CN117136238A

CN117136238A - Vector comprising a stuffer polynucleotide sequence

Info

Publication number: CN117136238A
Application number: CN202280025832.1A
Authority: CN
Inventors: C·P·古尔德; R·R·格雷厄姆; P·扬基; R·陈; E·格林
Original assignee: Metz Therapy
Current assignee: Metz Therapy
Priority date: 2021-02-05
Filing date: 2022-02-04
Publication date: 2023-11-28

Abstract

The present disclosure provides: vector-filled polynucleotides and compositions thereof, including expression constructs and vectors, such as viral vectors; and methods of delivering a therapeutic agent (e.g., an inhibitory nucleic acid) or treating a disease to a mammal.

Description

Vector comprising a stuffer polynucleotide sequence

Statement regarding sequence listing

The sequence listing relevant to the present application is provided in text format in place of paper copies and is incorporated herein by reference. The name of the text file containing the sequence listing is 630264_402WO_SEQUENCE_LISTING. The text file is 163KB, created at 2022, 2, 4 and submitted electronically via the EFS-Web.

Background

Adeno-associated virus (AAV) vector genomes are known to be limited in size, with naturally-sized vector sizes of about 4.7kb being successfully packaged, but the much larger vector genomes reduce the production of functional AAV vectors (Wu et al, molecular therapy (Molecular Therapy) 2010 18 (1): 80-86). On the other hand, vector genomes significantly smaller than the vector genome packaging limit may result in suboptimal packaging (Dong et al, human Gene therapy (Human gene therapy) 1996 7:2101-2112). Moreover, if the cis plasmid has a 'backbone' sequence, such as elements that generally encode sources of bacterial resistance and antibiotic resistance required to propagate the plasmid in bacteria, that is sized to approximate the intended AAV vector genome, the amount of the unexpected 'reverse packaging' sequence will increase (Hauck et al, molecular therapy (2009) 17:144-152). For these reasons, it may be beneficial to include a 'stuffer sequence' that does not itself confer the undesirable properties of packaged AAV as a material in the plasmid backbone and for use in situations where AAV payloads are expected to be significantly shorter than the native AAV package size. Keiser et al, (Nature Medicine) 27:1982-1989 underscores the importance of inert stuffer sequences, where it was shown that 'payload-free' AAV with stuffer sequences could induce significant toxicity in non-human primates.

There is a need for AAV vectors with improved packaging characteristics.

Drawings

Fig. 1: the selection is performed on a UCSC genome browser to identify expression regions within the base sequence, known or predicted regulatory elements, and the orbitals (tracks) of repetitive elements (including retroviruses and transposable elements).

FIGS. 2A-2B show liver enzyme function tests (FIG. 2A) for aspartate aminotransferase (AST; U/L) and (FIG. 2B) for alanine aminotransferase (ALT; U/L) measured 13 days post-administration using treated sera from mice intravenously dosed with vehicle or scaAAV 9 vector. None of the vectors containing stuffer alone had significant elevation of ALT or AST compared to control mice, whereas vector expression in the toxic positive control artificial miRNA did cause significant elevation of liver enzymes ALT and AST (one-way ANOVA), dannitt multiple comparison test (Dunnett's multiple comparisons test). All groups were n=5 except the toxicity positive control (n=3).

Fig. 3A-3H: a fragment analyzer (Fragment Analyzer) trace of the column purified scAAV9 vector DNA is shown. Using representative vectors, FIG. 3A shows a standard high molecular weight bimodal that upon enzymatic cleavage of mutITR (retention of bimodality; FIG. 3B) or wtITR (now unimodal; FIG. 3C) indicates that the bimodal represents a full length population. FIG. 3D 3 components of fragment analyzer trace of purified scAAV9 vector DNA: the full length vector represented by the two peaks at the highest molecular weight, the next peak or miR-centric truncation, and finally the truncation that occurs within the stuffer sequence. In fig. 3E are traces of scAAV 9H 1 MCS constructs psg11_v1 and psg11_v2, top and bottom traces, respectively. In the trace shown in fig. 3F, psg11_v2 has a higher concentration of truncations when overlapped than psg11_v1. In FIG. 3G are fragment analyzer traces of PSG11_V1 vector (top) and PSG11_V2 vector (bottom) embedded with artificial miRNAmiR-1-1XD-14792 (XD-14792 is also referred to herein as 1784). Fig. 3H shows the overlapping of the traces in fig. 3G. As demonstrated by the size of the miR-centric peak (arrow), the miR-centric truncation in psg11_v1 was reduced compared to psg11_v2.

Fig. 4A to 4B show the secondary structure of psg11_v1 (fig. 4A) and psg11_v2 (fig. 4B) predicted using Mfold web server, adjacent to artificial miRNA and terminator, of 200 nucleotides. As demonstrated by the smaller negative gibbs free energy value and the relative lack of secondary structure, psg11_v1 has a more desirable predicted DNA secondary structure adjacent to the artificial miRNA than psg11_v2.

FIGS. 5A-5B show liver enzyme values from vector structure studies (FIGS. 5A-aST; FIGS. 5B-ALT).

FIG. 6 shows bundled RNAseq traces from a representative vector with an H1 long promoter, amiRNA3330 in the miR-100 backbone and PSG11_V5 stuffer (left), and an H1 short promoter, amRNA1784 in the miR-1-1 backbone (also referred to herein as 14792) and PSG11_V5 stuffer (right).

Fig. 7: titers of 24 constructs tested in the vector structure study are shown. Vectors containing H1 long promoters (left column) always have higher titers than the H1 native promoters (middle) and H1 short promoters (right).

Figures 8A to 8C show miR and stuffer truncation of scAAV9 vectors with amely_v3 and psg11_v5 stuffer sequences as measured by a fragment analyzer parallel capillary electrophoresis system. In fig. 8A, miR100 has less miR truncation in both the amely_v3 and psg11_v5 vectors compared to miR1-1 across all 3 promoter contexts, H1 long (left panel), H1 natural (middle panel) and H1 short (right panel), and without (circles) and with (triangles) SV40 polyadenylation sequences. In FIG. 8B, vectors with H1 long promoters with and without SV40 polyadenylation sequences have fewer stuffer truncations than vectors with H1 native promoters and H1 short promoters. In FIG. 8C, vectors with H1 long promoters with and without SV40 polyadenylation sequences have fewer truncations in combination with the miR/stuffer truncations than vectors with H1 native promoters and vectors with H1 short promoters.

Detailed Description

Before explaining the present disclosure in more detail, an understanding of the present invention may be helpful in providing definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.

In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, as well as fractions thereof (e.g., one tenth and one hundredth of an integer) as appropriate, unless otherwise indicated. Moreover, unless otherwise indicated, any numerical ranges described herein relating to any physical feature, such as polymer subunits, dimensions, or thickness, should be understood to include any integer within the stated range. As used herein, the term "about" refers to ±20% of the specified range, value or structure, unless otherwise indicated. It should be understood that as used herein, the terms "a" and "an" refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. As used herein, "comprising," "having," and "including" are used synonymously, the terms and variations thereof are intended to be interpreted as non-limiting.

As used herein, the term "nucleic acid" or "polynucleotide" refers to any nucleic acid polymer composed of covalently linked nucleotide subunits, such as polydeoxyribonucleotides or polyribonucleotides. Examples of nucleic acids include RNA and DNA.

As used herein, "RNA" refers to a molecule that includes one or more ribonucleotides and includes double-stranded RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, and modified RNA that differs from natural RNA by the addition, deletion, substitution, and/or substitution of one or more nucleotides. The nucleotides of the RNA molecule may include standard nucleotides or nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.

As used herein, "DNA" refers to a molecule that includes one or more deoxyribonucleotides and includes double-stranded DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from natural DNA by the addition, deletion, substitution, and/or substitution of one or more nucleotides. The nucleotides of the DNA molecule may include standard nucleotides or nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.

"isolated" refers to a substance that has been isolated or artificially created from its natural environment. As used herein with respect to a cell, "isolated" refers to a cell that has been isolated from its natural environment (e.g., from a subject, organ, tissue, or body fluid). As used herein with respect to nucleic acids, "isolated" refers to nucleic acids that have been isolated or purified from their natural environment (e.g., from cells, organelles, or cytoplasm), recombinantly produced, amplified, or synthesized. In an embodiment, the isolated nucleic acid comprises a nucleic acid contained within a vector.

As used herein, the term "wild-type" or "non-mutated" form of a gene refers to a nucleic acid encoding a protein associated with normal or non-pathogenic activity (e.g., a protein without mutations such as an expansion of regions of repetitive sequences that result in a higher risk of developing, onset of, or progression of a neurodegenerative disease).

As used herein, the term "mutation" refers to any change in the structure of a gene (e.g., gene sequence) that results in an altered form of the gene that may or may not be transmitted to a progeny (genetic mutation). Genetic mutations include substitutions, insertions or deletions of a single base in DNA, or substitutions, insertions, deletions or rearrangements of multiple bases or larger parts of a gene or chromosome, including repeat sequence extensions.

As used herein, the term "inhibitory nucleic acid" refers to a nucleic acid that includes a guide strand sequence that hybridizes to and inhibits expression or activity of a target nucleic acid, e.g., at least a portion of a neurodegenerative disease target RNA, mRNA, mRNA precursor or mature mRNA. The inhibitory nucleic acid can 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., double-stranded duplex) or in the same strand (e.g., single-stranded, self-annealing duplex structure). In some embodiments, the inhibitory nucleic acid is an RNA molecule, such as siRNA, shRNA, miRNA or dsRNA.

As used herein, "microrna" or "miRNA" refers to a small non-coding RNA molecule capable of mediating target gene silencing by cleavage of target mRNA, translational inhibition of target mRNA, target mRNA degradation, or a combination thereof. Typically, mirnas are transcribed as hairpin or stem-loop (e.g., with a self-complementary single-stranded backbone) duplex structures, referred to as primary mirnas (pri-mirnas), that are enzymatically processed (e.g., by Drosha, DGCR8, pasha, etc.) to miRNA precursors (pre-mirnas). The miRNA precursor is exported into the cytoplasm where it is processed by Dicer enzymes to produce a miRNA duplex with a passenger strand, followed by a single-stranded mature miRNA molecule, which is then loaded into an RNA-induced silencing complex (RISC). The mirnas mentioned may comprise synthetic or artificial mirnas.

