Vectors containing stuffer polynucleotide sequences

Abstract

The present disclosure provides vector stuffer polynucleotides and compositions thereof, including expression constructs and vectors (e.g., viral vectors), and methods for delivering therapeutic agents (e.g., inhibitory nucleic acids) to mammals or treating diseases. The size of the adeno-associated virus (AAV) vector genome is known to be constrained, and while vector sizes of approximately the natural size of 4.7 kb have been successfully packaged, much larger vector genomes are prone to reduced production of functional AAV vectors.

Description

[Technical field]

[0001] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 630264_402WO_SEQUENCE_LISTING.txt. The text file is 163KB, was created on February 4, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0002] background It is known that there is a size constraint on the adeno-associated virus (AAV) vector genome; vector sizes of approximately the natural size of 4.7 kb have been successfully packaged, but much larger vector genomes are prone to reduced production of functional AAV vectors (Wu et al., Molecular Therapy 2010 18(1): 80-86). On the other hand, vector genomes substantially smaller than the vector genome packaging limit may result in suboptimal packaging (Dong et al., Human gene therapy 1996 7:2101-2112). Also, if a cis-plasmid has a "backbone" sequence (e.g., typically encoding bacterial resistance sources and antibiotic resistance elements required to propagate the plasmid in bacteria) close in size to its intended AAV vector genome, the amount of unintended "reverse packaging" sequence may be increased (Hauck et al., Molecular Therapy (2009) 17:144-152). For these reasons, it may be beneficial to include a "stuffer sequence" that does not confer undesirable properties on its own to the packaged AAV, both as material in the plasmid backbone and for use in situations where the intended AAV payload is significantly smaller than the native AAV packaging size. The importance of inactive stuffer sequences has been highlighted by Keiser et al., Nature Medicine (2021) 27:1982-1989, which showed that "payload-free" AAVs with stuffer sequences were capable of inducing substantial toxicity in non-human primates. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Wu et al., Molecular Therapy 2010 18(1): 80-86 [Non-Patent Document 2] Dong et al., Human gene therapy 1996 7:2101-2112 [Non-Patent Document 3] Hauck et al., Molecular Therapy (2009) 17:144-152 [Non-Patent Document 4] Keiser et al., Nature Medicine (2021) 27:1982-1989 Summary of the Invention [Means for solving the problem]

[0004] There is currently a need for AAV vectors with improved packaging characteristics. [Brief description of the drawings]

[0005] [Figure 1-1] Figure 1: Tracks selected in the UCSC Genome Browser to identify expressed regions, known or putative regulatory elements, and repetitive elements (including retroviral elements and transposable elements) within a sequence. [Figure 1-2] Same as above. [Figure 2A] Figures 2A-2B show liver enzyme function tests (Figure 2A) aspartate transaminase (AST; U / L) and (Figure 2B) alanine transaminase (ALT; U / L) measured 13 days after administration of treated serum from mice administered vehicle or scAAV9 vectors intravenously. When compared to control mice, the stuffer-only vector had no significant elevation in either ALT or AST, whereas vector expression in the toxicity positive control artificial miRNA induced a substantial and significant elevation in the liver enzymes ALT and AST (one-way ANOVA, Dunnett's multiple comparisons test). All groups were N=5 except for the toxicity positive control (N=3). [Figure 2B] Same as above. [Figure 3A]Figures 3A-3H show Fragment Analyzer traces of column-purified scAAV9 vector DNA. Using a representative vector, Figure 3A shows a standard high molecular weight double peak, clearly indicating that upon enzymatic cleavage of the mutITR (double peak is retained; Figure 3B) or wtITRs (now a single peak; Figure 3C), the double peak represents a full-length population. Figure 3D displays three components of the Fragment Analyzer trace of purified scAAV9 vector DNA: the full-length vector as represented by the two peaks at the highest molecular weight, the next peak, or miR-centered truncation, and finally the truncation occurring within the stuffer sequence. In Figure 3E, the traces for the scAAV9 H1 MCS constructs, PSG11_V1 and PSG11_V2, in the top and bottom traces, respectively. In the traces shown in Figure 3F, when overlaid, PSG11_V2 has a higher concentration of truncation compared to PSG11_V1. In Figure 3G, Fragment Analyzer traces of PSG11_V1 vector (top) and PSG11_V2 vector (bottom) embedded with artificial miRNA miR-1-1 XD-14792 (XD-14792 is also referred to herein as 1784). Figure 3H shows an overlay of the traces in Figure 3G. As evidenced by the size of the miR-centered peak (arrow), the shortening of the miR-centered is reduced in PSG11_V1 compared to PSG11_V2. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3H] Same as above. [Figure 4A-1]Figures 4A-4B show the predicted secondary structures of the 200 nucleotides adjacent to the artificial miRNA and terminator for PSG11_V1 (Figure 4A) and PSG11_V2 (Figure 4B) using the Mfold web server. PSG11_V1 has a more favorable predicted DNA secondary structure adjacent to the artificial miRNA compared to PSG11_V2, as evidenced by the lower negative Gibbs free energy values ​​and the relative lack of secondary structure. [Figure 4A-2] Same as above. [Figure 4B-1] Same as above. [Figure 4B-2] Same as above. [Figure 4B-3] Same as above. [Figure 5A] Figures 5A-5B show liver enzyme values ​​(Figure 5A - AST; Figure 5B - ALT) from the vector construction test. [Figure 5B] Same as above. [Figure 6] FIG. 6 shows standard RNAseq traces from a representative vector with an H1 long promoter, amiRNA 3330 in a miR-100 backbone and a PSG11_V5 stuffer (left), and a representative vector with an H1 short promoter, amRNA 1784 (also referred to herein as 14792) in a miR-1-1 backbone and a PSG11_V5 stuffer (right). [Figure 7] Figure 7 shows the titers of the 24 constructs tested in the vector construction study. Vectors containing the H1 long promoter (left column) consistently had higher titers compared to both the H1 native promoter (center) and the H1 short promoter (right). [Figure 8A]Figures 8A-8C show miR and stuffer truncation for scAAV9 vectors with AMELY_V3 and PSG11_V5 stuffer sequences as measured by Fragment Analyzer parallel capillary electrophoresis system. In Figure 8A, across all three promoter contexts, H1 long (left panel), H1 native (middle panel), and H1 short (right panel), and both without (circle) and with (triangle) SV40 polyadenylation sequences, miR100 has less miR truncation compared to miR1-1 in both AMELY_V3 and PSG11_V5 vectors. In Figure 8B, vectors with H1 long promoter, both with and without SV40 polyadenylation sequences, have less stuffer truncation compared to vectors with H1 native and H1 short promoters. In FIG. 8C, vectors with the H1 long promoter, both with and without the SV40 polyadenylation sequence, have less truncation in the combined miR / stuffer truncation compared to vectors with the H1 native promoter and vectors with the H1 short promoter. [Figure 8B] Same as above. [Figure 8C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description Before setting forth the present disclosure in more detail, it may be helpful to an understanding of the same to provide definitions of certain terms that will be used herein. Additional definitions are set forth throughout the disclosure.

[0007] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer within the stated range and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer) unless otherwise indicated. Also, any numerical range described herein for any physical characteristic (e.g., polymer subunits, size, or thickness) should be understood to include any integer within the stated range unless otherwise indicated. As used herein, the term "about" means ±20% of the stated range, value, or structure unless otherwise indicated. The terms "a" and "an" as used herein should be understood to refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean either one of the alternatives, both, or any combination thereof. As used herein, the terms "include," "have," and "comprise" are used interchangeably and it is intended that these terms and variations thereof be construed as non-limiting.

[0008] As used herein, the term "nucleic acid" or "polynucleotide" refers to any nucleic acid polymer composed of covalently linked nucleotide subunits (e.g., polydeoxyribonucleotides or polyribonucleotides). Examples of nucleic acids include RNA and DNA.

[0009] As used herein, "RNA" refers to a molecule that contains one or more ribonucleotides, and includes double-stranded RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of an RNA molecule can include standard nucleotides or non-standard nucleotides (e.g., non-naturally occurring nucleotides or chemically synthesized nucleotides).

[0010] As used herein, "DNA" refers to a molecule containing one or more deoxyribonucleotides, and includes double-stranded DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of a DNA molecule can include standard nucleotides or non-standard nucleotides (e.g., non-naturally occurring nucleotides or chemically synthesized nucleotides).

[0011] "Isolated" refers to a material that is isolated from its natural environment or that is artificially produced. As used herein with respect to a cell, "isolated" refers to a cell that is isolated from its natural environment (e.g., from a subject, organ, tissue, or bodily fluid). As used herein with respect to a nucleic acid, "isolated" refers to a nucleic acid that is isolated or purified from its natural environment (e.g., from a cell, organelle, or cytoplasm), recombinantly produced, amplified, or synthesized. In an embodiment, an isolated nucleic acid includes a nucleic acid contained within a vector.

[0012] As used herein, the term "wild-type" or "non-mutant" form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein that lacks a mutation (e.g., a repeat region expansion that results in a higher risk of development, onset, or progression of a neurodegenerative disease).

[0013] As used herein, the term "mutation" refers to any change in the structure of a gene, e.g., in the sequence of a gene, that results in an altered form of the gene. Mutations may or may not be passed on to the next generation (hereditary mutations) (somatic mutations). Genetic mutations include the substitution, insertion, or deletion of a single base in DNA, or the substitution, insertion, deletion, or rearrangement (including repeat expansion) of multiple bases or larger parts of a gene or chromosome.

[0014] As used herein, the term "inhibitory nucleic acid" refers to a nucleic acid that comprises a guide strand sequence that hybridizes to at least a portion of a target nucleic acid (e.g., a neurodegenerative disease target RNA, mRNA, pre-mRNA, or mature mRNA) and inhibits its expression or activity. The inhibitory nucleic acid may target a protein coding region (e.g., an exon) or a non-coding region (e.g., a 5'UTR, a 3'UTR, an intron, etc.) of the target nucleic acid. In some embodiments, the inhibitory nucleic acid is a single-stranded or double-stranded molecule. The inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., a double stranded duplex) or within the same strand (e.g., a single stranded self-annealing duplex structure). In some embodiments, the inhibitory nucleic acid is an RNA molecule (e.g., an siRNA, shRNA, miRNA, or dsRNA).

[0015] As used herein, "microRNA" or "miRNA" refers to a small non-coding RNA molecule that can mediate target gene silencing by target mRNA cleavage, target mRNA translational repression, target mRNA degradation, or a combination thereof. Typically, miRNA is transcribed as a hairpin or stem-loop (e.g., with a self-complementary single-stranded backbone) duplex structure, also called primary miRNA (pri-miRNA), which is enzymatically processed into pre-miRNA (e.g., by Drosha, DGCR8, Pasha, etc.). Pre-miRNA is exported to the cytoplasm, where it is enzymatically processed by Dicer to generate miRNA duplex with passenger strand, which then generates single-stranded mature miRNA molecule, which is then loaded into RNA-induced silencing complex (RISC). Reference to miRNA can include synthetic or artificial miRNA.

[0016] As used herein, "synthetic miRNA" or "artificial miRNA" or "amiRNA" refers to an endogenous, modified, or synthetic pri-miRNA or pre-miRNA (e.g., miRNA scaffold or scaffold) in which endogenous miRNA guide and passenger sequences within the stem sequence have been replaced with miRNA guide and passenger sequences that direct highly efficient RNA silencing of targeted genes (see, e.g., Eamens et al., Methods Mol. Biol. (2014) 1062:211-224). In some embodiments, the nature of the complementarity of the guide and passenger sequences (e.g., number of bases, location of mismatches, type of bulge, etc.) can be similar or different from the nature of the complementarity of the guide and passenger sequences in the endogenous miRNA scaffold from which the synthetic miRNA is constructed.

