CN116723868A - Methods of treating neurological disorders - Google Patents

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 methods of treating a neurological disease or disorder comprising administering a viral vector comprising an interfering nucleic acid (e.g., an artificial miRNA) and a viral vector comprising a CYP46A1 protein. In some embodiments, the disclosure provides methods of treating huntington's disease comprising administering a viral vector comprising an interfering nucleic acid (e.g., an artificial miRNA) targeting huntington's gene (HTT) and a viral vector comprising a CYP46A1 protein. In some embodiments, the viral vector comprises a modified viral capsid, e.g., for preferential targeting to cells in the CNS or PNS.

Description

Methods of treating neurological diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims the benefit of U.S. Provisional Application No. 63/080,925 filed on September 21, 2020, U.S. Provisional Application No. 63/121,152 filed on December 3, 2020, U.S. Provisional Application No. 63/139,410 filed on January 20, 2021, U.S. Provisional Application No. 63/140,440 filed on January 22, 2021, and U.S. Provisional Application No. 63/180,407 filed on April 27, 2021, the contents of each of which are incorporated herein by reference in their entirety.

Technical Field

The technology described herein relates to methods of treating a neurological disease or disorder, such as Huntington's disease.

Background Art

Huntington's disease (HD) is a devastating inherited neurodegenerative disorder caused by an expansion of the CAG repeat region in exon 1 of the Huntington gene. Although 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 suffer from mood symptoms (including depression and anxiety) accompanied by characteristic movement disorders and chorea. There is currently no cure for Huntington's disease; treatment options are limited to relieving disease symptoms.

Summary of the invention

One aspect provided herein describes a method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject having a neurological disease or disorder or at risk of developing a neurological disease or disorder a therapeutically effective amount of (a) a nucleic acid encoding at least one miRNA; and (b) a nucleic acid encoding a CYP46A1 protein.

In one aspect, compositions or combinations are described herein, comprising (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 aspect, compositions or combinations are 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.

In one aspect, described herein are methods for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject having the neurological disease or disorder or at risk of developing the neurological disease or disorder 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. In one aspect, described herein are methods for treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject having the neurological disease or disorder or at risk of developing the neurological disease or disorder 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.

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

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.

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.

In some embodiments, the CNS disease is Huntington's disease and at least one miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein at least one miRNA comprises a sequence of any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66, flanked by a miRNA backbone sequence. In some embodiments, the CNS disease is Huntington's disease and at least one miRNA comprises a sequence of any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66. In some embodiments, at least one of the miRNAs hybridizes with human Huntington and inhibits expression of human Huntington. In some embodiments, the subject comprises a Huntington gene having more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. In some embodiments, the subject is less than 20 years old.

In some embodiments, the recombinant viral vector is selected from the group consisting of: AAV vector, adenoviral vector, lentiviral vector, retroviral vector, herpes virus vector, alphavirus vector, poxvirus vector, baculovirus vector and chimeric virus vector.

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

In some embodiments, (a) and (b) are administered at substantially the same time. In some embodiments, (a) and (b) are administered at different time points. In some embodiments, the different time points are separated by at least 1 min, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year or longer. In some embodiments, (a) is administered before the administration of (b). In some embodiments, (b) is administered before the administration of (a). In some embodiments, the administration of (a), (b), or (a) and (b) is repeated at least once.

In some embodiments, the transgene comprises two miRNAs in tandem, the two miRNAs being 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.

In some embodiments, the transgene comprises a promoter. In some embodiments, the promoter is a synapsin (Syn1) promoter, or a promoter of Table 10-Table 13.

In some embodiments, the one or more miRNAs are located in the non-translated portion of transgenic. In some embodiments, the non-translated portion is an intron. In some embodiments, the non-translated portion is between the last codon and the poly-A tail sequence of the nucleic acid sequence encoding the protein, or between the last nucleotide base and the poly-A tail sequence of the promoter sequence. In some embodiments, the non-translated portion is a 5' non-translated region (5'UTR).

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.

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

In some embodiments, the administration is such that the viral vector or isolated nucleic acid is delivered to the central nervous system (CNS) of the subject. In some embodiments, the administration is by injection, optionally intravenous injection or intrastriatal injection.

In some embodiments, the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a heterologous chimera thereof. In some embodiments, the viral vector comprises a capsid protein from AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a heterologous 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).

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

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

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

In some embodiments of any of the aspects, at least one of the capsid modifications is preferentially targeted to cells in the CNS or PNS.

In some embodiments of any of the aspects, the chemical modification comprises a chemically modified tyrosine residue modified to comprise a covalently attached monosaccharide or polysaccharide moiety.

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.

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.

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

In some embodiments of any of the aspects, the modified viral capsid is a chimeric capsid or a haploid capsid.

In some embodiments of any of the aspects, the modified viral capsid is a haploid capsid.

In some embodiments of any of the aspects, the modified viral capsid is a chimeric or haploid capsid further comprising a modification.

In some embodiments of any aspect, the modified viral capsid is AAV serotype AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a mutant modified form, a chimera, a mosaic, or a reasonable haploid thereof.

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

In some embodiments of any of the aspects, the modified viral capsids are useful for repeated administration.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram showing the HD plasmid map of pJAL130-CYP46A1 (7314 bp), see, eg, SEQ ID NO: 111, and Table 16 showing the ITR to ITR sequence of CYP46 variant sequences from the plasmid (see, eg, SEQ ID NO: 110).

FIG2 shows the intracranial biodistribution of transgenic GFP under the control 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) and control promoter hSyn1 (see, e.g., SEQ ID NO: 152) delivered by intracerebroventricular (ICV) and intravenous (IV) injections in sagittal sections. Scale bar is 1 mm.

Figures 3A-3B show images of coronal brain sections. Figure 3A shows the intracranial biodistribution of transgenic GFP under the control 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), and CNS-4 (see, e.g., SEQ ID NO: 115) delivered by ICV in coronal sections. The scale bar is 1 mm. Figure 3B shows the intracranial biodistribution of transgenic GFP under the control of 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) and the control promoter hSyn1 (see, e.g., SEQ ID NO: 152) delivered by ICV in coronal sections. The scale bar is 1 mm.

Figure 4 shows the percentage of GFP immunoreactivity in different brain regions after ICV or IV delivery of GFP driven by CNS 1-8 (see, e.g., SEQ ID NO: 112-SEQ ID NO: 115, SEQ ID NO: 122-SEQ ID NO: 125) or Synapsin-1 (see, e.g., SEQ ID NO: 152). Data were obtained by quantitatively measuring 10 non-overlapping RGB images of GFP staining intensity via threshold analysis in the cortex, hippocampus, striatum, midbrain, and cerebellum (mean ± SEM). Images were taken at x40 magnification with constant settings through discrete brain regions. Foreground immunostaining was defined by averaging the highest and lowest signals. Data are expressed as the average percentage area of immunoreactivity per field for each region of interest (n = 3). In the case of ICV delivery, expression was highest in the cortical and hippocampal brain regions. CNS 1-8 (see, e.g., SEQ ID NO: 112-SEQ ID NO: 115, SEQ ID NO: 122-SEQ ID NO: 125) showed higher expression in the hippocampus than the hSyn1 control. In the case of ICV delivery, CNS-1 (see, e.g., SEQ ID NO: 112) showed higher expression in the hippocampus, midbrain, and cerebellum compared to hSyn1.

Figures 5A-5B show the tissue expression patterns of faf1 and pitx3 genes, from which CRE/proximal promoters from CNS-5, CNS-5_v2, CNS-2, CNS-3 and CNS-4 were designed. Figure 5A shows the expression pattern of the faf1 gene in mouse PNS neurons from single-cell transcriptome data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. faf1 is expressed in many PNS neurons. Figure 5B shows the expression pattern of the pitx3 gene in PNS neurons from single-cell transcriptome data (Zeisel et al., 2018). Dark gray indicates high expression, white indicates no expression, and light gray indicates low expression. Pixt3 is expressed in sympathetic PNS neurons. faf1 is expressed in many PNS neurons, so it is expected that synthetic promoters containing CRE or proximal promoters (such as CNS-5 and CNS-5_v2) designed from the faf1 gene have strong expression in the PNS. Pitx3 is expressed in sympathetic PNS neurons, so synthetic promoters containing CREs designed from the pitx3 gene (e.g., CNS-2, CNS-3, or CNS-4) are expected to have expression in PNS sympathetic neurons. Similar analysis of lmx1b and pitx2 showed no expression in the PNS above the cutoff score for the analysis (trinization score less than 0.95; data not shown), so CNS-1, CNS-6, CNS-6_v2, CNS-7, CNS-7_v2, CNS-8, and CNS-8_v2 are not expected to be active in PNS neurons.

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

Figure 6B shows the expression pattern of the CYP46A1 gene in coronal sections from adult mouse brain (taken from the Allen Mouse Brain Atlas; mouse.brain-map.org). CYP46A1 is widely expressed in the brain.

Fig. 7 A shows the median GFP expression of synthetic NS-specific promoters SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036 and control promoter Synapsin-1 relative to control promoter CAG in SH-SY5Y cells derived from neuroblastoma. NTC represents untransfected cells. Data are collected from three biological replicates, each of which is the average of two technical replicates. Error bars are standard errors.

Fig. 7 B shows when being transfected with synthetic NS specific promoter SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035, SP0036 or control promoter Synapsin-1 and CAG operably connected to GFP, the transfection efficiency in SH-SY5Y cells derived from neuroblastoma.NTC represents untransfected cells.Data are collected from three biological replicates, each of which is the mean value of two technical replicates.Error bars are standard errors.GFP positive % represents the percentage of GFP positive in all cells.

DETAILED DESCRIPTION

Aspects of the present invention relate to administering an interfering RNA (e.g., miRNA, such as an artificial miRNA) and a nucleic acid encoding a CYP46A1 protein that is effective in reducing the expression of a pathogenic gene in a subject when delivered to the subject. Thus, in some embodiments, the methods and compositions described herein are useful for treating a neurological disease or disorder.

Treatment

The present disclosure provides methods for delivering nucleic acids and/or transgenes (e.g., inhibitory RNA, such as miRNA and/or nucleic acids encoding CYP46A1) to a subject. The methods generally involve administering to a subject an effective amount of a nucleic acid encoding at least one interfering RNA/inhibitory nucleic acid and a nucleic acid encoding CYP46A1, wherein the at least one interfering RNA/inhibitory nucleic acid is capable of reducing the expression of a target gene (e.g., a pathogenic gene associated with a neurological disease or disorder (e.g., Huntington (htt) protein)). In some embodiments, one or both of the nucleic acids are provided in a viral vector and/or in a viral particle (e.g., rAAV).

As used herein, "neurological disease or disorder" may refer to any disease, disorder or condition that affects or is associated with the nervous system, i.e., a disease, disorder or condition that affects the central nervous system (brain and spinal cord), the peripheral nervous system (PNS; e.g., peripheral nerves and cranial nerves), and the autonomic nervous system (parts of which are located in the central nervous system and the peripheral nervous system). More than 600 neurological diseases have been identified in humans. As non-limiting examples, the neurological disease or disorder may be Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, spinocerebellar ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten's disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, Niemann Pick's disease, disease), muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger disease, trauma caused by spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Rett syndrome, neuropathic pain, Lesch-Nyhan disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusions, attention deficit disorder, sexual psychosis, sleep disorders, pain disorders and/or eating or weight disorders. In some embodiments, the nervous system disease or disorder is a central nervous system (CNS) disease or disorder, such as Huntington's disease, Parkinson's disease or Alzheimer's disease.

As used herein, "Huntington's disease" or "HD" refers to a neurodegenerative disease caused by a trinucleotide repeat expansion (e.g., CAG, which is translated into polyglutamine, or PolyQ, tracts) in the HTT gene, resulting in the production of pathogenic mutant Huntington (HTT, or mHTT), characterized by progressively worsening motor, cognitive, and behavioral changes.

As used herein, "HTT" or "Huntington" refers to the gene encoding the huntingtin protein. Normal huntingtin protein plays a role in nerve cells, and the normal HTT gene usually has about 7 to about 35 CAG repeats at the 5' end. In patients with Huntington's disease or patients at risk of developing Huntington's disease, the HTT gene is usually mutated. In some embodiments, the mutated huntingtin protein accelerates the rate of neuronal cell death in certain areas of the brain. In general, the severity of HD is related to the size of the trinucleotide repeat expansion in the subject. For example, a subject having a CAG repeat region containing between 36 and 39 repeats is characterized as having "reduced penetrance" HD, while a subject having greater than 40 repeats is characterized as having "full penetrance" HD. Therefore, in some embodiments, a subject suffering from HD or at risk of suffering from HD has an HTT gene containing between about 36 and about 39 CAG repeats (e.g., 36, 37, 38, or 39 repeats). In some embodiments, a subject with HD or at risk of HD has an HTT gene comprising 40 or more (e.g., 40, 45, 50, 60, 70, 80, 90, 100, 200 or more) CAG repeats. In some embodiments, a subject with an HTT gene comprising more than 100 CAG repeats develops HD earlier than a subject with less than 100 CAG repeats. In some embodiments, a subject with an HTT gene comprising more than 100 CAG repeats may develop HD symptoms before the age of about 20 and is referred to as having juvenile HD (also known as akinetic-rigid HD, or Westphal variant HD). The number of CAG repeats in the subject's HTT gene allele can be determined by any suitable means known in the art. For example, nucleic acid (e.g., DNA) can be isolated from a biological sample (e.g., blood) of the subject, and the number of CAG repeats of the HTT allele can be determined by a hybridization-based method (e.g., PCR or nucleic acid sequencing (e.g., Illumina sequencing, Sanger sequencing, SMRT sequencing, etc.)). The sequence of the HTT gene is known in some species, for example, the human HTT (NCBI Gene ID: 3064) mRNA sequence (NCBI Ref Seq: NM_002111.8, SEQ ID NO: 4) and protein sequence (NCBI Ref Seq: NP_0021012.4, SEQ ID NO: 5). Therefore, in some embodiments related to the treatment of Huntington's disease, the one or more inhibitory nucleic acids (e.g., miRNA) can hybridize with HTT and/or reduce the expression of HTT.

As used herein, "Alzheimer's disease" or "AD" refers to a neurodegenerative disease characterized by progressively worsening memory, disorientation, mood swings, and increasing difficulty with language, motivation, and self-care. Some genes may contribute to or increase the risk of AD, including amyloid precursor protein (APP; NCBI Gene ID: 351), presenilin 1 (PSEN1; NCBI Gene ID 5663), presenilin 2 (PSEN2; NCBI Gene ID 5664), ATP-binding cassette subfamily A member 7 (ABCA7; NCBI Gene ID 10347), and sortilin-related receptor 1 (SORL1; NCBI Gene ID 6653). The sequences of such AD-related genes are known in some species, for example, human mRNA and protein sequences can be obtained in the NCBI database using the provided Gene ID numbers. These AD-related genes and others, as well as their AD-related alleles (e.g., 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 related to the treatment of Alzheimer's disease, the one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with APP, PSEN1, PSEN2, ABCA7, and/or SORL1 and/or reduce the expression of APP, PSEN1, PSEN2, ABCA7, and/or SORL1.

As used herein, "Parkinson's disease" or "PD" refers to a neurodegenerative disease characterized by gradually worsening tremors and rigidity and increasing problems with balance, walking, and coordination. Several genes may contribute to or increase the risk of PD, including 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), parkinRBR E3 ubiquitin (PRKN; NCBI Gene ID 5071), PTEN-induced kinase 1 (PINK1; NCBI Gene ID 65018), Parkinson's disease-associated deglycosylation enzyme (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), and DnaJ heat shock protein family member C13 (DNAJC13; NCBI Gene ID 55737). 23317), coiled-coil-coil-coil-coil 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-related genes in some species are known, for example, human mRNA and protein sequences can be obtained in the NCBI database using the provided Gene ID numbers. These PD-associated genes and others, as well as their PD-associated alleles (e.g., mutations, SNPs, etc.) are known in the art and are further described, for example, in: 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 herein by reference in its entirety. Therefore, in some embodiments related to the treatment of Parkinson's disease, the one or more inhibitory nucleic acids (e.g., miRNAs) can hybridize with SNCA, LRRK2/PARK8, PRKN, PINK1, DJ1/PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1 and/or GBA1 and/or reduce the expression of SNCA, LRRK2/PARK8, PRKN, PINK1, DJ1/PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1 and/or GBA1.

An "effective amount" of a substance is an amount sufficient to produce the desired effect. In some embodiments, the effective amount of an isolated nucleic acid is sufficient to transfect (or infect, in the case of rAAV-mediated delivery) a sufficient number of target cells of 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., which can be delivered by 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), to extend the life span of a subject, to improve one or more disease symptoms in a subject (e.g., symptoms of Huntington's disease), etc. The effective amount will depend on various factors, such as, for example, species, age, weight, health of the subject, and tissue to be targeted, and may therefore vary between subjects and tissues, as described elsewhere in this disclosure.

Inhibitory RNA

In some aspects, the present disclosure provides inhibitory nucleic acids (e.g., miRNAs) that specifically bind to (e.g., hybridize to) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive bases of a target (e.g., human Huntington 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 to) at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive bases of exon 1 of human Huntington mRNA (e.g., SEQ ID NO: 3). As used herein, "contiguous bases" refers to two or more nucleotide bases covalently bound (e.g., by one or more phosphodiester bonds, etc.) to each other (e.g., as part of a nucleic acid molecule). In some embodiments, the at least one miRNA is 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 SEQ ID NO: 4). In some embodiments, the inhibitory RNA is a miRNA comprising a sequence shown in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66, or a miRNA encoded by a sequence shown in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66.

In one aspect, described herein are inhibitory RNAs that can be used to treat a neurological disease or disorder. In some embodiments of any aspect, the nucleic acid sequence of the inhibitory RNA comprises one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66, or a sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to at least one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66 and maintains the same function as SEQ ID NO: 3 or SEQ ID NO: 4 (e.g., HTT inhibition).

In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a sequence as set forth in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66. In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a sequence as set forth in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66, flanked by a miRNA backbone sequence.

In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a seed sequence complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49. In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a seed sequence complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49, flanked by miRNA backbone sequences. In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a seed sequence substantially complementary to one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 18-SEQ ID NO: 39, or SEQ ID NO: 46-SEQ ID NO: 49. In some embodiments, the vectors described herein comprise at least one miRNA, each miRNA comprising a seed sequence substantially complementary to one of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:18-SEQ ID NO:39, or SEQ ID NO:46-SEQ ID NO:49, flanked by a miRNA backbone sequence.

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

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

Table 3 Target sequences in exon 1 of the human HTT gene targeted by the miRNAs provided in Table 1 and Table 2

Target sequence SEQ ID NO: aaggacuuga gggacucgaa 18 tccaagatgg acggccgctc a 19 ccaagatgga cggccgctca g 20 agatggacgg ccgctcaggt t twenty one atggacggcc gctcaggttc t twenty two gacggccgct caggttctgc t twenty three cggccgctca ggttctgctt t twenty four gtgctgagcg gcgccgcgag t 25 cgccgcgagt cggcccgagg c 26 accgccatgg cgaccctgga a 27 ccgccatggc gaccctggaa a 28 gaaggccttc gagtccctca a 29 cttcgagtcc ctcaagtcct t 30 ccgccgccgc ctcctcagct t 31 gccgcctcct cagcttcctc a 32

tcagccgccg ccgcaggcac a 33 gccgcaggca cagccgctgc t 34 ggcacagccg ctgctgcctc a 35 gccgctgctg cctcagccgc a 36 cggcccggct gtggctgagg a 37 ctgtggctga ggagccgctg c 38 tgtggctgag gagccgctgc a 39

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

In some embodiments, the vector comprises a pre-miRNA having a sequence of SEQ ID NO: 40 or SEQ ID NO: 41. These pre-miRNAs include a scaffold comprising SEQ ID NO: 8. In either of SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 8 may be replaced with an alternative first RNA sequence disclosed herein.

In some embodiments, the vector comprises a pri-miRNA having a sequence of SEQ ID NO: 42 or SEQ ID NO: 43. The pri-miRNA of SEQ ID NO: 42 comprises a scaffold comprising SEQ ID NO: 8 and SEQ ID NO: 16. In SEQ ID NO: 42, SEQ ID NO: 8 and SEQ ID NO: 16 may be replaced with alternative RNA sequences disclosed herein. The pri-miRNA of SEQ ID NO: 43 and SEQ ID NO: 44 comprises a scaffold comprising SEQ ID NO: 8 and SEQ ID NO: 17. In either of SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 8 and SEQ ID NO: 17 may be replaced with alternative RNA sequences disclosed herein.