As used herein, "synthetic miRNA" or "artificial miRNA" or "amiRNA" refers to an endogenous, modified or synthetic primary miRNA or miRNA precursor (e.g., miRNA scaffold or scaffold), wherein the endogenous miRNA guide sequence and passenger sequence in the stem sequence have been replaced with the miRNA guide sequence and miRNA passenger sequence that cause efficient RNA silencing of the target gene (see, e.g., approaches et al, methods of molecular biology (2014) 1062:211-224). In some embodiments, the complementarity properties (e.g., number of bases, mismatch position, type of projections, etc.) of the guide sequence and the passenger sequence may be similar or different from the complementarity properties of the guide sequence and the passenger sequence in the endogenous miRNA backbone from which the synthetic miRNA is constructed.

As used herein, the terms "microrna scaffold," "miR scaffold," "microrna scaffold," or "miR scaffold" refer to a primary miRNA or miRNA precursor scaffold whose stem sequence is replaced by a miRNA of interest, and are capable of producing a functional mature miRNA that causes RNA silencing of a gene targeted by the miRNA of interest. The miR scaffold includes a 5 'flanking region (also referred to as a 5' miR context, > 9 nucleotides), a stem region comprising a miRNA duplex (guide and passenger strand sequences) and a basal stem (5 'and 3', each of about 4 to 13 nucleotides), at least one loop motif region comprising a terminal loop (for the terminal loop, >10 nucleotides), a 3 'flanking region (also referred to as a 3' miR context, > 9 nucleotides), and optionally one or more projections in the stem. The miR scaffold can be derived entirely or in part from a wild-type miRNA scaffold or a fully artificial sequence.

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 to 50 nucleotides (e.g., about 15 to 30, 16 to 25, 18 to 23, or 19 to 22 nucleotides) of the mRNA of the gene targeted for silencing. The antisense sequence is sufficiently complementary to the target mRNA sequence to cause target-specific silencing, e.g., to trigger destruction of the target mRNA by an RNAi mechanism or process. In some embodiments, the antisense sequence or guide strand sequence refers to the mature sequence remaining after cleavage by Dicer.

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

As used herein, "duplex" when used in reference to an inhibitory nucleic acid refers to two strands of nucleic acid (e.g., a guide strand and a passenger strand) that hybridize together to form a duplex structure. Duplex may be formed from two separate nucleic acid strands or from a single nucleic acid strand having a self-complementary region (e.g., hairpin or stem loop).

As used herein, the term "complementary" refers to the ability of polynucleotides to form base pairs with each other. Base pairs are typically formed by hydrogen bonding between nucleotide subunits in antiparallel polynucleotide strands or in a single self-annealing polynucleotide strand. The complementary polynucleotide strands may form base pairs in Watson-Crick manner (e.g., A and T, A and U, C and G) or in any other manner that allows duplex formation. As will be apparent to those skilled in the art, when RNA is used instead of DNA, the base complementary to adenosine is considered uracil instead of thymine. Furthermore, when "U" is expressed in the context of the present invention, its ability to replace "T" is understood unless otherwise indicated. Complementarity also encompasses Watson-Crick base pairing (e.g., 5-methylcytosine instead of cytosine) between an unmodified nucleobase and a modified nucleobase. Full complementarity, perfect complementarity, or 100% complementarity between two polynucleotide strands is where each nucleotide of one polynucleotide strand may form a hydrogen bond with a nucleotide unit of the second polynucleotide strand. Percent complementarity refers to the number of nucleotides in a nucleic acid molecule of consecutive nucleotide sequences complementary to an aligned reference sequence (e.g., target mRNA, passenger strand) divided by the total number of nucleotides and multiplied by 100. In such alignments, nucleobases/nucleotides that do not form base pairs are referred to as mismatches. Insertions and deletions are not allowed when calculating the percent complementarity of consecutive nucleotide sequences. It will be appreciated by those skilled in the art that chemical modifications to nucleobases are not considered in calculating complementarity so long as the Watson-Crick base pairing ability of the nucleobases is preserved (e.g., 5-methylcytosine is considered identical to cytosine in order to calculate percent complementarity).

"percent identity" between two or more nucleic acid sequences refers to the ratio of nucleotides of consecutive nucleotide sequences in a nucleic acid molecule shared by the reference sequences (i.e., percent identity = number of identical nucleotides/total number of nucleotides in the aligned region (e.g., consecutive nucleotide sequences) ×100). Insertions and deletions are not allowed when calculating the percent identity of consecutive nucleotide sequences. It will be appreciated by those skilled in the art that chemical modifications to nucleobases (e.g., 5-methylcytosine is considered identical to cytosine for the purpose of calculating percent identity) are not considered as long as the Watson-Crick base pairing ability of the nucleobases is preserved.

As used herein, the term "hybridization" refers to the hybridization of two nucleic acid strands inHydrogen bonds are formed between base pairs on the antiparallel strands, thereby forming a duplex. The hybridization intensity between two nucleic acid strands can be determined by the melting temperature (T _m ) To describe, the melting temperature is defined as the temperature at which 50% of the target sequence hybridizes to the complementary polynucleotide at a given ionic strength and pH.

As used herein, an "expression construct" refers to any type of nucleic acid-containing genetic construct (e.g., transgene) in which part or all of the nucleic acid coding sequence is capable of being transcribed. In some embodiments, expression comprises transcription of the nucleic acid, e.g., to produce a biologically active polypeptide product or inhibitory RNA (e.g., siRNA, shRNA, miRNA) from the transcribed gene. In some embodiments, the transgene is operably linked to an expression control sequence.

As used herein, the term "transgene" refers to an exogenous nucleic acid that has been transferred, either naturally or by genetic engineering means, into another cell and is capable of being transcribed and optionally translated.

As used herein, the term "gene expression" refers to the process by which nucleic acids are transcribed from a nucleic acid molecule and are typically translated into peptides or proteins. The process may comprise transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. Reference to measurement of "gene expression" may refer to measurement of transcription products (e.g., RNA or mRNA), translation products (e.g., peptides or proteins).

As used herein, the term "inhibiting expression of a gene" refers to reducing, down-regulating, inhibiting, blocking, reducing, or stopping expression of the gene. The expression product of a gene may be an RNA molecule (e.g., mRNA) transcribed from the gene or a polypeptide translated from an mRNA transcribed from the gene. In general, a decrease in mRNA levels results in a decrease in the level of polypeptide translated therefrom. Standard techniques for measuring mRNA or protein can be used to determine expression levels.

As used herein, a "vector" refers to a genetic construct capable of transporting a nucleic acid molecule (e.g., a transgene encoding an inhibitory nucleic acid) between cells and affecting expression of the nucleic acid molecule when operably linked to appropriate expression control sequences. Expression control sequences may 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; a sequence that stabilizes cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and, when desired, sequences that enhance secretion of the encoded product. The vector may be a plasmid, phage particle, transposon, cosmid, phagemid, chromosome, artificial chromosome, virus particle, or the like. Once transformed into a suitable host cell, the vector may replicate and function independently of the host genome, or may, in some cases, integrate into the genome itself.

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, such as a rodent cell (mouse or rat) or 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 from an established cell line or primary cell. In embodiments, the host cell is a cell of the CNS, such as a neuron, glial cell, astrocyte, and microglial cell.

As used herein, "neurodegenerative disease" or "neurodegenerative disorder" refers to a disease or disorder that exhibits neuronal cell death as a pathological condition. Neurodegenerative diseases may exhibit chronic neurodegeneration, e.g., slow progressive neuronal cell death over a period of 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 (SCA 2), frontotemporal dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Chronic neurodegenerative diseases include diseases characterized by TDP-43 proteinopathies characterized by mislocalization of the nucleus to the cytoplasm, ubiquitination and deposition of highly phosphorylated TDP-43 into inclusion bodies, protein truncation leading to the formation of toxic C-terminal TDP-43 fragments, and protein aggregation. TDP-43 proteinopathic diseases include ALS, FTD, primary lateral sclerosis, progressive muscular dystrophy, age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with lewy bodies, degeneration of corticobasal ganglia, progressive Supranuclear Palsy (PSP), dementia Parkinson's disease, ALS syndrome (G-PDC), pick's disease, hippocampal sclerosis, huntington's disease, parkinson's disease and Alzheimer's disease. Acute neurodegeneration may be caused by ischemia (e.g., stroke, traumatic brain injury), demyelination-induced axonal transection, or trauma (e.g., spinal cord injury or multiple sclerosis). Neurodegenerative diseases may exhibit death of a major type of neuron or types of neurons.

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, such as primates (monkeys, chimpanzees), cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, and transgenic species thereof.

Stuffer sequences and expression constructs

AAV preferentially packages full-length genomes, i.e., genomes that are about the same size as the native genome and that are not too large or too small. However, the expression cassette encoding the inhibitory nucleic acid sequence is significantly smaller than the full-length AAV genome. To avoid packaging fragmented genomes, stuffer sequences may be ligated to expression constructs comprising heterologous nucleic acid sequences and flanked by 5 'ITRs and 3' ITRs to expand the packagable genome, resulting in a genome with a near-standard length between ITRs. Typically, the packaging capacity of AAV is about 4.7kb between the 5'itr and the 3' itr. For a self-complementary AAV (scAAV) vector, the packaging capacity is about 2.4kb between the 5'itr and the 3' itr.

