. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical arts. This method essentially allows one skilled in the art to isolate a limb from the systemic circulation before administering rAAV virions. A variant of the isolated limb perfusion technique described in U.S. Pat. No. 6,177,403 can also be used by those skilled in the art to administer virions into the blood vessels of an isolated limb to potentially enhance transduction of muscle cells or tissues. In certain instances, it may also be desirable to deliver virions to the subject's CNS. "CNS" refers to all cells and tissues of the vertebrate brain and spinal cord. As such, this term includes, but is not limited to, neurons, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage, etc. Recombinant AAV can be delivered directly to the CNS or brain by injection into the ventricular region, and to the striatum (e.g., the caudate nucleus and / or putamen of the dorsal striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule using a needle, catheter, or related device, using neurosurgical techniques known in the art, such as by 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). In some embodiments, the rAAV described in the present disclosure is administered by intravenous injection. In some embodiments, rAAV is administered by intracerebral injection. In some embodiments, rAAV is administered by intrathecal injection. In some embodiments, rAAV is administered by intrastriatal injection. In some embodiments, rAAV is delivered by intracranial injection.In some embodiments, the rAAV is delivered by cisternal injection. In some embodiments, the rAAV is delivered by lateral ventricle injection of the brain.

[0454] In some embodiments, rAAV is administered by multiple injections along a single trajectory. For example, a cannula can be placed at a first location in a target structure (e.g., the putamen) through the brain, and a first injection is performed 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 is performed at the second location. Additional injections can be performed at additional locations within the target structure by extending or retracting the cannula along the same single trajectory. Targeting of brain structures can be performed by placing a cannula through the brain with trajectory-guided MRI. The trajectory can be anterior, for example, through the frontal bone, or posterior, for example, through the occipital bone. In other embodiments, multiple trajectories are used to target a single tissue or structure.

[0455] Aspects of the present disclosure relate to compositions comprising 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. In some embodiments, each miRNA comprises a sequence set forth in any one of SEQ ID NOS: 6-17, 40-44, 50-66, 158-185, or 217-260. In some embodiments, the nucleic acid further comprises AAV ITRs. In some embodiments, the ITRs are AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, or AAV13 ITRs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. Compositions of the present disclosure may comprise rAAV alone or in combination with one or more other viruses (e.g., a second rAAV carrying and encoding one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs, each carrying one or more different transgenes.

[0456] Suitable carriers can be easily selected by those skilled in the art, taking into account the indications for which rAAV is intended. For example, one suitable carrier includes saline, which may be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of carriers does not limit the present disclosure.

[0457] If necessary, the compositions of the present disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0458] The rAAV is administered in an amount sufficient to transfect cells of the desired tissue and provide sufficient 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 a selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parenteral routes of administration. Optionally, routes of administration may be combined. In some embodiments, all or at least one of the nucleic acid sequences disclosed herein are delivered via a non-viral DNA construct containing at least one DD-ITR. For example, the non-viral DNA constructs described in WO2019 / 246554 can be used to deliver one or more of the nucleic acids described herein. WO2019 / 246554 is incorporated herein by reference in its entirety.

[0459] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., a dose in genome copies per kilogram of body weight (GC / kg), will vary based on several factors, including, but not limited to, the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve the therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of ordinary skill in the art can readily determine the dose range of rAAV virions for treating a patient with a particular disease or disorder based on the aforementioned factors and other factors well known in the art.

[0460] The effective amount of rAAV is sufficient to target, infect, and target desired tissue in animals.In some embodiments, the effective amount of rAAV is sufficient to generate stable somatic transgenic animal models.Effective amount mainly depends on factors such as the species, age, weight, health, and tissue of the target subject, and therefore may vary between animals and tissues.For example, the effective amount of rAAV is generally about 109 ~10 16 In some cases, the volume of the solution containing the genome copies ranges from about 1 ml to about 100 ml. 11 ~10 13 In certain embodiments, a dosage of between 10 and 100 rAAV genome copies is appropriate. 12 or 10 13 rAAV genome copies are effective in targeting CNS tissue. In some cases, stable transgenic animals are generated by multiple doses of rAAV.