Table 4: pre-miRNA and pri-miRNA containing miRNAs provided in Table 1 and Table 2

In some embodiments, the inhibitory nucleic acid may comprise one or more of SEQ ID NO: 1-SEQ ID NO: 102 and/or SEQ ID NO: 103-SEQ ID NO: 249 of International Patent Publication WO2017/201258. In some embodiments, the inhibitory nucleic acid may comprise one or more of a duplex combination of SEQ ID NO: 1-SEQ ID NO: 249 selected from International Patent Publication WO2017/201258, which are provided in Tables 3-5 of International Patent Publication WO2017/201258. In some embodiments, the vector may comprise one or more of the pri-miRNA provided in Table 9 or the pri-raiRNA provided in Table 10 of International Patent Publication WO2017/201258. The contents of International Patent Publication WO2017/201258 are incorporated herein by reference in their entirety.

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

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

In some embodiments, the inhibitory nucleic acid can target a heterozygous SNP within a gene encoding a gain-of-function mutant huntingtin protein (e.g., comprising a sequence that is complementary or substantially complementary to a heterozygous SNP within a gene encoding a gain-of-function mutant huntingtin protein). In some embodiments, the SNP has an allele frequency of at least 10% in the sample population. In some embodiments, the SNP is present at a genomic locus selected from the group consisting of: RS362331, RS4690077, RS363125, RS363075, RS362268, RS362267, RS362307, RS362306, RS362305, RS362304, RS362303, and RS7685686. Such SNPs are described in more detail, for example, in U.S. Pat. No. 9,343,943, which is incorporated herein by reference in its entirety. In some embodiments, the target sequence is one of SEQ ID NO:45-SEQ ID NO:49. In some embodiments, the inhibitory nucleic acid sequence comprises one or more of SEQ ID NO:50-SEQ ID NO:61. In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:50 and SEQ ID NO:51 (e.g., in a duplex). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:52 and SEQ ID NO:53, (e.g., in a duplex). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:54 and SEQ ID NO:55 (e.g., in a duplex). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:56 and SEQ ID NO:57 (e.g., in a duplex). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:58 and SEQ ID NO:59 (e.g., in a duplex). In some embodiments, the inhibitory nucleic acid sequence comprises at least SEQ ID NO:60 and SEQ ID NO:61 (e.g., in a duplex).

Table 5

Target sequence SEQ ID NO: ccacgccugcucccucauccacugugugcacuucauccug 45 ccacgccugcucccucaucuacugugugcacuucauccug 46 uaagagaugg ggacaguaau ucaacgcuag aagaaca 47 uaagagaugg ggacaguacu ucaacgcuag aagaaca 48 cagatgcc atggcctgtgct gggccag 49

Table 6: Sense and antisense (or first and second RNA sequences) targeting SNPs in the human HTT gene

Justice Sequence SEQ ID NO: Antisense sequence SEQ ID NO: ucccucaucc acugugugaa c 50 gcacacagug gaugagggag c 51 ucccucaucu acugugugaa c 52 cgagggagua gaugacacac g 53 gggacaguaa uucaacgcgu c 54 agcguugaau uacugucccc a 55 gggacaguac uucaacgcgu c 56 accccuguca ugaaguugcg a 57 ugccauggcc ugugcugguc c 58 cccagcacag gccauggca c 59 ugccauggca ugugcugguc c 60 cccagcacau gccuaggcau c 61

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

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

Table 7. In some embodiments, capital letters contain 2'-O-(2-methoxy)ethyl modifications.

SEQ ID NO 5’-CTCAGtaacattgacACCAC-3’ 62 5′-CTCGActaaagcaggATITC-3 63 5’-CCTTCCcctgaaggttCCTCC-3’ 64 5’-GCAGGgttaccgccaTCCCC-3’ 65 5’-CGAGAcagtcgcttcCACTT-3’ 66

Further suitable sequences are known in the art, for example in U.S. Patent 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.

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

In some embodiments, the inhibitory RNA and/or the vector does not comprise a sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the inhibitory RNA and/or the vector does not comprise a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any one of SEQ ID NO: 67-SEQ ID NO: 73.

In some embodiments, the inhibitory RNA and/or vector does not comprise a sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the inhibitory RNA and/or vector does not comprise a sequence of any one of SEQ ID NO: 135-SEQ ID NO: 151. In some embodiments, the inhibitory RNA and/or vector does not comprise a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the inhibitory RNA and/or vector does not comprise a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any one of SEQ ID NO: 135-SEQ ID NO: 151.

In some embodiments, the inhibitory RNA and/or vector does comprise the sequence of any one of SEQ ID NO: 67-SEQ ID NO: 73. In some embodiments, the inhibitory RNA and/or vector does comprise a sequence having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98% sequence identity to any one of SEQ ID NO: 67-SEQ ID NO: 73.

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

Table 8

SEQ ID NO: CGAGGCCGGGGCGGGGCACA 67 CGGGGCGGGGCCGTGGAGGG 68 ACTGTGCCACTATGTTTTCA 69 GCCTTCATCAGCTTTTCCAG 70 GCTGCTGCTGCTGCTGCTGC 71 TGCTGGAAGGACTTGAGGGA 72 TGTTGCTGCTGCTGCTGCTG 73 TGCTGCTGCTGCTGCTGCTG 135 GGCGGCGGCGGCGGCGGCGGCG 136 GAGGGGTGGGGAGGCTGGGG 137 TCCTTGACCTGCTGCTGCAG 138 CCTTCCACTGGCCATGATGC 139 ACTGTGCCACTATGTTTTCA 140 TGAGGTATCAGATTGTCTAG 141 AAAttAATCTCTTACCTGAT 142 CCCAGGGCTAGCAAGGAACA 143 AATTCAGTAGCTTCCCTTAA 144 CTGGGCCCGCAGCGGAAGGG 145 TTATTGCTGTCTACTATCCG 146 TCAGTCCTTCCCAAAGCTCT 147 TAATCTCTTTACTGATATAA 148 TCAGCAGTGTTATTTCTTAC 149 AAACCGttACCAttACtGAGtt 150 AAAtCGCtGAtttGtGtAGtC 151

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

In some embodiments of any aspect, the agent for treating a neurological disease or disorder is an inhibitory nucleic acid or comprises an inhibitory nucleic acid. In some embodiments of any aspect, the inhibitor of expression of a specific 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.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conservative regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein may comprise an RNA strand (antisense strand) having a region of less than 30 nucleotides in length (i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length), which is substantially complementary to at least a portion of the targeted mRNA transcript. The use of these iRNAs makes it possible to degrade the targeted mRNA transcript, resulting in reduced expression and/or activity of the target.

As used herein, the term "iRNA" refers to an agent containing RNA (or a modified nucleic acid as described below) and mediating the targeted cleavage of an RNA transcript by an RNA-induced silencing complex (RISC) approach. In some embodiments of any aspect, an iRNA as described herein causes the expression of a target and/or the inhibition of activity. In some embodiments of any aspect, a cell is contacted with an inhibitor (e.g., iRNA) such that the target mRNA level in the cell is reduced by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in a cell without the presence of iRNA. In some embodiments of any aspect, administration of an inhibitor (e.g., an iRNA) to a subject reduces the level of target mRNA in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the level of target mRNA found in the subject in the absence of the iRNA.

In some embodiments of any aspect, iRNA can be dsRNA.DsRNA comprises two RNA chains that are sufficiently complementary to hybridize to form a duplex structure under the conditions of using dsRNA.One chain (antisense strand) of dsRNA comprises a complementary region, which is substantially complementary to the target sequence, and is generally fully complementary.The target sequence can be derived from the sequence of the mRNA formed during the expression of the target, for example, it can span the border of one or more introns.Another chain (sense strand) comprises a region complementary to the antisense strand so that the two chains hybridize and form a duplex structure when combined under suitable conditions.In general, the length of the duplex structure is between 15 (inclusive) and 30 (inclusive) base pairs, and the length is more generally between 18 (inclusive) and 25 (inclusive) base pairs, and the length is also more generally between 19 (inclusive) and 24 (inclusive) base pairs, and the length is most generally between 19 (inclusive) and 21 (inclusive) base pairs. Similarly, the length of the region complementary to the target sequence is between 15 (inclusive) and 30 (inclusive) base pairs, the length is more generally between 18 (inclusive) and 25 (inclusive) base pairs, the length is still more generally between 19 (inclusive) and 24 (inclusive) base pairs, and the length is most generally between 19 (inclusive) and 21 (inclusive) base pairs. In some embodiments of any aspect, the length of the dsRNA is between 15 (inclusive) and 20 (inclusive) nucleotides, and in other embodiments, the length of the dsRNA is between 25 (inclusive) and 30 (inclusive) nucleotides. As will be recognized by ordinary technicians, the targeted region of the RNA of targeted cutting will often be a part of a larger RNA molecule (usually an mRNA molecule). In relevant cases, "a portion" of an mRNA target is a continuous sequence of an mRNA target, which is long enough to be a substrate for RNAi directed cutting (i.e., cutting by the RISC approach). In some cases, dsDNA with a duplex as short as 9 base pairs can mediate RNA directed RNA cutting of RNAi. Most typically, the target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length. Exemplary embodiments of inhibitory nucleic acid types may include, for example, siRNA, shRNA, miRNA, and/or amiRNA, which are well known in the art.

In some embodiments of any aspect, the inhibitor is miRNA. MicroRNA (miRNA) is a small RNA of 17-25 nucleotides that functions as a regulator of gene expression in eukaryotic cells. "microRNA" or "miRNA" is a small non-coding RNA molecule that can mediate transcriptional gene silencing or post-translational gene silencing. Typically, miRNA is transcribed into a hairpin or stem-loop (e.g., a single-stranded backbone with self-complementarity) duplex structure, referred to as primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) to become pre-miRNA. The duplex structure comprises: a) a first RNA sequence, a complementary region that is substantially complementary to the target sequence and generally fully complementary; and b) a second RNA sequence region that is complementary to the first RNA sequence chain so that the two sequences hybridize and form a duplex structure when combined under appropriate conditions. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, for example, it can span the boundaries of one or more introns. Generally, the duplex structure is between 15 and 30 base pairs in length, more typically between 18 and 25 base pairs in length, still more typically between 19 and 24 base pairs in length, and most typically between 19 and 21 base pairs in length.

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

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

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

In the context of the present invention, a miRNA molecule or its equivalent or mimic or isomiR may be synthetic, or natural, or recombinant, or mature, or a portion of a mature miRNA, or a human miRNA, or derived from a human miRNA, as further defined in the section dedicated to general definitions. A human miRNA molecule is a miRNA molecule found in human cells, tissues, organs or body fluids (i.e., an endogenous human miRNA molecule). A human miRNA molecule may also be a human miRNA molecule derived from an endogenous human miRNA molecule by substitution, deletion and/or addition of nucleotides. A miRNA molecule or its equivalent or mimic may be a single-stranded or double-stranded RNA molecule. Preferably, the miRNA molecule or its equivalent or mimetic 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 more.

In a preferred embodiment, the miRNA molecule or its equivalent or mimic or isomiR comprises at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule or its equivalent or mimic or isomiR. Preferably, in this embodiment, the miRNA molecule or its equivalent or mimic or isomiR is 6 to 30 nucleotides in length, and more preferably comprises at least 6 of the 7 nucleotides present in the seed sequence of the miRNA molecule or its equivalent. Even more preferably, the miRNA molecule or its equivalent or mimic or isomiR is 15 to 28 nucleotides in length, and 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 nucleotides or more in length.

Therefore, preferred miRNA molecules or their equivalents or mimetics or isomiRs comprise at least 6 of the 7 nucleotides present in the seed sequence determined as SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44 or SEQ ID NO: 50-SEQ ID NO: 66 or determined in SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44 or SEQ ID NO: 50-SEQ ID NO: 66, and more preferably are 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 more.

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

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

The term substantial complementarity means that it is not required that the first and second RNA sequences are completely complementary, or that the first RNA sequence and a reference or target sequence (e.g., SEQ ID NO: 3 or SEQ ID NO: 4) are completely complementary. In one embodiment, the substantial complementarity between the RNA sequence and the target consists of: no mismatches, one mismatched nucleotide, or two mismatched nucleotides. It is understood that one mismatched nucleotide means that there is one nucleotide that is not base-paired with the target over the entire length of the RNA sequence that is base-paired with the target. No mismatches means that all nucleotides are base-paired with the target, and two mismatches means that there are two nucleotides that are not base-paired with the target.

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

Nucleic Acids

In some aspects, the present disclosure provides isolated nucleic acids that are useful for reducing (e.g., inhibiting) the expression of pathogenic genes (e.g., HTT) and/or encode 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" refers to artificial production. As used herein for nucleic acids, the term "isolated" refers to: (i) in vitro amplification by, for example, polymerase chain reaction (PCR); (ii) produced by cloning recombination; (iii) purified, such as by cutting and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is a nucleic acid that can be easily manipulated by recombinant DNA techniques well known in the art. Therefore, a nucleotide sequence contained in a vector in which the 5' and 3' restriction sites are known or its polymerase chain reaction (PCR) primer sequence has been disclosed is considered to be isolated, but a nucleic acid sequence that exists in its natural state in its natural host is not. An isolated nucleic acid can be substantially purified, but it does not have to be. For example, a nucleic acid isolated within a cloning or expression vector is not pure because it may contain only a very small percentage of the material in the cell in which it resides. However, such a nucleic acid is isolated, as the term is used herein, because it can be readily manipulated by standard techniques known to those of ordinary skill in the art. As used herein for a protein or peptide, the term "isolated" refers to a protein or peptide that has been separated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

Those skilled in the art will also recognize that conservative amino acid substitutions can be performed to provide functionally equivalent variants, or homologues of capsid proteins. In some aspects, the disclosure includes sequence changes that result in conservative amino acid substitutions. As used herein, conservative amino acid substitutions refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitutions have been performed. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as methods found in references for writing such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel et al., John Wiley & Sons, Inc. New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus, conservative amino acid substitutions can be made to the amino acid sequences of the proteins or polypeptides disclosed herein.

The isolated nucleic acid of the present invention can be a recombinant adeno-associated virus (AAV) vector (rAAV vector). In some embodiments, the isolated nucleic acid as described in the present disclosure comprises a region (e.g., a first region) containing a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. The isolated nucleic acid (e.g., a recombinant AAV vector) can be packaged in a capsid protein and administered to a subject and/or delivered to a selected target cell. A "recombinant AAV (rAAV) vector" is typically composed of at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). As disclosed elsewhere herein, a transgene may include one or more regions encoding one or more inhibitory RNAs (e.g., miRNAs), the inhibitory RNA comprising a nucleic acid targeting an endogenous mRNA of a subject. As described elsewhere in the present disclosure, a transgene may also include a region encoding, for example, a protein and/or an expression control sequence (e.g., a poly-A tail).

Generally, the length of the ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although some degree of slight modification of these sequences is permitted. The ability to modify these ITR sequences is within the skill of the art. (See, for example, Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd edition, 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 can be obtained from any known AAV, including currently identified mammalian AAV types. 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, AAV11, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

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, AAV11, and variants thereof. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal resolution site (TRS). The term "lack of a terminal resolution site" may refer to an AAV ITR comprising a mutation (e.g., a sense mutation, such as a non-synonymous mutation, or a missense mutation) that abolishes the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR (e.g., an ATRS ITR) lacking a nucleic acid sequence encoding a functional TRS. Without wishing to be bound by any particular theory, rAAV vectors comprising ITRs lacking functional TRSs are generated from complementary rAAV vectors, such as described by McCarthy (2008) Molecular Therapy 16(10): 1648-1656. In some embodiments of any aspect disclosed herein, at least one or more ITRs are less than 145 bp in length, such as 130 bp in length.

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

As used herein, when a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence 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, they can be referred to as being operably linked. If the desired nucleic acid sequence is translated into a functional protein, if the induction of the promoter in the 5' regulatory sequence causes the coding sequence to be transcribed, and if the nature of the connection between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein, then the two DNA sequences are referred to as being operably linked. Therefore, if the promoter region is able to affect the transcription of the DNA sequence so that the resulting transcript may be translated into the desired protein or polypeptide, the promoter region will be operably linked to the nucleic acid sequence. Similarly, when two or more coding regions are operably linked so that the result of their transcription from a common promoter is to express two or more proteins that have been translated in frame, the two or more coding regions are operably linked. In some embodiments, the operably linked coding sequence produces a fusion protein. In some embodiments, the operably linked coding sequence produces a functional RNA (e.g., miRNA).

In some aspects, the disclosure provides an isolated nucleic acid comprising a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more microRNAs (eg, miRNAs).

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

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 endogenous pri-miRNA or pre-miRNA (e.g., miRNA backbone, which is a precursor miRNA capable of producing a functional mature miRNA), wherein the miRNA and miRNA* (e.g., the passenger strand of the miRNA duplex) sequences have been replaced with corresponding amiRNA/amiRNA* sequences, which guide efficient RNA silencing of the targeted gene, such as described by Eamens et al. (2014), Methods Mol. Biol. 1062: 211-224. For example, in some embodiments, the artificial miRNA comprises a miR-155pri-miRNA backbone, into which a sequence encoding a mature HTT-specific miRNA (e.g., any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66) is inserted to replace the endogenous miR-155 mature miRNA coding sequence. In some embodiments, the miRNA (e.g., artificial miRNA) described by the present disclosure comprises a miR-155 backbone sequence, a miR-30 backbone sequence, a mir-64 backbone sequence, or a miR-122 backbone sequence.

The region (e.g., second region, third region, fourth region, etc.) comprising the transgene can be located at any suitable position of the isolated nucleic acid. The region can be located at any untranslated part of the nucleic acid, including, for example, introns, 5' or 3' untranslated regions, etc.

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

In some cases, the region can be positioned at the downstream of the last base of the poly-A tail of the transgenic. The region can be between the position of the last base of the poly-A tail and 2000 nucleotides downstream of the last base. The region can be between the position of the last base of the poly-A tail and 1000 nucleotides downstream of the last base. The region can be between the position of the last base of the poly-A tail and 500 nucleotides downstream of the last base. The region can be between the position of the last base of the poly-A tail and 250 nucleotides downstream of the last base. The region can be between the position of the last base of the poly-A tail and 150 nucleotides downstream of the last base.

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

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

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

For protein-encoding nucleic acids, a polyadenylation sequence is generally inserted after the transgenic sequence and before the 3'AAV ITR sequence. The rAAV constructs useful in the present disclosure may also contain introns, which are desirably located between the promoter/enhancer sequence and the transgenic. A possible intron sequence is from SV-40 and is referred to as the SV-40T intron sequence. Another vector element that can be used is an internal ribosome entry site (IRES). The IRES sequence is used to produce more than one polypeptide from a single gene transcript. The IRES sequence will be used to produce proteins containing more than one polypeptide chain. It is conventional to select these and other common vector elements, and many such sequences are available [see, for example, Sambrook et al., and references cited therein, for example, 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 polypeptide; this is a small peptide (about 18 amino acids in length) that has been shown to mediate cleavage of polyproteins (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459). The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems, including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6:198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11:1921-1931; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

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.

Inducible promoters allow for regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors (e.g., temperature), or specific physiological states (e.g., acute phase, specific differentiation states of cells, or in cells that are only replicating). Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be easily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-induced sheep metallothionein (MT) promoter, the dexamethasone (Dex)-induced mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 2000). 98/10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4: 432-441 (1997)), and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100: 2865-2872 (1997)). Other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological states (e.g., temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells).

In another embodiment, the native promoter of the transgenic gene will be used. When it is desired that the expression of the transgenic gene should mimic the native expression, the native promoter may be preferred. When the expression of the transgenic gene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus, the native promoter may be used. In further embodiments, other native expression control elements (e.g., enhancer elements, polyadenylation sites, or Kozak consensus sequences) may also be used to mimic the native expression.