[0017] As used herein, the terms "microRNA backbone", "miR backbone", "microRNA scaffold", or "miR scaffold" refer to a pri-miRNA or pre-miRNA scaffold whose stem sequence can be replaced by a miRNA of interest to generate a functional mature miRNA that directs RNA silencing in a gene targeted by the miRNA of interest. The miR backbone includes a 5' flanking region (also referred to as the 5' miR context, ≧9 nucleotides), a stem region that includes the miRNA duplex (guide strand sequence and passenger strand sequence), and a basal stem (5' and 3', about 4-13 nucleotides each), at least one loop motif region (≧10 nucleotides for the terminal loop) that includes a terminal loop, a 3' flanking region (also referred to as the 3' miR context, ≧9 nucleotides), and optionally one or more bulges in the stem. The miR backbone may be derived entirely or partially from a wild-type miRNA scaffold, or may be a fully artificial sequence.

[0018] As used herein, the term "antisense strand sequence" or "guide strand sequence" of an inhibitory nucleic acid refers to a sequence that is substantially complementary (e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary) to a region of about 10-50 nucleotides (e.g., about 15-30 nucleotides, 16-25 nucleotides, 18-23 nucleotides, or 19-22 nucleotides) of the mRNA of a gene targeted for silencing. The antisense sequence is sufficiently complementary to the target mRNA sequence to direct target-specific silencing, e.g., to induce destruction of the target mRNA by the RNAi mechanism or process. In some embodiments, the antisense sequence or guide strand sequence refers to the mature sequence remaining after cleavage by Dicer.

[0019] As used herein, the term "sense sequence" or "passenger strand sequence" of inhibitory nucleic acid refers to a sequence that is homologous to target mRNA and partially or completely complementary to the above-mentioned antisense strand sequence or guide strand sequence of inhibitory nucleic acid. The antisense strand sequence and the sense strand sequence of inhibitory nucleic acid are hybridized to form a duplex structure (e.g., form a double-stranded duplex or a single-stranded self-annealing duplex structure). In some embodiments, the above-mentioned sense sequence or passenger strand sequence refers to the mature sequence remaining after Dicer cleavage.

[0020] As used herein, "duplex," when used in reference to an inhibitory nucleic acid, refers to two nucleic acid strands (e.g., a guide strand and a passenger strand) that hybridize together to form a duplex structure. A duplex can be formed by two separate nucleic acid strands, or by a single nucleic acid strand that has a region of self-complementarity (e.g., a hairpin or stem-loop).

[0021] As used herein, the term "complementary" refers to the ability of polynucleotides to base pair with each other. Base pairs are typically formed by hydrogen bonds between nucleotide subunits in antiparallel polynucleotide strands or in a single self-annealing polynucleotide strand. Complementary polynucleotide strands may base pair in Watson-Crick fashion (e.g., A and T, A and U, C and G), or in any other fashion that allows for the formation of a duplex. As will be apparent to those skilled in the art, when using RNA as opposed to DNA, the base considered to be complementary to adenosine is uracil, not thymine. Furthermore, where "U" is indicated in the context of the present invention, it is understood that it can be substituted for "T" unless otherwise stated. Complementary also encompasses Watson-Crick base pairing between unmodified and modified nucleobases (e.g., 5-methylcytosine used instead of cytosine). Full complementarity, perfect complementarity or 100% complementarity between two polynucleotide strands is when each nucleotide of one polynucleotide strand can form hydrogen bonds with the nucleotide units of the second polynucleotide strand. % complementarity refers to the number of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule that is complementary to an aligned reference sequence (e.g., target mRNA, passenger strand), divided by the total number of nucleotides, multiplied by 100. In such an alignment, the nucleobase / nucleotide that does not form a base pair is called a mismatch. Insertions and deletions are not allowed in calculating the % complementarity of a contiguous nucleotide sequence. It is understood by those skilled in the art that in calculating complementarity, chemical modifications to nucleobases are not taken into account as long as the Watson-Crick base pairing ability of the nucleobase is maintained (e.g., 5-methylcytosine is considered the same as cytosine for the purpose of calculating % complementarity).

[0022] "Percent identity" between two or more nucleic acid sequences refers to the percentage of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule that is shared by a reference sequence (i.e., % identity = number of identical nucleotides / total number of nucleotides in the aligned region (e.g., contiguous nucleotide sequence) x 100). Insertions and deletions are not allowed when calculating the % identity of a contiguous nucleotide sequence. It is understood by those skilled in the art that, when calculating identity, chemical modifications to nucleobases are not taken into account, so long as the Watson-Crick base pairing ability of the nucleobase is maintained (e.g., 5-methylcytosine is considered the same as cytosine for the purpose of calculating % identity).

[0023] As used herein, the terms "hybridizing" or "hybridizes" refer to two nucleic acid strands that form hydrogen bonds between base pairs on antiparallel strands, thereby forming a duplex. The strength of hybridization between two nucleic acid strands is measured by the melting temperature (T m ), which is defined as the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide at a defined ionic strength and pH.

[0024] As used herein, "expression construct" refers to any type of genetic construct that includes a nucleic acid (e.g., a transgene) from 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 an inhibitory RNA (e.g., siRNA, shRNA, miRNA) from the transcribed gene. In some embodiments, the transgene is operably linked to an expression control sequence.

[0025] As used herein, the term "transgene" refers to an exogenous nucleic acid that can be transferred, either naturally or by genetic engineering means, into another cell and transcribed and optionally translated.

[0026] As used herein, the term "gene expression" refers to the process in which nucleic acid is transcribed from a nucleic acid molecule and often translated into a peptide or protein. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. Reference to measuring "gene expression" may refer to measuring the transcription product (e.g., RNA or mRNA), the translation product (e.g., peptide or protein).

[0027] As used herein, the term "inhibit expression of a gene" means to reduce, downregulate, suppress, block, decrease, or stop the expression of a gene. The expression product of a gene can be an RNA molecule (e.g., mRNA) transcribed from the gene or a polypeptide translated from the mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom. The level of expression can be determined using standard techniques for measuring mRNA or protein.

[0028] As used herein, "vector" refers to a genetic construct that can transport a nucleic acid molecule (e.g., a transgene encoding an inhibitory nucleic acid) between cells and can result in the expression of the nucleic acid molecule when operably linked to an appropriate expression control sequence. Expression control sequences can include: transcription initiation sequences, termination sequences (also referred to herein as terminator sequences), promoter sequences and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance secretion of the encoded product, if desired. The vector can be a plasmid, phage particle, transposon, cosmid, phagemid, chromosome, artificial chromosome, virus, virion, etc. Once transformed into a suitable host cell, the vector can replicate and function independently of the host genome, or in some cases, can be integrated into the genome itself.

[0029] As used herein, "host cell" refers to any cell that contains or is capable of containing a composition of interest (e.g., an inhibitory nucleic acid). In embodiments, the host cell is a mammalian cell (e.g., a rodent cell (mouse or rat) or a primate cell (monkey, chimpanzee, or human). In embodiments, the host cell may be in vitro or in vivo. In embodiments, the host cell may be derived from an established cell line or a primary cell. In embodiments, the host cell is a cell of the CNS (e.g., a neuron, a glial cell, an astrocyte, and a microglial cell).

[0030] As used herein, "neurodegenerative disease" or "neurodegenerative disorder" refers to a disease or disorder that exhibits neuronal cell death as a pathological condition. Neurodegenerative disease can exhibit chronic neurodegeneration, e.g., slow progressive neuronal cell death over a period of several years, or acute neurodegeneration, e.g., sudden onset or neuronal cell death. Examples of chronic neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia type 2 (SCA2), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). Chronic neurodegenerative diseases include diseases characterized by TDP-43 proteinopathy (characterized by nuclear to cytoplasmic mislocalization, deposition of ubiquitinated and hyperphosphorylated TDP-43 into inclusion bodies, protein truncation leading to the formation of toxic C-terminal TDP-43 fragments, and protein aggregation). TDP-43 proteinopathy diseases include ALS, FTD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Acute neurodegeneration can be caused by ischemia (e.g., stroke, traumatic brain injury), demyelination, or traumatic axonal transection (e.g., spinal cord injury or multiple sclerosis). Neurodegenerative diseases can show the death of mainly one type of neuron or multiple types of neurons.

[0031] As used herein, "subject," "patient," and "individual" are used interchangeably herein and refer to a living organism (e.g., a mammal) selected for treatment or therapy. Examples of subjects include humans and non-human mammals (e.g., primates (monkeys, chimpanzees), cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, pigs, and transgenic species thereof).

[0032] Stuffer sequences and expression constructs AAV preferentially packages full-length genomes, i.e., those that are approximately the same size as the natural genome, and are neither too large nor too small. However, the expression cassettes that code for inhibitory nucleic acid sequences are substantially smaller than the AAV full-length genome. To avoid packaging of fragmented genomes, stuffer sequences can be linked to the expression constructs that contain heterologous nucleic acid sequences and flanked by 5' and 3' ITRs to expand the packageable genome, resulting in a genome that is approximately normal in size in the length between the ITRs. In general, the packaging capacity of AAV is about 4.7 kb between 5' and 3' ITRs. For self-complementary AAV (scAAV) vectors, the packaging capacity is about 2.4 kb between 5' and 3' ITRs.

[0033] Preferably, the starting sequence for obtaining the vector stuffer sequence is of mammalian origin (e.g., human origin). The length of the stuffer sequence can be adjusted so that the vector genome is at or near the (natural) packaging limit of the AAV capsid. Furthermore, the vector stuffer sequence can be designed to minimize adverse effects in the context of in vivo gene therapy. For example, a region of the human genome can be identified as a source of vector stuffer sequences by identifying sequences that have minimal impact if integration in the genome occurs and minimal risk of initiating unpredicted transcription. Thus, the region of the genome can be examined in which: i) deletions and duplications are common in the population and are not associated with disease-related phenotypes (no evidence of evolutionary pressure), and / or ii) RNA expression across human tissues was low or undetectable (lacking strong intrinsic enhancer / promoter elements). Furthermore, the vector stuffer sequence can be designed to reduce, minimize, remove, or lack one or more elements to render the vector sequence inactive or safe. In some embodiments, vector stuffer sequences are modified to: reduce or remove expressed regions (e.g., exons +10 bp on either side, human ESTs); reduce or remove regulatory elements (e.g., promoter sequences, enhancer sequences, repressor sequences, splicing donors or acceptors, or other cis-acting elements found in the human genome that may potentially affect transcription of the transgene); reduce or remove repetitive elements (e.g., microsatellite repeats, dinucleotide repeats, trinucleotide repeats); reduce, remove, or modify ATG codons to reduce or eliminate the possibility that peptides will be generated from filler or stuffer sequences due to latent start codons; reduce or eliminate CpG dinucleotides to decrease the likelihood of unmethylated CpG dinucleotides (from cis plasmids generated in bacteria) inducing an innate immune response; or any combination thereof.The present disclosure provides vector stuffer sequences having one or more of the aforementioned characteristics and one or more of the following additional characteristics: high titer; low toxicity; and reduced truncations in the miRNA and / or stuffer sequences.

[0034] In one aspect, the disclosure provides a vector stuffer sequence comprising a nucleic acid having at least 75% identity to the following, the nucleic acid being about 1300 to about 2300 nucleotides in length: nucleotides 489 to 2185 of any one of SEQ ID NO:13-16; nucleotides 342 to 2222 of SEQ ID NO:17; nucleotides 488 to 2177 of SEQ ID NO:18; nucleotides 489 to 2177 of SEQ ID NO:19; nucleotides 711 to 2187 of SEQ ID NO:20; nucleotides 711 to 2187 of SEQ ID NO:21; nucleotides 488 to 2177 of SEQ ID NO:22; nucleotides 711 to 2187 of SEQ ID NO:23; nucleotides 711 to 2187 of SEQ ID NO:24; nucleotides 252 to 2132 of SEQ ID NO:25; nucleotides 252 to 2132 of SEQ ID NO:26; nucleotides 252 to 2132 of SEQ ID NO:27; nucleotides 252 to 2132 of SEQ ID NO:28; Nucleotides 357 to 2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; Nucleotides 580 to 2187 of SEQ ID NO:31; Nucleotides 580 to 2196 of SEQ ID NO:32; Nucleotides 357 to 2237 of SEQ ID NO:33; Nucleotides 358 to 2195 of SEQ ID NO:34; Nucleotides 580 to 2187 of SEQ ID NO:35; Nucleotides 580 to 2196 of SEQ ID NO:36; Nucleotides 348 to 2228 of SEQ ID NO:37; Nucleotides 349 to 2229 of SEQ ID NO:38; Nucleotides 349 to 2186 of SEQ ID NO:39; Nucleotides 571 to 2178 of SEQ ID NO:40; Nucleotides 571 to 2178 of SEQ ID NO:41; Nucleotides 348 to 2228 of SEQ ID NO:42; Nucleotides 349 to 2186 of SEQ ID NO:43; Nucleotides 571 to 2178 of SEQ ID NO:44; Nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358 to 2238 of SEQ ID NO:79; nucleotides 342 to 2222 of SEQ ID NO:80; nucleotides 342 to 2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51.