[0461] In other cases, the administered dose depends on the rAAV titer and the volume of tissue injected. For example, rAAV (or rAAV composition) can be administered in an amount of about 0.1 x 10 9 vg / μL ~ approx. 1.5×10 9 Titer of vg / µL, e.g., about 0.1 x 10 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 x 10 9The amount of injected agent may be in the range of 0.15 to 1.5 vg / µL. The amount of injected agent depends independently on the volume of at least one target tissue or structure as determined by MRI. For example, the volume of the injected tissue may be about 1% to about 90% of the total volume of the tissue or structure, e.g., 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 volume of the tissue or structure. Similarly, the volume of the tissue or structure 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 volume of the tissue or structure. When two or more tissues or structures are targeted (i.e., injected), the volumes of each tissue or structure injected can be the same or different. For example, in some embodiments, the titer is injected into the left and right putamen in separate volumes, e.g., equivalent to about 25% of the total volume of each putamen. In other embodiments, the titer is injected into the left and right caudate nuclei in separate volumes, e.g., equivalent to about 25% of the total volume of each caudate nucleus. In yet other embodiments, both the left and right putamen and both the left and right caudate nuclei receive injections of, for example, about 25% strength based on the total volume size of the respective putamen and caudate nuclei.

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

[0463] In some embodiments, the rAAV composition may be particularly useful in cases where a high concentration of rAAV is present (e.g., -10 13 The composition is formulated to reduce aggregation of AAV particles in the composition when the concentration of rAAV is less than 100 ng / ml (GC / ml or more). Methods for reducing aggregation of rAAV are well known in the art and include, for example, adding surfactants, adjusting pH, adjusting salt concentration, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178). (See, for example, US Pat. No. 6,229,999, the contents of which are incorporated herein by reference).

[0464] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable administration and treatment regimens for use with the particular compositions described herein in various treatment regimens.

[0465] Typically, these formulations may contain at least about 0.1% or more of the active compound, although the percentage of active ingredient may, of course, vary and may conveniently be between about 1% or 2% and about 70% or 80% or more of the total formulation weight or volume. Essentially, the amount of active compound in each therapeutically useful composition may be prepared in such a way that a suitable dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations; as such, various dosage and treatment regimens may be desirable.

[0466] In certain circumstances, it may be desirable to deliver the rAAV-based therapeutic construct in a suitably formulated pharmaceutical composition disclosed herein subcutaneously, intrapancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the rAAV may be delivered using the administration modalities described in U.S. Patent Nos. 5,543,158, 5,641,515, and 5,399,363 (each of which is specifically incorporated herein by reference in its entirety). In some embodiments, the preferred mode of administration is via portal vein injection.

[0467] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, this form is sterile and fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), a suitable mixture thereof, and / or vegetable oils. For example, proper fluidity can be maintained by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

[0468] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermic fluid or injected at the planned site of infusion (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0469] Sterile injectable solutions are prepared by incorporating the active rAAV with various other ingredients listed herein in the required amount into a suitable solvent, and then optionally sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0470] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), such as inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution is administered in a manner compatible with the dosage formulation and in such an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, etc.

[0471] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agent...

Claims

1. 1. A method for treating a neurological disease or disorder in a subject in need of treatment, comprising administering a therapeutically effective amount of (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) an isolated nucleic acid encoding a CYP46A1 protein; to a subject having or at risk of developing said neurological disease or disorder.

2. 1. A method for treating a neurological disease or disorder in a subject in need of treatment, comprising administering a therapeutically effective amount of (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof, and (ii) a second region comprising a transgene encoding one or more miRNAs; and (b) a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein. to a subject having or at risk of developing said neurological disease or disorder.

3. The method according to any one of claims 1 to 2, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinal cerebral ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten disease, Refsum disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, delusion, 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 neurological 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. 6. The method of any of claims 1-5, wherein the CNS disease or disorder is Alzheimer's disease and at least one miRNA comprises a seed sequence complementary to amyloid precursor protein (APP), presenilin 1, presenilin 2, ABCA7, SORL1, and disease-associated alleles thereof.

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

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

9. 9. The method of any of claims 1-8, wherein the CNS disease is Huntington's disease and at least one miRNA comprises the sequence of any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260.

10. The method according to any one of claims 8 to 9, wherein at least one of the miRNAs hybridizes to human huntingtin and inhibits its expression.