In some embodiments, regulatory sequences confer tissue-specific gene expression ability. In some cases, tissue-specific regulatory sequences are combined with 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, 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 (mammalian desmin, DES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters include the β-actin promoter; the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)); the osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)); the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); the immunoglobulin heavy chain promoter (Hansal et al., promoter; T cell receptor α-chain promoter; neuronal promoters, such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13: 503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 5611-5 (1991)), and neuron-specific vgf gene promoter ( Piccioli et al., Neuron, 15:373-84 (1995), as well as other promoters that are apparent to the skilled artisan. NS-specific promoters contemplated for use in the present methods and compositions also include promoters described in patent application GB2013940.8 filed on September 4, 2020 and patent application GB2005732.9 filed on April 20, 2020, which are incorporated herein by reference in their entirety. In some embodiments, the NS-specific promoter is a promoter of Table 10, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 10. In some embodiments, the NS-specific promoter is a promoter of Table 10, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 10 and retaining the NS-specific promoter activity of the promoter of Table 10.

CNS-specific promoters contemplated for use in the present methods and compositions also include promoters described in International Patent Application PCT/GB2021/050939 filed on April 19, 2021, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the CNS-specific promoter is a promoter of Table 11-Table 13, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 11-Table 13. In some embodiments, the CNS-specific promoter is a promoter of Table 11-Table 13, or a promoter having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with a promoter of Table 11-Table 13 and retaining a CNS-specific promoter activity of a promoter of Table 11-Table 13.

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 that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to a CRE of Tables 13-15. In some embodiments, the CRE is a CRE of Tables 13-15, or a CRE that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identical to a CRE of Tables 13-15 and retains the activity of a CRE of Tables 13-15.

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

Table 10 – NS-specific promoters

Table 11 – CNS-specific promoters

Table 12 - Minimal/proximal promoters included in the promoters of Table 11

Table 13 - Overview of synthetic CNS-specific promoters

Table 14 - Exemplary CREs

Table 15. Cis-regulatory elements (CRE) contained in the promoters of Table 11

Aspects of the present disclosure relate to isolated nucleic acids comprising more than one promoter (e.g., 2, 3, 4, 5 or more promoters). For example, in the context of a transgenic construct having a first region comprising a protein encoding region and a second region encoding an inhibitory RNA (e.g., miRNA), it may be desirable to use a first promoter sequence (e.g., a first promoter sequence operably connected to a protein coding region) to drive the expression of the protein coding region, and to use a second promoter sequence (e.g., a second promoter sequence operably connected to an inhibitory RNA coding region) to drive the expression of the inhibitory RNA coding region. In general, 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., a promoter driving the expression of the protein coding region) is an RNA polymerase III (polIII) promoter sequence. Non-limiting examples of polIII promoter sequences include U6 and HI promoter sequences. In some embodiments, the second promoter sequence (e.g., a promoter sequence driving the expression of the inhibitory RNA) is an RNA polymerase II (polII) promoter sequence. Non-limiting examples of polII promoter sequences include T7, T3, SP6, RSV, and cytomegalovirus promoter sequences. In some embodiments, the polIII promoter sequence drives expression of an inhibitory RNA (e.g., miRNA) coding region. In some embodiments, the polII promoter sequence drives expression of a protein coding region.

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%, 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, such as 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 13023-016-0405-3. In some embodiments, the therapeutic protein is wild-type huntingtin protein (e.g., huntingtin protein having a PolyQ repeat region containing less than 36 repeats).

CYP46A1

Cholesterol 24-hydroxylase is a neuronal enzyme encoded by the CYP46A1 gene. It converts cholesterol to 24-hydroxycholesterol and plays a key role in the outflow of cholesterol from the brain (Dietschy, J.M. et al., 2004). Brain cholesterol is basically produced in situ, but cannot be degraded, and the intact blood-brain barrier limits the direct transport of cholesterol from the brain (Dietschy, J.M. et al., 2004). 24-hydroxycholesterol can pass through the plasma membrane and the blood-brain barrier and reach the liver, where it is degraded.

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

In the early stages of AD, the concentration of 24-hydroxycholesterol in CSF and peripheral circulation is high. In the late stages of AD, the concentration of 24-hydroxycholesterol may decrease, which reflects neuronal loss (Kolsch, H. et al., 2004). CYP46A1 is expressed around the amyloid core of neuritic plaques in the brains of AD patients (Brown, J. et al., 3rd edition, 2004).

Activation of cholesterol 24-hydroxylase encoded by CYP46A1 provides a significant reduction in neuropathology and improvement of cognitive deficits in mouse models of CNS disease. For example, in a Huntington's disease model, co-expression of CYP46A1 and ExpHtt promoted a strong and significant reduction in the formation of ExpHtt aggregates (58% vs. 27.5%) (WO2012/049314). (See also International Patent Publication WO2009/034127; which is incorporated herein by reference in its entirety). The methods described herein involve the combination of activating CYP46A1 with the administration of miRNAs targeting certain other targets. For example, the methods may involve the administration of a viral vector to treat a neurological disease or disorder, wherein the vector expresses CYP46A1 in cells of the central nervous system.

In some embodiments, described herein are viral vectors 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 can be an adeno-associated virus (AAV) vector.

Further description of CY46A1 and its therapeutic uses (e.g., for Alzheimer's disease, ALS and ataxia) is described in the art (e.g., in WO 2012/049314, WO 2009/034127, WO 2018/138371 and WO2020/089154). The sequences, methods and compositions described therein can be used 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, which is capable of encoding a specific polypeptide or protein after being transcribed or translated.

The term "coding sequence" or "sequence encoding a specific protein" refers 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' (carboxyl) terminus. The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.

The cDNA sequence of CYP46A1 is disclosed in Genbank Accession No. NM_006668 (SEQ ID NO: 1). The amino acid sequence is shown in SEQ ID NO: 2. The present invention utilizes a nucleic acid construct comprising the sequence SEQ ID NO: 1 or a variant thereof to treat a neurological disease or disorder. The variants include naturally occurring variants due to, for example, allelic variants (e.g., polymorphisms) between individuals, alternative splicing forms, etc. 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 SEQ ID NO: 2, i.e., exhibit a nucleotide sequence that generally has 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 SEQ ID NO: 2. In some embodiments, the nucleic acid construct comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1 and retaining the activity of SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., the ability to convert cholesterol into 24-hydroxycholesterol). Variants of the CYP46A1 gene also include nucleic acid sequences that hybridize to the sequence as defined above (or its complementary strand) under stringent hybridization conditions. Typical stringent hybridization conditions include a temperature above 30°C, preferably above 35°C, more preferably above 42°C, and/or a salinity below about 500 mM, preferably below 200 mM. Hybridization conditions can be adjusted by a skilled artisan by modifying the temperature, salinity and/or the concentration of other reagents (e.g., SDS, SSC, etc.).

Exemplary CYP46A1 variants contemplated for use herein are provided in SEQ ID NO: 109 and SEQ ID NO: 110. In some embodiments, the viral vector comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 109. In some embodiments, the viral vector comprises a nucleic acid sequence encoding an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity 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.

In one aspect, provided herein are compositions comprising an isolated nucleic acid comprising a sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 110. In one aspect, provided herein are compositions comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 110. In some embodiments, the isolated nucleic acid encoding the 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 compared to SEQ ID NO: 1. In some embodiments, the mutation comprises a deletion and/or addition and/or substitution of at least one nucleic acid compared to the sequence shown in SEQ ID NO: 1. The mutation may result in, for example, removal of bacterial sequences, and/or removal of alternate reading frames, and/or removal of CpGs, and/or removal of restriction enzyme sites. In several embodiments, the aforementioned compositions may be used to treat a neurological disease or disorder described herein, for example, in the absence of an administered miRNA. In various embodiments, the aforementioned compositions may be used to treat a neurological disease or disorder described herein, for example, in the presence of an administered miRNA. In some embodiments, a recombinant viral vector (e.g., a recombinant AAV comprising an isolated nucleic acid as shown in SEQ ID NO: 110) is administered to a subject in need for expression of a CYP46A1 protein and/or for treatment of a neurological disease or disorder as described herein. In some embodiments, a recombinant viral vector (e.g., a recombinant AAV comprising an isolated nucleic acid sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 110) is administered to a subject in need thereof for expression of a CYP46A1 protein and/or for treatment of a neurological disease or disorder described herein. In some embodiments, a recombinant viral vector (e.g., a recombinant AAV comprising an isolated nucleic acid sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 111) is administered to a subject in need thereof for expression of a CYP46A1 protein and/or for treatment of a neurological disease or disorder described herein.

SEQ ID NO: 1CYP46A1 mRNA

SEQ ID NO: 2 CYP46A1 amino acid sequence

Carrier

Without wishing to be bound by any particular theory, allele-specific silencing of a pathogenic gene, such as mutant Huntington (HTT), may provide improved safety in a subject due to retention of wild-type expression and function in the cell compared to non-allele-specific silencing (e.g., silencing of wild-type and mutant HTT alleles). For example, aspects of the present invention relate to the inventors' recognition and understanding that isolated nucleic acids and vectors that introduce one or more inhibitory RNA (e.g., miRNA) sequences that target the HTT gene in a non-allele-specific manner, while driving expression of a hardened wild-type HTT gene (a wild-type HTT gene that is not targeted by the miRNA), can achieve concomitant knockdown of mutant HTT (e.g., in CNS tissues), and increased expression of wild-type HTT. Generally, the sequence of the nucleic acid encoding the endogenous wild-type and mutant HTT mRNA and the nucleic acid encoding the transgenic "hardened" wild-type HTT mRNA are sufficiently different that the "hardened" wild-type HTT transgenic mRNA is not targeted by the one or more inhibitory RNAs (e.g., miRNAs). For example, this can be achieved by introducing one or more silent mutations into the HTT transgenic sequence so that it encodes the same protein as the endogenous wild-type HTT gene but has a different nucleic acid sequence. In this case, the exogenous mRNA can be referred to as "hardened". Alternatively, the inhibitory RNA (e.g., miRNA) can target the 5' and/or 3' untranslated regions of the endogenous wild-type HTT mRNA. These 5' and/or 3' regions can then be removed or replaced in the transgenic mRNA so that the transgenic mRNA is not targeted by one or more inhibitory RNAs.

The reporter sequence (e.g., a nucleic acid sequence encoding a reporter protein) that can be provided in the transgene includes, but is not limited to, a DNA sequence encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other items well known in the art. When associated with a regulatory element that drives their expression, the reporter sequence provides a signal that can be detected by conventional means (including enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic determinations), fluorescence activated cell sorting determinations, and immunological determinations (including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunohistochemistry). For example, when the marker sequence is a LacZ gene, the presence of a vector carrying the signal is detected by determining the activity of β-galactosidase. When the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be visually measured by the generation of color or light in a photometer. For example, such reporters can be used to verify the tissue-specific targeting ability of nucleic acids and the regulatory activity of tissue-specific promoters. Recombinant adeno-associated viruses (rAAVs).

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. As used herein for AAV, the term "isolated" refers to an AAV that has been artificially produced or obtained. An isolated AAV can be produced using a recombinant method. Such AAVs are referred to herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities so that the transgenes and/or nucleases of the rAAVs will be specifically delivered to one or more predetermined tissues. The AAV capsid is an important element for determining the targeting capabilities of these tissue specificities. Therefore, rAAVs with capsids suitable for targeted tissues can be selected.

Methods for obtaining recombinant AAV with desired capsid proteins are well known in the art. (See, e.g., US 2003/0138772, the contents of which are incorporated herein by reference in their entirety). Generally, the method involves culturing a host cell containing a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector consisting of an AAV inverted terminal repeat (ITR) and a transgene; and sufficient auxiliary functions to allow the recombinant AAV vector to be packaged within the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. AAV contains three capsid proteins, virion proteins 1 to 3 (named VP1, VP2, and VP3), all of which are transcribed from a single cap gene by alternative splicing. In some embodiments, the molecular weights of VP1, VP2, and VP3 are about 87 kDa, about 72 kDa, and about 62 kDa, respectively. In some embodiments, when translated, the capsid protein forms a spherical 60-mer protein shell around the viral genome. In some embodiments, the function of the capsid protein is to protect the viral genome, deliver the genome and interact with the host. In some aspects, the capsid protein delivers the viral genome to the host in a tissue-specific manner.

In some embodiments, the recombinant AAV (rAAV) comprises an AAV capsid protein selected from the group consisting of AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8, AAVrh10, AAV 2G9, AAV 2.5G9, AAV9, and AAV10. In some embodiments, the recombinant AAV capsid (rAAV) protein is a serotype derived from a non-human primate, such as the AAVrh10 serotype. In some embodiments, rAAV is AAV PhP.eB or AAV PhP.B, as described in U.S. Publication Nos. and U.S. Granted Patents US20170166926A1, US9585971, US10301360, US9957303, US10202425, US10519198, US20190292230A1, US20200087353A1, which are incorporated herein by reference in their entirety. In some embodiments, rAAV comprises AAV, which comprises a surface-binding peptide, such as PB5-3, PB5-5, PB5-14 described in International Publication WO201912635, which are incorporated herein by reference in their entirety. In some embodiments, rAAV is AAV9 serotype. In some embodiments, rAAV is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 serotype or its heterologous chimera. In some embodiments, 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 its heterologous chimera. In certain embodiments, the rAAV comprises a chemically modified capsid disclosed in WO 2017/212019 (e.g., mannose ligand chemically coupled to AAV2). The rAAV with chemically modified capsid disclosed in WO 2017/212019 is incorporated herein by reference in its entirety. As a further embodiment, the rAAV comprises the AAV capsid protein of the present invention, which can be polyploid (also referred to as haploid, or reasonable haploid, or reasonable polyploid), because they can contain VP1, VP2 and VP3 capsid proteins from more than one AAV serotype in a single AAV virion, as described in PCT/US18/22725, PCT/US2018/044632 or US 10,550,405, which are incorporated herein by reference. In some embodiments, the rAAV comprises a capsid protein selected from the AAV serotypes listed in Table 17.

Table 17: AAV serotypes and exemplary published corresponding capsid sequences

The components to be cultured in the host cell to package the rAAV vector in the AAV capsid can be provided to the host cell in trans. Alternatively, any one or more required components {e.g., recombinant AAV vector, rep sequence, cap sequence and/or auxiliary function) can be provided by a stable host cell, which has been engineered to contain one or more of the required components using methods known to those skilled in the art. Most suitably, such stable host cells will contain the required components under the control of an inducible promoter. However, the required components may be under the control of a constitutive promoter. In the discussion of regulatory elements suitable for use with transgenics, examples of suitable inducible and constitutive promoters are provided herein. In another optional manner, 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, a stable host cell can be generated, the cell being derived from a 293 cell (which contains an E1 auxiliary function under the control of a constitutive promoter), but which contains rep and/or cap proteins under the control of an inducible promoter. Other stable host cells can be generated by those skilled in the art. In some embodiments, the disclosure relates to a host cell containing a nucleic acid comprising a coding sequence encoding a protein (e.g., wild-type huntingtin protein, optionally "hardened" wild-type huntingtin protein). In some embodiments, the disclosure relates to a composition comprising the above host cell. In some embodiments, the composition comprising the above host cell further comprises a cryoprotectant.

Any suitable genetic element (vector) can be used to deliver the recombinant AAV vector, rep sequence, cap sequence and auxiliary functions required for producing the rAAV of the present disclosure to the packaging host cell. The selected genetic element can be delivered by any suitable method (including those described herein). The method for constructing any embodiment of the present disclosure is known to those skilled in the art of nucleic acid manipulation and includes 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, N.Y.. Similarly, methods for generating rAAV virions are well known, and the selection of suitable methods is not a limitation of the present disclosure. See, for example, K. Fisher et al., J. Virol., 70: 520-532 (1993) and U.S. Patent No. 5,478,745.

In some embodiments, recombinant AAV can be produced using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV auxiliary function vector, and an accessory function vector. The AAV auxiliary function vector encodes an "AAV auxiliary function" sequence (i.e., rep and cap), which acts in trans for efficient AAV replication and encapsidation. Preferably, the AAV auxiliary 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 pHLP19 (described in U.S. Patent No. 6,001,650) and pRep6cap6 vectors (described in U.S. Patent No. 6,156,303), the entire contents of which are incorporated herein by reference. The accessory function vector encodes a nucleotide sequence of a non-AAV-derived viral and/or cellular function (i.e., "accessory function") on which AAV replication depends. Accessory functions include those required for AAV replication, including but not limited to those involved in: activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

In some aspects, the present disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art. See, for example, Graham et al. (1973) Virology, 52: 456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986), Basic Methods in Molecular Biology; Elsevier, and Chu et al. (1981), Gene 13: 197. Such techniques can be used to introduce one or more exogenous nucleic acids (e.g., nucleotide integration vectors and other nucleic acid molecules) into suitable host cells.

"Host cell" refers to any cell that harbors or is capable of harboring a substance of interest. Typically, the host cell is a mammalian cell. The host cell can be used as a recipient of the following: AAV helper constructs, AAV minigene plasmids, accessory function vectors or other transfer DNAs associated with the production of recombinant AAV. The term includes the descendants of the transfected original cells. Therefore, "host cell" as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the descendants of a single parent cell may not necessarily be exactly the same as the original parent in morphology or in the genome or total DNA complement due to natural, accidental or deliberate mutations.

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

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

As used herein, the term "vector" includes any genetic element, such as plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which can be replicated when associated with appropriate control elements, and it can transfer gene sequences between cells. Therefore, the term includes cloning tools and expression tools, and viral vectors. One type of vector is "plasmid", which refers to a circular double-stranded DNA loop, to which other DNA segments are connected. Another type of vector is a viral vector, in which other DNA segments are connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). In addition, some vectors can guide the expression of genes operably connected to them. Such vectors are referred to as "expression vectors" in this article. In general, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this application document, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. 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 the same functions.

A cloning vector is a vector capable of autonomous replication or integration in the genome of a host cell, further characterized by one or more endonuclease restriction sites, at which the vector can be sheared in a determinable manner, and the desired DNA sequence can be connected thereto so that the new recombinant vector maintains its ability to replicate in the host cell. In the case of a plasmid, as the number of copies of the plasmid increases in a host cell (e.g., a host bacterium), the replication of the desired sequence can occur many times, or the replication of the desired sequence before the host is propagated by mitosis is only once in each host. In the case of a phage, replication can occur actively during the lysis phase or passively during the lysogenic phase.

An expression vector is a vector into which a desired DNA sequence can be inserted by restriction and connection so that it can be operably joined to a regulatory sequence and can be expressed as an RNA transcript. The vector may further contain one or more marker sequences suitable for identifying cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds; genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase); and genes that visually affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vector used herein is capable of autonomous replication and expression of structural gene products present in the DNA segment to which it is operably connected.

In some embodiments, useful vectors are considered to be those in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. If the coding sequence is desired to be translated into a functional protein, if induction of the promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the connection 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, then the two DNA sequences are said to be operably joined. Therefore, if the promoter region is able to effect transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide, then the promoter region will be operably joined to the coding sequence.

"Promoter" refers to a DNA sequence that is recognized by the synthetic apparatus of the cell or the synthetic apparatus introduced and is required to initiate specific transcription of a gene. When the nucleic acid molecule encoding any polypeptide 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 natural promoter, i.e., a promoter of a gene in its endogenous environment, which provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter is unregulated, allowing continuous transcription of its associated gene. Various conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule.

The precise nature of the regulatory sequences required for gene expression may vary between species or cell types, but may generally include 5' non-transcribed sequences and 5' non-translated sequences associated with transcription initiation and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, etc., if necessary. In particular, such 5' non-transcribed regulatory sequences will include a promoter region comprising a promoter sequence for transcriptional control of an operably joined gene. Depending on the expectation, the regulatory sequence may also include an enhancer sequence or an upstream activator sequence. The vector of the present invention may optionally include a 5' leader or signal sequence. The selection and design of an appropriate vector is within the ability and discretion of those of ordinary skill in the art.

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. The heterologous DNA (RNA) is placed under the operable control of transcriptional elements to allow the heterologous DNA to be expressed in the host cell.

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

The aforementioned methods for packaging a recombinant vector into a desired AAV capsid to produce the rAAV of the present disclosure are not meant to be limiting, and other suitable methods will be apparent to those skilled in the art.

In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequences provided herein (including the sequences provided in the sequence table) can be replaced by any other nucleotides suitable for base pairing with adenosine nucleotides (e.g., by Watson-Crick base pairs). For example, in some embodiments, any one or more thymidine (T) nucleotides in the sequences provided herein (including the sequences provided in the sequence table) can be appropriately replaced by uridine (U) nucleotides, and vice versa.