[0035] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 75% identity to the following, with a length of about 1500-2000 nucleotides: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; Nucleotides 357 to 2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; Nucleotides 580 to 2187 of SEQ ID NO:31; Nucleotides 580 to 2196 of SEQ ID NO:32; Nucleotides 357 to 2237 of SEQ ID NO:33; Nucleotides 358 to 2195 of SEQ ID NO:34; Nucleotides 580 to 2187 of SEQ ID NO:35; Nucleotides 580 to 2196 of SEQ ID NO:36; Nucleotides 348 to 2228 of SEQ ID NO:37; Nucleotides 349 to 2229 of SEQ ID NO:38; Nucleotides 349 to 2186 of SEQ ID NO:39; Nucleotides 571 to 2178 of SEQ ID NO:40; Nucleotides 571 to 2178 of SEQ ID NO:41; Nucleotides 348 to 2228 of SEQ ID NO:42; Nucleotides 349 to 2186 of SEQ ID NO:43; Nucleotides 571 to 2178 of SEQ ID NO:44; Nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358 to 2238 of SEQ ID NO:79; nucleotides 342 to 2222 of SEQ ID NO:80; nucleotides 342 to 2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51.

[0036] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 75% identity to the following, having a length of about 1600-1900 nucleotides: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; Nucleotides 357 to 2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; Nucleotides 580 to 2187 of SEQ ID NO:31; Nucleotides 580 to 2196 of SEQ ID NO:32; Nucleotides 357 to 2237 of SEQ ID NO:33; Nucleotides 358 to 2195 of SEQ ID NO:34; Nucleotides 580 to 2187 of SEQ ID NO:35; Nucleotides 580 to 2196 of SEQ ID NO:36; Nucleotides 348 to 2228 of SEQ ID NO:37; Nucleotides 349 to 2229 of SEQ ID NO:38; Nucleotides 349 to 2186 of SEQ ID NO:39; Nucleotides 571 to 2178 of SEQ ID NO:40; Nucleotides 571 to 2178 of SEQ ID NO:41; Nucleotides 348 to 2228 of SEQ ID NO:42; Nucleotides 349 to 2186 of SEQ ID NO:43; Nucleotides 571 to 2178 of SEQ ID NO:44; Nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358 to 2238 of SEQ ID NO:79; nucleotides 342 to 2222 of SEQ ID NO:80; nucleotides 342 to 2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51.

[0037] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 80% identity to: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; nucleotides 342-2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51. In some embodiments, the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 nucleotides in length.

[0038] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 85% identity to: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; nucleotides 342-2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51. In some embodiments, the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 nucleotides in length.

[0039] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 90% identity to: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; nucleotides 342-2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51. In some embodiments, the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 nucleotides in length.

[0040] In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 95% identity to: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; nucleotides 342-2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51. In some embodiments, the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 nucleotides in length.In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 97% identity to: nucleotides 489-2185 of any one of SEQ ID NO:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; nucleotides 342-2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51. In some embodiments, the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 nucleotides in length.

[0041] In some embodiments, the vector stuffer sequence comprises or consists of: nucleotides 489 to 2185 of any one of SEQ ID NO:13-16; nucleotides 342 to 2222 of SEQ ID NO:17; nucleotides 488 to 2177 of SEQ ID NO:18; nucleotides 489 to 2177 of SEQ ID NO:19; nucleotides 711 to 2187 of SEQ ID NO:20; nucleotides 711 to 2187 of SEQ ID NO:21; nucleotides 488 to 2177 of SEQ ID NO:22; nucleotides 711 to 2187 of SEQ ID NO:23; nucleotides 711 to 2187 of SEQ ID NO:24; nucleotides 252 to 2132 of SEQ ID NO:25; nucleotides 252 to 2132 of SEQ ID NO:26; nucleotides 252 to 2132 of SEQ ID NO:27; nucleotides 252 to 2132 of SEQ ID NO:28; nucleotides 357 to 2237 of SEQ ID NO:29; Nucleotides 358 to 2195 of SEQ ID NO:30; nucleotides 580 to 2187 of SEQ ID NO:31; nucleotides 580 to 2196 of SEQ ID NO:32; nucleotides 357 to 2237 of SEQ ID NO:33; nucleotides 358 to 2195 of SEQ ID NO:34; nucleotides 580 to 2187 of SEQ ID NO:35; nucleotides 580 to 2196 of SEQ ID NO:36; nucleotides 348 to 2228 of SEQ ID NO:37; nucleotides 349 to 2229 of SEQ ID NO:38; nucleotides 349 to 2186 of SEQ ID NO:39; nucleotides 571 to 2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; Nucleotides 358 to 2238 of SEQ ID NO:79; nucleotides 342 to 2222 of SEQ ID NO:80; nucleotides 342 to 2222 of SEQ ID NO:81; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49, SEQ ID NO:50; or SEQ ID NO:51.

[0042] In some embodiments, the vector stuffer sequence comprises or consists of SEQ ID NO:48, or nucleotides 489 to 2185 of any one of SEQ ID NOs:13-16.

[0043] In some embodiments, the vector is an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is self-complementary.

[0044] In some embodiments, the vector stuffer sequence is located adjacent (e.g., 5' or 3') to an expression construct that includes a heterologous nucleic acid sequence. In some embodiments, the heterologous nucleic acid sequence encodes a therapeutic agent. In some embodiments, the therapeutic agent comprises a nucleic acid encoding a therapeutic protein or an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises an siRNA, miRNA, shRNA, or dsRNA. In some embodiments, the inhibitory nucleic acid comprises an siRNA, miRNA, shRNA, or (of) dsRNA targeting a neurodegenerative disease-associated gene. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia type 2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-parkinsonism ALS complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease. In some embodiments, the neurodegenerative disease is polyglutamine repeat disease. In some embodiments, the inhibitory nucleic acid (e.g., siRNA, miRNA, shRNA, or dsRNA) targets ATXN2. ATXN2 refers to the protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) tract. ATXN2 genes or transcripts may refer to normal alleles of ATXN2, which typically have 22 or 23 repeats, or mutated alleles with moderate (about 24-32 repeats) or longer repeat expansions (about 33 to >100 repeats). In some embodiments, ATXN2 refers to mammalian ATNX2, including human ATXN2.

[0045] In some embodiments, the expression construct comprises a heterologous nucleic acid encoding an artificial miRNA targeting ATXN2. Examples of guide and passenger sequences, as well as heterologous nucleic acids encoding an artificial miRNA targeting ATXN2, are provided in Table 5. RNA formats of the encoded guide, passenger, and artificial miRNA sequences are provided, as well as DNA formats for insertion into cis plasmids and rAAV vectors. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA targeting ATXN2 and comprises a guide sequence selected from SEQ ID NOs: 1-4 and 71. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA comprising a guide sequence provided by SEQ ID NO: 1 and a passenger sequence provided by SEQ ID NO: 5. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 2 and a passenger sequence provided by SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 3 and a passenger sequence provided by SEQ ID NO: 7. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 4 and a passenger sequence provided by SEQ ID NO: 8. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 71 and a passenger sequence provided by SEQ ID NO: 72. In some embodiments, the heterologous nucleic acid comprises a guide sequence and a passenger sequence embedded in a miRNA backbone (or scaffold). In some embodiments, the miRNA backbone is miR-100 or miR-1-1. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA sequence provided by any one of SEQ ID NOs: 9-12 and 73. Further examples of ATXN2-targeting guide sequences, passenger sequences, miR backbones, and artificial miRNA sequences are provided in PCT Publication WO2021 / 159008, which is incorporated by reference in its entirety.

[0046] In some embodiments, the expression construct further comprises one or more expression control sequences (regulatory sequences) operably linked to the transgene (e.g., nucleic acid encoding an artificial miRNA). An "operably linked" sequence includes an expression control sequence that is contiguous with the transgene or that acts in trans or at a distance from the transgene to control expression of the transgene. Examples of expression control sequences include transcription initiation sequences, termination sequences, promoter sequences, enhancer sequences, repressor sequences, splice site sequences, polyadenylation (polyA) signal sequences, or any combination thereof.

[0047] In some embodiments, the promoter is an endogenous promoter, a synthetic promoter, a constitutive promoter, an inducible promoter, a tissue-specific promoter (e.g., CNS-specific), or a cell-specific promoter (neuron, glial cell, or astrocyte). Examples of constitutive promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, and the dihydrofolate reductase promoter. Examples of inducible promoters include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system. Further examples of promoters that can be used include, for example, chicken β-actin promoter (CBA promoter), CAG promoter, H1 promoter, CD68 promoter, JeT promoter, synapsin promoter, RNA pol II promoter, or RNA pol III promoter (e.g., U6, H1, etc.). In some embodiments, the promoter is a tissue-specific RNA pol II promoter. In some embodiments, the tissue-specific RNA pol II promoter is derived from a gene that exhibits neuron-specific expression. In some embodiments, the neuron-specific promoter is a synapsin 1 promoter or a synapsin 2 promoter.

[0048] In some embodiments, the promoter is an H1 promoter. In some embodiments, the H1 promoter is what is referred to herein as a "native" H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:53). In some embodiments, the promoter is what is referred to herein as a "short" H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:54 or nucleotides 113-203 of any one of SEQ ID NOs:17, 25-28, and 37-44). In some embodiments, the promoter is what is referred to herein as a "long" H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:52 or nucleotides 113-343 of any one of SEQ ID NOs:13-16 and 18-24).

[0049] In some embodiments, the termination sequence is an SV40 termination sequence. Examples of SV40 termination sequences are set forth in SEQ ID NO:77; nucleotides 489-710 of any one of SEQ ID NOs:20, 21, 23, and 24; nucleotides 358-579 of any one of SEQ ID NOs:31, 32, 35, and 36; and nucleotides 349-570 of any one of SEQ ID NOs:40, 41, and 44.

[0050] In some embodiments, the sequence encoding the inhibitory nucleic acid of the disclosure is placed in an untranslated region of the expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the disclosure is placed in an intron, 5' untranslated region (5'UTR) or 3' untranslated region (3'UTR) of the expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the disclosure is placed in an intron downstream of the promoter and upstream of the gene to be expressed.

[0051] Vectors and host cells In another aspect, a vector is provided that includes the vector stuffer sequence of the present disclosure. The vector can be a plasmid, a cosmid, a phagemid, a bacterial artificial chromosome (BAC) or a viral vector. Examples of viral vectors include herpes virus (HSV) vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, baculovirus vectors, and the like. In some embodiments, the retroviral vector is a mouse stem cell virus, a murine leukemia virus (e.g., Moloney murine leukemia virus vector), a feline leukemia virus, a feline sarcoma virus, or an avian reticuloendotheliosis virus vector. In some embodiments, the lentiviral vector is an HIV (human immunodeficiency virus (including HIV type 1 and HIV type 2)), an equine infectious anemia virus, a feline immunodeficiency virus (FIV), a bovine immunodeficiency virus (BIV), and a simian immunodeficiency virus (SIV), an equine infectious anemia virus, or a Maedi-Visna virus vector.

[0052] In some embodiments, the vector is an adeno-associated virus (AAV) vector (e.g., a recombinant AAV (rAAV) vector), which is produced by recombinant methods. In some embodiments, the rAAV vector comprises an expression construct comprising a vector stuffer sequence of the present disclosure and a heterologous nucleic acid sequence (e.g., encoding an inhibitory nucleic acid). The vector stuffer sequence can be located adjacent to either the 5' or 3' side of the expression construct comprising the heterologous nucleic acid.