Preferably, the starting sequence for obtaining the vector filling sequence is of mammalian origin, such as of human origin. The length of the stuffer sequence may be adjusted so that the vector genome is at or near the (natural) packaging limit of the AAV capsid. In addition, the vector filling sequences may be designed to minimize adverse effects in the context of in vivo gene therapy. For example, if integration occurs in the genome and the risk of initiating accidental transcription is minimal, a region of the human genome can be identified as the source of the vector-filled sequence by identifying the sequence that affects the least. Thus, regions of the genome can be examined, where i) deletions and duplications are common in the population and are not associated with disease-related phenotypes (no evidence of evolutionary stress) and/or ii) RNA expression across human tissues is low or undetectable (lack of powerful intrinsic enhancer/promoter elements). In addition, the vector fill sequence may be designed to reduce, minimize, eliminate, or lack one or more elements to render the vector sequence inert or safe. In some embodiments, the vector stuffer sequence is modified to: reducing or removing expression regions (e.g., exons +10bp on either side, human ESTs); reducing or removing regulatory elements (e.g., promoter sequences, enhancer sequences, repressor sequences, splice donors or acceptors, or other cis-acting elements found in the human genome that may potentially affect transcription of the transgene); reducing or removing repeat elements (e.g., microsatellite repeat, dinucleotide repeat, trinucleotide repeat); reducing, removing or modifying the ATG codon to reduce or eliminate the possibility of peptide production from the stuffer or stuffer sequence due to the potential start codon; reducing or eliminating CpG dinucleotides to reduce the likelihood that unmethylated CpG dinucleotides (from cis-plasmids produced in bacteria) induce an innate immune response; or any combination thereof. The present disclosure provides vector filling sequences having one or more of the above features, with the further advantage of one or more of the following: high titer; low toxicity; and reduced truncations in miRNA and/or stuffer sequences.

In one aspect, the present disclosure provides a vector stuffer sequence comprising a nucleic acid having a length of about 1300 to about 2300 nucleotides and at least 75% identity to: 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 1500-2000 nucleotides in length and at least 75% identity to: 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 1600 to 1900 nucleotides in length and at least 75% identity to: 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 having at least 80% identity to: 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 nucleic acids about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.

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 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 nucleic acids about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.

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 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 nucleic acids about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.

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 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 nucleic acids about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 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 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 nucleic acids about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.

In some embodiments, the vector filling sequence comprises or consists of: 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; 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 or consists of nucleotides 489-2185 of any one of SEQ ID NOs 48 or SEQ ID NOs 13-16.

In some embodiments, the vector is an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is self-complementing.

In some embodiments, the vector stuffer sequence is positioned near (e.g., 5 'or 3') to an expression construct comprising 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 siRNA, miRNA, shRNA or dsRNA. In some embodiments, the inhibitory nucleic acid comprises siRNA, miRNA, shRNA or dsRNA targeting a gene associated with a neurodegenerative disease. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, edge-dominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, lewy body dementia (dementia with Lewy bodies), corticobasal degeneration, progressive Supranuclear Palsy (PSP), dementia-parkinsonism-ALS syndrome (dementia Parkinsonism ALS complex of guam, G-PDC), pick's disease (Pick's disease), hippocampus sclerosis, huntington's disease (Huntington's disease), parkinson's disease (Parkinson's disease), or Alzheimer's disease (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.ATXN2 refers to a protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) chain. An ATXN2 gene or transcript may refer to a normal allele of ATXN2, which typically has 22 or 23 repeats, or a mutant allele with a moderate (-24 to 32 repeats) or longer repeat expansion (-33 to >100 repeats). In some embodiments, ATXN2 refers to mammalian ATNX2, including human ATXN2.

In some embodiments, the expression construct comprises a heterologous nucleic acid encoding an artificial miRNA targeting ATXN 2. Examples of heterologous nucleic acids encoding guide and passenger sequences and artificial mirnas targeting ATXN2 are provided in table 5. RNA formats encoding guide, passenger and artificial miRNA sequences are provided, as are DNA formats for insertion into cis-plasmids and rAAV vectors. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA that targets ATXN2 and includes a guide sequence selected from the group consisting of 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 scaffold (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. Additional examples of guide sequences, passenger sequences, miR frameworks, and artificial miRNA sequences that target ATXN2 are provided in PCT publication WO 2021/159538, which is incorporated by reference in its entirety.

In some embodiments, the expression construct further comprises one or more expression control sequences (regulatory sequences) operably linked to the transgene (e.g., a nucleic acid encoding an artificial miRNA). "operably linked" sequences comprise expression control sequences that function adjacent to or in trans or away from the transgene to control its expression. 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.

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 (neuronal, glial, or astrocyte). Examples of constitutive promoters include the rous sarcoma virus (Rous sarcoma virus, RSV) LTR promoter (optionally with an RSV enhancer), the Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter and the dihydrofolate reductase promoter. Examples of inducible promoters include zinc inducible sheep Metallothionein (MT) promoter, dexamethasone (Dex) inducible Mouse Mammary Tumor Virus (MMTV) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline (tetracyclic) suppression system, tetracycline induction system, RU486 induction system, and rapamycin (rapamycin) induction system. Other examples of promoters that may be used include, for example, chicken β -actin promoter (CBA promoter), CAG promoter, H1 promoter, CD68 promoter, jeT promoter, synaptotagin 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 RNApol II promoter is derived from a gene that exhibits neuronal specific expression. In some embodiments, the neuron-specific promoter is a synapsin 1 promoter or a synapsin 2 promoter.

In some embodiments, the promoter is an H1 promoter. In some embodiments, the H1 promoter is a promoter referred to herein as a "native" H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO. 53. In some embodiments, the promoter is a promoter referred to herein as a "short" H1 promoter, such as 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 NO. 17, 25-28 and 37-44. In some embodiments, the promoter is a promoter referred to herein as a "long" H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO. 52 or nucleotides 113-343 of any of SEQ ID NO. 13-16 and 18-24.

In some embodiments, the termination sequence is an SV40 termination sequence. An example of an SV40 termination sequence is shown 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; nucleotides 349-570 of any of SEQ ID NOs 40, 41 and 44.

In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is located in the untranslated region of an expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is located in an intron, 5 'untranslated region (5' utr), or 3 'untranslated region (3' utr) of an expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is located in an intron downstream of the promoter and upstream of the expressed gene.

Vectors and host cells

In another aspect, a vector comprising the vector stuffer sequence of the present disclosure is provided. The vector may be a plasmid, cosmid, phagemid, bacterial Artificial Chromosome (BAC) or viral vector. Examples of viral vectors include herpes virus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, lentiviral 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 (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 HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV)), equine infectious anemia virus, or a Wirnner-MEDIV vector (Maedi-Visna viral vector).

In some embodiments, the vector is an adeno-associated virus (AAV) vector, such as a recombinant AAV (rAAV) vector, which is produced by recombinant methods. In some embodiments, the rAAV vectors include vector stuffer sequences of the present disclosure and expression constructs comprising heterologous nucleic acid sequences (e.g., encoding inhibitory nucleic acids). The vector stuffer sequence may be positioned 5 'or 3' adjacent to an expression construct comprising a heterologous nucleic acid.

AAV is a single-stranded, non-enveloped DNA virus whose genome encodes a protein for replication (rep) and a capsid (Cap), flanked by two ITRs, which serve as origins of viral genome replication. AAV also contains packaging sequences, allowing packaging of the viral genome into AAV capsids. Recombinant AAV vectors (rAAV) (also referred to as rAAV vector genomes or rAAV genomes) can be obtained from wild-type genomes of AAV by removing all or part of the wild-type genome (e.g., rep, cap) from the AAV using molecular methods, and replacing it with a non-native nucleic acid such as a heterologous nucleic acid sequence (e.g., a nucleic acid molecule encoding an inhibitory nucleic acid). Typically, for AAV, one or both Inverted Terminal Repeat (ITR) sequences remain in the rAAV vector. In some embodiments, the rAAV vector includes 5 'Inverted Terminal Repeats (ITRs) and 3' ITRs flanking the expression construct and the vector stuffer sequence. Functional ITR sequences are necessary for rescue, replication and packaging of AAV viral particles. Thus, rAAV vectors are defined herein to include at least those sequences (e.g., functional ITRs) required to replicate and package a virus in cis. All other viral sequences may be provided in trans. In some embodiments, the rAAV retains only the 5 'itrs and 3' itrs from the AAV genome in order 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., about 145bp in length and contains a functional Rep Binding Site (RBS) and a terminal dissociation site (trs)). In some embodiments, one or both of the ITRs are modified, e.g., by insertion, deletion, or substitution, provided that the ITRs provide functional rescue, replication, and packaging. In some embodiments, the modified ITRs lack functional terminal dissociation sites (trs) and are used to generate self-complementing AAV vectors (scAAV vectors). In some embodiments, the rAAV vector is a self-complementary AAV vector comprising a mutant ITR on the 5 'side (lacking a terminal dissociation site) and a wild-type AAV ITR on the 3' side. Examples of mutants 5' ITRs lacking terminal cleavage sites are shown in nucleotides 1-106 of SEQ ID NO. 57 or any of SEQ ID NO. 13-24, 29-44 and 78-82. In some embodiments, the modified ITR is a truncated version of the AAV2 ITR known as AITR (D sequence and TRS deletion).