In some embodiments of any aspect, the nucleic acid (e.g., miRNA) is chemically modified to enhance stability or other beneficial properties. The nucleic acids described herein can be synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse connection, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse connection, etc.); (b) base modifications, such as replacement with stable bases, destabilizing bases, or bases that base pair with an extended partner library, base removal (abasic nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars; and (d) backbone modifications, including modification or replacement of phosphodiester bonds. The specific examples of nucleic acid compounds useful in the embodiments described herein include but are not limited to the backbone comprising modification or the nucleic acid not containing natural internucleosides connected. The nucleic acid with modified backbone is especially included in the nucleic acid without phosphorus atom in the backbone. For the purpose of the present application document, and as mentioned sometimes in the art, the nucleic acid without the modification of phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside. In some embodiments of any aspect, the nucleic acid of modification will have phosphorus atom in its internucleoside backbone.

For example, the modified nucleic acid backbone may include thiophosphates, chiral thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), borophosphates with normal 3'-5' linkages, thionophosphoramidates, thionoalkylphosphonates, and thionoalkylphosphotriesters, their 2'-5' connection analogs, and those with opposite polarity, wherein adjacent nucleoside unit pairs are connected with 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 a phosphorus atom have backbones formed from short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These backbones include backbones having: morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformyl and thioformyl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; other backbones having mixed N, O, S and _CH2 components, and oligonucleosides having heteroatom backbones, particularly --CH2 _- NH-- _CH2-- , _--CH2- N( _CH3 )--O-- _CH2-- [referred to as a methylene(methylimino) or MMI backbone], _--CH2 --O--N( _CH3 )-- _CH2-- , _--CH2 --N(CH3)--N( _CH3 )-- _CH2-- and --N( _{CH 3} )--CH ₂ --CH ₂ --[wherein the natural phosphodiester skeleton is represented by --O--P--O--CH ₂ --].

In other nucleic acid mimics, the sugar of nucleotide unit and internucleoside connection (i.e., backbone) are both replaced by new groups. Keep base unit for hybridization with suitable nucleic acid target compound. A kind of such oligomeric compound (RNA mimics with excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by the backbone containing amides, particularly aminoethylglycine backbone. Core base is retained and directly or indirectly bonded to the aza nitrogen atom of the amide part of the backbone.

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

The modified nucleic acid may also comprise one or more substituted sugar moieties. The nucleic acids described herein may comprise 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, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ and O(CH _{2 ) n} ON[(CH ₂ ) _n CH ₃ )] ₂ , wherein n and m are 1 to about 10. In some embodiments of any aspect, the nucleic acid comprises one of the following at the 2' position: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, _SCH3 , OCN, Cl _, Br, CN, _CF3 , _OCF3 , _SOCH3 , _SO2CH3 , _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group that improves the pharmacokinetic properties of nucleic acid, or group that improves the pharmacodynamic properties of nucleic acid, and other substituents with similar properties. In some embodiments of any aspect, the modification includes 2'methoxyethoxy (2'-O--CH ₂ CH ₂ OCH ₃ , 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., an O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH ₂ --O--CH ₂ --N(CH ₂ ) ₂ , also described in the Examples below.

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

Nucleic acids can also include modifications or substitutions of nucleobases (often 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 can include other synthetic and natural nucleobases, including but not limited to 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6 ... Azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine (7-daazaadenine), and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the present invention. These nucleobases 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 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. In some embodiments of any aspect, the modified nucleobase may include d5SICS and dNAM, which are non-limiting examples of non-natural nucleobases that can be used alone 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 aspect, the oligonucleotide tag (e.g., Oligopaint) comprises any modified nucleobase known in the art, i.e., any nucleobase modified from an unmodified and/or natural nucleobase.

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

Another modification of the nucleic acids featured in the invention involves chemically linking the nucleic acid to 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, lipid moieties, such as a cholesterol moiety (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. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); a fatty chain, such as, for example, dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54); phospholipids, such as di-hexadecyl-racemic glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-racemic-glycero-3-phosphonate/salt (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14: 969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783); Lett., 1995, 36:3651-3654); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments of any aspect, the vector is pEMBL. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCG intron only. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x control pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-CYP46A1-hCGin-2x artificial pre-miR. In some embodiments of any aspect, the vector is pEMBL-D(+)Syn1-luc-HTT-3'UTR/mutant. In some embodiments of any aspect, the vector comprises at least one of: at least one (e.g., 2) ITR; Synl promoter; at least one (e.g., 2) hCG intron; at least one (e.g., 2) copies of a premiR (e.g., a control pre-miR, an artificial pre-miR; SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66); 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 another embodiment, the miRNA targets both wild-type and mutant HTT alleles. In yet another embodiment, the miRNA targets any HTT mRNA.

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

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

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 that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 152. In one aspect, the compositions provided herein comprise a recombinant viral vector comprising a promoter comprising a nucleic acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 152.

Synapsin-1 (SEQ ID NO: 152)

In one aspect, provided herein is a composition comprising an isolated nucleic acid comprising a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 111. In one aspect, provided herein is a composition comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 111. In some embodiments, the vector (e.g., rAAV) comprises a promoter (e.g., a synthetic nervous system-specific promoter; see, e.g., Tables 10-13) or a fragment thereof, and/or an enhancer, and/or a cis-regulatory element (CRE; see, e.g., Tables 13-15) that replaces the promoter/enhancer of SEQ ID NO: 111. In some embodiments, the vector (e.g., rAAV) 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 further comprises a promoter (e.g., a synthetic nervous system-specific promoter; see, e.g., Tables 10-13) or a fragment thereof, and/or an enhancer, and/or a cis-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, e.g., SEQ ID NO: 111, Table 16. In several embodiments, the aforementioned compositions can be used, e.g., in the absence of administered miRNA, to treat a nervous system disease or disorder described herein. In various embodiments, the aforementioned compositions can be used, for example, in the presence of an administered miRNA to treat a neurological disease or disorder described herein. In some embodiments, a recombinant viral vector (e.g., a recombinant AAV) comprising an isolated nucleic acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 111 is administered to a subject in need thereof for expression of a CYP46A1 protein, and/or for treatment of a neurological disease or disorder described herein. In some embodiments, a recombinant viral vector (e.g., recombinant AAV) comprising the isolated nucleic acid sequence SEQ ID NO: 111 is administered to a subject in need thereof for expression of CYP46A1 protein and/or for treatment of a nervous system disease or disorder described herein; and wherein the CMV enhancer and/or ACTB proximal promoter and/or chimeric ACTB-HBB2 intron of SEQ ID NO: 111 is replaced by one or more of a synthetic nervous system-specific promoter or fragment thereof, and/or enhancer selected from Tables 10-13, and/or a cis-regulatory element (CRE) selected from Tables 13-15.

SEQ ID NO: 111, 4036 bp, comprising the ITR to ITR sequence of the CYP46A1 variant sequence (see, e.g., SEQ ID NO: 110;).

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

Italic text (eg, nt 182-436 of SEQ ID NO: 111) indicates an enhancer.

Bold italic text (eg, nt 550-804 of SEQ ID NO: 111) indicates a promoter.

Double underlined text (eg, nt 824-1892 of SEQ ID NO: 111) indicates introns.

Bold double underlined text (eg, nt 1966-3465 of SEQ ID NO: 111) indicates the coding sequence (CDS) of a CYP46A1 variant sequence (see, eg, SEQ ID NO: 110).

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

Bold italic double underlined text (eg, nt3907-4036 of SEQ ID NO: 111) indicates the right ITR.

Table 16

In one aspect, provided herein is a composition comprising an isolated nucleic acid comprising a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 153. In one aspect, provided herein is a composition comprising a recombinant viral vector comprising an isolated nucleic acid comprising a sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 153. 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, and/or an enhancer, and/or a cis-regulatory element (CRE; see, e.g., Tables 13-15) that replaces the promoter and/or enhancer of SEQ ID NO: 153.

SEQ ID NO: 153

In some aspects provided herein, the nucleic acid sequence as shown in SEQ ID NO: 153 is used to make rAAV lacking bacterial sequences. In some embodiments, the rAAV is made from a plasmid DNA template (e.g., as shown in SEQ ID NO: 111). In some embodiments, the rAAV is made from a closed-end linear duplex DNA (e.g., as shown in SEQ ID NO: 153 or SEQ ID NO: 111).

Modified capsid

In one embodiment, the capsid described herein is further modified to increase tropism for the CNS. The compositions provided herein contain a modified viral capsid comprising a payload, wherein the payload comprises a nucleic acid sequence flanked by an inverted terminal repeat (ITR) targeting a disorder of the central nervous system and a regulatory sequence, and wherein the modification is a chemical, non-chemical or amino acid modification. In some embodiments, the nucleic acid sequence of the payload comprises (a) an isolated nucleic acid encoding a transgenic, the transgenic 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 transgenic, the transgenic encoding one or more miRNAs. In some embodiments, the nucleic acid sequence of the payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

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 nucleic acid sequence flanked by an inverted terminal repeat (ITR) targeting a central nervous system disorder and a regulatory sequence, wherein the first modified viral capsid and at least the second modified viral capsid are identical, and the first payload and the second payload are different, and wherein the modification is a chemical, non-chemical or amino acid modification. In some embodiments, the nucleic acid sequence of the first payload or the 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 payload or the second payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

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 nucleic acid sequence flanked by an inverted terminal repeat (ITR) targeting a central nervous system disorder and a regulatory sequence, wherein the first modified capsid and at least the second modified capsid are different, and the first payload and the second payload may be the same or different, and wherein the modification is a chemical, non-chemical or amino acid modification. In some embodiments, the nucleic acid sequence of the first payload or the second payload comprises an isolated nucleic acid encoding a transgenic one or more miRNAs. In some embodiments, the nucleic acid sequence of the first payload or the second payload comprises an isolated nucleic acid encoding a CYP46A1 protein.

In certain embodiments, the modified viral capsid comprises modifications that allow it to preferentially target the CNS or PNS. For example, the modified viral capsid has an increased tropism for the CNS, and/or a tropism that is reduced for at least a second position (e.g., liver). The preferential targeting of the CNS does not exclude targeting of other sites, but rather indicates that it is more highly targeted to the CNS than other sites.

In one embodiment, the modified viral capsid comprises a modification that enables it to target the CNS or PNS. For example, modification of a capsid that is normally targeted to a non-CNS site (e.g., liver) can redirect the capsid to now target the CNS and non-CNS sites. In such embodiments, CNS targeting need not be a priority.

In one embodiment, the modification of the capsid is an amino acid modification, e.g., a deletion, insertion or substitution of an amino acid. 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.

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

In one embodiment, the modified viral capsid is an AAV capsid protein comprising one or more amino acid substitutions, wherein the substitution introduces a new glycan binding site 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 the corresponding amino acid positions in AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV10. Such AAV capsid proteins are further described in, for example, U.S. Patent Application No. 16/110,773; the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the modified viral capsid is an AAV2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT/US2020/029493), an AAV2.5 capsid protein (SEQ ID NO: 1 of International Patent Application No. PCT/US2020/029493), or an AAV 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), wherein the AAV2.5 capsid protein comprises 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 (e.g., astrocytes). In embodiments of the capsid protein, capsid, viral vector and method described in International Patent Application No. PCT/US2020/029493, the one or more amino acid substitutions include A267S, SQAGASDIRDQSR464-476SX ₁ AGX ₂ SX ₃ X ₄ X ₅ X ₆ QX ₇ R (wherein X _1-7 can be any amino acid), and EYSW 500-503EX ₈ X ₉ W, wherein X _8-9 can be any amino acid. In embodiments of the capsid protein, capsid, viral vector and method described herein, X ₁ is V or a conservative substitution thereof; X ₂ is P or a conservative substitution thereof; X ₃ is N or a conservative substitution thereof; X ₄ is M or a conservative substitution thereof; X ₅ is A or a conservative substitution thereof; X ₆ is V or a conservative substitution thereof; X ₇ is G or a conservative substitution thereof; X ₈ is F or a conservative substitution thereof; and/or X ₉ is A or a conservative substitution thereof. In embodiments of the capsid proteins, capsids, viral vectors, and methods described herein, _X1 is V, _X2 is P, _X3 is N, _X4 is M, _X5 is A, _X6 is V, X7 is G, _X8 is F, _X9 is A, wherein the novel glycan binding site is a galactose binding site. Such AAV capsid proteins are further described, for example _, in International Patent Application No. PCT/US2020/029493; the contents of which are incorporated herein by reference in their entirety.

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

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 comprises 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 positions corresponding to: one or more, or each, of Y705, Y731 and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. patent application Ser. No. 16/565,191; the contents of which are incorporated herein by reference in their entirety); one or more, or each, of Y436, Y693 and Y719 of a wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. patent application Ser. No. 16/565,191); or one or more, or each, of Y705, Y731 and T492 of a wild-type AAV6 capsid protein (e.g., SEQ ID NO: 3 of U.S. patent application Ser. No. 16/565,191). This AAV capsid targets neurons and astrocytes.

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

In one embodiment, the modified viral capsid is an AAV capsid protein (e.g., AAV1, AAV5, or AAV6 capsid protein), wherein the VP3 region of the capsid protein comprises 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 positions corresponding to: one or more or each of Y705, Y731, and T492 of a wild-type AAV1 capsid protein (e.g., SEQ ID NO: 1 of U.S. Patent Application No. 16/565,191); one or more or each of Y436, Y693, and Y719 of a wild-type AAV5 capsid protein (e.g., SEQ ID NO: 2 of U.S. Patent Application No. 16/565,191); or one or more or each of Y705, Y731, and T492 of 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.

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

In one embodiment, the amino acid modification allows the modified capsid to escape neutralizing antibodies, such as neutralizing antibodies produced against a viral vector (e.g., a viral vector of the same serotype). In one embodiment, the amino acid modification allows the modified capsid to be used for repeated administration, for example, the modification will cause the capsid to have a therapeutic effect when it is administered again.

In one embodiment, the modified viral capsid is a chimeric capsid. As used herein, a "chimeric" capsid protein refers to an AAV capsid protein (e.g., any one or more of VP1, VP2, or VP3) that has been modified by substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to the wild type, and insertion or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to the wild type. In some embodiments, a complete or partial domain, functional region, epitope, etc. from one AAV serotype can replace the corresponding wild-type domain, functional region, epitope, etc. of a different AAV serotype in any combination to produce 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 large number of chimeric capsid proteins that can be included in the capsid of the present invention are described in the literature and herein.

In one embodiment, the modified viral capsid is a haploid capsid. As used herein, the term "haploid AAV" shall refer to the AAV described in International Application WO2018/170310 or U.S. Application US2018/037149, which is incorporated herein by reference in its entirety. In some embodiments, the group of virions is a haploid AAV group, in which virion particles can be constructed, in which at least one viral protein from a group consisting of AAV capsid protein, 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 (VP1, VP2 and/or VP3) present, the protein is of the same type (e.g., all AAV2VP1). In one example, at least one of the viral proteins is a chimeric viral protein, and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric, and only VP3 is non-chimeric. For example, the viral particle consists only of: VP1/VP2 from chimeric AAV2/8 (N-terminus of AAV2 and C-terminus of AAV8) paired with VP3 from AAV2 alone; or only chimeric VP1/VP2 28m-2P3 (N-terminus from AAV8 and C-terminus from AAV2 without VP3 start codon mutation) paired with VP3 from AAV2 alone. In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-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 is paired with VP3 from AAV3 alone. In another example, no chimeric protein is present.

In some embodiments of the technology described herein, the modified viral capsid comprises one or more modifications, such as chemical modifications, non-chemical modifications or amino acid modifications to the capsid. Such modifications can, for example, modify the tissue tropism or cell tropism of the modified capsid and others.

Modifications may directly alter the properties of the capsid (including biochemical properties, such as receptor binding), such that the modification itself alters the behavior of the capsid, or may allow for further modifications (such as the attachment of a ligand, which in turn modifies the behavior of the capsid in a desired manner).

In one embodiment, chemical modification of cysteine residues (which may be naturally occurring or introduced by genetic modification of the capsid polypeptide encoding sequence) allows for covalent attachment of ligands via disulfide bond formation (see, e.g., WO 2005/106046, the contents of which are incorporated herein by reference).

A variety of ligands are contemplated, including but not limited to, for example, antibodies or antigen-binding fragments thereof that target cell surface proteins expressed by target cells (see, for example, WO 2000/002654, which is incorporated herein by reference).

WO 2015/062516 (the contents of which are also incorporated herein by reference) describes the insertion of an amino acid comprising an azido group by genetic modification of the capsid gene, followed by chemical coupling of a ligand via the azido group.

Horowitz et al., Bioconjugate Chem., 22:529-532 (2011) describe the tropism for modifying AAV capsids by glycosylation or chemical coupling of sugar moieties. This method and similar methods are contemplated for modification of capsids as described herein.

In other embodiments, coating of the viral capsid with a polymer 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.

In other embodiments, carbodiimide coupling is specifically contemplated. See, for example, Joo et al., ACS Nano 5, entitled "Enhanced Real-time Monitoring of Adeno-Associated Virus Trafficking by Virus-Quantum Dot Conjugates" (2011).

In other embodiments, the viral capsid can be modified, for example, as described in WO 2017/212019, see also US National Phase USSN 16/308,740, the contents of which are each incorporated herein by reference. The method described therein couples the viral capsid with a ligand by a bond comprising -CSNH- and an aromatic moiety. Although the genetically modified viral capsid can be further modified by this method, the modification described therein does not require the genetic modification of the viral capsid. The ligand described therein includes, for example, a targeting agent, a spatial shielding agent for avoiding neutralizing antibody interactions, a labeling agent or a magnetic agent. The targeting ligand described therein includes, for example, cell type-specific ligands, proteins, monosaccharides or polysaccharides, steroid hormones, RGD motif peptides (e.g., Arg-Gly-Asp, a cell adhesion motif that can simulate cell adhesion proteins and bind to integrins), vitamins and small molecules.

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

In one embodiment, the chemical modification of the present invention is a modification described in international patent application PCT/EP2020/069554, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the capsid has at least one chemically modified tyrosine residue in its capsid, wherein the chemically modified tyrosine residue is of formula (I):

in:

_-X1 is selected from the group consisting of:

-Ar is an optionally substituted aryl or heteroaryl moiety.

In one embodiment, the capsid has at least one chemically modified tyrosine residue, which is of formula (Ia):

in:

-X _i , and Ar are as defined above,

- The spacer is a group used to connect the "Ar" group to the functional part "M", which is preferably

contains up to 1000 carbon atoms and which is preferably in the form of a chemical chain optionally containing heteroatoms and/or cyclic moieties,

-n is 0 or 1; and

-M is a functional moiety comprising a steric hindering agent, a labeling agent, a cell type specific ligand, or a drug moiety,

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

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

in:

-X ₂ is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, 0-(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-.

_-X2 is in the para, meta or ortho position of the phenyl group, preferably in the para position,

- Spacer, n and M are as defined above.

In one embodiment, when present, the "spacer" is selected from the group consisting of a saturated or unsaturated, linear or branched _C2 - _C40 hydrocarbon chain, optionally substituted polyethylene glycol, polypropylene glycol, pHPMA (polymer of N-(2-hydroxypropyl)methacrylamide), polylactic-co-glycolic acid (PLGA), polymers of alkyl diamines, and combinations thereof, and/or

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

In one embodiment, the "spacer" (when present) is selected from the group consisting of: a linear or branched _C2 - _C20 alkyl chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, a polymer of alkyldiamine, and combinations thereof, wherein the polymer having 2 to 20 monomers and/or "M" comprises or consists of a cell type-specific ligand, wherein the cell type-specific ligand is derived from: a protein selected from transferrin, epidermal growth factor (EGF) and basic fibroblast growth factor 13FGF; a monosaccharide or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, a GalNac bridge or mannose-6-phosphate; an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7; and a vitamin (e.g., folic acid).

In one embodiment, the capsid further has at least one additional chemically modified amino acid residue in the capsid, which is different from the tyrosine residue, and the amino acid residue preferably has an amino group chemically modified with a group of formula (V):

in:

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

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

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

In one embodiment, the capsid is incubated with a chemical reagent of Formula VId to obtain at least one chemically modified tyrosine residue in the capsid of Formula Ic.