[0053] AAV is a single-stranded, non-enveloped DNA virus with a genome that encodes proteins for replication (rep) and capsid (Cap) and is flanked by two ITRs, which serve as origins of replication for the viral genome. AAV also contains packaging sequences, which allow packaging of the viral genome into the AAV capsid. Recombinant AAV vectors (rAAV) (also referred to as rAAV vector genomes, or rAAV genomes) can be derived from the wild-type genome of AAV by using molecular methods to remove all or part of the wild-type genome (e.g., Rep, Cap) from AAV and replacing it with a non-naturally occurring nucleic acid (e.g., a heterologous nucleic acid sequence, e.g., a nucleic acid molecule encoding an inhibitory nucleic acid). Typically, for AAV, one or both of the inverted terminal repeat (ITR) sequences are retained in the rAAV vector. In some embodiments, the rAAV vector contains 5' inverted terminal repeats (ITRs) and 3' ITRs flanking the expression construct and vector stuffer sequences. ITRs. Functional ITR sequences are necessary for rescue, replication and packaging of AAV viral particles. Thus, rAAV vectors are defined herein to contain at least those sequences (e.g., functional ITRs) required in cis for viral replication and packaging. All other viral sequences may be supplied in trans. In some embodiments, the rAAV only retains the 5' and 3' ITRs from the AAV genome to maximize the size of the transgene that can be efficiently packaged by the vector. In some embodiments, each AAV ITR is a full-length ITR (e.g., approximately 145 bp in length and includes a functional Rep binding site (RBS) and terminal resolution site (trs)). In some embodiments, one or both of the ITRs are modified, e.g., by insertion, deletion, or substitution, provided that the ITR provides functional rescue, replication, and packaging. In some embodiments, the modified ITR lacks a functional terminal resolution site (trs) and is used for the generation of self-complementary AAV vectors (scAAV vectors).In some embodiments, the rAAV vector is a self-complementary AAV vector comprising a mutant ITR (lacking a terminal resolution site) at the 5' end and a wild-type AAV ITR at the 3' end. An example of a mutant 5' ITR lacking a terminal resolution site is shown in SEQ ID NO:57, or nucleotides 1-106 of any one of SEQ ID NOs:13-24, 29-44, and 78-82. In some embodiments, the modified ITR is a truncated version of the AAV2 ITR, referred to as an AITR (lacking the D-sequence and TRS).

[0054] In some embodiments, the rAAV vector comprises a 5' ITR that comprises or consists of nucleotides 1-106 of SEQ ID NO:57, or any one of SEQ ID NOs:13-24, 29-44, and 78-82. In some embodiments, the rAAV vector comprises a 3' ITR comprising or consisting of: SEQ ID NO:58, nucleotides 2192-2358 of any one of SEQ ID NOs:13-16, nucleotides 2214-2358 of any one of SEQ ID NOs:13-16, nucleotides 2229-2395 of SEQ ID NO:17, nucleotides 2251-2395 of SEQ ID NO:17, nucleotides 2184-2350 of SEQ ID NO:18, nucleotides 2206-2350 of SEQ ID NO:18; nucleotides 2206-2350 of SEQ ID NO:19; nucleotides 2216-2360 of SEQ ID NO:20; nucleotides 2216-2360 of SEQ ID NO:21; nucleotides 2206-2350 of SEQ ID NO:22; nucleotides 2216-2360 of SEQ ID NO:23; nucleotides 2216-2360 of SEQ ID NO:24; nucleotides 2161-2305 of SEQ ID NO:25; Nucleotides 2161 to 2305 of SEQ ID NO:26; Nucleotides 2161 to 2305 of SEQ ID NO:27; Nucleotides 2161 to 2305 of SEQ ID NO:28; Nucleotides 2266 to 2410 of SEQ ID NO:29; Nucleotides 2224 to 2368 of SEQ ID NO:30; Nucleotides 2216 to 2360 of SEQ ID NO:31; Nucleotides 2225 to 2369 of SEQ ID NO:32; Nucleotides 2266 to 2410 of SEQ ID NO:33; Nucleotides 2224 to 2368 of SEQ ID NO:34; Nucleotides 2216 to 2360 of SEQ ID NO:35; Nucleotides 2225 to 2369 of SEQ ID NO:36; Nucleotides 2257 to 2401 of SEQ ID NO:37; Nucleotides 2258 to 2402 of SEQ ID NO:38; Nucleotides 2215 to 2359 of SEQ ID NO:39; Nucleotides 2207 to 2351 of SEQ ID NO:40; Nucleotides 2207 to 2351 of SEQ ID NO:41; nucleotides 2257 to 2401 of SEQ ID NO:42; nucleotides 2215 to 2359 of SEQ ID NO:43; nucleotides 2207 to 2351 of SEQ ID NO:44; nucleotides 2258 to 2402 of SEQ ID NO:78; nucleotides 2267 to 2411 of SEQ ID NO:79;Nucleotides 2251 to 2395 of SEQ ID NO:80; nucleotides 2251 to 2395 of SEQ ID NO:81; nucleotides 2187 to 2331 of SEQ ID NO:82. In some embodiments, the rAAV vector comprises: a 5' ITR comprising or consisting of SEQ ID NO:57, and a 3' ITR comprising or consisting of SEQ ID NO:58; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:13 and a 3' ITR comprising or consisting of nucleotides 2192 to 2358 of SEQ ID NO:13; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:14 and a 3' ITR comprising or consisting of nucleotides 2192 to 2358 of SEQ ID NO:14; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:15 and a 3' ITR comprising or consisting of nucleotides 2192 to 2358 of SEQ ID NO:15; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:16 and a 3' ITR comprising or consisting of nucleotides 2192 to 2358 of SEQ ID NO:16; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:17. a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 18 and a 3' ITR comprising or consisting of nucleotides 2184 to 2350 of SEQ ID NO: 18; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 13 and a 3' ITR comprising or consisting of nucleotides 2214 to 2358 of SEQ ID NO: 13; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 14 and a 3' ITR comprising or consisting of nucleotides 2214 to 2358 of SEQ ID NO: 14; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 15 and a 3' ITR comprising or consisting of nucleotides 2214 to 2358 of SEQ ID NO: 15; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 16 and a 3' ITR comprising or consisting of nucleotides 2214 to 2358 of SEQ ID NO: 16; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:17 and a 3' ITR comprising or consisting of nucleotides 2251 to 2395 of SEQ ID NO:17;a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 18 and a 3' ITR comprising or consisting of nucleotides 2206 to 2350 of SEQ ID NO: 18; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 19 and a 3' ITR comprising or consisting of nucleotides 2206 to 2350 of SEQ ID NO: 19; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 20 and a 3' ITR comprising or consisting of nucleotides 2216 to 2360 of SEQ ID NO: 20; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 21 and a 3' ITR comprising or consisting of nucleotides 2216 to 2360 of SEQ ID NO: 21; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 22 and a 3' ITR comprising or consisting of nucleotides 2206 to 2350 of SEQ ID NO: 22; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 23 a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:24 and a 3' ITR comprising or consisting of nucleotides 2216 to 2360 of SEQ ID NO:24; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:25 and a 3' ITR comprising or consisting of nucleotides 2161 to 2305 of SEQ ID NO:25; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:26 and a 3' ITR comprising or consisting of nucleotides 2161 to 2305 of SEQ ID NO:26; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:27 and a 3' ITR comprising or consisting of nucleotides 2161 to 2305 of SEQ ID NO:27; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:28 and a 3' ITR comprising or consisting of nucleotides 2161 to 2305 of SEQ ID NO:28; a 5' ITR comprising, or consisting of, nucleotides 1 to 106 of SEQ ID NO:29 and a 3' ITR comprising, or consisting of, nucleotides 2266 to 2410 of SEQ ID NO:29;a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:30 and a 3' ITR comprising or consisting of nucleotides 2224 to 2368 of SEQ ID NO:30; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:31 and a 3' ITR comprising or consisting of nucleotides 2216 to 2360 of SEQ ID NO:31; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:32 and a 3' ITR comprising or consisting of nucleotides 2225 to 2369 of SEQ ID NO:32; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:33 and a 3' ITR comprising or consisting of nucleotides 2266 to 2410 of SEQ ID NO:33; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:34 and a 3' ITR comprising or consisting of nucleotides 2224 to 2368 of SEQ ID NO:34; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO:35 a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 36 and a 3' ITR comprising or consisting of nucleotides 2225 to 2369 of SEQ ID NO: 36; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 37 and a 3' ITR comprising or consisting of nucleotides 2257 to 2401 of SEQ ID NO: 37; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 38 and a 3' ITR comprising or consisting of nucleotides 2258 to 2402 of SEQ ID NO: 38; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 39 and a 3' ITR comprising or consisting of nucleotides 2215 to 2359 of SEQ ID NO: 39; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 40 and a 3' ITR comprising or consisting of nucleotides 2207 to 2351 of SEQ ID NO: 40; a 5' ITR comprising, or consisting of, nucleotides 1 to 106 of SEQ ID NO:41 and a 3' ITR comprising, or consisting of, nucleotides 2207 to 2351 of SEQ ID NO:41;a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 42 and a 3' ITR comprising or consisting of nucleotides 2257 to 2401 of SEQ ID NO: 42; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 43 and a 3' ITR comprising or consisting of nucleotides 2215 to 2359 of SEQ ID NO: 43; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 44 and a 3' ITR comprising or consisting of nucleotides 2207 to 2351 of SEQ ID NO: 44; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 78 and a 3' ITR comprising or consisting of nucleotides 2258 to 2402 of SEQ ID NO: 78; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 79 and a 3' ITR comprising or consisting of nucleotides 2267 to 2411 of SEQ ID NO: 79; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 80 a 3' ITR comprising or consisting of nucleotides 2251 to 2395 of SEQ ID NO: 80; a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 81 and a 3' ITR comprising or consisting of nucleotides 2251 to 2395 of SEQ ID NO: 81; or a 5' ITR comprising or consisting of nucleotides 1 to 106 of SEQ ID NO: 82 and a 3' ITR comprising or consisting of nucleotides 2187 to 2331 of SEQ ID NO: 82.

[0055] In some embodiments, the rAAV vector is a mammalian serotype AAV vector (e.g., an AAV genome and ITRs from a mammalian serotype AAV), including a primate serotype AAV vector or a human serotype AAV vector. In some embodiments, the rAAV vector is a chimeric AAV vector. In some embodiments, the ITRs are selected from the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PHP.A, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP. B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GG T-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, A AVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B- STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A 15 / G2A3, AAVG2B4, AAVG2B5, or variants thereof.

[0056] In some embodiments, the heterologous nucleic acid sequence encodes a therapeutic agent. In some embodiments, the therapeutic agent comprises a nucleic acid encoding a therapeutic protein or an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid comprises an siRNA, miRNA, shRNA, or dsRNA. In some embodiments, the inhibitory nucleic acid comprises an siRNA, miRNA, shRNA, or (of) dsRNA targeting a neurodegenerative disease-related gene. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia type 2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-parkinsonism ALS complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease. In some embodiments, the neurodegenerative disease is a polyglutamine repeat disease. In some embodiments, the inhibitory nucleic acid (e.g., siRNA, miRNA, shRNA, or dsRNA) targets ATXN2. In some embodiments, ATXN2 refers to mammalian ATNX2 (including human ATXN2).