In some embodiments, the rAAV vector comprises a 5' itr comprising or consisting of: SEQ ID NO. 57; or nucleotides 1 to 106 of any one of SEQ ID NOS 13 to 24, 29 to 44 and 78 to 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 2161-2305 of SEQ ID NO. 26; nucleotides 2161-2305 of SEQ ID NO. 27; nucleotides 2161-2305 of SEQ ID NO. 28; nucleotides 2266 to 2410 of SEQ ID NO. 29; nucleotides 2224-2368 of SEQ ID NO. 30; nucleotides 2216-2360 of SEQ ID NO. 31; nucleotides 2225-2369 of SEQ ID NO. 32; nucleotides 2266 to 2410 of SEQ ID NO. 33; nucleotides 2224-2368 of SEQ ID NO. 34; nucleotides 2216-2360 of SEQ ID NO. 35; nucleotides 2225-2369 of SEQ ID NO. 36; nucleotides 2257-2401 of SEQ ID NO. 37; nucleotides 2258-2402 of SEQ ID NO. 38; nucleotides 2215-2359 of SEQ ID NO. 39; nucleotides 2207-2351 of SEQ ID NO. 40; nucleotides 2207-2351 of SEQ ID NO. 41; nucleotides 2257-2401 of SEQ ID NO. 42; nucleotides 2215-2359 of SEQ ID NO. 43; nucleotides 2207-2351 of SEQ ID NO. 44; nucleotides 2258-2402 of SEQ ID NO. 78; nucleotides 2267-2411 of SEQ ID NO. 79; nucleotides 2251-2395 of SEQ ID NO. 80; nucleotides 2251-2395 of SEQ ID NO. 81; nucleotides 2187-2331 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; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 13 and nucleotides 2192 to 2358 comprising or consisting of 3' ITR of SEQ ID NO. 13; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 14 and nucleotides 2192 to 2358 comprising or consisting of 3' ITR of SEQ ID NO. 14; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 15 and a 5' ITR comprising or consisting of 2192 to 2358 of SEQ ID NO. 15; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 16 and 5' ITR comprising or consisting of 2192 to 2358 of SEQ ID NO. 16; 5'ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO. 17 and 3' ITR comprising or consisting of nucleotides 2229-2395 of SEQ ID NO. 17; nucleotides 1 to 106 comprising or consisting of nucleotide 1 to 106 of SEQ ID NO. 18 and nucleotide 2184 to 2350 comprising or consisting of nucleotide 1 to 2350 of SEQ ID NO. 18; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 13 and nucleotides 2214 to 2358 comprising or consisting of SEQ ID NO. 13; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 14 and nucleotides 2214 to 2358 comprising or consisting of 3' ITR of SEQ ID NO. 14; 1-106 or a 5'ITR consisting of nucleotide 1-106 comprising SEQ ID NO. 15 and 2214-2358 or a 3' ITR consisting of SEQ ID NO. 15; 1-106 or a 5'ITR consisting of nucleotide 1-106 comprising SEQ ID NO. 16 and 2214-2358 or a 3' ITR consisting of SEQ ID NO. 16; 5'ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO. 17 and 3' ITR comprising or consisting of nucleotides 2251-2395 of SEQ ID NO. 17; nucleotides 1-106 comprising or consisting of nucleotide 1-106 of SEQ ID NO. 18 and nucleotides 2206-2350 comprising or consisting of nucleotide 1-2350 of SEQ ID NO. 18; nucleotides 1-106 comprising or consisting of nucleotide 1-106 of SEQ ID NO. 19 and nucleotides 2206-2350 comprising or consisting of nucleotide 1-2350 of SEQ ID NO. 19; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 20 and nucleotides 2216 to 2360 comprising or consisting of 3' ITR comprising or consisting of SEQ ID NO. 20; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 21 and nucleotides 2216 to 2360 comprising or consisting of SEQ ID NO. 21; nucleotides 1-106 comprising or consisting of SEQ ID NO. 22 and nucleotides 2206-2350 comprising or consisting of 3' ITR of SEQ ID NO. 22; nucleotides 1-106 comprising or consisting of nucleotide 1-106 of SEQ ID NO. 23 and nucleotides 2216-2360 comprising or consisting of nucleotide 1-2360 of SEQ ID NO. 23; nucleotides 1-106 comprising or consisting of nucleotide 1-106 of SEQ ID NO. 24 and nucleotides 2216-2360 comprising or consisting of nucleotide 1-2360 of SEQ ID NO. 24; 5'ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO. 25 and 3' ITR comprising or consisting of nucleotides 2161-2305 of SEQ ID NO. 25; 5'ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO. 26 and 3' ITR comprising or consisting of nucleotides 2161-2305 of SEQ ID NO. 26; nucleotides 1-106 comprising or consisting of SEQ ID NO. 27 and nucleotides 2161-2305 comprising or consisting of 3' ITR of SEQ ID NO. 27; nucleotides 1-106 comprising or consisting of nucleotide No. 28 and nucleotide No. 2161-2305 comprising or consisting of nucleotide No. 28; nucleotides 1 to 106 comprising or consisting of nucleotide No. 29 and nucleotide No. 2266 to 2410 comprising or consisting of nucleotide No. 29; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 30 and nucleotides 2224 to 2368 comprising or consisting of 3' ITR of SEQ ID NO. 30; nucleotides 1 to 106 comprising or consisting of nucleotide 1 to 106 of SEQ ID NO. 31 and nucleotides 2216 to 2360 comprising or consisting of nucleotide 1 to 2360 of SEQ ID NO. 31; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 32 and nucleotides 2225 to 2369 comprising or consisting of 3' ITR of SEQ ID NO. 32; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 33 and nucleotides 2266 to 2410 comprising or consisting of 3' ITR of SEQ ID NO. 33; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 34 and nucleotides 2224 to 2368 comprising or consisting of 3' ITR of SEQ ID NO. 34; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 35 and nucleotides 2216 to 2360 comprising or consisting of SEQ ID NO. 35; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 36 and nucleotides 2225 to 2369 comprising or consisting of 3' ITR of SEQ ID NO. 36; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 37 and nucleotides 2257 to 2401 comprising or consisting of SEQ ID NO. 37; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 38 and nucleotides 2258 to 2402 comprising or consisting of nucleotide 1 to nucleotide 4 of SEQ ID NO. 38; nucleotides 1-106 comprising or consisting of SEQ ID NO. 39 and nucleotides 2215-2359 comprising or consisting of SEQ ID NO. 39; nucleotides 1-106 comprising or consisting of SEQ ID NO. 40 and nucleotides 2207-2351 comprising or consisting of SEQ ID NO. 40; nucleotides 1-106 comprising or consisting of nucleotide 1-106 of SEQ ID NO. 41 and nucleotides 2207-2351 comprising or consisting of nucleotide 1-2351 of SEQ ID NO. 41; nucleotides 1 to 106 comprising or consisting of nucleotide numbers 1-106 of SEQ ID NO. 42 and nucleotides 2257-2401 comprising or consisting of nucleotide numbers 2257-2401 of SEQ ID NO. 42; nucleotides 1 to 106 comprising or consisting of SEQ ID NO. 43 and nucleotides 2215 to 2359 comprising or consisting of SEQ ID NO. 43; nucleotides 1-106 comprising or consisting of nucleotide numbers 1-106 of SEQ ID NO. 44 and nucleotides 2207-2351 comprising or consisting of nucleotide numbers 2207-2351 of SEQ ID NO. 44; nucleotides 1 to 106 comprising or consisting of nucleotide numbers 1 to 106 of SEQ ID NO. 78 and nucleotides 2258 to 2402 comprising or consisting of nucleotide numbers 2258 to 2402 of SEQ ID NO. 78; nucleotides 1 to 106 comprising or consisting of nucleotide No. 79 and nucleotide No. 2267 to 2411 comprising or consisting of nucleotide No. 79; nucleotides 1 to 106 comprising or consisting of SEQ ID No. 80 and nucleotides 2251 to 2395 comprising or consisting of nucleotide No. 80; nucleotides 1 to 106 comprising or consisting of nucleotide 1 to 106 of SEQ ID NO. 81 and nucleotides 2251 to 2395 comprising or consisting of nucleotide 2251 to 2395 of SEQ ID NO. 81; or 5'ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO. 82 and 3' ITR comprising or consisting of nucleotides 2187-2331 of SEQ ID NO. 82.

In some embodiments, the rAAV vector is a mammalian serotype AAV vector (e.g., AAV genome and ITRs derived from mammalian serotype AAV), comprising 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 ITR is selected from the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PHP.A, AAV-PHP.B, AAV PHP.B2, AAV PHP.B3, AAV PHP.N/PHP.B-DGT, AAV PHP.B-EST, AAV PHP.B-GGT, AAV PHP.B-ATP, AAV PHP.B-ATT-GGT, AAV PHP.B-DGT, AAV PHP.B-GGT-SGS, AAV PHP.B-SGS AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.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-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3, AAVG2B4, AAVG2B5, or variants thereof.

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 siRNA, miRNA, shRNA or dsRNA. In some embodiments, the inhibitory nucleic acid comprises siRNA, miRNA, shRNA or dsRNA targeting a gene associated with a neurodegenerative disease. In some embodiments, the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, edge-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, lewy body dementia, corticobasal degeneration, progressive Supranuclear Palsy (PSP), dementia parkinsonism of the guan island type (G-PDC), pick's disease, hippocampus 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.

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, a termination sequence, a promoter sequence, an enhancer sequence, a repressor sequence, a splice site sequence, a polyadenylation (polyA) signal sequence, or any combination thereof.

In some embodiments, the rAAV vector comprises an H1 promoter. In some embodiments, the H1 promoter is a native H1 promoter, such as 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, such as a promoter comprising or consisting of the sequence set forth in nucleotide 113-203 of SEQ ID NO. 54 or any of SEQ ID NO. 17, 25-28 and 37-44. In some embodiments, the promoter is a long H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO 52 or nucleotides 113-343 of any of SEQ ID NO 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 cleavage site.

In some embodiments, the rAAV vector includes an SV40 termination sequence. An example of an SV40 termination sequence is shown 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; nucleotides 349-570 of any of SEQ ID NOs 40, 41 and 44.

The rAAV vector may have all or a portion of one or more AAV wild type genes deleted. In some embodiments, the rAAV vector is replication defective. In some embodiments, the rAAV vector lacks a functional Rep protein and/or a capsid protein. In some embodiments, the rAAV vector is a self-complementary AAV (scAAV) vector.

In some embodiments, the rAAV vector comprises: a 5'itr, a promoter operably linked to a heterologous nucleic acid encoding an ATXN2 specific artificial miRNA, a vector stuffer sequence, and a 3' itr. In some embodiments, the 5' itr is modified to lack a terminal dissociation site. In some embodiments, the promoter is oriented in the 5 'to 3' direction.