Application

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

rAAV can be delivered to a mammalian subject by, for example, intramuscular injection or by administration to the bloodstream of a mammalian subject. Administration to the bloodstream can be by injection into a vein, an artery or any other vascular conduit. In some embodiments, the rAAV is administered into the bloodstream by means of isolated limb perfusion (a technique well known in the surgical field), and the method is mainly to isolate the limb from the systemic circulation by the technician before administering the rAAV virion. Variants of isolated limb perfusion techniques (described in U.S. Patent No. 6,177,403) can also be used by technicians to administer virions to the vascular system of isolated limbs to potentially enhance transduction into muscle cells or tissues. In addition, in some cases, it may be desirable to deliver virions to the CNS of a subject. "CNS" refers to all cells and tissues of the brain and spinal cord of vertebrates. Therefore, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), intercellular spaces, bones, cartilage, etc. Recombinant AAV can be delivered directly to the CNS or brain by injection with a needle, catheter, or related device using neurosurgical techniques known in the art (e.g., by stereotactic injection, see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000) into, for example, the ventricular region and the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule. In some embodiments, rAAV as 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, the rAAV is delivered by intracranial injection. In some embodiments, the rAAV is delivered by cisterna magna injection. In some embodiments, the rAAV is delivered by intracerebroventricular injection.

Delivery of the composition to a mammalian subject can be by, for example, any known means of delivery to a desired site (e.g., CNS). It may be desirable to deliver the composition to the CNS of a subject. "CNS" refers to all cells and tissues of the brain and spinal cord of vertebrates. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bones, cartilage, and the like. Any composition described herein can be delivered by injection using a needle, catheter, or related device, using neurosurgical techniques known in the art (e.g., by stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11: 2315-2329, 2000) is injected into, for example, the ventricular region and the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobule for direct delivery to the CNS or brain. In some embodiments, the composition as described herein is administered by intravenous injection. In some embodiments, the composition as described herein is administered by intraspinal injection. In some embodiments, the composition as described herein is administered by intraventricular injection of the brain. In some embodiments, the composition is administered by intracerebral injection. In some embodiments, the composition is administered by intrathecal injection. In some embodiments, the composition is administered by intrastriatal injection. In some embodiments, the composition is delivered by intracranial injection. In some embodiments, the composition is delivered by injection into the cerebellomedullary cisterna magna. In some embodiments, the composition is delivered by injection into the lateral cerebral ventricle.

The CNS includes, but is not limited to, certain regions of the CNS, neural pathways, somatosensory system, visual system, auditory system, nerves, neuroendocrine system, neurovascular system, brain neurotransmitter system, and duralmeningeal system.

Exemplary regions of the CNS include, but are not limited to, the myelencephalon; medulla oblongata; medullary pyramids; olives; inferior olivary nucleus; rostral ventrolateral medulla; caudal ventrolateral medulla; nucleus tractus solitarius (nucleus of the tractus solitarius); respiratory center-respiratory groups dorsal respiratory group; ventral respiratory group or apneustic centre pre-Bauchinger complex; complex; Bautzinger complex; Retrotrapezoid nucleus; Retrofacial nucleus; Nucleus retroambiguus; Nucleus para-ambiguus; Paramedian reticular nucleus; Giant cell reticular nucleus; Parafacial zone; Cuneate nucleus; Gracilis nucleus; Perihypoglossal nuclei; Intercalated nucleus; Prepositus nucleus; Hypoglossal nucleus; Area postrema; Medullary cranial nerve nuclei; Inferior salivatory nucleus; Nucleus ambiguus; Dorsal vagal nucleus; Hypoglossal nucleus; Chemoreceptor trigger zone; Metencephalon; Pons; Pontine nuclei; Pontine nuclei cranial nerve nuclei); main or pontine nucleus of the trigeminal sensory nucleus; motor nucleus of the trigeminal nerve; abducens nucleus (VI); facial nucleus (VII); vestibulocochlear nuclei (vestibular and cochlear nuclei) (VIII); superior salivatory nucleus; pontine tegmentum; pontine micturition center (Barrington's nucleus); locus coeruleus; pedunculopontine nucleus; lateral tegmental nucleus; reticular nucleus of the pontine tegmentum; nucleus incertus; parabrachial area; medial parabrachial nucleus; lateral parabrachial nucleus; subparabrachial nucleus ( -Fuse nucleus; Pontine respiratory group; Superior olivary complex; Medial superior olive; Lateral superior olive; Medial trapezoid nucleus; Paramedian pontine reticular formation; Parvocellular reticular nucleus; Caudal pontine reticular nucleus; Cerebellar peduncles; Superior cerebellar peduncle; Middle cerebellar peduncle; Inferior cerebellar peduncle; Fourth ventricle; Cerebellum vermis; Cerebellar hemispheres; Anterior lobe; Posterior lobe; Flocculonodular lobe; Cerebellar nuclei; Fastigial nucleus; Interposed nucleus; Globular nucleus; Emboliform nucleus nucleus; dentate nucleus; midbrain (mesencephalon); corpus quadrigemina (TectumCorpora quadrigemina); inferior colliculi; superior colliculi; anterior tectum; tegmentum periaqueductal gray; rostral interstitial nucleus of medial longitudinal fasciculus; midbrain reticular formation; dorsal raphe nucleus; red nucleus; ventral tegmental area; parabrachial pigmented nucleus; paranigral nucleus; rostromedial tegmental nucleus; caudal linear nucleus; rostral linear nucleus of the raphe; interfascicular nucleus; substantia nigra nigra; Pars compacta; Pars reticulata; Nucleus interpedunculi; Cerebral peduncle; Crus cerebri; Mesencephalic cranial nerve nuclei; Oculomotor nucleus (III); Edinger-Westphal nucleus; Trochlear nucleus (IV); Mesencephalic canal (aqueduct of the cerebral aqueduct, the aqueduct of the midbrain); Forebrain (prosencephalon); Diencephalon; Epithalamus; Pineal body (pinealgland); Habenular nuclei; Stria medullaris; Taenia thalami; Third ventricle; Subcommissural organ organ; thalamus; anterior nuclear group; anteroventral nucleus (also called ventral anterior nucleus); anterior dorsal nucleus; anterior medial nucleus; medial nuclear group; dorsomedial nucleus; midline nuclear group; paratenial nucleus; reuniensnucleus; rhomboidal nucleus; intralaminar nuclear group; central nucleus of thalamus; parafascicular nucleus; central nucleus; central lateral nucleus; lateral nuclear group; dorsolateral nucleus; lateral posterior nucleus; pulvinar; ventral nuclear group; ventral anterior nucleus; ventral lateral nucleus; ventral posterior nucleus; ventral posterolateral nucleus; ventral posteromedial nucleus; metathalamus; medial geniculate body; lateral geniculate body; reticular nucleus of thalamus; hypothalamus (limbic system) (HPA axis); anteromedial part of preoptic area; medial preoptic nucleus INAH INAH 1; INAH 2; INAH 3; INAH 4; median preoptic nucleus; suprachiasmatic nucleus; paraventricular nucleus; supraoptic nucleus (main); anterior hypothalamic nucleus; lateral area; part of preoptic area; lateral preoptic nucleus; anterior part of lateral nucleus; part of supraoptic nucleus; other nuclei of preoptic area; median preoptic nucleus; periventricular preoptic nucleus; tuberal medial area; dorsomedial nucleus of hypothalamus; ventromedial nucleus; arcuate nucleus; lateral area tuberal part of lateral nucleus; lateral tuberal nucleus; posteromedial area of mammillary nucleus (part of mammillary bodies); posterior nucleus; posterior part of lateral area posterior of lateral nucleus; surface median eminence eminence; Mammillary body; Pituitary stalk (infundibulum); Optic chiasm; Subfornical organ; Periventricularnucleus; Tuber cinereum; Tuberomammillarynucleus; Tuberous region; Mammillary nucleus; Subthalamus (HPA axis); Subthalamic nucleus; Zona incerta; Pituitary gland (HPA axis); Neurohypophysis; Parsintermedia (Intermediate Lobe); Adenohypophysis; Telencephalon (cerebrum); Cerebral hemispheres; White matter; Centrum semiovale; Corona radiata; Internal capsule; External capsule; Extreme capsule capsule; Subcortical; Hippocampus (medial temporal lobe); Dentate gyrus; Cornu ammonia (CA area); Cornu amygdala (limbic system) (limbic lobe); Central nucleus (autonomic nervous system); Medial nucleus (accessory olfactory system); Cortical and basomedial nuclei (primary olfactory system); Lateral and basolateral nuclei (frontotemporal cortical system); Extended amygdala; Bed nucleus of the stria terminalis; Claustrum; Basal ganglia; Striatum Dorsal striatum (also called neostriatum); Putamen; Caudate nucleus; Ventral striatum; Nucleus accumbens; Olfactory tubercle tubercle; globus pallidus (which forms the lenticular nucleus with the putamen); ventral globus pallidus; subthalamic nucleus; basal forebrain; anteriorperforated substance; substantia innominate; basal nucleus; oblique band of Broca; septal nucleus; medial septalnuclei; lamina terminalis; vascular organ of lamina terminalis; olfactory brain (paleocortex); olfactory bulb; olfactory tract; anterior olfactory nucleus; piriform cortex; anterior commissure; uncus; periamygdaloid cortex; cerebral cortex (neocortex); frontal lobe; cortex primary motor cortex (precentral gyrus, M1); supplementary motor cortex; premotor cortex; prefrontal cortex; orbitofrontal cortex; dorsolateral prefrontal cortex cortex; Gyri: Superior frontal gyrus; Middle frontal gyrus; Inferior frontal gyrus; Brodmannarea: 4, 6, 8, 9, 10, 11, 12, 24, 25, 32, 33, 44, 45, 46, 47; Parietal cortex: Primary somatosensory cortex (S1); Secondary somatosensory cortex (S2); Posterior parietal cortex; Postcentral gyrus (primary somatosensory area); Brodmannarea 1, 2, 3 (primary somatosensory area); 5, 7, 23, 26, 29, 31, 39, 40; Occipital cortex: Primary visual cortex (V1), V2, V3, V4, V5/MT; Lateral occipital gyrus (S2) =Occipitalgyrus; Brodmann areas 17 (V1, primary visual cortex); 18, 19; Temporal lobe cortex Primary auditory cortex (A1); Secondary auditory cortex (A2); Inferior temporal cortex; Posterior inferior temporal cortex; Gyri: superior temporal gyrus; Middle temporal gyrus; Inferior temporal gyrus; Entorhinal cortex; Perirhinal cortex; Parahippocampal gyrus; Fusiform gyrus; Brodmann areas: 20, 21, 22, 27, 34, 35, 36, 37, 38, 41, 42; Insular cortex; Cingulate cortex Anterior cingulate; Posterior cingulate; Retrosplenial cortex cortex); Indusium griseum; Subgenual area 25; Brodmann's zones 23, 24; 26, 29, 30 (posterior compression zone); 31 and 32.

Exemplary neural pathways include, but are not limited to: Superiorlongitudinal fasciculus Arcuate fasciculus; Uncinate fasciculus; Perforant pathway; Thalamocortical radiation; Corpuscallosum; Anterior commissure; Amygdalofugal pathway; Interthalamic adhesion; Posterior commissure; Habenular commissure; Fornix; Mammillotegmental; Fasciculus; Incertohypothalamic pathway pathway); cerebral peduncle; medial forebrain fasciculus; medial longitudinal fasciculus; myoclonus triangle; solitary fasciculus; major dopaminergic pathway from dopaminergic cell populations; mesocortical pathway; mesolimbic pathway; nigrostriatal pathway; tuberoinfundibular pathway; serotonergic pathway raphe nuclei; norepinephrine pathway locus coeruleus and other noradrenergic cell populations; adrenaline pathway from adrenergic cell populations; glutamate and acetylcholine pathways from mesopontinenuclei; motor system/descending fibers; extrapyramidal system; pyramidal tract; corticospinal tract; or cerebrospinal fibers; lateral corticospinal tract; anterior corticospinal tract; corticopontine fibers; frontopontine fibers; temporopontine fibers; corticobulbar tract; corticomesencephalic tract tract); tectospinal tract; interstitial tract; rubrospinal tract; rubrospinal tract; olivocerebellar tract; olivospinal tract; vestibulospinal tract; lateral vestibulospinal tract; medial vestibulospinal tract; reticulospinal tract; lateral reticulospinal tract; alpha system; and gamma system.

Exemplary somatosensory systems include, but are not limited to, the dorsal column-medial lemniscal pathway; the gracile fasciculus; the cuneate fasciculus; the medial lemniscus; the spinothalamic tract; the lateral spinothalamic tract; the anterior spinothalamic tract; the spinomesencephalic tract; the spinocerebellar tract; the spinoolivary tract; and the spinoreticular fasciculus.

Exemplary visual systems include, but are not limited to, the optic tract; the optic radiation; and the retinohypothalamic tract.

Exemplary auditory systems include, but are not limited to, the stria medulla of the fourth ventricle; the trapezoidal body; and the lateral lemniscus.

Exemplary nerves include, but are not limited to, brainstem cranial nerve endings (O); olfactory (I); visual (II); oculomotor (III); trochlear (IV); trigeminal (V); abducens (VI); facial (VII); vestibulocochlear (VIII); glossopharyngeal (IX); vagus (X); accessory (XI); and hypoglossal (XII).

Exemplary neuroendocrine systems include, but are not limited to, hypothalamic-pituitary hormones; the HPA axis; the HPG axis; the HPT axis; and GHRH-GH.

Exemplary neurovascular systems include, but are not limited to, the middle cerebral artery; the posterior cerebral artery; the anterior cerebral artery; the vertebral artery; the basilar artery; the circle of Willis (arterial system); the blood-brain barrier; the glymphatic system; the venous system; and the periventricular organ.

Exemplary brain neurotransmitter systems; norepinephrine system; dopamine system; serotonin system; cholinergic system; GABA; neuropeptide opioid peptides; endorphins; enkephalins; dynorphins; oxytocin; and substance P.

Exemplary dura mater systems include, but are not limited to, the brain-CSF barrier; meningeal covering, the dura mater; arachnoid mater; pia mater; epidural space; subdural space; subarachnoid space arachnoid septum; superior cistern; lamina terminalis cistern; chiasmatic cistern; interpeduncular cistern; pontine cistern; cerebellomedullar cistern; spinal cord subarachnoid space; ventricular system; cerebrospinal fluid; third ventricle; fourth ventricle; angular bundle; anterior horn; body of lateral ventricle; inferior horn; calcar avis; and subventricular zone.

In one embodiment, AAV is administered to the PNS. "PNS" refers to the nerves and ganglia outside the brain and spinal cord. The main function of the PNS is to connect the CNS to the limbs and organs, essentially acting as a relay between the brain and spinal cord and the rest of the body. Unlike the CNS, the PNS is not protected by the spine and skull or the blood-brain barrier, which exposes it to, for example, toxins and mechanical injury.

The PNS is divided into the somatic nervous system and the autonomic nervous system. In the somatic nervous system, the cranial nerves are part of the PNS, except for the optic nerve (cranial nerve II) and the retina. The second cranial nerve is not a true peripheral nerve, but a bundle in the diencephalon. The cranial nerve ganglia originate from the CNS. However, the remaining ten cranial nerve axons extend outside the brain and are therefore considered part of the PNS. The autonomic nervous system exercises involuntary control over smooth muscles and glands. The connection between the CNS and the organs allows the system to be in two different functional states: sympathetic and parasympathetic.

The somatic nervous system is under voluntary control and transmits signals from the brain to end organs (e.g., muscles). The sensory nervous system is part of the somatic nervous system and transmits signals from the senses (e.g., taste and touch (both fine and gross touch)) to the spinal cord and brain. The autonomic nervous system is a "self-regulating" system that affects the function of organs outside of voluntary control (e.g., heart rate), or the function of the digestive system.

The PNS can be described in different parts, including the cervical spinal nerves (C1-C4). The first four cervical nerves, C1 to C4, split and recombine to create the various nerves that serve the neck and back of the head. Spinal nerve C1 is called the suboccipital nerve, which provides motor innervation to the muscles at the base of the skull. C2 and C3 form many nerves in the neck, providing sensation and motor control. They include the greater occipital nerve, which provides sensation to the back of the head, the lesser occipital nerve, which provides sensation to the area behind the ear, the greater auricular nerve, and the lesser auricular nerve. The phrenic nerve is a nerve that is essential for our survival and arises from the nerve roots C3, C4, and C5. It supplies the thoracic diaphragm, allowing breathing to occur. If the spinal cord is transected above C3, then spontaneous breathing is impossible. Brachial plexus (C5-T1). The last four cervical spinal nerves, C5 to C8, and the first thoracic spinal nerve, T1, combine to form the brachial plexus or plexus brachialis (a tangled array of nerves), which split, combine, and recombine to form nerves that serve the upper limbs and upper back. Although the brachial plexus may appear tangled, it is highly organized and predictable, with little variation between people. Lumbar Sacral Plexus (L1-Co1). The anterior strands of the lumbar, sacral, and coccygeal nerves form the lumbar plexus, with the first lumbar nerve frequently connected by branches of the twelfth thoracic nerve. For descriptive purposes, this plexus is often divided into three parts: the lumbar plexus, the sacral plexus, and the pudendal plexus. Autonomic nervous system. Exemplary autonomic nervous systems include the sympathetic nervous system; the parasympathetic nervous system; and the enteric nervous system.

In one embodiment, administration causes the modified capsid to be delivered to the CNS or PNS of the subject. In one embodiment, administration causes the payload to be delivered to the CNS or PNS of the subject. In one embodiment, administration causes the modified viral capsid to be delivered to a CNS or PNS cell group. In one embodiment, administration causes the payload to be delivered to a CNS or PNS cell group. Exemplary CNS cell groups include, but are not limited to, neurons, oligodendrocytes, astrocytes, microglia, ependymal cells, radial glial cells, and pituitary cells. Those skilled in the art can use standard techniques to identify specific CNS cell groups, such as known cell markers for evaluating cell groups. In one embodiment, administration causes the modified capsid to be delivered to a cell type derived from the CNS, such as a cell (e.g., nerve) derived from the CNS but extending away from the CNS. In one embodiment, administration causes the payload to be delivered to a cell type derived from the CNS, such as a cell (e.g., nerve) derived from the CNS but extending away from the CNS.

In one embodiment, when a composition of the invention is topically administered to the CNS or PNS (e.g., by catheter, cannula, etc.), the administration results in distribution of the composition extending at least 0.5 inches from the initial site of administration. In one embodiment, the administration results in distribution of the composition extending at least 1 inch, at least 1.5 inches, at least 2 inches, at least 2.5 inches, at least 3 inches, at least 3.5 inches, at least 4 inches, at least 4.5 inches, at least 5 inches, at least 5.5 inches, at least 6 inches, at least 6.5 inches, at least 7 inches, at least 7.5 inches, at least 8 inches, at least 8.5 inches, at least 9 inches, at least 9.5 inches, at least 10 inches, or more from the initial site of administration. In other words, the modified viral capsid of the composition is detectable in (i.e., it has transduced) cells at least 0.5 inches, at least 1 inch, at least 1.5 inches, at least 2 inches, at least 2.5 inches, at least 3 inches, at least 3.5 inches, at least 4 inches, at least 4.5 inches, at least 5 inches, at least 5.5 inches, at least 6 inches, at least 6.5 inches, at least 7 inches, at least 7.5 inches, at least 8 inches, at least 8.5 inches, at least 9 inches, at least 9.5 inches, at least 10 inches, or more from the initial site of administration.

In one embodiment, when the composition of the present invention is topically administered to the CNS or PNS (e.g., by catheter, cannula, etc.), the administration results in expression of the modified capsid, viral vector, and/or payload in at least one cell type of the CNS or PNS. In one embodiment, when the composition of the present invention is topically administered to the CNS or PNS (e.g., by catheter, cannula, etc.), the administration results in expression of the modified capsid, viral vector, and/or payload in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more cell types of the CNS or PNS. In certain embodiments, the at least 2 cell types are adjacent to each other in the CNS or PNS. Alternatively, the at least cell types do not need to be adjacent to each other.

Aspects of the present disclosure relate to compositions comprising recombinant AAV comprising a capsid protein and a nucleic acid encoding a transgene, wherein the transgene comprises a nucleic acid sequence encoding one or more miRNAs. In some embodiments, each miRNA comprises a sequence shown in any one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66. In some embodiments, the nucleic acid further comprises an AAV ITR. In some embodiments, the ITR is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 ITR. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The composition of the present disclosure may comprise rAAV alone, or rAAV in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each with one or more different transgenes.