[0057] In some embodiments, the expression construct comprises a heterologous nucleic acid encoding an artificial miRNA targeting ATXN2. Examples of heterologous nucleic acids comprising guide and passenger sequences and an artificial miRNA targeting ATXN2 are provided in Table 5. RNA formats of the encoded guide, passenger, and artificial miRNA sequences are provided as well as DNA formats for insertion into cis plasmids and rAAV vectors. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA targeting ATXN2 and comprises a guide sequence selected from SEQ ID NOs: 1-4 and 71. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA comprising a guide sequence provided by SEQ ID NO: 1 and a passenger sequence provided by SEQ ID NO: 5. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 2 and a passenger sequence provided by SEQ ID NO: 6. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 3 and a passenger sequence provided by SEQ ID NO: 7. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 4 and a passenger sequence provided by SEQ ID NO: 8. In some embodiments, the heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO: 71 and a passenger sequence provided by SEQ ID NO: 72. In some embodiments, the heterologous nucleic acid comprises a guide sequence and a passenger sequence embedded in a miRNA backbone (or scaffold). In some embodiments, the miRNA backbone is miR-100 or miR-1-1. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA sequence provided by any one of SEQ ID NOs: 9-12 and 73. Further examples of guide sequences, passenger sequences, miR backbones, and artificial miRNA sequences targeting ATXN2 are provided in PCT Publication WO2021 / 159008, which is incorporated by reference in its entirety.

[0058] Other expression control sequences may be present in the rAAV vector operably linked to the heterologous nucleic acid (e.g., encoding an inhibitory nucleic acid), including one or more of a transcription initiation sequence, termination sequence, promoter sequence, enhancer sequence, repressor sequence, splice site sequence, polyadenylation (polyA) signal sequence, or any combination thereof.

[0059] In some embodiments, the rAAV vector comprises an H1 promoter. In some embodiments, the H1 promoter is a native H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:53). In some embodiments, the promoter is a short H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:54, or nucleotides 113-203 of any one of SEQ ID NOs:17, 25-28, and 37-44). In some embodiments, the promoter is a long H1 promoter (e.g., a promoter comprising or consisting of the sequence set forth in SEQ ID NO:52, or nucleotides 113-343 of any one of SEQ ID NOs:13-16, and 18-24). In some embodiments, the H1 promoter is oriented in the 5' to 3' direction in the expression construct, particularly when the 5' ITR lacks a terminal resolution site.

[0060] In some embodiments, the rAAV vector comprises an SV40 termination sequence. Examples of SV40 termination sequences are set forth in SEQ ID NO:77; nucleotides 489-710 of any one of SEQ ID NOs:20, 21, 23, and 24; nucleotides 358-579 of any one of SEQ ID NOs:31, 32, 35, and 36; and nucleotides 349-570 of any one of SEQ ID NOs:40, 41, and 44.

[0061] The rAAV vector may have one or more AAV wild-type genes deleted in whole or in part. In some embodiments, the rAAV vector is replication-deficient. In some embodiments, the rAAV vector lacks functional Rep protein and / or capsid protein. In some embodiments, the rAAV vector is a self-complementary AAV (scAAV) vector.

[0062] In some embodiments, the rAAV vector comprises a 5' ITR, a promoter operably linked to the heterologous nucleic acid encoding ATXN2-specific artificial miRNA, a vector stuffer sequence, and a 3' ITR.In some embodiments, the 5' ITR is modified to lack terminal resolution site.In some embodiments, the promoter is oriented in the 5' to 3' direction.

[0063] In some embodiments, the rAAV vector comprises, from the 5' ITR to the 3' ITR, the nucleotide sequence shown in any one of SEQ ID NO: 13-24, 29-44, and 78-80. In some embodiments, the rAAV vector comprises, from the 5' ITR to the 3' ITR, the nucleotide sequence shown in SEQ ID NO: 13, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises, from the 5' ITR to the 3' ITR, the nucleotide sequence shown in SEQ ID NO: 14, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises, from the 5' ITR to the 3' ITR, the nucleotide sequence shown in SEQ ID NO: 15, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 16, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 17, where nucleotides 204-335 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 18, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 19, from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 20, from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest.In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 21 from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 22 from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 23 from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 24 from the 5' ITR to the 3' ITR, where nucleotides 344-481 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 29 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 30 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 31 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 32 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 33 from the 5' ITR to the 3' ITR, wherein nucleotides 213 to 350 are replaced with a sequence encoding the artificial miRNA of interest.In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 34 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 35 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 36 from the 5' ITR to the 3' ITR, where nucleotides 213-350 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence shown in SEQ ID NO: 37 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 38 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 39 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 40 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 41 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO:42 from the 5' ITR to the 3' ITR, wherein nucleotides 204 to 341 are replaced with a sequence encoding the artificial miRNA of interest.In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 43 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 44 from the 5' ITR to the 3' ITR, where nucleotides 204-341 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 78 from the 5' ITR to the 3' ITR, where nucleotides 204-342 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 79 from the 5' ITR to the 3' ITR, where nucleotides 213-351 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 80 from the 5' ITR to the 3' ITR, where nucleotides 204-335 are replaced with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO: 81 from the 5' ITR to the 3' ITR, where nucleotides 204-335 are replaced with a sequence encoding an artificial miRNA of interest.

[0064] The recombinant AAV vector of the present disclosure can be encapsulated by one or more AAV capsid proteins to form rAAV particles. "rAAV particle" or "rAAV virion" refers to an infectious, replication-defective virus that contains an AAV protein shell and encapsulates a rAAV vector containing a transgene of interest, which is flanked on each side by 5' and 3' AAV ITRs. rAAV particles are produced in a suitable host cell into which the rAAV vector, AAV helper functions, and sequences that specify accessory functions are introduced, allowing the host cell to encode the AAV polypeptides required to package the rAAV vector (including the transgene sequence of interest) into an infectious rAAV particle for subsequent gene delivery.

[0065] Methods for packaging recombinant AAV vectors into AAV capsid proteins using host cell culture are known in the art. In some embodiments, one or more of the components required for packaging the rAAV vector (e.g., Rep sequence, cap sequence, and / or accessory functions) can be provided by a stable host cell engineered (e.g., by a vector) to contain one or more of the required components. The expression of the required components for AAV packaging can be under the control of an inducible or constitutive promoter in the host packaging cell. AAV helper vectors are commonly used to provide transient expression of AAV rep and / or cap genes that function in trans to complement missing AAV functions required for AAV replication. In some embodiments, AAV helper vectors lack AAV ITRs and cannot replicate or package themselves. AAV helper vectors can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.

[0066] In some embodiments, rAAV particles can be produced using a triple transfection method (see, e.g., U.S. Patent No. 6,001,650, which is incorporated by reference in its entirety). In this approach, the rAAV particles can be produced by transfecting a host cell with an rAAV vector (containing a transgene) for packaging into an rAAV particle, an AAV helper vector, and an accessory function vector. In some embodiments, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). The accessory function vector encodes nucleotide sequences for non-AAV derived viral and / or cellular functions on which AAV depends for replication (e.g., "accessory functions"). The accessory functions include those functions required for AAV replication, including, but not limited to, those portions involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses (e.g., adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus). In some embodiments, a double transfection method (where AAV helper and accessory functions are cloned onto a single vector) is used to generate rAAV particles.

[0067] The AAV capsid is an important element in determining the tissue specificity of rAAV particles. Thus, rAAV particles having capsid tissue specificity can be selected. In some embodiments, the rAAV particles are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PHP.A, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAV PHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AA The capsid protein is selected from an AAV serotype of VPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAV2A 15 / G2A3, AAVG2B4, AAVG2B5, and variants thereof. In some embodiments, the AAV capsid is selected from a serotype capable of crossing the blood-brain barrier, such as AAV9, AAVrh.10, AAV-PHP-B, or variants thereof. An example of AAV9 capsid sequence is provided in U.S. Patent No. 7,906,111 (incorporated by reference in its entirety). In some embodiments, the AAV capsid is a chimeric AAV capsid. In some embodiments, the AAV particle is a pseudotyped AAV having a capsid and genome from different AAV serotypes.

[0068] In some embodiments, the rAAV particles are capable of transducing cells of the CNS. In some embodiments, the rAAV particles are capable of transducing non-neuronal or neuronal cells of the CNS. In some embodiments, the CNS cells are neurons, glial cells, astrocytes, or microglial cells.

[0069] In another aspect, the present disclosure provides a host cell transfected with an rAAV particle comprising the vector stuffer sequence described herein.In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.In some embodiments, the host cell is a mammalian cell (e.g., HEK293T, COS cell, HeLa cell, KB cell), a bacterial cell (E.coli), a yeast cell, an insect cell (Sf9, Sf21, Drosophila, mosquito), etc.

[0070] How to use In another aspect, the present disclosure provides a method of delivering a therapeutic agent to a subject, the method comprising administering a composition of the present disclosure (e.g., an rAAV particle comprising an rAAV vector comprising an expression construct comprising a vector stuffer sequence provided herein and a heterologous nucleic acid sequence encoding a therapeutic agent). In some embodiments, the cell is a CNS cell. In some embodiments, the cell is a non-neuronal or neuronal cell of the CNS. In some embodiments, the non-neuronal cell of the CNS is a glial cell, an astrocyte, or a microglial cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is derived from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease.

[0071] As used herein, the term "treating" refers to preventing or delaying the onset of a neurodegenerative disease (e.g., ALS / FTD, Alzheimer's disease, Parkinson's disease, etc.); reducing the severity of a neurodegenerative disease; reducing or preventing the onset of symptoms characteristic of a neurodegenerative disease; preventing the worsening of symptoms characteristic of a neurodegenerative disease, or any combination thereof.

[0072] In some embodiments, the subject has neurodegenerative disease or is at risk of developing neurodegenerative disease.Examples of neurodegenerative disease include spinocerebellar ataxia type 2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-parkinsonism ALS complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease.

[0073] In some embodiments, the method for treatment of the present disclosure reduces, prevents, or delays the occurrence or progression of one or more symptoms characteristic of neurodegenerative disease. Examples of symptoms characteristic of neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional / behavioral dysfunction, or any combination thereof. Paralysis, tremors, unsteadiness, rigidity, spasms, muscle weakness, muscle spasms, muscle rigidity, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slow movement, difficulty walking, dementia, depression, anxiety, or any combination thereof.

[0074] In some embodiments, the method for treatment of the present disclosure includes administration as a monotherapy for the treatment of neurodegenerative disease or in combination with one or more additional therapies.Combination therapy can mean administration of the composition of the present disclosure to a subject prior to, subsequent to, and in conjunction with one or more therapies.Concurrent administration of combination therapy can mean that the composition of the present disclosure and the additional therapies are formulated for administration in the same dosage form or are administered in separate dosage forms.

[0075] In some embodiments, the subject treated in any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or a human.

[0076] In any of the methods described herein, the compositions of the present disclosure may be administered to a subject by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular, intraventricular, intralumbar, subcutaneous, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (such as by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) and / or the condition of the subject. In some embodiments, the composition is injected directly into the CNS of the subject. In some embodiments, the compositions are injected intrathecally, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular, intravenous, intralumbar, or any combination thereof.

[0077] In some embodiments, the composition of the present disclosure is directly injected into the CNS of subject.In some embodiments, the direct injection into the CNS is intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof.In some embodiments, the direct injection into the CNS is directly injected into the cerebrospinal fluid (CSF) of subject, and optionally, said direct injection is intracisternal injection, intravenous injection, intralumbar injection, or any combination thereof. EXAMPLES

[0078] Working Example Example 1: Design of vector "stuffer" sequences Selection of "non-essential" human genomic DNA for base stuffer sequences To minimize any undesired effects of stuffer sequences, we identified "non-essential" regions of the human genome that have two intended properties: (a) sequences that have minimal impact if integration in the genome occurs (although this is thought to be very rare for AAV); and (b) sequences that have minimal risk of unpredicted initiation of transcription. Thus, we searched for regions of the genome where i) deletions and duplications are common in the population and not associated with disease-related phenotypes (no evidence of evolutionary pressure); and / or ii) RNA expression across human tissues is low or undetectable (lacking strong intrinsic enhancer / promoter elements). From this search, we prioritized two regions:

[0079] Region I: AMELY gene region (chrY:6,865,918-6,874,027, HG38) Multiple lines of evidence suggest that the AMELY gene region is not under evolutionary selection. First, the AMELY region arose from an ancient duplication event and has a functional homolog on the X chromosome that is under selection (Lahn, & Page, Science (1999) 286, 964-967). Second, the AMELY gene has more protein-altering variants (including putative loss-of-function variants) than expected without evidence of recent positive selection (Nature (2020) 581, 434-443; https: / / gnomad.broadinstitute.org / gene / ENSG00000099721?dataset=gnomad_r2_1). Third, genetic variants in the AMELY region have been shown to have no phenotypic effect in humans with respect to deletions or duplications. AMELY duplications are common and have no phenotype (Hum Genet. (2015) 134:789-800). Fourth, deletions are common and have no detectable phenotype (Hum Mol Genet (2007) 16:307-16). Furthermore, AMELY has not been associated with any rare disease in humans (Online Mendelian Inheritance in Man, OMIM.org.).