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

The recombinant AAV vectors of the present disclosure may be packaged with one or more AAV capsid proteins to form rAAV particles. "rAAV particle" or "rAAV virion" refers to an infectious, replication-defective virus comprising an AAV protein capsid that encapsulates an rAAV vector comprising a transgene of interest flanked by a 5'AAV ITR and a 3' AAV ITR, respectively. The rAAV particles are produced in a suitable host cell having the sequence of the specified rAAV vector into which AAV helper functions (helper functions) and accessory functions (acessory function) are introduced to enable the host cell to encode AAV polypeptides required for packaging the rAAV vector (containing the transgene sequence of interest) into the infectious rAAV particles for subsequent gene delivery.

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 components (e.g., rep sequences, cap sequences, and/or accessory functions) required to package a rAAV vector can be provided by a stable host cell that has been engineered to contain one or more of the required components (e.g., by the vector). Expression of the components required for AAV packaging may be under the control of inducible or constitutive promoters in the host packaging cells. AAV helper vectors are typically used to provide transient expression of trans-acting AAV rep and/or cap genes to complement the deleted AAV functions necessary for AAV replication. In some embodiments, the AAV helper vector lacks AAV ITRs and is neither replication nor self-packaging. AAV helper vectors may be in the form of plasmids, phages, transposons, cosmids, viruses or virions.

In some embodiments, rAAV particles can be produced using a triple transfection method (see, e.g., U.S. patent No. 6,001,650, incorporated herein by reference in its entirety). In this method, the rAAV particle is produced by transfecting a host cell with a rAAV vector (including a transgene), an AAV helper vector, and an accessory functional vector to be packaged into the rAAV particle. 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 a nucleotide sequence of a non-AAV-derived virus and/or cellular function upon which AAV replicates (e.g., an "accessory function"). Such accessory functions include functions required for AAV replication, including but not limited to those involving activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. The viral-based accessory function may be derived from any known helper virus, such as adenovirus, herpes virus (other than herpes simplex virus type 1) and vaccinia virus. In some embodiments, a double transfection method in which the AAV helper functions and accessory functions are cloned on a single vector is used to generate rAAV particles.

The AAV capsids are important elements in determining these tissue specificities of the rAAV particles. Thus, rAAV particles with capsid tissue specificity can be selected. In some embodiments, the rAAV particle comprises a capsid protein selected from the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu 14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV-PHP.A, AAV-PHP.B, AAV PHP.B2, AAV PHP.B3, AAV PHP.N/PHP.B-DGT, AAV PHP.B-EST, AAV PHP.B-GGT, AAV PHP.B-ATP, AAV PHP.B-ATT-GGT, AAV PHP.B-DGT, AAV PHP.B-GGT-SGS, AAV PHP.B-SGS AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.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-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A 15/G2A3, AAVG2B4, AAVG2B5, and variants thereof. In some embodiments, the AAV capsid is selected from serotypes capable of crossing the blood brain barrier, e.g., AAV9, aavrh.10, AAV-PHP-B, or variants thereof. Examples of AAV9 capsid sequences are provided in U.S. patent No. 7,906,111, which is 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 capsids and genomes from different AAV serotypes.

In some embodiments, the rAAV particle is capable of transducing cells of the CNS. In some embodiments, the rAAV particle is capable of transducing non-neuronal cells or neuronal cells of the CNS. In some embodiments, the CNS cell is a neuron, a glial cell, an astrocyte, or a microglial cell.

In another aspect, the disclosure provides host cells transfected with rAAV particles comprising the vector stuffer sequences 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), bacterial cell (e.coli), yeast cell, insect cell (Sf 9, sf21, drosophila, mosquito), etc.

Application method

In another aspect, the disclosure provides methods of delivering a therapeutic agent to a subject, the methods comprising administering a composition of the disclosure (e.g., a rAAV particle comprising a rAAV vector comprising a vector stuffer sequence provided herein and an expression construct comprising 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 cell or a neuronal cell of the CNS. In some embodiments, the non-neuronal cells of the CNS are glial cells, astrocytes or microglial cells. In some embodiments, the cell is in vitro. In some embodiments, the cells are from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease.

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 neurodegenerative disease; reducing or preventing the development of symptomatic properties of neurodegenerative diseases; preventing deterioration of the symptomatic characteristics of neurodegenerative diseases or any combination thereof.

In some embodiments, the subject has or is at risk of developing a neurodegenerative disease. Examples of neurodegenerative diseases include spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscle atrophy, age-related edge-predominant TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with lewy bodies, corticobasal degeneration, progressive Supranuclear Palsy (PSP), dementia-related parkinsonism (G-PDC), pick's disease, hippocampal sclerosis, huntington's disease, parkinson's disease and alzheimer's disease.

In some embodiments, the methods of treatment of the present disclosure reduce, prevent, or slow the development or progression of one or more symptomatic characteristics of a neurodegenerative disease. Examples of symptomatic characteristics of neurodegenerative diseases include motor dysfunction, cognitive dysfunction, mood/behavioral dysfunction, or any combination thereof. Paralysis, tremor, instability, stiffness, twitch, muscle weakness, muscle spasms, muscle stiffness, muscle atrophy, dysphagia, dyspnea, speech and language difficulties (e.g., aphtha), slow motion, difficulty walking, dementia, depression, anxiety, or any combination thereof.

In some embodiments, the methods of treatment of the present disclosure include administration as monotherapy or in combination with one or more additional therapies for treating a neurodegenerative disease. Combination therapy may mean that the composition of the present disclosure is administered to a subject concurrently with, before, and after one or more additional therapies. Simultaneous administration of a combination therapy may mean formulating the compositions of the present disclosure and additional therapies for administration in the same dosage form or in separate dosage forms.

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 human.

In any of the methods described herein, the compositions of the present disclosure can be administered to a subject by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular, intraventricular, intravertebral, subcutaneous, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., 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. The most appropriate route of administration will generally 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 composition is injected by intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intraventricular, intraocular, intraventricular, intravitreal administration, or any combination thereof.

In some embodiments, the compositions of the present disclosure are injected directly into the CNS of a subject. In some embodiments, the direct injection into the CNS is an intra-brain injection, an intra-brain parenchymal injection, an intrathecal injection, an intrastriatal injection, an subpial injection, or any combination thereof. In some embodiments, the direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracisternal injection, intraventricular injection, or any combination thereof.

Examples

Example 1: design of vector "stuffer" sequences

Selection of "nonessential" human genomic DNA for base filling sequences

To minimize any undesired effects of the stuffer sequence, the "nonessential" region of the human genome is identified as having two desirable properties: (a) Sequences that have minimal impact if integration occurs in the genome (although this is considered very rare for AAV); and (b) a sequence that minimizes the risk of initiating accidental transcription. Thus, regions of the genome were examined for i) deletions and duplications that are common in the population and that are not associated with disease-associated phenotypes (no evidence of evolutionary stress); and/or ii) low or undetectable (lack of powerful intrinsic enhancer/promoter elements) RNA expression across human tissues. From this search, two regions are prioritized:

Zone I: AMELY Gene region (chrysY: 6,865,918-6,874,027, HG38)

Several lines of evidence suggest that the AMELY gene region is not under evolutionary selection. First, the AMELY region originates from an ancient replication event and has selected functional homologs on the X chromosome (Lahn and Page, (Science) (1999) 286, 964-967). Second, the AMELY gene has more protein change variants than expected (including predicted loss of function variants) without evidence of recent positive selection (Nature) (2020) 581,434-443; https:// gnomad. Broadinstitute. Org/gene/ENSG00000099721dataset = gnomad_r2_1). Third, genetic variants of the AMELY region have been demonstrated to have no phenotypic effect on humans by deletions or duplications. Repetition of AMELY is common and has no phenotype (human genet.) (2015) 134:789-800). Fourth, deletions are common and have no detectable phenotype (human molecular Genet) (2007) 16:307-16. Furthermore, AMELY is not associated with any rare human disease (online human mendelian inheritance (Online Mendelian Inheritance in Man), omim. Org)

AMELY mRNA is not detectable in most human tissues (GTEX, GTEx alliance (The GTEx Consortium)) (science (2020) 369:1318-1330) and is only present in Y chromosome carriers (men). Further, AMELX (a functional homolog of AMELY) is associated with dental enamel during development (physiological Front (Front Physiol.) (2017) 8:435).

Zone II: PSG gene region (chr 19:43,511,809-43,530,631, HG38)

Similarly, several lines of evidence suggest that Pregnancy Specific Glycoproteins (PSGs) are not under clear evolutionary selection. First, PSG clusters are derived from segment duplicates (Dumont and Eichler, public science library complex (PLoS One) (2013) 8:e75949). Second, there is no evidence that the region is evolutionarily limited, with predicted loss of function variants being more than expected (Nature 2020) 581:434-443; https:// gnomad. Broadenstitute. Org/gene/ENSG00000243130dataset = gnomad_r2_1). Third, the PSG cluster is not associated with any rare human disease (online human mendelian inheritance, omim. Org).

The PSG region is normally highly expressed by the placenta only during pregnancy, while it is not/is not expressed in other tissues (GTEX, GTEx alliance) ("science (2020) 369:1318-1330).

Modification of base filling sequences

The DNA sequences from the AMELY region (chrY: 6,865,918-6,874,027, HG38) and the PSG gene region (chr 19:43,511,809-43,530,631, HG38) are modified in order to render the sequences "inert" or lack any expression regions, known or predicted regulatory and repeat elements (including retroviruses and transposable elements). These changes are most strictly performed for the filling sequences expected to be used in the region containing the packaging vector genome.