11. The method of any of claims 8 to 10, wherein the subject comprises a huntingtin gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

12. The method according to any one of claims 8 to 11, wherein the subject is under 20 years of age.

13. 13. The method of any of claims 1 to 12, wherein the recombinant viral vector is selected from the group consisting of an AAV vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes viral vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric viral 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 of any 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 of 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) are administered at different times.

19. 20. The method of claim 18, wherein the different time points are separated by at least one minute, at least one hour, at least one day, at least one week, at least one month, at least one year, or more.

20. The method of any of claims 18-19, wherein (a) is administered prior to administration of (b).

21. The method of any of claims 18-19, wherein (b) is administered prior to administration of (a).

22. 22. The method of any of claims 1-21, wherein said administration of (a), (b), or (a) and (b) is repeated at least one time.

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

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

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

26. 24. The method of claim 23, wherein the adjacent intron is an hCG intron.

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

28. 28. The method of claim 27, wherein the promoter is a synapsin (Syn1) promoter or a promoter of Tables 10-13.

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

30. 30. The method of claim 29, wherein the untranslated portion is an intron.

31. 31. The method of claim 30, wherein the untranslated portion is between the last codon of the nucleic acid sequence encoding a protein and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence.

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

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

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

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

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

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

38. 38. The method of claim 37, wherein the capsid protein is an 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 of claims 2 to 39, wherein the viral vector is formulated for delivery to the central nervous system (CNS).

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

42. (a) a recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (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. A composition or combination comprising at least one of:

43. 43. A composition or combination according to any of claims 41 to 42 for use in a method for treating a neurological disease or disorder in a subject in need of treatment, said method comprising administering a therapeutically effective amount of said composition or combination to a subject having or at risk of developing said neurological disease or disorder.

44. 44. The composition or combination of claim 43, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinal cerebral ataxia, Krabbe disease, polyglutamine repeat spinocerebellar ataxia, Batten disease, Refsum disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord and head injury, eye diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug addiction, neurosis, psychosis, dementia, delusion, attention deficit disorder, psychosexual disorder, sleep disorder, pain disorder, eating or weight disorder.

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

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

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

49. 47. The composition or combination of any of claims 41-46, wherein 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 NOs: 6-17, 40-44, 50-66, 158-185, or 217-260 adjacent to a miRNA scaffold sequence.

50. 47. The composition or combination of any of claims 41 to 46, wherein at least one miRNA comprises the sequence of any one of SEQ ID NOs: 6-17, 40-44, 50-66, 158-185, or 217-260.

51. A composition or combination according to any of claims 49 to 50, wherein at least one of said miRNAs hybridizes to human huntingtin and inhibits its expression.

52. 52. The composition or combination of any of claims 49 to 51, wherein the subject comprises a huntingtin gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats.

53. 53. The composition or combination of any of claims 49 to 52, wherein the subject is under 20 years of age.

54. The composition or combination of any of claims 42 to 53, wherein the recombinant viral vector is selected from the group consisting of an AAV vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a herpes viral vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric viral vector.

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

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

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

58. The composition or combination of any of claims 41 to 57, wherein (a) and (b) are administered at substantially the same time.

59. The composition or combination of any of claims 41 to 54 and 56, wherein (a) and (b) are administered at different times.

60. 60. The composition or combination of claim 59, 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 more.

61. The composition or combination of any of claims 59-60, wherein (a) is administered prior to the administration of (b).

62. The composition or combination of any of claims 59-60, wherein (b) is administered prior to the administration of (a).

63. 61. The composition or combination of any of claims 59 to 60, wherein administration of (a), (b), or (a) and (b) is repeated at least one time.

64. The composition or combination of any one of claims 41 to 63, wherein the transgene comprises two miRNAs in tandem flanked by introns.

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

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

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

68. 68. The composition or combination of any one of claims 41 to 67, wherein the transgene comprises a promoter.

69. 69. The composition or combination of claim 68, wherein the promoter is a synapsin (Syn1) promoter or a promoter of Tables 10-13.

70. 70. The composition or combination of any of claims 41 to 69, wherein the one or more miRNAs are located in an untranslated portion of the transgene.

71. 71. The composition or combination of claim 70, wherein the untranslated portion is an intron.