Those skilled in the art can easily select a suitable carrier according to the indication for which the rAAV is intended. For example, a suitable carrier includes saline, which can be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not a limitation of the present disclosure.

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

rAAV is administered in sufficient amounts 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 the selected organ (e.g., intraportal delivery to the liver), oral administration, inhalation administration (including intranasal and intratracheal delivery), intraocular administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, and other parenteral administration routes. If desired, the route of administration can be combined. In some embodiments, all or at least one nucleic acid sequence disclosed herein is delivered via a non-viral DNA construct comprising at least one DD-ITR. For example, one or more of the nucleic acids described herein can be delivered using a non-viral DNA construct as described in WO 2019/246554. WO 2019/246554 is incorporated herein by reference in its entirety.

The dosage of rAAV virions required to achieve a particular "therapeutic effect" (e.g., dosage units in terms of 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. Based on the above factors and other factors well known in the art, one skilled in the art can readily determine the dosage range of rAAV virions to treat a patient with a particular disease or disorder.

The effective amount of rAAV is an amount sufficient to target infected animals and target desired tissues. In some embodiments, the effective amount of rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health status, and tissue to be targeted of the subject, and therefore may vary from animal to tissue. For example, the effective amount of the rAAV is generally in the range of about 1 mL to about 100 mL of a solution containing about 10 ⁹ to 10 ¹⁶ genome copies. In some cases, a dose between about 10 ¹¹ to 10 ¹³ rAAV genome copies is suitable. In certain embodiments, 10 ¹² or 10 ¹³ rAAV genome copies are effective for targeting CNS tissues. In some cases, stable transgenic animals are produced by multiple doses of rAAV.

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

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

Dosage forms of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for use of the specific compositions described herein in a variety of treatment regimens.

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

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

The pharmaceutical form suitable for injectable use includes sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under general storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and flowing, reaching the extent of easy injection. It must be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures and/or vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of microbial action can be brought about by a variety of antibacterial and antifungal agents (such as parabens, trichlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents (such as sugars or sodium chloride) will preferably be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For example, for the use of injectable aqueous solutions, if necessary, the solution can be appropriately buffered, and the liquid diluent is first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous medium that can be used will be known to those skilled in the art. For example, a dose can be dissolved in 1mL isotonic NaCl solution, and added to the subcutaneous perfusion fluid of 1000mL or injected at the recommended site of infusion (see, for example, " Remington ' s Pharmaceutical Sciences " the 15th edition, 1035-1038 pages and 1570-1580 pages). According to the situation of the host, some changes in dosage will definitely occur. In any case, the personnel responsible for administration will determine the appropriate dosage for individual hosts.

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

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

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

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

Such dosage forms may be preferably used for introducing a pharmaceutically acceptable dosage form of a nucleic acid or rAAV construct disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved serum stability and circulation half-life have been developed (U.S. Patent No. 5,741,516). In addition, various methods of liposomes and liposome-like dosage forms as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been successfully used with many cell types that are usually resistant to other program transfections. In addition, liposomes are not limited by DNA length (typical in viral-based delivery systems). Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into various cultured cell lines and animals. In addition, several successful clinical trials have been completed to detect the effectiveness of liposome-mediated drug delivery.

Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles, also known as multilamellar vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 μm. Ultrasonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 Å to 500 Å, containing an aqueous solution in their core.

Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules can generally capture substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are considered.

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

In some embodiments, the methods described herein relate to treating a subject suffering from or diagnosed with a neurological disease or disorder (e.g., Huntington's disease) with a nucleic acid described herein. Subjects suffering from a neurological disease or disorder (e.g., Huntington's disease) can be identified by a physician using current methods for diagnosing such diseases and disorders. For example, symptoms and/or complications of Huntington's disease that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, depression and anxiety and characteristic movement disorders and chorea. Tests that may aid in the diagnosis of Huntington's disease include, for example, but are not limited to, genetic testing. A family history of Huntington's disease may also be helpful in determining whether a subject may suffer from Huntington's disease or in making a Huntington's disease diagnosis.

Compositions and methods as described herein can be applied to subjects suffering from or diagnosed with a nervous system disease or disorder. In some embodiments, the methods described herein include applying an effective amount of compositions as described herein (e.g., nucleic acids as described herein) to subjects to alleviate the symptoms of nervous system diseases or disorders. As used herein, "relieve symptoms" is to alleviate any condition or symptom associated with a nervous system disease or disorder. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, measured by any standard technique.

Effective amount, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, to determine the minimum effective dose and/or the maximum tolerated dose. Dosage can be different according to the dosage form used and the route of administration utilized. The therapeutically effective dose can be estimated initially by cell culture assay. In addition, dosage can be formulated in animal models to reach a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any particular dose can be monitored by suitable bioassays (e.g., for neuronal degeneration and/or functional assays, etc.). Dosage can be determined by a physician and adjusted (if necessary) to adapt to the observed therapeutic effect.

Immunomodulators

In some embodiments, the methods and compositions for treating a neurological disease or disorder as described herein further comprise administering an immunomodulator. In some embodiments, the immunomodulator can be administered at the time of administering the rAAV vector, before administering the rAAV vector, or after administering the rAAV vector.

In some embodiments, the immunomodulator is an immunoglobulin degrading enzyme, such as IdeS, IdeZ, IdeS/Z, Endo S, or a functional variant thereof. Non-limiting examples of references to such immunoglobulin degrading enzymes and their uses are described in US 7,666,582, US 8,133,483, US 20180037962, US 20180023070, US20170209550, US 8,889,128, WO2010057626, US 9,707,279, US 8,323,908, US20190345533, US 20190262434, and WO2020016318, each of which is incorporated by reference in its entirety.

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

In alternative embodiments, the immunomodulator is an inhibitor of the NF-kB pathway. In certain aspects of the embodiments, the immunomodulator is rapamycin or a functional variant. The non-limiting examples of references describing rapamycin and its uses described below are incorporated in their entirety: US 10,071,114, US 20160067228, US20160074531, US 20160074532, US 20190076458, US 10,046,064. In other aspects of the embodiments, the immunomodulator is a synthetic nanocarrier comprising an immunosuppressant. Non-limiting examples of references to immunosuppressants, immunosuppressants coupled to synthetic nanocarriers, synthetic nanocarriers comprising rapamycin and/or tolerogenic synthetic nanocarriers, their dosages, administration and uses are in US20150320728, US 20180193482, US20190142974, US 20150328333, US20160243253, US 10,039,822, US 20190076522, US20160022650, US 10,441,651, US 10,420,835, US 20150320870, US2014035636, US 10,434,088, US 10,335,395, US 20200069659, US 10,357,483, US 20140335186, US 10,668,053, US 10,357,482, US 20160128986, US 20160128987, US 20200038462, US 20200038463, each of which is incorporated by reference in its entirety.

In some embodiments, the immunomodulator is a synthetic nanocarrier (ImmTOR ^TM nanoparticle) comprising rapamycin (Kishimoto et al., 2016, Nat Nanotechnol, 11 (10): 890-899; Maldonado et al., 2015, PNAS, 112 (2): E156-165), as disclosed in US20200038463, U.S. Patent 9,006,254, each of which is incorporated herein in its entirety. In some embodiments, the immunomodulator is an engineered cell, for example, an immune cell modified using the SQZ technology disclosed in WO2017192786 (incorporated herein in its entirety by reference).

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

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

In some embodiments, the rAAV vector may also encode a negative regulator of innate immunity (e.g., NLRX1). Thus, in some embodiments, the rAAV vector may also optionally encode one or more of NLRX1, NS1, NS3/4A, or A46R, or any combination. In addition, in some embodiments, the composition comprising at least one rAAV vector as disclosed herein may also comprise a synthetic, modified RNA encoding inhibitor of the innate immune system to avoid an innate immune response generated by a tissue or subject.

In some embodiments, the immunomodulator used in the method for administration disclosed herein is an immunosuppressant. As used herein, the term "immunosuppressive drug or agent" is intended to include agents that inhibit or interfere with normal immune function. Examples of immunosuppressants suitable for use with the methods disclosed herein include agents that inhibit T-cell/B-cell costimulatory pathways (e.g., agents that interfere with T-cells and B-cells coupling through CTLA4 and B7 pathways), as disclosed in U.S. Patent Publication No. 2002/0182211. In one embodiment, the immunosuppressant is cyclosporin A. Other examples include myophenylate mofetil, rapamycin, and antithymocyte globulin. In one embodiment, the immunosuppressant is administered in a composition comprising at least one rAAV vector as disclosed herein, or may be administered in a separate composition, but simultaneously, or before, or after the administration of a composition comprising at least one rAAV vector according to the method for administration disclosed herein. The immunosuppressant is administered in a formulation compatible with the route of administration, and is administered to the subject in a dose sufficient to achieve the desired therapeutic effect. In some embodiments, the immunosuppressive drug is administered transiently for a sufficient period of time to induce tolerance to the rAAV vectors disclosed herein.

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

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

Reagent test kit

In one aspect, the present disclosure relates to a nucleic acid or recombinant viral vector comprising: (i) one or more inhibitory nucleic acids (e.g., miRNA); and (ii) a nucleic acid encoding a CYP46A1 protein. In one aspect, the present disclosure relates to a combination of: (i) one or more inhibitory nucleic acids (e.g., miRNA); and (ii) a nucleic acid encoding a CYP46A1 protein. In the combination of (i) and (ii), the two or more elements may be provided in a mixture or a single formulation. Alternatively, the two or more elements may be provided in separate formulations that are packaged as a set or kit or provided as a set or kit.

In some embodiments, the agents described herein (e.g., viral vectors) can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in treatment, diagnosis, or research applications. The kit can include one or more containers and instructions for use of components of the present disclosure. Specifically, such kits can include one or more reagents described herein, as well as instructions describing the intended application and proper use of these reagents. In certain embodiments, the reagents in the kit can be pharmaceutical dosage forms and dosages suitable for specific applications and methods of administering the reagents. Kits for research purposes can include components of appropriate concentrations or amounts to run various experiments.

In some embodiments, the present disclosure relates to a kit for producing rAAV, the kit comprising a container containing one or more of:

a) an isolated nucleic acid comprising a miRNA, for example, the miRNA comprises a sequence as shown in any one of SEQ ID NO: 6 to SEQ ID NO: 17, SEQ ID NO: 40 to SEQ ID NO: 44, or SEQ ID NO: 50 to SEQ ID NO: 66, or is encoded by a sequence as shown in any one of SEQ ID NO: 6 to SEQ ID NO: 17, SEQ ID NO: 40 to SEQ ID NO: 44, or SEQ ID NO: 50 to SEQ ID NO: 66, or the miRNA comprises a seed sequence complementary to SEQ ID NO: 4, SEQ ID NO: 18 to SEQ ID NO: 39, or SEQ ID NO: 46 to SEQ ID NO: 49;

b) a recombinant viral vector comprising an isolated nucleic acid comprising a transgene encoding one or more miRNAs,

For example, wherein each miRNA comprises a seed sequence complementary to SEQ ID NO:4, or wherein each miRNA comprises a sequence as set forth in any one of SEQ ID NO:6-SEQ ID NO:17, SEQ ID NO:40-SEQ ID NO:44, or SEQ ID NO:50-SEQ ID NO:66, flanked by a miRNA backbone sequence;

c) a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein; and/or

d) a recombinant viral vector comprising a nucleic acid containing a transgene encoding one or more miRNAs,

For example, wherein each miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein each miRNA comprises a sequence as set forth in any one of SEQ ID NO: 6 to SEQ ID NO: 17, SEQ ID NO: 40 to SEQ ID NO: 44, or SEQ ID NO: 50 to SEQ ID NO: 66, flanked by a miRNA backbone sequence; and

Nucleic acid encoding CYP46A1 protein.

In some embodiments, the kit further comprises a container containing an isolated nucleic acid encoding an AAV capsid protein (eg, an AAV9 capsid protein).

The kit can be designed to facilitate researchers to use the methods described herein and the kit can take many forms. Where applicable, each of the compositions of the kit can be provided in liquid form (e.g., in solution) or in solid form (e.g., dry powder). In some cases, some compositions can be configurable or otherwise processable (e.g., forming an active form), for example, by adding a suitable solvent or other substance (e.g., water or cell culture medium), which may or may not be provided with the kit. As used herein, "instructions" can define the components of instructions and/or promotion, and are generally related to written instructions on or associated with the packaging of the present disclosure. Instructions can also include any oral or electronic instructions provided in any manner so that the user will clearly recognize that the instructions are associated with the kit, such as audio-visual (e.g., videotapes, DVDs, etc.), the Internet, and/or network-based communications, etc. The written instructions can be in the form of government agencies that regulate the manufacture, use, or sale of drugs or biological products, and the instructions can also reflect the approval of the agency for the manufacture, use, or sale of animal administration.

The kit may include any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include one or more components and/or separation and mixing samples for mixing the kit and instructions for use in a subject. The kit may include a container for holding reagents as described herein. The reagent may be in the form of a liquid, gel or solid (powder). The reagent may be aseptically prepared, packaged in a syringe and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have other reagents prepared aseptically. Alternatively, the kit may include an activating agent premixed and shipped in a syringe, vial, tube or other container.

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

definition

For convenience, the meanings of some terms and phrases used in this specification, examples and appended claims are provided below. Unless otherwise specified or implied in the context, the following terms and phrases include the meanings provided below. The definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the invention belongs. If there is a clear difference between the use of a term in the art and its definition provided herein, the definition provided in this specification shall prevail.

For convenience, certain terms used herein in the specification, examples, and appended claims are collected here.

The terms "decrease", "reduced/reduction", or "inhibit" are all used herein to indicate a statistically significant amount of reduction. In some embodiments, "decrease" or "reduction" or "inhibit" generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more reduction. As used herein, "decrease" or "inhibit" does not include complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. The reduction may preferably be down to a level that is acceptable as being within the normal range for individuals not having a given disorder.

The terms "increased/increase", "enhance" or "activate" are all used herein to indicate an increase in a statistically significant amount. In some embodiments, the terms "increase", "enhance" or "activate" may represent an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or up to and including a 100% increase or any increase between 10%-100%, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 and 10 times or more compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in the level.

As used herein, "subject" means a person or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches) and fish (e.g., trout, catfish and salmon). In some embodiments, a subject is a mammal, such as a primate (e.g., a human). The terms "individual," "patient," and "subject" are used interchangeably herein.

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

The subject may be a subject who has been previously diagnosed with or identified as suffering from or having a condition (e.g., Huntington's disease) or one or more complications associated with such a condition that requires treatment, and optionally has undergone treatment for the condition or one or more complications associated with the condition. Alternatively, the subject may also be a subject who has not been previously diagnosed with the condition or one or more complications associated with the condition. For example, the subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition or a subject who does not exhibit such risk factors.

A "subject in need" of treatment for a particular disorder may be a subject suffering from the disorder, diagnosed as having the disorder, or at risk of developing the disorder.

As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino groups and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to small polypeptides, but the usage of these terms in the art overlaps. When it comes to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments and analogs described above.

Variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the native or reference sequence. For example, the degree of homology (percent identity) between the native sequence and the mutant sequence can be determined by comparing the two sequences using a commonly used freely available computer program for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

The change of the native amino acid sequence can be achieved by any of a variety of techniques known to those skilled in the art. Mutations can be introduced at a specific locus, for example, by synthesizing an oligonucleotide containing the mutant sequence and flanking it with restriction sites to enable connection to a fragment of the native sequence. After connection, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis program can be used to provide a changed nucleotide sequence with a specific codon that is changed according to the desired substitution, deletion or insertion. The technology for making such changes is well established and includes, for example, the technology disclosed by Walder et al., (Gene 42: 133, 1986); Bauer et al., (Gene 37: 73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al., (Genetic Engineering: Principles and Methods, Plenum Press, 1981) and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine residue that is not involved in maintaining the correct conformation of the polypeptide may also be replaced, usually with serine, to increase the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine bonds may be added to a polypeptide to increase its stability or promote oligomerization.

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

In some embodiments of any aspect, a polypeptide, nucleic acid or cell as described herein may be engineered. As used herein, "engineered" refers to aspects that have been manipulated by human hands. For example, a polypeptide is considered "engineered" when at least one aspect of the polypeptide (e.g., its sequence) has been manipulated by human hands to be different from that found in nature. According to common practice and as understood by those skilled in the art, the descendants of engineered cells are still generally referred to as "engineered", even if the actual operations were performed on a previous entity.

Variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the native or reference sequence. For example, the degree of homology (percent identity) between the native sequence and the mutant sequence can be determined by comparing the two sequences using a commonly used freely available computer program for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

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

The term "exogenous" refers to a substance present in a cell that is not its natural source. The term "exogenous" when used herein may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced into a biological system (e.g., a cell or an organism) in which it is not normally present through a process involving the hand of a person, and people hope to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that has been introduced into a biological system (e.g., a cell or an organism) in which it is present in a relatively low amount through a process involving the hand of a person, and people hope to increase the amount of the nucleic acid or polypeptide in the cell or organism (e.g., to produce ectopic expression or level). In contrast, the term "endogenous" refers to a natural substance of a biological system or cell. As used herein, "ectopic" refers to a substance that exists in an unusual position and/or amount. Ectopic substances may be substances that are normally present in a given cell, but in much less amount and/or at different times. Ectopic also includes substances, such as polypeptides or nucleic acids that are not naturally present or expressed in a given cell in its natural environment.

As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral. The term "vector" includes any genetic element that can replicate and transfer a gene sequence to a cell when associated with appropriate control elements. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, cosmids, chromosomes, viruses, virions, etc.

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

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

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

As used herein, the term "viral vector" refers to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be packaged into a viral vector particle. The viral vector may comprise a nucleic acid encoding a polypeptide as described herein instead of a non-essential viral gene. The vector and/or particle may be used for the purpose of transferring any nucleic acid to a cell in vitro or in vivo. Various forms of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, herpes virus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and therapies. In some embodiments, the vector is an additional type. Using a suitable additional type vector provides a method for maintaining high copy number extrachromosomal DNA of nucleotides of interest in a subject, thereby eliminating the potential impact of chromosome integration.

As used herein, the terms "treat/treatment/treating" or "amelioration" refer to therapeutic treatment, wherein the purpose is to reverse, alleviate, relieve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., Huntington's disease). The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. If one or more symptoms or clinical markers are reduced, the treatment is generally "effective". Alternatively, if the progression of the disease is reduced or stopped, the treatment is "effective". That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviating one or more symptoms, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, delaying or slowing the progression of the disease, alleviating or alleviating the disease state, regressing (whether partially or completely) and/or reducing mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief of symptoms or side effects of the disease (including palliative care).

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

As used herein, the term "administering" refers to placing a compound as disclosed herein into a subject by a method or approach that causes at least a portion of the medicament to be delivered at a desired site. Pharmaceutical compositions comprising compounds disclosed herein can be administered by any appropriate approach that produces effective treatment in a subject. In some embodiments, administration includes physical activity of a person, such as injection, ingestion action, smearing action, and/or operation of a delivery device or machine. Such activities can be performed, for example, by a medical professional and/or a subject being treated.

As used herein, "contacting" refers to any suitable means for delivering or exposing an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to a cell culture medium, perfusion, injection, or other delivery methods known to those skilled in the art. In some embodiments, contacting includes physical activity of a person (e.g., injection; the act of dispensing, mixing, and/or decanting); and/or operating a delivery device or machine.

The term "statistically significant" or "significantly" refers to statistical significance and generally means a difference of two standard deviations (2SD) or greater.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ±1%.

As used herein, the term "includes/comprising" means that there may be additional elements other than the defined elements presented. The use of "includes/comprising" means inclusion rather than limitation.

The term "consisting of refers to the compositions, methods, and respective components thereof as described herein, excluding any elements not recited in the description of the embodiment.

As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

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

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

Unless the context clearly indicates otherwise, the singular terms "a/an" and "the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "e.g." is synonymous with the term "for example".

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

Unless otherwise defined herein, scientific and technical terms related to the present application should have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs. It should be understood that the present invention is not limited to the specific methodology, schemes and reagents described herein, etc., and therefore can be varied. The terms used herein are only used to describe the purpose of specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims. Definitions of commonly used terms in immunology and molecular biology can be found in: The Merck Manual of Diagnosis and Therapy, 20th edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (Eds.), W.W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (Eds.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (Eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737); WO 2018/057855A; US 10,457,940; the contents of each of which are all incorporated herein by reference in their entirety.