Specifically, the base 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) Removing the expression region: the exons +10bp flanking (exons are defined using the gene model in GENCODEv 19), the sequence tag expressed by humans (EST: according to Genbank data, using the "including unspliced human EST" track definition on the UCSC genome browser: "Nucleic Acids research (Nucleic Acids Res.)" (2004) 32 (database stage): D23-6).

ii) removal of regulatory elements: cpG islands (journal of molecular biology (J Mol biol.) (1987) 196 (2): 261-82), regions identified by the CHiP-seq (sequence bound by the transcription factor) transcription factor CHIP-seq peak (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 site orbitals), the VISTA enhancer (VISTA HMR conserved non-coding human enhancer from LBNL orbitals), and the open regulatory annotation "OReganno" (regulatory elements from OReganno orbitals) ("nucleic acid research (2016) 44 (D1): D126-32.) were defined using the following orbitals on the UCSC genome browser.

iii) Removing the duplicate element: the repeat masking element (including SINE, LINE, LTR, DNA elements) of the repeat element (Repeating Elements), the microsatellite repeat sequence defined by the microsatellite-dinucleotide and trinucleotide repeat sequence orbits (nucleic acids research (1999) 27 (2): 573-80), and the interrupt and simple repeat sequences defined by the repeat mask (http:// www.repeatmasker.org) are used by the repeat mask orbit (http:// www.repeatmasker.org).

iv) to reduce the likelihood of accidental translation, ATG residues are modified to reduce the frequency of ATG sequences.

v) editing of CpG dinucleotides. To reduce the likelihood that unmethylated CpG dinucleotides in cis-plasmids produced in bacteria will trigger innate immune activation when packaged in AAV, the presence of CG dinucleotides is reduced by editing the bases.

Example 2: safety assessment of AAV vector genomes using filled polynucleotide packaging

The safety of vectors incorporating the filling sequences described in example 1, without any additional active elements (such as mirnas), was tested in vivo. A set of 4 vectors was designed, each with two variants of AMELY and PSG11 derived stuffer (SEQ ID NOS: 25-28). Each vector contained an H1 short promoter on the 5' side immediately downstream of the left ITR (SEQ ID NO: 57), and included a non-miRNA control sequence instead of a miRNA downstream of the H1 short promoter. The Pol III terminator is located 3' to the control sequence. Three controls were tested in parallel: a vehicle; a vector encoding an artificial miRNA sequence targeting ATXN2 (scaav_h1_mir 16-2-1479_amely_v1 (SEQ ID NO: 81)), previously demonstrated to show toxicity when tested in lentivirus format on HeLa and U2OS cells under the control of the H1 promoter; and a vector encoding an Atxn 2-specific miRNA and GFP (scAAV_H2_miR 1-1-XD-14792_CBh_GFP_SV40p (SEQ ID NO: 82)). Artificial mirnas that have previously demonstrated toxicity are selected as positive controls for toxicity, which may be caused by off-target effects of specific artificial miRNA sequences.

Vectors were intravenously administered to 16 week old wild type C57Bl/6 mice via the tail vein as the total viral genome (vg) of 1.3E11. The titer was determined based on the DNA concentration of the column purified vector. This concentration was compared to the concentration of similarly purified vector which was also subjected to titre determination by vector genome qPCR to calculate the corresponding titres of the filled vector described in this example. Blood was treated and the concentrations of the liver enzymes alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum were assessed as an indicator of any liver toxicity. Vectors expressing the toxic positive control artificial miRNA resulted in significant increases in liver enzymes ALT and AST. In contrast, none of the vectors with the filling sequence caused an increase in liver enzymes (fig. 2A to 2B).

Pathologists perform histological evaluation of the liver to assess any toxicity. At 28 days post-dose, animals were sacrificed (n=5 animals each filled with vehicle treatment) and the livers were immersed in 4% paraformaldehyde for a fixed period of 24 hours. Two sections of each liver were embedded in paraffin, sectioned at a thickness of 5 microns, and stained with hematoxylin and eosin. Photomicrographs were taken on an Olympus BX60 microscope. Animals dosed with toxic positive control mirnas exhibit a number of lesions including oval cell proliferation, hepatocyte fusion and multinuclear, lymphocytic hepatitis, sinus fibrosis, tissue cytochrome, and rare single cell apoptosis/necrosis. In vectors containing stuffer fragments, histological findings were limited to sporadic background lesions that did not follow treatment-related trends and were visible across multiple treatment groups. Variations including hepatoglycogen storage disease (hepatocellular glycogen accumulation) and rare granulomas were found. These findings are not believed to reflect the treatment toxicity associated with the compounds.

Example 3: assessment of vector genome truncation in specific filling designs

rAAV vector genome formulations are known to contain truncated vector genomes that are most pronounced at hairpin structures (e.g., mirnas), but also at other regions of DNA secondary structure. Thus, the presence of truncated genomes in rAAV formulations containing different forms of stuffer sequences was assessed.

Fig. 3A to 3C show DNA traces from fragment analyzer analysis. After production of the rAAV vector (scadAAW9_H2_Long_miR-100_3330_PSG11_V5 (SEQ ID NO: 16)), the vector DNA was extracted and subjected to column purification (Zymo, inc. (Zymo), P/N D3015) and run on an Agilent fragment analyzer (HS NGS fragment 1-6000 bp). Bimodal traces can be seen (fig. 3A), and these bimodal are determined to contain the full length structure of the ITR, as they are broken down by restriction digestion at the site where the right-hand wild-type ITR is removed. A smaller shoulder to the left of the full length product, indicating a shorter, truncated genome, is also expected to be present. An amiRNA-centered side peak was found in many formulations, which represents the vector genome truncated at the amiRNA (fig. 3D). Similar peaks exist if a control sequence is used instead of amiRNA but contains secondary structures such as palindromic sequences.

FIGS. 3E to 3F compare two rAAV formulations (scAAW9_H2_MCS_PSG11_V1 (SEQ ID NO: 27) and scAAW9_H2_MCS_PSG11_V2 (SEQ ID NO: 28)) derived from cis plasmids containing one of two versions of stuffer sequences derived from PSG11 (PSG11_V1 (SEQ ID NO: 45) and PSG11_V2 (SEQ ID NO: 46)). These sequences are similar in structure, but the ATG site was edited in PSG11_V2 to eliminate the ATG sequence. There were some structural differences in the 5' portion of the stuffer sequence adjacent to the expression cassette (H1 promoter and amiRNA or in the vector shown in fig. 3E, control sequence). The trace shows that the 'shoulder' on the left of the full-length product is smaller in the vector genome formulation containing psg11_v1 (fig. 3F). In these vector formulations, amiRNA was not used.

FIGS. 3G through 3H compare a similar set of two rAAV preparations (scadARV9_H2_miR-1-1-XD-14792_PSG11_V1 (SEQ ID NO: 78) and scadARV9_H2_Natural_miR-1-XD-14792_PSG11_V2 (SEQ ID NO: 79)) derived from a cis plasmid containing either the stuffer sequence PSG11_V1 (SEQ ID NO: 45) or PSG11_V2 (SEQ ID NO: 46), in which case both vectors contain an amiRNA element downstream of the H1 promoter (3 nucleotides are inserted in the promoter sequence on the PSG11_V2-containing vector compared to the V1-containing vector, but the 5' sequence is otherwise identical). By examining the fragment analyzer trace (fig. 3H), it can be seen that the truncation at the amiRNA site is significantly greater in the vector containing the psg11_v2 filling sequence.

Replication from a complementary AAV (scAAV) vector (such as the vectors described herein) involves replication starting from the wild-type ITR. In the sequences described herein, this is located at the 3' side of the cis plasmid. Thus, the polymerase will initially pass from 3 'to 5' through the stuffer sequence to the amiRNA. The potential explanation for the increased truncated product is that the secondary structure that the polymerase may encounter just prior to the amiRNA hairpin promotes strand switching activity, resulting in increased truncation. Consistent with this hypothesis, examination of the 200 nucleotide fold of the DNA sequence within the PSG11_V1 stuffer sequence (SEQ ID NO:55; FIG. 4A) and PSG11_V2 stuffer sequence (SEQ ID NO:56; FIG. 4B) immediately 3' of the amiRNA revealed a predicted region of increased secondary structure in the PSG11_V2 stuffer fragment. Therefore, the PSG11_V1 sequence structure is favored. The "PSG11_V5" stuffer sequence (SEQ ID NO: 48) is generated by using the advantageous sequence structure from the PSG11_V1 sequence. This is envisaged to produce a vector formulation with a reduced truncated vector genome. The_v5 stuffer incorporates editing of CG and ATG sequences to reduce the likelihood of hypomethylated CpG dinucleotides or accidental open reading frames.

Example 4: further evaluation of the safety of stuffer sequences when combined with the ATXN2 AMIRNA expression cassette

An experiment was performed in which the in vivo safety of 24 different vector designs covering various stuffer sequences was assessed. Three different variants of the H1 promoter (H1 short (SEQ ID NO: 54), H1 natural (SEQ ID NO: 53), H1 long (SEQ ID NO: 52)), two different artificial ATXN 2-targeting miRNAs (3330 or 14792 (also referred to herein as 1784))/miR scaffold (miR1.1. Or miR 100) combinations (miR 100_3330 (encoding SEQ ID NO: 12), miR1.1_14792 (encoding SEQ ID NO: 73)), the presence or absence of Pol-II transcription terminator SV40 polyadenylation sequences, and different versions of the filler sequences (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 from 5 'to 3' in a cis plasmid intended for packaging as a self-complementing AAV vector, with the mutant ITR (lacking the terminal cleavage site) on the 5 'side (e.g., nucleotide 1-106 of SEQ ID NO:57 or any of SEQ ID NO:13-24, 29-44, and 78-82) and the wild-type AAV ITR on the 3' side (e.g., nucleotide 2214-2358 of any of SEQ ID NO:58 or SEQ ID NO: 13-16). The reason for using this promoter orientation is that miR-centric truncation is possible in vectors incorporating ITRs lacking terminal cleavage sites (self-complementing AAV vectors). In the 5 'to 3' promoter orientation, vectors with miR-centric truncation will not include the promoter and should therefore be inert. If the promoter is in the 3 'to 5' orientation, the promoter may remain in the vector with the miR-centric truncation.

rAAV vectors were generated with these cis plasmids and the vectors were intravenously administered to 12 week old wild-type C57Bl/6 mice by tail vein injection at a dose of 3.21E9 vector genomes per gram of mice, which was adjusted by weight (average total dose 8.5E10 vector genomes). Vehicle control animals were dosed with PBS containing 0.001% PF-68. Blood was collected 2 weeks after administration by submandibular hemorrhage, and liver enzyme concentrations (aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT)) were measured in animals designed for administration with various carriers (fig. 5A-5B). Almost all data points of animals treated with the vector containing the stuffer fragment were within normal reference ranges. Although two data points showed that the AST signal had risen above the reference value, hemolysis was found in these samples and one sample was from the vehicle treated animal. At this time point, none of the AST or ALT increased above the reference range in other samples from animals dosed with any vector form, indicating that there is no evidence that administration of rAAV with vectors encoding these stuffer sequences would result in acute liver injury.