72. 71. The composition or combination of claim 70, wherein the untranslated portion is between the last codon of the nucleic acid sequence encoding the protein and the polyA tail sequence, or between the last nucleotide base of the promoter sequence and the polyA tail sequence.

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

74. 74. A composition or combination according to any one of claims 41 to 73, wherein the ITR variant lacks a functional terminal separation site (TRS), optionally wherein the ITR variant is an ATRS ITR.

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

76. A composition or combination according to any of claims 41 to 75, wherein said administration is by injection, optionally intravenous or intrastriatal injection.

77. The composition or combination of any of claims 42 to 76, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, or a chimera thereof.

78. 78. The composition of any 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, AAVrhlO, AAV11, or AAV12, or a chimera thereof.

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

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

81. The composition or combination of any of claims 42 to 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, said nucleic acid comprising a sequence at least 80% identical to SEQ ID NO:

110.

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

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

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

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

89. The method of any of claims 84-88, wherein the administration is repeated at least one time.

90. 90. The method of any of claims 84-89, wherein said administering 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 of claims 84 to 90, wherein the administration is by injection, optionally intravenous or intrastriatal injection.

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

93. 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, AAVrhlO, AAV11, or AAV12, or a chimera thereof.

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

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

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

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

110.

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

110.

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

110.

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

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

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

103. 102. The composition or method of claim 100 or 101, wherein at least one of the capsid modifications preferentially targets cells in the CNS or PNS.

104. 102. The composition or method of claim 100 or 101, wherein the chemical modification comprises a chemically modified tyrosine residue that has been modified to include a covalently linked monosaccharide or polysaccharide moiety.

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

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

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

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

109. 108. The composition or method of any of claims 100-107, wherein the modified viral capsid is a monoploid capsid.

110. 108. The composition or method of any of claims 100-107, wherein the modified viral capsid is a chimeric or monomelic capsid further comprising the modification.

111. 11. The composition or method of any of claims 100-110, wherein the modified viral capsid is derived from AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, or a mutated, chimeric, mosaic, or rational monomorphic form thereof.

112. 112. The composition or method of any of claims 100-111, wherein the modification alters the antigenic profile of the modified viral capsid compared to an unmodified viral capsid.

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

114. A synthetic CNS-specific promoter comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 187-189, or a functional variant thereof.

115. 115. The synthetic CNS-specific promoter of claim 114, wherein the functional variant is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 187-189.

116. A CRE comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 191 or 192, or a functional variant thereof.

117. The CRE of claim 116, comprising an array that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 191 or 192.

118. A CRM comprising a CRE according to claim 116 or 117.

119. A minimal promoter comprising or consisting of the sequence set forth in SEQ ID NO: 211 or a functional variant thereof.

120. A CNS-specific promoter comprising a CRE according to claim 116 or 117, a CRM according to claim 118, or a minimal promoter according to claim 119.

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

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

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

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

126. 127. 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 virion of claim 125.

127. 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 virion of claim 125 or the pharmaceutical composition of claim 126 for use in therapy.

128. 128. The synthetic CNS-specific promoter, expression cassette, vector, virion, or pharmaceutical composition of claim 127 for use in gene therapy, preferably wherein the gene therapy comprises expression of a therapeutic expression product in the CNS.

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

130. 127. 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 virion of claim 125.

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

132. 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 or the virion of claim 125 for use in the manufacture of a pharmaceutical composition for the treatment of a medical condition or disease, optionally wherein the disease is a CNS-related disease.

133. 122. A method for producing an expression product, the method comprising: introducing a synthetic CNS-specific expression cassette described in claim 121 into a cell, optionally a CNS cell; and expressing a gene present in the synthetic CNS-specific expression cassette.

134. 126. A method for expressing a therapeutic transgene in a cell, optionally a CNS cell, said method comprising introducing into said cell an expression cassette according to claim 121, a vector according to any one of claims 122 to 124, or a virion according to claim 125.

135. 127. A method of treatment of a subject in need of treatment, said method comprising administering to a subject in need of treatment an expression cassette according to claim 120, a vector according to any one of claims 122-124, or a virion according to claim 125, or a pharmaceutical composition according to claim 127 or 128, comprising a sequence encoding an operably linked therapeutic product.

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