[0080] AMELY mRNA is not detectably expressed in most human tissues (GTEX, The GTEx Consortium. (Science (2020) 369:1318-1330) and is present only in Y chromosome carriers (males). Furthermore, AMELX, a functional homolog of AMELY, is involved in dental enamel during development (Front Physiol. (2017) 8:435).

[0081] Region II: PSG gene region (chr19:43,511,809-43,530,631, HG38) Similarly, multiple lines of evidence suggest that pregnancy-specific glycoproteins (PSGs) are not under clear evolutionary selection. First, the PSG cluster arose from a segmental duplication (Dumont and Eichler PLoS One (2013) 8:e75949). Second, there is no evidence that the region is under evolutionary constraint with more predicted loss-of-function variants (Nature (2020) 581:434-443; https: / / gnomad.broadinstitute.org / gene / ENSG00000243130?dataset=gnomad_r2_1). Third, the PSG cluster has not been associated with any rare disease in humans (Online Mendelian Inheritance in Man, OMIM.org).

[0082] PSG regions are typically highly expressed only by the placenta during pregnancy, with no / low expression in other tissues (GTEX, The GTEx Consortium. (Science (2020) 369: 1318-1330).

[0083] Modification of base stuffer array DNA sequences from the AMELY region (chrY:6,865,918-6,874,027, HG38) and the PSG gene region (chr19:43,511,809-43,530,631, HG38) were modified with the goal of "inactivating" the sequences or lacking any expressed regions, known or putative regulatory elements, and repetitive elements, including retroviral elements and transposable elements. These changes were most rigorously performed with respect to the stuffer sequences intended to be used in the region containing the packaged vector genome.

[0084] Specifically, the nucleotide sequences (chrY:6,865,918-6,874,027, HG38) and (chr19:43,511,809-43,530,631, HG38) were modified in the following manner (see FIG. 1): i) Remove the following expressed regions: exon + 10bp on either side (exons were defined using gene models in GENCODEv19), human expressed sequence tags (EST's: defined based on Genbank data using the "Human ESTs Including Unspliced" track on the UCSC genome browser: (Nucleic Acids Res. (2004) 32(Database issue):D23-6.). ii) Remove regulatory elements: The following tracks on the UCSC Genome Browser were used to define: CpG Islands (J Mol Biol. (1987) 196(2):261-82), regions identified by CHiP seq (sequences bound by transcription factors) Transcription Factor ChIP-seq Peaks (340 factors in 129 cell types) from ENCODE 3 (Nature(2012) 489(7414):57-74.), conserved transcription factor binding sites (HMR Conserved Transcription Factor Binding Sites track), VISTA enhancers (Vista HMR-Conserved Non-coding Human Enhancers from LBNL track), and open regulatory annotations. Annotation) "ORegAnno" (Regulatory elements from the ORegAnno track; (Nucleic Acids Res. (2016) 44(D1):D126-32.). iii) Remove repetitive elements: Use the Repeating Elements by RepeatMasker track (http: / / www.repeatmasker.org) to remove repetitive mask elements (including SINE, LINE, LTR, DNA elements), microsatellite repeats defined by the Microsatellites - Di-nucleotide and Tri-nucleotide Repeats track (Nucleic Acids Res. (1999) 27(2):573-80), and interrupted and simple repeats defined by RepeatMasker (http: / / www.repeatmasker.org). iv) To reduce the likelihood of unpredicted translations, ATG residues were modified to reduce the frequency of ATG sequences. v) CpG dinucleotide editing. To reduce the likelihood of unmethylated CpG dinucleotides from cis plasmids generated in bacteria triggering innate immune activation when packaged into AAV, the presence of CG dinucleotides was reduced by base editing.

[0085] Example 2: Evaluation of the safety of AAV vector genomes packaged using stuffer polynucleotides Vectors incorporating the stuffer sequence described according to Example 1 without the inclusion of any additional active elements (e.g., miRNA) were tested for safety in vivo. A set of four vectors was designed with two variants each of the stuffers from AMELY and PSG11 (SEQ ID NO:25-28). Each vector contained an H1 short promoter immediately 5' downstream of the left ITR (SEQ ID NO:57) and a non-miRNA regulatory sequence was included downstream of the H1 short promoter in place of the miRNA. A Pol III terminator was placed 3' to the regulatory sequence. In parallel, three controls were tested: vehicle; a vector encoding an artificial miRNA sequence targeting ATXN2, previously shown to be toxic when tested in lentiviral format on HeLa and U2OS cells under the control of the H1 promoter (scAAV_H1_miR16-2-1479_AMELY_V1 (SEQ ID NO: 81)); and a vector encoding an Atxn2-specific miRNA and GFP (scAAV_H1_miR1-1-XD-14792_CBh_GFP_SV40p (SEQ ID NO: 82)). An artificial miRNA previously shown to be toxic was selected to serve as a positive control for toxicity, possibly through off-target effects of the specific artificial miRNA sequence.

[0086] Vectors were administered intravenously via the tail vein to 16-week-old wild-type C57Bl / 6 mice with 1.3E11 whole viral genome (vg). Titers were determined based on DNA concentration of column-purified vector. This concentration was compared to that of a similarly purified vector, which was also titrated by vector genome qPCR, to calculate the equivalent titer for the stuffer vector described in this example. Blood was processed and serum was assessed for concentrations of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), as surrogates for any liver toxicity. Vectors expressing the positive control artificial miRNA for toxicity induced substantial elevations in liver enzymes, ALT and AST. In contrast, none of the vectors carrying the stuffer sequence induced elevations in liver enzymes (Figures 2A-2B).

[0087] Histological evaluation of the liver was performed by a pathologist to assess any toxicity. 28 days after dosing, animals (n=5 animals / stuffer vector treatment) were sacrificed and livers were immersion fixed in 4% paraformaldehyde for 24 hours. Two cross sections per liver were embedded in paraffin, sectioned at 5 micron thickness, and stained with hematoxylin and eosin. Photomicrographs were taken with an Olympus BX60 microscope. Animals administered the toxicity positive control miRNA showed numerous lesions including oval cell hyperplasia, hepatocyte fusion and multinucleation, lymphohistiocytic hepatitis, sinusoidal fibrosis, intrahistocytic pigmentation, and rare single cell apoptosis / necrosis. In the stuffer-containing vector, histological findings were limited to incidental background lesions, did not follow treatment-related trends, and were seen across multiple treatment groups. Findings included variations in hepatic glycogen storage disease (hepatocyte glycogen accumulation) and rare microgranulomas. Findings were not considered to reflect compound-related treatment toxicity.

[0088] Example 3: Evaluation of vector genome shortening in specific stuffer designs rAAV vector genome preparations are known to contain truncated vector genomes, most notably in hairpin structures such as miRNAs, but also in other regions of DNA secondary structure. Therefore, the presence of truncated genomes in preparations of rAAV containing different forms of stuffer sequences was evaluated.

[0089] Figures 3A-3C show DNA traces from Fragment Analyzer analysis. After rAAV vector (scAAV9_H1_long_miR-100_3330_PSG11_V5 (SEQ ID NO: 16)) generation, vector DNA was extracted and column purified (Zymo, P / N D3015) and run on an Agilent Fragment Analyzer (HS NGS Fragment 1-6000bp). Double peak traces can be seen (Figure 3A), and these double peaks were determined to be full-length structures containing ITRs because they are resolved by restriction digestion at the site that removes the wild-type ITRs on the right. There is also a smaller shoulder (indicating a shorter truncated genome) to the left of the intended full-length product. A subpeak centered around the amiRNA (representative of the vector genome truncated in the amiRNA) is found in many preparations (Figure 3D). A similar peak is present when a control sequence is used in place of the amiRNA but contains secondary structure such as a palindromic sequence.

[0090] Figures 3E-3F compare two rAAV preparations (scAAV9_H1_MCS_PSG11_V1 (SEQ ID NO: 27) and scAAV9_H1_MCS_PSG11_V2 (SEQ ID NO: 28)) derived from cis plasmids containing one of two versions of the stuffer sequence derived from PSG11 (PSG11_V1 (SEQ ID NO: 45) and PSG11_V2 (SEQ ID NO: 46)). These sequences are structurally similar, but the ATG site was edited to eliminate the ATG sequence in PSG11_V2. There were some structural differences in the 5' portion of the stuffer sequence adjacent to the expression cassette (either the H1 promoter and amiRNA or control sequences in the vector shown in Figure 3E). The traces show that the "shoulder" to the left of the full-length product is smaller in the PSG11_V1-containing vector genome preparation (Figure 3F). In these vector preparations, no amiRNA was used.

[0091] Figures 3G-3H compare a similar set of two rAAV preparations (scAAV9_H1_miR-1-1-XD-14792_PSG11_V1 (SEQ ID NO: 78) and scAAV9_H1_native_miR-1-1-XD-14792_PSG11_V2 (SEQ ID NO: 79)) derived from cis plasmids containing either the stuffer sequence PSG11_V1 (SEQ ID NO: 45) or PSG11_V2 (SEQ ID NO: 46). In this case, both vectors contain an amiRNA element downstream of the H1 promoter (there is a three nucleotide insertion in the promoter sequence of the PSG11_V2-containing vector compared to the V1-containing vector, but otherwise the 5' sequences are identical). By inspection of the fragment analyzer traces (Figure 3H), it can be seen that the truncation at the amiRNA site is much larger in the vector containing the PSG11_V2 stuffer sequence.

[0092] Replication of self-complementary AAV (scAAV) vectors such as those described herein involves initiation of replication from the wild-type ITR. In the sequences described herein, this is present on the 3' side in a cis plasmid. Thus, the polymerase first traverses through the stuffer sequence from 3' to 5' toward the amiRNA. A potential explanation for the increase in truncated products is that secondary structures that the polymerase may encounter just before the hairpin of the amiRNA promote strand-switching activity that results in exacerbated truncation. Consistent with this hypothesis, examination of the folding of 200 nucleotides of DNA sequence within the PSG11_V1 stuffer sequence (SEQ ID NO: 55; FIG. 4A) versus the PSG11_V2 stuffer sequence (SEQ ID NO: 56; FIG. 4B), immediately 3' to the amiRNA, revealed an area of ​​increased putative secondary structure in the PSG11_V2 stuffer. Thus, the PSG11_V1 sequence structure was favored. The "PSG11_V5" stuffer sequence (SEQ ID NO: 48) was generated by using a convenient sequence structure derived from the PSG11_V1 sequence. It is assumed that this will result in a vector preparation with reduced shortened vector genomes. In order to reduce the potential for hypomethylated CpG dinucleotides or unintended open reading frames, the _V5 stuffer incorporates edits to the CG and ATG sequences.