Table 1: summary of animals (liver histology) for each vehicle administered intravenously evaluated

Carrier body	Promoters	miR	Terminator	Stuffer segments	N
						scaav_h1_short_mir 100_3330_SV40_AMELY_V3[SEQ ID NO:44]	H1. Short length	miR100–3330	SV40	AMELY_V3	1
scaav_h1_short_mir 100_3330_AMELY_V3[SEQ ID NO:42]	H1. Short length	miR100-3330	Without any means for	AMELY_V3	1
						scaav_h1_short_mir 1-1_1784_SV40_AMELY_V3[SEQ ID NO:40]	H1. Short length	miR1.1–1784	SV40	AMELY_V3	1
scaav_h1_short_mir 1-1_1784_AMELY_V3[SEQ ID NO:37]	H1. Short length	miR1.1-1784	Without any means for	AMELY_V3	1
						scaav_h1_natural_mir 100_3330_SV40_AMELY_V3[SEQ ID NO:35]	H1. Natural material	miR100.3330	SV40	AMELY_V3	1
scaav_h1_natural_mir 100_3330_AMELY_V3[SEQ ID NO:33]	H1. Natural material	miR100.3330	Without any means for	AMELY_V3	1
						scaav_h1_natural_mir 1-1_1784_SV40_AMELY_V3[SEQ ID NO:31]	H1. Natural material	miR1.1.1784	SV40	AMELY_V3	2
scaav_h1_natural_mir 1-1_1784_AMELY_V3[SEQ ID NO:29]	H1. Natural material	miR1.1.1784	Without any means for	AMELY_V3	1
						scaav_h1_long_mir 100_3330_SV40_AMELY_V3[SEQ ID NO:23]	H1. Long length	miR100.3330	SV40	AMELY_V3	1
scaav_h1_long_mir 100_3330_AMELY_V3[SEQ ID NO:22]	H1. Long length	miR100.3330	Without any means for	AMELY_V3	1
						scAAV_H2_Length_miR 1-1_1784_SV40_AMELY_V3[SEQ ID NO:20]	H1. Long length	miR1.1.1784	SV40	AMELY_V3	1
scAAV_H2_Length_miR 1-1_1784_AMELY_V3[SEQ ID NO:18]	H1. Long length	miR1.1.1784	Without any means for	AMELY_V3	2
						scaav_h1_short_mir 1-1_1784_SV40_PSG11_V3[SEQ ID NO:41]	H1. Short length	miR1.1.1784	SV40	PSG11_V3	1
scaav_h1_short_mir 1-1_1784_PSG11_V3[SEQ ID NO:38]	H1. Short length	miR1.1.1784	Without any means for	PSG11_V3	1
						scAAV_H2_Length_miR 1-1_1784_PSG11_V2[SEQ ID NO:19]	H1. Long length	miR1.1.1784	Without any means for	PSG11_V2	1
scaav_h1_short_mir 100_3330_PSG11_V5[SEQ ID NO:43]	H1. Short length	miR100.3330	Without any means for	PSG11_V5	1
						scaav_h1_short_mir 1-1_1784_PSG11_V5[SEQ ID NO:39 ]	H1. Short length	miR1.1.1784	Without any means for	PSG11_V5	1
scaav_h1_natural_mir 100_3330_SV40_PSG11_V5[SEQ ID NO:36]	H1. Natural material	miR100.3330	SV40	PSG11_V5	1
						scAAV_H2_Length_miR 1-1_1784_SV40_PSG11_V5[SEQ ID NO:21]	Length of H1	miR1.1.1784	SV40	PSG11_V5	11
scaav_h1_natural_mir 100_3330_PSG11_V5[SEQ ID NO:34]	H1. Natural material	miR100.3330	Without any means for	PSG11_V5	1
						scaav_h1_natural_mir 1-1_1784_SV40_PSG11_V5[SEQ ID NO:32]	H1. Natural material	miR1.1.1784	SV40	PSG11_V5	1
scaav_h1_natural_mir 1-1_1784_PSG11_V5[SEQ ID NO:30]	H1. Natural material	miR1.1.1784	Without any means for	PSG11_V5	1
						scaav_h1_long_mir 100_3330_SV40_PSG11_V5[SEQ ID NO:24]	H1. Long length	miR100.3330	SV40	PSG11_V5	1
scaav_h1_long_mir 100_3330_PSG11_V5[SEQ ID NO:16]	H1. Long length	miR100.3330	Without any means for	PSG11_V5	1

Animals dosed intravenously with the vector containing the stuffer fragment were also assessed histologically for hepatotoxicity. Three weeks after dosing, two consecutive sections of about 0.5cm liver lobes were collected per animal, embedded in paraffin, sectioned to 5 microns, and stained with hematoxylin and eosin. Slides were assessed by a trained pathologist. As shown in table 1, n=1-2 animals per group were evaluated. After evaluation, intravenous administration of the test article was not interpreted as being related to changes in liver examination sections for all of the evaluated vectors.

Striatal in vivo testing

Table 2: summary of animals assessed for each vehicle administered intrastriatally (striatal histology)

These vectors were also administered to 8 week old wild type C57Bl/6 mice at a dose of 7.5E9 vector genomes per striatum by direct injection into the striatum, and the toxicity of a subset of the vectors was assessed by histological observation (table 2 above). At 3 weeks post-dose, the striatal sections of the animals were paraffin embedded, wherein four serial sections cut into 5 micron blocks were mounted on separate slides in advance. Including a set of 2 slides that include step sections from three levels. Sections were immunohistochemical with hematoxylin and eosin, glial Fibrillary Acidic Protein (GFAP), ionocalcareous binding adapter molecule 1 (IBA-1) and floro-jade B stained. The latter three stains can be used to assess astrocyte reactivity, microglial cell reactivity and neuronal death, respectively.

As with liver sections of animals treated with the vehicle administered intravenously, no effect of the test preparation was identified in the brain sections examined. Processing artifacts limits interpretation in some tissues. Typically, changes are observed at the injection site and include slight tissue destruction, with slight gliosis (identified on H & E stained slides), slight microgliasis (identified on GFAP labeled slides) and slight ferrioxacin. Ferrioxacin is a common variation of intraparenchymal injection. In any of the examination sections at the basal core/striatum level of any animal, the test article was not interpreted as causing deterioration or change associated with the experimental procedure.

Transcriptional analysis

To further evaluate the safety of the vector, a sequencing analysis was performed on a striatal puncture biopsy of animals dosed with the vector. The striatal tissue was column purified using the Qiagen AllPrep DNA/RNA/protein Mini kit (Qiagen, P/N80004) to extract RNA (after DNase treatment). A library of chain RNA-seq was prepared by the chain Total RNAPrep Ligation using the Ribo-Zero Plus kit (Ennomina, illumina, P/N20040525) and paired-end 2X 100bp sequencing was performed on the Illumina Novaseq 6000 system. These reads were then aligned with the corresponding cis-plasmids used to package the rAAV administered to the animal. DNA was purified separately from the same puncture biopsy used to extract sequenced RNA (qiagen, P/N80004) and vector exposure was confirmed. Table 3 lists the exposure and carrier for each test sample.

Table 3: assessment of vector biodistribution in samples by chain RNA-seq

FIG. 6 shows the stacking of reads from a representative vector. Surprisingly, the use of the H1 short promoter and miR 1.1 backbone, as well as the PSG 11V 5 stuffer sequence, resulted in an increased number of reads aligned with the cis plasmid downstream of the intended transcription of the primary miRNA. In contrast, the use of the H1 long promoter with the PSG 11V 5 stuffer enables beneficial minimal amounts of unintended transcription. Thus, the combination of such elements produces a vector structure with the desired low frequency of non-miRNA transcription.

Example 5: display of the function of stuffer sequences in different RAAV vector forms when combined with the ATXN2 AMIRNA expression cassette

Animals dosed with a set of 24 vector combinations described in example 4 were also assessed for the function of all combinations of stuffer, promoter and ATXN2 targeted artificial miRNA. Knockdown of Atxn2 was assessed by extracting RNA from liver tissue 3 weeks after intravenous administration (dose: 3.21E9 vg/gram mice) followed by RT-ddPCR with Atxn2 and control probe Hprt. Atxn2 levels were calculated as an average of these Atxn 2/control transcript ratios and were further normalized to the ratios obtained for vehicle dosed animals. Vector genome exposure was also measured using ddPCR with probes for the vector genome and a mouse genome probe (Tert). As can be seen in table 4, robust Atxn2 knockdown occurred in animals treated with all combinations of stuffer sequences and miRNA vector components.

Table 4: knock-down of liver Atxn2 by various vectors containing stuffer sequences

Example 6: production yield and truncation further evaluation of stuffer sequences in different RAAV vector forms when combined with the ATXN2 AMIRNA expression cassette.

Example 4 shows that when the "H1 long" variant of the promoter is inserted upstream of the psg11_v5 stuffer sequence, there is an unexpected benefit in reducing transcriptional activity downstream of the artificial miRNA sequence compared to the "H1 short" variant of the promoter. The production yield of the combination of H1 long and H1 short promoters and AMELY and PSG11 derived stuffer sequences was also assessed. Production yield was assessed by ddPCR of the vector genome using primer/probe sets specific for the stuffer region. In general, the use of a combination of cis plasmids with H1 long form promoter (SEQ ID NO: 52) 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 (FIG. 7). Analysis of vector truncations (figures 8A to 8C) further showed that truncations were less when the H1 long promoter version was included upstream than the H1 short promoter. In summary, there are many surprising benefits to including an H1 long promoter upstream of these stuffer sequences, which may be due to local DNA secondary structural elements that may affect rAAV replication and packaging.