In some embodiments of any aspect, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic characteristics of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic characteristics of animals that may cause suffering to the animals without any substantial medical benefit to humans or animals, and animals produced by such processes.

Other terms are defined herein within the description of various aspects of the invention.

For the purpose of description and disclosure, all patents and other publications (including references, issued patents, published patent applications and co-pending patent applications) cited in the entire application are expressly incorporated herein by reference, and the methodology described in such publications may be used in combination with the technology described herein. These disclosures are provided only because they are disclosed before the filing date of the present application. Any content in this regard should not be interpreted as admitting that the inventor has no right to precede such disclosure due to prior invention or any other reason. All statements about dates or representations about the contents of these documents are based on information available to the applicant and do not constitute any recognition of the correctness of the dates or contents of these documents.

The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present disclosure as will be appreciated by those skilled in the relevant art. For example, although method steps or functions are presented in a given order, alternative embodiments may perform functions in different orders, or may perform functions substantially simultaneously. The teachings of the present disclosure provided herein may be appropriately applied to other programs or methods. Various embodiments described herein may be combined to provide further embodiments. If desired, aspects of the present disclosure may be modified to provide further embodiments of the present disclosure using the compositions, functions and concepts of the above references and applications. These and other changes may be made to the present disclosure according to the detailed description. All of these modifications are intended to be included within the scope of the appended claims.

The specific elements of any of the foregoing embodiments may be combined or substituted for elements in other embodiments. In addition, although the advantages associated with certain embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the present disclosure.

The technology described herein is further illustrated by the following examples, which should in no way be construed as further limiting.

Some implementations of the technology described herein may be defined according to any of the following numbered paragraphs:

Example

Example 1

One aspect described herein is an inhibitory RNA that can be used to treat Huntington's disease. In some embodiments of any aspect, the nucleic acid sequence of the inhibitory RNA comprises one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66, or a sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to at least one of SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66 and retaining the same function (e.g., HTT inhibition) as SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66.

Described herein are constructs containing artificial miRNAs. pEMBL-D(+)-Syn1-hCGintron is a control vector with an empty human chorionic gonadotropin (hCG) intron (hCGin) inserted and driven by the synapsin promoter. Two copies of the control miRNA precursor (random sequence or non-functional mutation) are inserted into the hCGin in the vector pEMBL-D(+)-Syn1-hCGin-2xcontrolpre-miR. Two copies of the artificial pre-miR (perfect match to the 3'-UTR targeting sequence, containing about 100bp-150bp flanking upstream and downstream sequences) are cloned between the hCG introns. The vector pEMBL-D(+)-Syn1-CYP46A1-hCGin-2xartificialpre-miR is a combo construct that can produce CYP46A1 and artificial miRNA simultaneously. To identify whether the pre-miRNA can be processed into mature miRNA and combined with a HTT targeting sequence containing a CAG expansion (which is perfectly complementary to the mature miRNA), it was inserted behind the luciferase gene. Due to packaging size limitations, a small poly A was used in the construct. Syn1 can be replaced by any of the following: CMV enhancer and/or ACTB proximal promoter and/or chimeric ACTB-HBB2 intron, and a synthetic nervous system-specific promoter or fragment thereof selected from Tables 10-13, and/or an enhancer, and/or one or more of the cis-regulatory elements (CRE) selected from Tables 13-15.

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

Synapsin1 (Synl) is a member of the synapsin gene family. Synapsin encodes a neuronal phosphoprotein associated with the cytoplasmic surface of synaptic vesicles. Family members are characterized by common protein domains, and they are involved in the regulation of synaptogenesis and neurotransmitter release, indicating a potential role in several neuropsychiatric diseases. Syn1 plays a role in the regulation of axonogenesis and synaptogenesis. The Syn1 protein acts as a substrate for several different protein kinases, and phosphorylation may play a role in the regulation of this protein in nerve terminals. Mutations in this gene may be associated with X-linked disorders with primary neuronal degeneration, such as Rett syndrome. Alternatively, spliced transcript variants encoding different isoforms have been identified. In some embodiments of any of the aspects, the Syn1 promoter can comprise the human promoter Syn1 (see, e.g., the Syn1 promoter associated with NCBI Reference Nos. NG_008437.1 RefSeqGene Range 5001-52957; NM_006950.3; NP_008881.2; NM_133499.2; NP_598006.1).

CYP46A1 is a member of the cytochrome P450 enzyme superfamily. Cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and the synthesis of cholesterol, steroids and other lipids. This endoplasmic reticulum protein is expressed in the brain, where it converts cholesterol into 24S-hydroxycholesterol. Although cholesterol cannot pass through the blood-brain barrier, 24S-hydroxycholesterol can be secreted into the circulation in the brain and returned to the liver for catabolism. In some embodiments of any aspect, CYP46A1 may comprise human CYP46A1 (see, e.g., NCBI ref number NG_007963.1RefSeqGene Range4881-47884; NM_006668.2; NP_006659.1). CYP46A1 is the rate-limiting enzyme for cholesterol degradation and is neuroprotective in Huntington's disease (see, e.g., Boussicault et al., CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease, Brain, 2016 Mar; 139(Pt 3):953-70; Kacher et al., CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington's disease, Brain, 2019 Aug 1; 142(8):2432-2450; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments of any of the aspects, the miRNA comprises a sequence that is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive bases of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 flanked by a miRNA backbone sequence. In some embodiments of any of the aspects, the miRNA comprises a sequence that is complementary to at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive bases of a sequence of an untranslated region (e.g., 5'UTR, 3'UTR), an exon, a CAG repeat, or a CAG-jumping region (e.g., a CAG 5'-jumping region, a CAG 3'-jumping region) flanked by a miRNA backbone sequence associated with HTT (see, e.g., NCBI Gene ID: 3064; e.g., SEQ ID NO: 4).

When administered to the same patient, an isolated nucleic acid encoding a transgene of one or more miRNAs and an isolated nucleic acid encoding a CYP46A1 protein can provide an improved therapeutic effect compared to the administration of either alone. When administered to the same patient, an isolated nucleic acid encoding a transgene of one or more miRNAs and an isolated nucleic acid encoding a CYP46A1 protein can provide a synergistic (rather than additive) improved therapeutic effect compared to the administration of either alone. An isolated nucleic acid encoding a transgene of one or more miRNAs and an isolated nucleic acid encoding a CYP46A1 protein can be administered to a subject sequentially or simultaneously according to any of the methods described herein. It is expected that rAAV comprising a CYP46A1 variant CDS (as described in SEQ ID NO: 110) will provide a better therapeutic effect to treat neurological diseases (e.g., Huntington's disease) than when an rAAV comprising a CYP46A1 non-variant sequence (as described in SEQ ID NO: 1) is administered. Similarly, it is expected that rAAV comprising a miRNA (e.g., selected from one or more of SEQ ID NO: 6-SEQ ID NO: 17, or SEQ ID NO: 40-SEQ ID NO: 44, or SEQ ID NO: 50-SEQ ID NO: 66) when administered with a CYP46A1 variant CDS (such as described in SEQ ID NO: 110) will provide better therapeutic effects for treating neurological diseases (e.g., Huntington's disease) than when administered with a CYP46A1 non-variant sequence (e.g., as described in SEQ ID NO: 1).

SEQ ID NO: 3 Exon 1 of human HTT gene

SEQ ID NO: 4: Human HTT mRNA sequence

SEQ ID NO: 5 Human HTT protein sequence

SEQ ID NO: 109 CYP46A1 variant

SEQ ID NO: 110 CYP46A1 variant CDS

Example 2

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

During the design of these promoters, the specific shortcomings of known NS-specific promoters were considered. First, known NS-specific promoters (such as Synapsin-1, CAMKIIa and GFAP) that are specific to NS cell types are not expressed in all cell populations (such as not expressed in all neurons/astrocytes). (Zhang et al., 2019) have demonstrated the situation for GFAP, and can be seen from the distribution of Syn-1 in neurons in the Allen brain atlas. Secondly, most known CREs, promoter elements and promoters are too large to be included in self-complementary AAV vectors (scAAV) (depending on the size of the transgenic, the size of the promoter may need to be less than 1000bp, preferably less than 900bp, more preferably less than 800bp, and most preferably less than 700bp). In addition, expression in a specific cell type or a combination of cell types across the entire NS (suitably, the entire CNS or the entire brain) may be required.

Currently known promoters cannot address these shortcomings, and there is a need to develop short, cell type NS-specific promoters with targeted local expression and also expression across the entire NS in gene therapy. For example, the expression pattern of HTT (Huntington) and CYP46A1 genes in the adult mouse brain shown in Figures 6A and 6B highlights the requirement for expression across the entire NS (such as the entire brain). Since the HTT (Huntington) gene is expressed throughout the brain, it may be advantageous to express any potential expression product that inhibits the defective Huntington gene and/or counteracts or mitigates the harmful effects of defective Huntington. Similarly, since the CYP46A1 gene is expressed throughout the brain, it may be advantageous to express throughout the brain for any potential supplemental CYP46A1.

Gene expression in all neurons as well as astrocytes and/or oligodendrocytes across the CNS may be desirable in the treatment of some diseases such as Huntington's disease. Because glial cells have been implicated in Huntington's disease (Shin et al., 2005), expression in astrocytes and oligodendrocytes may be advantageous.

Therefore, the present invention sets out to design tandem NS promoters that are active in multiple NS cell types while addressing some of the shortcomings listed above. For example, the design of the promoter involves combining one or more CREs with promoter elements to broaden the cell tropism compared to a single CRE/promoter element to create a promoter that is active in multiple NS cell types and address the shortcomings of known promoters that are not expressed throughout the cell population. In addition, to address the shortcomings of known CREs, promoter elements, and promoters that are too large to be included in AAV vectors (such as self-complementary AAV vectors (scAAV)), some of the CRE and promoter elements of the present invention have been shortened using bioinformatics analysis, literature searches, and publicly available genomic databases, but they are still expected to be active CRE and promoter elements.

The synthetic NS-specific promoter according to the present invention is operably connected to the nucleic acid sequence encoding the CYP46A1 transgenic and human influenza hemagglutinin (HA) tag, and is experimentally tested in wild-type C57BL6/J mice. The synthetic NS-specific promoter according to the present invention that is operably connected to the nucleic acid sequence encoding the CYP46A1 transgenic and HA tag is administered intravenously in a viral vector. The vector copy number will be assessed in brain and spinal cord tissue sections by qPCR analysis (normalized to internal genomic DNA copy number control) of the viral transgenic CYP46A1 to confirm the equivalent injection dose. Western blotting will be performed to assess the protein expression of the transgenic of the HA tag in brain and spinal cord tissue. Finally, immunofluorescence staining will be performed to brain and spinal cord tissue sections to assess the expression of the transgenic in CNS cell types. Similarly, immunofluorescence staining can be performed on PNS tissue sections to assess the expression of the transgenic in PNS cell types. Specifically, double staining will be performed using the HA tag for marking CYP46A1 gene expression and standard markers for neurons, astrocytes, oligodendrocytes and microglia.

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

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

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

Example 3

This article describes a method for making viral vectors from Pro10/HEK293 cells that have been engineered to stably integrate the CYP46A1 gene.

As described in U.S. Pat. No. 9,441,206, the stable cell line Pro10/HEK293 is ideal for scalable production of AAV vectors. This cell line can be contacted with an expression vector comprising a CYP46A1 gene operably linked to any NS-specific promoter described herein (e.g., as described in Tables 10-15) or variants thereof. A clonal population with CYP46A1 integrated into its genome is selected using methods well known in the art. Pro10/HEK293 cells stably containing the CYP46A1 gene are transfected with a packaging plasmid encoding Rep2 and serotype-specific Cap2: AAV-Rep/Cap, and an Ad-Helper plasmid (XX680: encoding adenovirus helper sequences).

Transfection. On the day of transfection, cells were counted using a ViCell XR Viability Analyzer (Beckman Coulter) and diluted for transfection. To mix the transfection mix, the following reagents were added to the conical tube in this order: plasmid DNA, I (Gibco) or OptiPro SFM (Gibco), or other serum-free compatible transfection medium, followed by a transfection reagent in a specific ratio with the plasmid DNA. The blend is inverted to mix, and then incubated at room temperature. The transfection blend is pipetted into a flask and then placed back into a shaker/incubator. All optimization studies were performed at 30 mL culture volume and then verified at a larger culture volume. Cells were harvested 48 hours after transfection.

rAAV was produced using the Wave Bioreactor system. The wave bags were inoculated 2 days prior to transfection. Two days after inoculating the wave bags, the cell culture was counted and then expanded/diluted prior to transfection. The wave bioreactor cell cultures were then transfected. The cell cultures were harvested from the wave bioreactor bags at least 48 hours after transfection.

Titer. AAV titer was calculated using qPCR relative to a standard curve (AAV ITR specific) and primers specific to the CYP46A1 gene after DNase digestion. Suspension cells were harvested from shake flasks and 60Wave bioreactor bags. 48 hours after transfection, the cell culture was collected into a 500mL polypropylene conical tube (Corning) by pouring from the shake flask or pumping from the wave bioreactor bag. The cell culture was then centrifuged at 655xg for 10min using a Sorvall RC3C plus centrifuge and H6000A rotor. The supernatant was discarded, the cells were resuspended in 1xPBS, transferred to a 50mL conical tube, and centrifuged at 655xg for 10min. At this point, the pellet can be stored at NLT-60°C or continued with purification.

rAAV was titrated from cell lysate using qPCR. 10 mL of cell culture was removed and centrifuged at 655 x g for 10 min using a Sorvall RC3C plus centrifuge and H6000A rotor. The supernatant was decanted from the cell pellet. The cell pellet was then resuspended in 5 mL of DNase buffer (5 mM CaCl ₂ , 5 mM MgCl ₂ , 50 mM Tris-HCl pH 8.0) and then sonicated to effectively lyse the cells. 300 μL was then removed and placed in a 1.5 mL microcentrifuge tube. 140 units of DNase I were then added to each sample and incubated at 37°C for 1 hour. To determine the effectiveness of DNase digestion, 4-5 mg of CYP46A1 plasmid was incorporated into non-transfected cell lysate with and without DNase added. 50 μL of EDTA/Sarkosyl solution (6.3% Sarcosyl, 62.5 mM EDTA pH 8.0) was added to each tube and incubated at 70°C for 20 minutes. Then 50 μL of proteinase K (10 mg/mL) was added and incubated at 55°C for at least 2 hours. The samples were boiled for 15 minutes to inactivate proteinase K. An aliquot was removed from each sample for analysis by qPCR. Two qPCR reactions were performed to effectively determine how many rAAV vectors were produced per cell. The first qPCR reaction was constructed using a set of primers designed as follows: the primers were designed to bind to homologous sequences on the backbones of plasmids XX680, pXR2, and CYP46A1. The second qPCR reaction was constructed using a set of primers that bind to and amplify regions within the CYP46A1 gene. qPCR was performed using Light cycler 480 and Sybr green reagents from Roche. The samples were denatured at 95°C for 10 min, followed by 45 cycles (90°C for 10 sec, 62°C for 10 sec, 72°C for 10 sec) and a melting curve (1 cycle of 99°C for 30 sec, 65°C for 1 min, continuous).

rAAV was purified from the crude lysate. Each cell pellet was adjusted to a final volume of 10 mL. The pellet was briefly vortexed and sonicated for 4 minutes with a pulse of one second on and one second off at 30% output. After sonication, 550U of DNase was added and incubated at 37°C for 45 minutes. The pellet was then centrifuged at 9400xg using a Sorvall RCSB centrifuge and HS-4 rotor to precipitate the cell debris, and the clarified lysate was transferred to a Type70Ti centrifuge tube (Beckman361625). For the separation of harvesting and lysing suspension HEK293 cells for rAAV, those skilled in the art can use mechanical methods (e.g., microfluidization) or chemical methods (e.g., detergents), etc., followed by a purification step using depth filtration or tangential flow filtration (TFF).

AAV vector purification. Clarified AAV lysate was purified by column chromatography as would be appreciated by one skilled in the art and described in the following manuscripts (Allay et al., Davidoff et al., Kaludov et al., Zolotukhin et al., Zolotukin et al., etc.), which are incorporated herein by reference in their entirety.

Example 4

Selected NS-specific promoters according to the invention were tested in SH-SY5Y cells derived from neuroblastoma.

Materials and methods

Cell maintenance and transfection.SH-SY5Y cells are cultivated in HAM F12 culture medium, and the culture medium has 1mM L-glutamine (Gibco 11765-054), 15% heat-inactivated FBS (ThermoFisher 10500064), 1% non-essential amino acids (Merck M1745-100ML) and 1% penicillin/streptomycin (ThermoFisher 15140122).Cells are passaged twice a week between 1:3 and 1:4 to maintain a healthy cell density between 70%-80%.Cells are maintained under passage number 20.For transfection, cells are seeded into 48-well plates with 10 ⁵ cells/well.24 hours after inoculation, 300ng plasmid is transfected into cells using Lipofectamine3000 reagent (ThermoFisher L3000008).

The plasmid transfected into the SHSY5Y cell line included SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0033, SP0011, SP0034, SP0035 or SP0036 operably linked to GFP.

Flow cytometry. 48 hours after transfection, SH-SY5Y cells were washed with PBS and then dissociated with 0.05% trypsin. Cells were collected and resuspended in a solution of 90% PBS, 10% FBS. The GFP expression of cells was evaluated by flow cytometry using the Attune Nxt acoustic focusing cytometer. The cell viability dye 7-AAD (ThermoFisher 00-6993-50) was mixed with the control cell population to identify and exclude dead cells. The expression of GFP was measured in a live single cell population using a blue 488nm laser at a bandpass filter 510/10nm. Untransfected cells were used to set the gating of GFP-negative and GFP-positive cells. The number of GFP-positive single cells and the median GFP fluorescence of all GFP-positive cells were calculated by the Attune Nxt software.

result

The result of this experiment is shown in Fig. 7 A and Fig. 7 B. The median GFP expression and the percentage of GFP positive cells of the SH-SY5Y cell deriving from neuroblastoma comprising the following expression cassette were transfected by flow cytometry assessment: SP0013, SP0014, SP0030, SP0031, SP0032, SP0019, SP0020, SP0021, SP0022, SP0011, SP0034, SP0035 or SP0036 operably connected to GFP. The expression cassette comprising the known promoter synapsin-1 and CAG operably connected to GFP was used as a control. The promoters of all tests have suitable transfection efficiency and median GFP expression (referring to Fig. 7 A and Fig. 7 B) with neuron-specific control promoter synapsin-1. The control promoter CAG showed 2- to 3-fold higher transfection efficiency ( FIG. 7B ) and about 2.5-fold higher median GFP expression compared to the control promoter synapsin-1 and the tested synthetic NS-specific promoters ( FIG. 7A ).

The synthetic NS-specific promoter SP0028 (SEQ ID NO: 86) is of similar design to the synthetic NS-specific promoter SP0019 (SEQ ID NO: 82), and both contain the same elements. Based on this, it is expected that SP0028 (SEQ ID NO: 86) and SP0019 (SEQ ID NO: 82) behave similarly.

The synthetic NS-specific promoter SP0029 (SEQ ID NO: 87) is of similar design to the synthetic NS-specific promoter SP0021 (SEQ ID NO: 84), and both contain the same elements. Based on this, it is expected that SP0029 (SEQ ID NO: 87) and SP0021 (SEQ ID NO: 84) behave similarly.

The synthetic NS-specific promoter SP0026 (SEQ ID NO: 76) is of similar design to the synthetic NS-specific promoter SP0013 (SEQ ID NO: 74), and both contain the same elements. Based on this, it is expected that SP0026 (SEQ ID NO: 76) behaves similarly to SP0013 (SEQ ID NO: 74).

The synthetic NS-specific promoter SP0027 (SEQ ID NO: 77) is of similar design to the synthetic NS-specific promoter SP0014 (SEQ ID NO: 75), and both contain the same elements. Based on this, it is expected that SP0027 (SEQ ID NO: 77) behaves similarly to SP0014 (SEQ ID NO: 75).

The synthetic NS-specific promoter SP0033 (SEQ ID NO: 81) is of similar design to SP0021 (SEQ ID NO: 84), both containing identical and similar elements. Thus, SP0033 (SEQ ID NO: 81) is a shorter version of SP0021 (SEQ ID NO: 84), and as such, they are expected to behave similarly.