[0093] Example 4: Further evaluation of the safety of the stuffer sequence when combined with the Atxn2 amiRNA expression cassette A study was performed to evaluate 24 different vector designs containing various stuffer sequences for their safety in vivo. Three different variants of the H1 promoter (H1 short (SEQ ID NO: 54), H1 native (SEQ ID NO: 53), H1 long (SEQ ID NO: 52)), two different ATXN2-targeting artificial miRNA (3330 or 14792 (also referred to herein as 1784)) / miR scaffold (miR1.1. or miR100) combinations (miR100_3330 (encoding SEQ ID NO: 12); miR1.1_14792 (encoding SEQ ID NO: 73)), the presence or absence of the Pol-II transcription terminator SV40 polyadenylation sequence, and different versions of the stuffer sequence (AMELY_V3 (SEQ ID NO: 51), PSG11_V2 (SEQ ID NO: 46), PSG11_V3 (SEQ ID NO: 47), PSG11_V5 (SEQ ID NO: 48)) were assembled in various combinations (see Table 1). As in the previous examples, in these vectors, the H1 promoter is oriented 5' to 3' in the cis plasmid intended for packaging as a self-complementary AAV vector, where the mutant ITR (lacking a terminal resolution site) is at the 5' side (e.g., nucleotides 1-106 of SEQ ID NO:57 or any one of SEQ ID NOs:13-24, 29-44, and 78-82), and the wild-type AAV ITR is at the 3' side (e.g., nucleotides 2214-2358 of SEQ ID NO:58 or any one of SEQ ID NOs:13-16). The reason for using this promoter orientation is that a truncation of the miR core is possible in vectors incorporating an ITR lacking a terminal resolution site (self-complementary AAV vector). In a 5' to 3' promoter orientation, a vector with a truncation of the miR core would not contain a promoter and should therefore be inactive. The promoter could be retained in a vector with a truncation of the miR core if it were in a 3' to 5' orientation.

[0094] rAAV vectors were generated with these cis plasmids and vectors were administered iv by tail vein injection to 12-week-old wild-type C57Bl / 6 mice at a dose of 3.21E9 vector genomes / g mouse (mean total dose 8.5E10 vector genomes) adjusted by body weight. Vehicle control animals received PBS with 0.001% PF-68. Blood was collected by submandibular bleeding two weeks after administration and liver enzyme concentrations (aspartate transaminase (AST) and alanine transaminase (ALT)) (Figures 5A-5B) were measured in animals administered the various vector designs. Nearly all data points for animals treated with stuffer-containing vectors were within the normal reference range. Two data points show that the AST signal was elevated above the reference, but hemolysis was noted in these samples, and one sample was from a vehicle-treated animal. None of the other samples from animals administered any of the above vector formats at this time point had elevated AST or ALT above the reference range, suggesting that there was no evidence of acute liver injury in response to rAAV administration with vectors encoding these stuffer sequences. [Table 1-1] [Table 1-2]

[0095] Animals administered the stuffer-containing vectors intravenously were also evaluated for hepatotoxicity by histology. Three weeks after administration, two consecutive 0.5 cm left liver lobe sections were harvested per animal, paraffin embedded, sectioned to 5 microns, and stained with hematoxylin and eosin. Slides were evaluated by a trained pathologist. N=1-2 animals per group were evaluated, as shown in Table 1. After evaluation, intravenous administration of the test article was not interpreted as associated with changes in the liver sections examined for all vectors evaluated.

[0096] Intrastriatal testing [Table 2]

[0097] These vectors were also administered via direct striatal injection to 8-week-old wild-type C57Bl / 6 mice at a dose of 7.5E9 vector genomes per striatum, and a subset of the vectors (Table 2, above) was evaluated for toxicity by histology. Slices of striatum from animals 3 weeks post-administration were paraffin embedded and four serial sections cut with a 5 micron block advance were mounted on separate slides. Two sets of slides containing step sections from three levels were included. Sections were stained with hematoxylin and eosin, glial fibrillary acidic protein (GFAP) immunohistochemistry, ionized calcium-binding adaptor molecule 1 (IBA-1) immunohistochemistry, and floro-jade B. The latter three stains can be used to assess astrocytic reactivity, microglial reactivity, and neuronal death, respectively.

[0098] Similar to liver sections from animals treated with iv-administered vector, no effects of the test article were identified in the brain sections examined. In some tissues, handling artifacts limited interpretation. In general, changes were observed at the injection site and included mild gliosis (identified in H&E stained slides), slight microgliosis (identified in GFAP-labeled slides), and slight destruction of tissue with subtle hemosiderin pigmentation, a common change associated with intraparenchymal injections. The test article was not interpreted as causing any exacerbations or changes associated with the experimental procedure in any of the sections examined at the level of the basal ganglia / striatum from any animal.

[0099] Transcriptional analysis To further evaluate the safety of the vectors, sequencing analysis was performed on striatal punch biopsies from animals administered the vectors described above. Striatal tissue was column purified and RNA was extracted (after DNase treatment) using Qiagen's AllPrep DNA / RNA / Protein Mini Kit (Qiagen, P / N 80004). Stranded RNA-seq libraries were prepared with Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit (Illumina, P / N 20040525) followed by paired-end 2x100bp sequencing on an Illumina Novaseq 6000 system. These reads were then aligned against the respective cis plasmids used to package the rAAV administered to the animals. Separately, DNA was purified (Qiagen, P / N 80004) from the same punch biopsies used to extract the sequenced RNA to confirm vector exposure. Table 3 lists the exposure for each sample tested along with the vector. [Table 3]

[0100] Figure 6 shows the pileup of reads from a representative vector. Surprisingly, the use of the H1 short promoter and miR 1.1 backbone with the PSG11 V5 stuffer sequence resulted in an increased number of reads aligning to the cis plasmid downstream of the predicted transcription of the pri-miRNA. In contrast, the use of the H1 long promoter with the PSG11 V5 stuffer resulted in a beneficial minimal amount of unintended transcription. Thus, this combination of elements resulted in a vector construct with a desirable low frequency of non-miRNA transcription.

[0101] Example 5: Demonstration of the functionality of the stuffer sequence when combined with the Atxn2 amiRNA expression cassette in different rAAV vector formats Animals administered the set of 24 vector combinations described in Example 4 were also evaluated for the functionality of all of the combinations of stuffer, promoter, and ATXN2-targeting artificial miRNA. Atxn2 knockdown was evaluated by extracting RNA from liver tissue 3 weeks after iv administration (dose: 3.21E9 vg / gram mouse) and then performing RT-ddPCR using Atxn2 and control probe Hprt. Atxn2 levels were calculated as the average of these ATXN2 / control transcript ratios and further normalized to the ratio obtained for animals administered vehicle. Vector genome exposure was also measured using ddPCR using a probe for the vector genome and a mouse genome probe (Tert). As seen in Table 4, strong Atxn2 knockdown occurred in animals treated with all combinations of stuffer sequence and miRNA vector components. [Table 4-1] [Table 4-2]

[0102] Example 6: Further evaluation of production yield and truncation of the stuffer sequence when combined with the ATXN2 amiRNA expression cassette in different rAAV vector formats Example 4 shows the unexpected advantage of the "H1 long" variant of the promoter when inserted upstream of the PSG11_V5 stuffer sequence compared to the "H1 short" variant of the promoter, which reduces the transcriptional activity downstream of the artificial miRNA sequence. The production yield of the combination of H1 long and H1 short promoters and stuffer sequences from AMELY and PSG11 was also evaluated. The production yield was evaluated by ddPCR of the vector genome using a primer / probe set specific for the stuffer region. In general, the use of cis plasmids with H1 long format promoter (SEQ ID NO: 52) combinations with the stuffer sequences evaluated, including AMELY_V3 (SEQ ID NO: 51), PSG11_V5 (SEQ ID NO: 48), PSG11_V3 (SEQ ID NO: 47), and PSG11_V2 (SEQ ID NO: 46), resulted in higher production yields (Figure 7). Analysis of vector truncation (Figures 8A-8C) further suggested that there was less truncation when the H1-long version of the promoter was included upstream compared to the H1-short promoter. Taken together, the inclusion of the H1-long promoter upstream of these stuffer sequences has a number of surprising benefits, likely due to local DNA secondary structure elements that may affect rAAV replication and packaging.

[0103] Example 7: Generation of vectors incorporating stuffer sequences with additional combinations of artificial miRNAs Stuffer sequences comprising combinations of promoters and specific stuffer sequences as prepared according to Example 1 and described in Example 4 were evaluated for their ability to be packaged in rAAV as part of the AAV vector genome. These stuffer sequences were also tested for their ability to consistently achieve good productivity in the context of different payloads in the vector.

[0104] The stuffer sequence derived from the PSG11 intron region and modified according to the design rules in Example 1, including the optimal H1 long promoter variant, was packaged in a set of vectors containing various ATXN2-targeting artificial miRNA packaging cassettes. The cis plasmid sequences from 5' ITR to 3' ITR are shown below: SEQ ID NO: 13 (scAAV_H1_long_miR100_1755_PSG11_V5_ITR_to_ITR), SEQ ID NO: 14 (scAAV_H1_long_miR100_2586_PSG11_V5_ITR_to_ITR), SEQ ID NO: 15 (scAAV_H1_long_miR100_2945_PSG11_V5_ITR_to_ITR), and SEQ ID NO: 16 (scAAV_H1_long_miR100_3330_PSG11_V5_ITR_to_ITR). The sequences of the amiRNA guide, passenger, and expression cassettes are listed in Table 5. [Table 5-1] [Table 5-2]

[0105] The H1 promoter ("H1 long") (nucleotides 113-343 of SEQ ID NO:52 or any one of SEQ ID NOs:13-16) was incorporated into the vector. Yields from AAV production of these vectors are listed in Table 6. [Table 6]

[0106] The stuffer sequence from AMELY was packaged in several vectors. The vector sequences from 5' ITR to 3' ITR, including the ATXN2-targeting artificial miRNA cassette and the AMELY stuffer sequence, are provided in "H1_short_miR16-2-1755_AMELY_V1" (SEQ ID NO: 17) and "H1 - miR1-1_1784_AMELY_V3" (SEQ ID NO: 18). In these vectors, the annotation "_V1" or "_V3" for AMELY refers to different versions of the AMELY stuffer sequence, where the "_V3" version has an edited ATG sequence and an edited CpG dinucleotide (nucleotides 488-2177 of SEQ ID NO: 51 or SEQ ID NO: 18), and "_V1" has not been so modified (nucleotides 342-2222 of SEQ ID NO: 49 or SEQ ID NO: 17). H1_short refers to a 91 bp shortened form of the H1 promoter (nucleotides 113-203 of SEQ ID NO: 54 or SEQ ID NO: 17). The yields from the generation of these vectors are listed in Table 7. [Table 7]

[0107] Example 8: Demonstration of functionality of AAV vector genomes packaged using a stuffer polynucleotide in combination with additional amiRNAs When the vector genome is integrated with the stuffer sequence and additional amiRNA described herein, the experiment is carried out to test the functionality of the payload.This further establishes the compatibility of stuffer sequence with amiRNA expression cassette.All the vectors described above express the artificial miRNA that targets ATXN2; the functionality is determined by knockdown of ATXN2 target.

[0108] In one experiment, the vectors listed in Table 8 were evaluated for knockdown of ATXN2 mRNA in human stem cell-derived motor neurons. The vectors included: scAAV_H1_long_miR100_1755_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO: 13), scAAV_H1_long_miR100_2586_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO: 14), scAAV_H1_long_miR100_2945_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO: 15), and scAAV_H1_long_miR100_3330_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO: 16). Stem cell-derived motor neurons were treated with the indicated vectors packaged in AAV-DJ, and 7 days after transduction, RNA was harvested and assessed for knockdown using RT-ddPCR with a probe for ATXN2 and housekeeping probes GUSB and B2M. The average ATXN2 mRNA signal (normalized to GUSB and B2M probes and untransduced cells) was measured. Data are listed for cells treated with a dose of 3.16E4 vector genomes / cell. [Table 8]

[0109] In another experiment, a vector containing the AMELY_V3 stuffer (nucleotides 488-2177 of SEQ ID NO:51 or SEQ ID NO:18) and an ATXN2-targeting amiRNA under the control of the H1 long promoter was tested by intravenous administration to adult male C57Bl / 6 mice and knockdown of the target ATXN2 measured 3 weeks after administration. Knockdown of Atxn2 was assessed by extracting RNA from liver tissue and performing RT-ddPCR with Atxn2 and control probes Hprt and Gusb. Table 9 lists the average percent knockdown in liver tissue assessed from three animals administered the vector compared to animals administered vehicle (PBS+0.001% PF-68). The dose administered averaged 8.5E10 in this study and was adjusted for mouse weight. As can be seen in Table 9, there was substantial knockdown in livers from animals administered this vector, indicating functionality of AAV packaged with the stuffer. [Table 9]

[0110] The various embodiments described above can be combined to provide further embodiments. The U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications mentioned herein and / or listed in the application data sheets, including U.S. Patent Application No. 63 / 146,522, filed February 5, 2021, and PCT Application No. PCT / US2021 / 016939, filed February 5, 2021, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to provide still further embodiments using the concepts of the various patents, applications and publications.