Example 5

The modified vectors containing CYP46A1 or GFP and covalent mannosylation of the vector will be compared with the parent unmodified rAAV. Delivery of CYP46A1 by rAAV promotes the secretion of CYP46A1 from transduced neurons, which can be detected by immunohistochemistry visualization and quantified by ELISA of tissue extracts. After injection of the modified AAV-CYP46A1 into the thalamus (e.g., by convection-enhanced delivery described in U.S. patent application 13/146,640 or catheter delivery in monkeys), the extent of CYP46A1 immunopositive staining will be assessed in the frontal cortex ipsilateral to the infusion site. Expression of CYP46A1 delivered with the modified vector will be significantly enhanced compared to the unmodified vector and extend significantly from the prefrontal association cortex area (cortical areas 9 and 10) through the frontal eye field (area 8), premotor cortex (area 6), primary somatosensory cortex areas (areas 3, 1 and 2) to the primary motor cortex (area 4), and include expression in the cingulate cortex (areas 23, 24, 32) and Broca's areas (areas 44 and 45). In addition to intense staining of individual neuronal cell bodies and cell processes, CYP46A1 staining will be observed in multiple layers across the frontal cortex, with the highest intensity gradients in cortical layers III and IV, compared to the same dose of the unmodified vector.

As described in U.S. Patent Application 13/146,640, the delivery of the modified vector comprising GFP compared with the parent will also be tested in the monkey model. The relative amount of the modified vector in the anterior nucleus of AN, the medial dorsal nucleus of MD, the ventral anterior nucleus of VA, the ventrolateral nucleus of VL, the ventroposterior nucleus of VP will be significantly higher than that of the unmodified vector. In addition, compared with the unmodified vector, the modified vector is widely distributed and more effective in the whole cortex. Compared with the parental vector, the percentage of positive cells in each district and region is significantly higher. It is expected to be more effective transduction to cortex 1-6 layers. It can be achieved to deliver all in multiple lobes of the cerebral cortex or cortex 1-4,6 and 8-10 regions.

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

Immunostaining was performed with antibodies against CYP46A1 (1:500, AF-212-NA, R&D Systems) and GFP (1:500, AB3080, Chemicon) on Zamboni-fixed 40 μm coronal sections covering the entire frontal cortex and extending posteriorly to the level of the thalamus. The localization of CYP46A1 and GFP immunopositive neurons was analyzed with reference to stereotaxic coordinate maps of the rhesus monkey brain to determine the specific areas of immunostaining in the cortex and thalamus.

CYP46A1 protein ELISA. Tissue aspiration of 3 mm coronal blocks of fresh frozen tissue was performed from some cortex and thalamus. Methods and Materials and the striatum region of monkeys perfused with modified vectors. Expression of human CYP46A1 clDNA or GFP cDNA was quantified by ELISA detection using a commercial ELISA kit (Emax ELISA, Promega, Wis.) following surgical delivery.

Example 6

Next, to determine whether changing the modification of the capsid can achieve repeated administration, the modified vector containing -CYP46A1 of Example 5 was redesigned to have different chemical modifications, but composed of the same capsid and containing the same payload as the capsid of Example 5 (i.e., CYP46A1). A first modified vector containing -CYP46A1 of Example 2 was administered to adult rhesus monkeys, and a second dose of the same vector, or the redesigned modified capsid, was administered 14 days after administration. The expression of CYP46A1 was evaluated using the ELISA assay described in Example 5 above. Repeated administration of the same vector was found to have significantly reduced expression, possibly due to neutralizing antibodies against the vector generated after the first administration. Surprisingly, the expression of the redesigned vector was high and widespread, indicating that changes in the modification of the capsid can enable the redesigned vector to be expressed.

Claims (113)

1. A method of treating a neurological disease or disorder in a subject in need thereof, said method comprising administering to a subject suffering from or at risk of developing said neurological disease or disorder. The subject is administered a therapeutically effective amount of: (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) Isolated nucleic acid encoding CYP46A1 protein. 2. A method of treating a neurological disease or disorder in a subject in need thereof, said method comprising administering to a subject suffering from or at risk of developing said neurological disease or disorder. The subject is administered a therapeutically effective amount of: (a) A recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) ) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs; and (b) A recombinant viral vector comprising an isolated nucleic acid encoding said CYP46A1 protein. 3. The method of any one of claims 1-2, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal encephalopathy ataxia, polyglutamine repeat spinocerebellar ataxia, Krabbe disease, Batten disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive myocardial infarction Atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan Disease, epilepsy, cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusion, attention deficit disorder, psychosexuality disorders, sleep disorders, pain disorders, eating or weight disorders. 4. The method of any one of claims 1-3, wherein the neurological disease or disorder is a central nervous system (CNS) disease or disorder. 5. The method of any one of claims 1-4, wherein the CNS disease or disorder is selected from the group consisting of Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, myocardial infarction, Amyotrophic lateral sclerosis and Parkinson's disease. 6. The method of any one of claims 1-5, wherein the CNS disease or disorder is Alzheimer's disease, and the at least one miRNA comprises a protein related to amyloid precursor protein (APP), Progeria Complementary seed sequences for protein 1, presenilin 2, ABCA7, SORL1, and their disease-associated alleles. 7. The method of any one of claims 1-5, wherein the CNS disease or disorder is Parkinson's disease, and the at least one miRNA comprises a combination with SNCA, LRRK2/PARK8, PRKN, PINK1, DJ1/ Complementary seed sequences of PARK7, VPS35, EIF4G1, DNAJC13, CHCHD2, UCHL1, GBA1, and their disease-associated alleles. 8. The method of any one of claims 1-5, wherein the CNS disease is Huntington's disease and the at least one miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein the At least one miRNA comprises the sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 17, SEQ ID NO: 40 to SEQ ID NO: 44, or SEQ ID NO: 50 to SEQ ID NO: 66 flanked by There are miRNA backbone sequences. 9. The method of any one of claims 1-8, wherein the CNS disease is Huntington's disease and the at least one miRNA comprises SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40 - The sequence of any one of SEQ ID NO: 44 or SEQ ID NO: 50 - SEQ ID NO: 66. 10. The method of any one of claims 8-9, wherein at least one of the miRNAs hybridizes to human huntingtin and inhibits the expression of human huntingtin. 11. The method of any one of claims 8-10, wherein the subject contains a huntingtin gene with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. 12. The method of any one of claims 8-11, wherein the subject is less than 20 years old. 13. The method of any one of claims 1-12, wherein the recombinant viral vector is selected from the group consisting of: AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, herpes vectors Viral vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors. 14. The method of any one of claims 2-13, wherein the recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (b). 15. The method of any one of claims 1-13, wherein the isolated nucleic acids of (a) and (b) are comprised in separate recombinant viral vectors. 16. The method of any one of claims 1-14, wherein the isolated nucleic acids of (a) and (b) are comprised in the same recombinant viral vector. 17. The method of any one of claims 1-16, wherein (a) and (b) are administered at substantially the same time. 18. The method of any one of claims 1-13 and 15, wherein (a) and (b) are administered at different time points. 19. The method of claim 18, wherein the different time points are separated by at least 1 min, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year, or more. 20. The method of any one of claims 18-19, wherein (a) is administered prior to the administration of (b). 21. The method of any one of claims 18-19, wherein (b) is administered prior to the administration of (a). 22. The method of any one of claims 1-21, wherein administration of (a), (b), or (a) and (b) is repeated at least once. 23. The method of any one of claims 1-22, wherein the transgene comprises two miRNAs in tandem flanked by introns. 24. The method of claim 23, wherein the flanking introns are identical. 25. The method of claim 23, wherein the flanking introns are from the same species. 26. The method of claim 23, wherein the flanking intron is an hCG intron. 27. The method of any one of claims 1-26, wherein the transgene comprises a promoter. 28. The method of claim 27, wherein the promoter is a synapsin (Syn1) promoter or a promoter from Table 10 to Table 13. 29. The method of any one of claims 1-28, wherein the one or more miRNAs are located in the untranslated portion of the transgene. 30. The method of claim 29, wherein the untranslated portion is an intron. 31. The method of claim 30, wherein the untranslated portion is between the last codon of the nucleic acid sequence encoding the protein and the poly-A tail sequence, or between the last nucleotide base of the promoter sequence. between the base and poly-A tail sequences. 32. The method of any one of claims 1-31, further comprising a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof. body. 33. The method of any one of claims 1-33, wherein the ITR variant lacks a functional terminal resolution site (TRS), optionally, wherein the ITR variant is an ATRS ITR. 34. The method of any one of claims 1-33, wherein said administering results in delivery of said viral vector or isolated nucleic acid to said central nervous system (CNS) of said subject. 35. The method of any one of claims 1-34, wherein said administration is by injection, optionally intravenous injection or intrastriatal injection. 36. The method of any one of claims 2-35, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or Its heterochimerism. 37. The method of any one of claims 2-36, the viral vector comprising AAV from AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 , or the capsid protein of a heterologous chimera thereof. 38. The method of claim 37, wherein the capsid protein is AAV9 capsid protein. 39. The method of any one of claims 2-38, wherein the viral vector is a self-complementary AAV (scAAV). 40. The method of any one of claims 2-39, wherein the viral vector is formulated for delivery to the central nervous system (CNS). 41. A composition or combination comprising: (a) an isolated nucleic acid encoding a transgene encoding one or more miRNAs; and (b) Isolated nucleic acid encoding CYP46A1 protein. 42. A composition or combination comprising: (a) A recombinant viral vector comprising an isolated nucleic acid comprising (i) a first region, and (ii) a second region, the first region comprising a first adeno-associated virus (AAV) inverted terminal repeat ( ITR) or a variant thereof, the second region comprising a transgene encoding one or more miRNAs; and (b) A recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein. 43. The composition or combination of any one of claims 41-42, for use in a method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to a subject suffering from the disease or disorder. A therapeutically effective amount of the composition or combination is administered to a subject who has the neurological disease or disorder, or is at risk of developing the neurological disease or disorder. 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's disease, Leigh's disease, spinal cerebral ataxia disorders, Krabbe's disease, polyglutamine repeat spinocerebellar ataxia, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive amyotrophic atrophy, cutaneous Chrysanthemum disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy , cerebral infarction, depression, bipolar disorder, persistent affective disorder, secondary mood disorder, schizophrenia, drug dependence, neurological disease, psychosis, dementia, delusion, attention deficit disorder, psychosexual disorder, sleep disorders, pain disorders, eating or weight disorders. 45. The composition or combination of claim 44, wherein the neurological disease or disorder is a central nervous system (CNS) disease or disorder. 46. The composition or combination of claim 45, wherein the CNS disease or disorder is selected from Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, amyotrophic lateral disease Sclerosis and Parkinson's disease. 47. The composition or combination of any one of claims 41-46, wherein the at least one miRNA comprises a combination with amyloid precursor protein (APP), Presenilin 1, Presenilin 2, ABCA7, SORL1 , and the complementary seed sequences of their disease-associated alleles. 48. The composition or combination of any one of claims 41-46, wherein the at least one miRNA comprises a combination with SNCA, LRRK2/PARK8, PRKN, PINK1, DJ1/PARK7, VPS35, EIF4G1, DNAJC13, Complementary seed sequences for CHCHD2, UCHL1, GBA1, and their disease-associated alleles. 49. The composition or combination of any one of claims 41-46, wherein the at least one miRNA comprises a seed sequence complementary to SEQ ID NO: 4, or wherein the at least one miRNA comprises The sequence of any one of SEQ ID NO: 6 to SEQ ID NO: 17, SEQ ID NO: 40 to SEQ ID NO: 44, or SEQ ID NO: 50 to SEQ ID NO: 66 flanked by a miRNA backbone sequence . 50. The composition or combination of any one of claims 41-46, wherein the at least one miRNA comprises SEQ ID NO: 6-SEQ ID NO: 17, SEQ ID NO: 40-SEQ ID NO :44 or the sequence of any one of SEQ ID NO:50-SEQ ID NO:66. 51. The composition or combination of any one of claims 49-50, wherein at least one of the miRNAs hybridizes to human huntingtin and inhibits the expression of human huntingtin. 52. The composition or combination of any one of claims 49-51, wherein the subject comprises huntingtin with more than 36 CAG repeats, more than 40 repeats, or more than 100 repeats. Gene. 53. The composition or combination of any one of claims 49-52, wherein the subject is less than 20 years old. 54. The composition or combination of any one of claims 42-53, wherein the recombinant viral vector is selected from the group consisting of: AAV vectors, adenoviral vectors, lentiviral vectors, retroviruses vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors and chimeric virus vectors. 55. The composition or combination of any one of claims 42-54, wherein the recombinant viral vector comprising (a) is the same as the recombinant viral vector comprising (b). 56. The composition or combination of any one of claims 41-54, wherein the isolated nucleic acids of (a) and (b) are comprised in separate recombinant viral vectors. 57. The composition or combination of any one of claims 41-55, wherein the isolated nucleic acids of (a) and (b) are comprised in the same recombinant viral vector. 58. The composition or combination of any one of claims 41-57, wherein (a) and (b) are administered at substantially the same time. 59. The composition or combination of any one of claims 41-54 and 56, wherein (a) and (b) are administered at different time points. 60. The composition or combination of claim 59, wherein the different time points are separated by at least 1 min, at least 1 hour, at least 1 day, at least 1 week, at least 1 month, at least 1 year or more . 61. The composition or combination of any one of claims 59-60, wherein (a) is administered prior to the administration of (b). 62. The composition or combination of any one of claims 59-60, wherein (b) is administered prior to the administration of (a). 63. The composition or combination of any one of claims 59-60, wherein administration of (a), (b), or (a) and (b) is repeated at least once. 64. The composition or combination of any one of claims 41-65, wherein the transgene comprises two miRNAs in tandem flanked by introns. 65. The composition or combination of claim 64, wherein the flanking introns are identical. 66. The composition or combination of claim 64, wherein the flanking introns are from the same species. 67. The composition or combination of claim 64, wherein the flanking intron is an hCG intron. 68. The composition or combination of any one of claims 41-67, wherein the transgene comprises a promoter. 69. The composition or combination of claim 68, wherein the promoter is a synapsin (Syn1) promoter or a promoter of Tables 10-13. 70. The composition or combination of any one of claims 41-69, wherein the one or more miRNAs are located in the untranslated portion of the transgene. 71. The composition or combination of claim 70, wherein the untranslated portion is an intron. 72. The composition or combination of claim 70, wherein the untranslated portion is between the last codon of the protein-encoding nucleic acid sequence and the poly-A tail sequence, or at the end of a promoter sequence Between one nucleotide base and the poly-A tail sequence. 73. The composition or combination of any one of claims 41-72, further comprising a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR) or its variants. 74. The composition or combination of any one of claims 41-73, wherein the ITR variant lacks a functional terminal resolution site (TRS), optionally, wherein the ITR variant is ATRS ITR. 75. The composition or combination of any one of claims 41-74, wherein said administering results in delivery of said viral vector or isolated nucleic acid to said central nervous system (CNS) of said subject ). 76. The composition or combination of any one of claims 41-75, wherein said administration is by injection, optionally intravenous injection or intrastriatal injection. 77. The composition or combination of any one of claims 42-76, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 , or heterologous chimeras thereof. 78. The composition of any one of claims 42-77, the viral vector comprising AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or Capsid protein of AAV12, or a heterologous chimera thereof. 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 42-79, wherein the viral vector is a self-complementing AAV (scAAV). 81. The composition or combination of any one of claims 42-80, wherein the viral vector is formulated for delivery to the central nervous system (CNS). 82. A composition comprising an isolated nucleic acid encoding a CYP46A1 protein, said nucleic acid comprising at least 80% identity to SEQ ID NO: 110, or at least 80% identity to SEQ ID NO: 111, or to SEQ ID NO: 111. ID NO: 153 has a sequence with at least 80% identity. 83. A composition comprising a recombinant viral vector comprising an isolated nucleic acid encoding a CYP46A1 protein, said nucleic acid comprising at least 80% identity to SEQ ID NO: 110, or to SEQ ID NO: 111 A sequence that is at least 80% identical, or that is at least 80% identical to SEQ ID NO: 153. 84. A method of treating a neurological disease or disorder in a subject in need thereof, said method comprising administering to a subject suffering from or at risk of developing said neurological disease or disorder. The subject is administered a therapeutically effective amount of the composition of claim 82 or 83. 85. The method of claim 84, wherein the neurological disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, spinal cerebral ataxia, multiple Polyglutamine repeat spinocerebellar ataxia, Krabbe's disease, Batten's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, Muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, neuropathic pain, trauma due to spinal cord or head injury, ophthalmic diseases and disorders, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction , depression, bipolar disorder, persistent affective disorder, secondary mood disorders, schizophrenia, drug dependence, neurosis, psychosis, dementia, delusions, attention deficit disorder, psychosexual disorders, sleep disorders, pain disorder, eating or weight disorder. 86. The method of any one of claims 84-85, wherein the neurological disease or disorder is a central nervous system (CNS) disease or disorder. 87. The method of any one of claims 84-86, wherein the CNS disease or disorder is selected from the group consisting of Huntington's disease, Alzheimer's disease, polyglutamine repeat spinocerebellar ataxia, myocardial infarction, Amyotrophic lateral sclerosis and Parkinson's disease. 88. The composition or method of any one of claims 83-87, wherein the recombinant viral vector is selected from the group consisting of: AAV vectors, adenoviral vectors, lentiviral vectors, retroviruses vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors and chimeric virus vectors. 89. The method of any one of claims 84-88, wherein said administering is repeated at least once. 90. The method of any one of claims 84-89, wherein said administering results in delivery of said viral vector or isolated nucleic acid to the central nervous system (CNS) of said subject. 91. The method of any one of claims 84-90, wherein said administration is by injection, optionally intravenous injection, or intrastriatal injection. 92. The composition or method of any one of claims 83-91, wherein the viral vector is AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or its heterologous chimera. or Capsid protein of AAV12, or a heterologous chimera thereof. 94. The composition or method of claim 93, wherein the capsid protein is an AAV9 capsid protein. 95. The composition or method of any one of claims 83-94, wherein the viral vector is a self-complementing AAV (scAAV). 96. The composition or method of any one of claims 83-95, wherein the viral vector is formulated for delivery to the central nervous system (CNS). 97. The composition or method of any one of claims 82-96, wherein the nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NO: 110. 98. The composition or method of any one of claims 82-96, wherein the nucleic acid comprises a sequence that is at least 95% identical to SEQ ID NO: 110. 99. The composition or method of any one of claims 82-96, wherein the nucleic acid comprises the same sequence as SEQ ID NO: 110. 100. The composition or method of any one of claims 2-40, 42-81, 83-99, wherein the viral vector comprises a modified viral capsid. 101. The composition or method of any one of claims 2-40, 42-81, 83-99, wherein the viral vector comprises modifications to the viral capsid. 102. The composition or method of claim 100 or 101, wherein the modification is a chemical, non-chemical or amino acid modification of the viral capsid. 103. The composition or method of claims 100 or 101, wherein at least one of the capsid modifications preferentially targets cells in the CNS or PNS. 104. The composition or method of claim 100 or 101, wherein the chemical modification comprises a chemically modified tyrosine residue modified to comprise a covalently linked monosaccharide or polysaccharide part. 105. The composition or method of claim 104, wherein the chemically modified tyrosine residue comprises a group selected from the group consisting of galactose, mannose, N-acetylgalactosamine, GalNac bridge, and mannose-6- Monosaccharide of phosphate. 106. 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. The composition or method of claim 106, wherein the ligand comprises an arylene or heteroarylene radical covalently bound to the ligand. 108. The composition or method of any one of claims 100-107, wherein the modified viral capsid is a chimeric capsid or a haploid capsid. 109. The composition or method of any one of claims 100-107, wherein the modified viral capsid is a haploid capsid. 110. The composition or method of any one of claims 100-107, wherein the modified viral capsid further comprises a modified chimeric capsid or a haploid capsid. 111. The composition or method of any one of claims 100-110, wherein the modified viral capsid is from AAV serotypes AAV1, AAV2, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV10, AAV11, AAV12, or mutant modified forms, heterochimeras, homologous chimeras or reasonable haplotypes thereof. 112. The composition or method of any one of claims 100-111, wherein the modification alters the antigenic profile of the modified viral capsid compared to an unmodified viral capsid. 113. The composition or method of any one of claims 100-112, wherein the modified viral capsid is available for repeated administration.