[0111] These and other changes can be made to the above-described embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope of the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents entitled to such claims. Thus, the claims are not limited by this disclosure.

Claims

1. A vector comprising a stuffer sequence comprising a nucleic acid of about 1300 to about 2300 nucleotides in length, having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% identity to any one of the following: SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; nucleotides 489-2185 of any one of SEQ ID NOs:13-16, nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; Nucleotides 252-2132 of SEQ ID NO:25; Nucleotides 252-2132 of SEQ ID NO:26; Nucleotides 252-2132 of SEQ ID NO:27; Nucleotides 252-2132 of SEQ ID NO:28; Nucleotides 357-2237 of SEQ ID NO:29; Nucleotides 358-2195 of SEQ ID NO:30; Nucleotides 580-2187 of SEQ ID NO:31; Nucleotides 580-2196 of SEQ ID NO:32; Nucleotides 357-2237 of SEQ ID NO:33; Nucleotides 358-2195 of SEQ ID NO:34; Nucleotides 580-2187 of SEQ ID NO:35; Nucleotides 580-2196 of SEQ ID NO:36; Nucleotides 348-2228 of SEQ ID NO:37; Nucleotides 349-2229 of SEQ ID NO:38; Nucleotides 349-2186 of SEQ ID NO:39; Nucleotides 571-2178 of SEQ ID NO:40; nucleotides 571 to 2178 of SEQ ID NO:41; nucleotides 348 to 2228 of SEQ ID NO:42; nucleotides 349 to 2186 of SEQ ID NO:43; nucleotides 571 to 2178 of SEQ ID NO:44; nucleotides 349 to 2229 of SEQ ID NO:78; nucleotides 358 to 2238 of SEQ ID NO:79; nucleotides 342 to 2222 of SEQ ID NO:80; and nucleotides 342 to 2222 of SEQ ID NO:

81.

2. 2. The vector of claim 1, wherein the stuffer sequence is about 1500-2000, or about 1600-1900 nucleotides in length.

3. The vector according to claim 1 or 2, wherein the nucleic acid comprises or consists of any one of the following: SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:50; SEQ ID NO:51; Nucleotides 489 to 2185 of any one of SEQ ID NOs:13 to 16; Nucleotides 342 to 2222 of SEQ ID NO:17; Nucleotides 488 to 2177 of SEQ ID NO:18; Nucleotides 711 to 2187 of SEQ ID NO:21; Nucleotides 488 to 2177 of SEQ ID NO:22; Nucleotides 711 to 2187 of SEQ ID NO:23; Nucleotides 711 to 2187 of SEQ ID NO:24; Nucleotides 252 to 2132 of SEQ ID NO:25; Nucleotides 252 to 2132 of SEQ ID NO:26; Nucleotides 252 to 2132 of SEQ ID NO:27; Nucleotides 252 to 2132 of SEQ ID NO:28; Nucleotides 357 to 2237 of SEQ ID NO:29; nucleotides 358-2195 of SEQ ID NO:30; nucleotides 580-2187 of SEQ ID NO:31; nucleotides 580-2196 of SEQ ID NO:32; nucleotides 357-2237 of SEQ ID NO:33; nucleotides 358-2195 of SEQ ID NO:34; nucleotides 580-2187 of SEQ ID NO:35; nucleotides 580-2196 of SEQ ID NO:36; nucleotides 348-2228 of SEQ ID NO:37; nucleotides 349-2229 of SEQ ID NO:38; nucleotides 349-2186 of SEQ ID NO:39; nucleotides 571-2178 of SEQ ID NO:40; nucleotides 571-2178 of SEQ ID NO:41; nucleotides 348-2228 of SEQ ID NO:42; nucleotides 349-2186 of SEQ ID NO:43; nucleotides 571-2178 of SEQ ID NO:44; nucleotides 349-2229 of SEQ ID NO:78; Nucleotides 358-2238 of SEQ ID NO:79; nucleotides 342-2222 of SEQ ID NO:80; and nucleotides 342-2222 of SEQ ID NO:

81.

4. The vector of any one of claims 1 to 3, wherein the stuffer sequence flanks an expression construct comprising a heterologous nucleic acid sequence.

5. A vector described in any one of claims 1 to 4, which is an adeno-associated virus (AAV) vector.

6. The vector described in claim 5, wherein the AAV vector is a self-complementary AAV (scAAV) vector.

7. The vector of any one of claims 4 to 6, wherein the heterologous nucleic acid sequence encodes a therapeutic agent.

8. The vector of claim 7 , wherein the therapeutic agent comprises a nucleic acid encoding an inhibitory nucleic acid.

9. The vector of claim 8 , wherein the inhibitory nucleic acid comprises an siRNA, miRNA, shRNA, or dsRNA.

10. The vector of claim 8 , wherein the inhibitory nucleic acid comprises an miRNA that targets a neurodegenerative disease-associated gene.

11. The vector of claim 10, wherein the neurodegenerative disease is spinocerebellar ataxia type 2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-parkinsonism ALS complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, or Alzheimer's disease.

12. The vector according to claim 10 , wherein the neurodegenerative disease is a polyglutamine repeat disease.

13. The vector of any one of claims 8 to 12, wherein the inhibitory nucleic acid targets ATXN2.

14. The inhibitory nucleic acid is (a) a guide sequence selected from SEQ ID NOs: 1-4 and 71; (b) a guide sequence of SEQ ID NO:1 and a passenger sequence of SEQ ID NO:5; a guide sequence of SEQ ID NO:2 and a passenger sequence of SEQ ID NO:6; a guide sequence of SEQ ID NO:3 and a passenger sequence of SEQ ID NO:7; a guide sequence of SEQ ID NO:4 and a passenger sequence of SEQ ID NO:8; or a guide sequence of SEQ ID NO:71 and a passenger sequence of SEQ ID NO:72; or (c) any one of SEQ ID NOs: 9 to 12 and 73; The vector according to any one of claims 8 to 12, which encodes an artificial miRNA comprising:

15. The vector of any one of claims 4 to 14, wherein the expression construct comprises a promoter, a polyadenylation signal, a termination signal, or any combination thereof.

16. 16. The vector of claim 15, wherein the promoter is an H1 promoter. (a) the H1 promoter is an H1 long promoter comprising nucleotides 113 to 343 of any one of SEQ ID NO:52, or SEQ ID NOs:13-16, and 18-24; (b) the H1 promoter is an H1 promoter comprising SEQ ID NO:53; or (c) the H1 promoter is an H1 short promoter comprising nucleotides 113-203 of SEQ ID NO:54 or any one of SEQ ID NOs:17, 25-28, and 37-44; and / or The termination signal is an SV40 termination signal. The vector described in claim 16.

18. The vector described in claim 17, wherein the SV40 termination signal comprises an array provided by sequence number 77.

19. The vector of any one of claims 4 to 18, comprising a 5' inverted terminal repeat (ITR) and a 3' ITR adjacent to the expression construct and the stuffer sequence.

20. said 5' and 3' ITRs are obtained from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11, and variants thereof; and / or one of the 5' and 3' ITRs lacks a functional terminal resolution site; The vector described in claim 19.

21. 21. The vector of claim 20, wherein the 5' ITR lacks a functional terminal resolution site.

22. (a) the nucleotide sequence of any one of SEQ ID NOs: 13-24, 29-44, and 78-80; (b) the nucleotide sequence of SEQ ID NO: 13, in which nucleotides 344-481 are replaced with a sequence encoding a miRNA of interest; (b) the nucleotide sequence of SEQ ID NO: 14, in which nucleotides 344-481 are replaced with a sequence encoding a miRNA of interest; (c) the nucleotide sequence of SEQ ID NO: 15, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (d) the nucleotide sequence of SEQ ID NO: 16, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (e) the nucleotide sequence of SEQ ID NO: 17, wherein nucleotides 204-335 are replaced with a sequence encoding a miRNA of interest; (f) the nucleotide sequence of SEQ ID NO: 18, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (g) the nucleotide sequence of SEQ ID NO: 19, wherein nucleotides 344-481 are replaced with a sequence encoding a miRNA of interest; (h) the nucleotide sequence of SEQ ID NO:20, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (i) the nucleotide sequence of SEQ ID NO:21, in which nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (j) the nucleotide sequence of SEQ ID NO:22, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (k) the nucleotide sequence of SEQ ID NO:23, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (l) the nucleotide sequence of SEQ ID NO:24, wherein nucleotides 344 to 481 are replaced with a sequence encoding a miRNA of interest; (m) the nucleotide sequence of SEQ ID NO:29, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (n) the nucleotide sequence of SEQ ID NO: 30, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (o) the nucleotide sequence of SEQ ID NO:31, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (p) the nucleotide sequence of SEQ ID NO: 32, in which nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (q) the nucleotide sequence of SEQ ID NO: 33, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (r) the nucleotide sequence of SEQ ID NO: 34, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (s) the nucleotide sequence of SEQ ID NO:35, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (t) the nucleotide sequence of SEQ ID NO: 36, wherein nucleotides 213-350 are replaced with a sequence encoding a miRNA of interest; (u) the nucleotide sequence of SEQ ID NO: 37, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (v) the nucleotide sequence of SEQ ID NO: 38, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (w) the nucleotide sequence of SEQ ID NO:39, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (x) the nucleotide sequence of SEQ ID NO: 40, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (y) the nucleotide sequence of SEQ ID NO:41, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (z) the nucleotide sequence of SEQ ID NO:42, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (aa) the nucleotide sequence of SEQ ID NO:43, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (bb) the nucleotide sequence of SEQ ID NO:44, wherein nucleotides 204 to 341 are replaced with a sequence encoding a miRNA of interest; (cc) the nucleotide sequence of SEQ ID NO: 78, in which nucleotides 204 to 342 are replaced with a sequence encoding an artificial miRNA of interest; (dd) the nucleotide sequence of SEQ ID NO: 79, in which nucleotides 213 to 351 are replaced with a sequence encoding an artificial miRNA of interest; (ee) the nucleotide sequence shown in SEQ ID NO: 80, in which nucleotides 204 to 335 are replaced with a sequence encoding an artificial miRNA of interest; or (ff) comprising the nucleotide sequence set forth in SEQ ID NO: 81, in which nucleotides 204 to 335 are replaced with a sequence encoding an artificial miRNA of interest; The vector according to any one of claims 1 to 21.

23. An rAAV particle comprising the vector of any one of claims 1 to 22.

24. The rAAV particle of claim 23, wherein the rAAV particle comprises a capsid protein.

25. 25. The rAAV particle of claim 24, wherein the capsid protein is capable of crossing the blood-brain barrier and / or the capsid protein is an AAV9 capsid protein.

26. A composition comprising the rAAV particles of any one of claims 23 to 25 for use in a method of delivering a therapeutic agent to a subject.

27. The subject has or is at risk of developing a neurodegenerative disease; or The subject has a neurodegenerative disease selected from the group consisting of spinocerebellar ataxia type 2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), Guam dementia-parkinsonism ALS complex (G-PDC), Pick's disease, hippocampal sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, and polyglutamine repeat diseases.

27. A composition for use according to claim 26.

28. A composition for use as described in claim 26 or 27, characterized in that the rAAV particles are administered to the subject.

29. 30. The composition for use of claim 28, wherein said administration comprises direct injection into the CNS of said subject.

30. The direct injection is an intracerebral injection, an intraparenchymal injection, an intrathecal injection, an intrastriatal injection, a subpial injection, or any combination thereof; or The direct injection is a direct injection into the cerebrospinal fluid (CSF) of the subject.

30. A composition for use according to claim 29.

31. The composition for use according to claim 29, wherein the direct injection is an intracisternal injection, an intraventricular injection, and / or an intralumbar injection.

32. The composition for use according to claim 28, wherein the administration comprises parenteral or intravenous injection of the subject.

Images