Example 7: production of vectors incorporating additional combinations of stuffer sequences with artificial MIRNAs

The stuffer sequences generated according to example 1 and comprising a combination of promoter and specific stuffer sequences as described in example 4 were evaluated for their ability to be packaged in rAAV as part of the AAV vector genome. The ability to continue to achieve good productivity with different payloads in the carrier was also tested for these filling sequences.

Filling sequences derived from the PSG11 intron region, modified according to the design rules in example 1 and containing the optimal H1 long promoter variant were packaged in a set of vectors containing various ATXN2 targeting artificial miRNA packages. The 5'ITR to 3' ITR cis plasmid sequences are shown in SEQ ID NO:13 (scAAV_H2_Long_miR 100_1755_PSG11_V5_ITR_to_ITR), SEQ ID NO:14 (scAAV_H2_Long_miR 100_2586_PSG11_V5_ITR_to_ITR), SEQ ID NO:15 (scAAV_H2_Long_miR 100_2945_PSG11_V5_ITR_to_ITR) and SEQ ID NO:16 (scAAV_H2_Long_miR 100_3330_PSG11_V5_ITR_to_ITR). The sequences of the amiRNA guide, passenger and expression cassette are described in table 5.

Table 5: artificial miRNA sequences in RNA and DNA forms

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The H1 promoter ("H1 long") (nucleotide 113-343 of either SEQ ID NO:52 or SEQ ID NO: 13-16) was incorporated into the vector. Table 6 lists the yields of AAV production from these vectors.

Table 6: AAV production yield of vectors containing H1 Long-PSG11_V5 stuffer sequences

The filling sequence derived from AMELY is packaged in several vectors. Vector sequences from 5'ITR to 3' ITR containing artificial miRNA cassettes targeting ATXN2 and AMELY filling sequences are provided for "H1_short_miR 16-2-1755_AMELY_V1" (SEQ ID NO: 17) and "H1-miR1-1_1784_AMELY_V3" (SEQ ID NO: 18). In these vectors, the notation "_v1" or "_v3" of AMELY refers to different versions of AMELY stuffer sequence, wherein in the "_v3" version, the ATG sequence is edited and the CpG dinucleotides are edited (nucleotides 488-2177 of SEQ ID NO:51 or SEQ ID NO: 18), and "_v1" is not so modified (nucleotides 342-2222 of SEQ ID NO:49 or SEQ ID NO: 17). H1-short refers to a 91bp truncated form of the H1 promoter (SEQ ID NO:54 or nucleotides 113-203 of SEQ ID NO: 17). The yields from these vector productions are listed in table 7.

Table 7: AAV production yield of vectors containing AMELY stuffer sequences

Example 8: functional display of AAV vector genome packaged using a combination of filler polynucleotide and additional amiRNA

Experiments were performed to test the function of the payload when incorporated into the vector genome incorporating the stuffer sequences and additional amirnas described herein. This further establishes the compatibility of the stuffer sequence with the amira expression cassette. The vectors all express artificial miRNA targeting ATXN 2; the function is determined by knockdown of the ATXN2 target.

In one experiment, knockdown of ATXN2mRNA in human stem cell derived motor neurons was assessed for the vectors listed in table 8. The vectors included scaav_h1_long_mir 100_1755_psg11_v5_itr_to_itr.gb (SEQ ID NO: 13), scaav_h1_long_mir 100_2586_psg11_v5_itr_to_itr.gb (SEQ ID NO: 14), scaav_h1_long_mir 100_2945_psg11_v5_itr_to_itr.gb (SEQ ID NO: 15), and scaav_h1_long_mir 100_3330_psg11_v5_itr_to_itr.gb (SEQ ID NO: 16). Stem cell-derived motor neurons were treated with indicator vector packaged in AAV-DJ and, 7 days after transduction, RNA was harvested and knockdown was assessed using RT-ddPCR with probes for ATXN2 and housekeeping probes GUSB and B2M. Average ATXN2mRNA signals normalized to GUSB and B2M probes and to untransfected cells were measured. Data for cells treated with 3.16E4 vector genome/cell dose are listed.

Table 8: ATXN2 knockdown in amiRNA/AAV vector treated human stem cell derived motor neurons

Carrier body	miR	Average_atxn2_31600	SD	N
					H1-miR-PSG11_V5	miR100_1755	13.96853215	2.13651083	6
H1-miR-PSG11_V5	miR100_2586	29.25180424	1.03259645	6
					H1-miR-PSG11_V5	miR100_2945	26.83870303	2.60157362	6
H1-miR-PSG11_V5	miR100_3330	32.17946149	2.69980734	6

In another experiment, vectors comprising AMELY_V3 stuffer (SEQ ID NO:51 or nucleotides 488-2177 of SEQ ID NO: 18) and an amiRNA targeting ATXN2 under the control of the H1 long promoter were tested by intravenous administration to adult male C57Bl/6 mice and measuring knockdown of the target ATXN2 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 3 animals dosed with vehicle relative to animals dosed with vehicle (PBS+0.001% PF-68). In this study, the dose administered was 8.5E10 on average and was adjusted according to the mouse body weight. As can be seen in table 9, there was a significant knockdown in the liver of animals dosed with the vector, indicating the function of AAV packaged with the stuffer.

Table 9: ATXN2 knockdown in liver of AAV vector treated mice

The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications (including U.S. patent application Ser. No. 63/146,522 filed on month 5 of 2021 and PCT application Ser. No. PCT/US2021/016939 filed on month 5 of 2021) cited in this specification and/or listed in the application data sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit 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 to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

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<400> 9

cccaaaagag agaagauauu gaggccuguu gccacaucgg guugaaaucu gaaguguggu 60

auuaguccgc acacuccagc uuucaaaccg uugugucugu uaggcaaucu cacggaccug 120

gggcuuugcu uauaugcc 138

<210> 10

<211> 138

<212> RNA

<213> artificial sequence

<220>

<223> miR100_2945 (RNA)

<400> 10

cccaaaagag agaagauauu gaggccuguu gccacaugua guagaaggcu uuggcugagu 60

auuaguccgu cagcccaaga cuucuaauac uugugucugu uaggcaaucu cacggaccug 120

gggcuuugcu uauaugcc 138

<210> 11

<211> 138

<212> RNA

<213> artificial sequence

<220>

<223> miR100_2586 (RNA)

<400> 11

cccaaaagag agaagauauu gaggccuguu gccacauaga uucagaagua gaacuugggu 60

auuaguccgc caaguccuaa uucugacucu uugugucugu uaggcaaucu cacggaccug 120

gggcuuugcu uauaugcc 138

<210> 12

<211> 138

<212> RNA

<213> artificial sequence

<220>

<223> miR100_3330 (RNA)

<400> 12

cccaaaagag agaagauauu gaggccuguu gccacauaug cugagacuga uaaugugggu 60

auuaguccgc cacaucaucc gucucaacau uugugucugu uaggcaaucu cacggaccug 120

gggcuuugcu uauaugcc 138

<210> 13

<211> 2358

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_1755_psg11_v5_itr_to_itr

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_1755

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(2185)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2214)..(2358)

<223> 3' ITR

<400> 13

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat cgggttgaaa tctgaagtgt ggtattagtc cgcacactcc 420

agctttcaaa ccgttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttac caggtcagtt gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag 540

aaggtggaag ggatttcact ctgtttttag agataggatt ggaactaaga attttaagtc 600

tatttgggtg aaggggcagg gaagtgtagg aagacagttc taattaggaa ggaggaatta 660

tactaggaag tagaggtaaa ggaagtgaga aggcttagta aatagaagaa atctgaactt 720

atcaaatagg ctagtgtata cccctggaga ttcttgatta agtgaattaa ctagatttta 780

gaaaggtatt gaggcagtta tttgcaagga acaattcaaa gtctggagac aagaagtcaa 840

gcaatacttt gagaagtggt tgagcttttt gggattggag agaattacac tattgagatt 900

tgttaaaaat attgaagaag gtttcacctt gtgaaattgt gtttctcaat tgaatctgag 960

aaacaattga gcagaggctg gagataattt tgtctaaaag acagtggggt acagggagca 1020

aaaaggccag ggagaaaggg aactgcagga attagtgctg agaagcagga gttagtggtt 1080

gaaggaggag atccaggccc agatacaggc aaataagtca attccctctc ccaagcttgg 1140

cagtcagccc tgcaggaacc aggataagag aaattgtttg gtaaaaacta acaaaccagg 1200

cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag tagccttgtt 1260

ctagtgtttt ttgtgttacc tctttttcct gaaaaaggaa ggtactgttg agttattgtt 1320

gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt gtttcccacc 1380

taaattgcac acttagaaac agttattggt aagccttata ttagatatat atagtgtctg 1440

gtagtcttac cctctctata attacacact ttttggcact gcccctttcc tgccttgcag 1500

agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt actcagtgct 1560

gtgtcccacc tgctgtgccc acagcctcaa acagccagtg acttcagagc caggaaacag 1620

ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg gccaagggga 1680

ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac caggactctt 1740

tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca ccagaggagc 1800

ccagagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg ggcttcctcc 1860

tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct tgtagttctt 1920

ggaagaggta caagaaaaca aagacctgac agaaggggtt gaggtggtga cagtgagaag 1980

caactggttg ttcagcactc tccttcttgt ccctctgtga agcctcttct accacaaagg 2040

gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag gtcagtcttg 2100

agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggagg gttcaggagg 2160

agaatttggg atttgcctgt gcccagctag cgtagataag tagcatggcg ggttaatcat 2220

taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 2280

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2340

gagcgagcga gcgcgcag 2358

<210> 14

<211> 2358

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_2586_psg11_v5_itr_to_itr

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_2586

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(2185)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2214)..(2358)

<223> 3' ITR

<400> 14

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat agattcagaa gtagaacttg ggtattagtc cgccaagtcc 420

taattctgac tctttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttac caggtcagtt gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag 540

aaggtggaag ggatttcact ctgtttttag agataggatt ggaactaaga attttaagtc 600

tatttgggtg aaggggcagg gaagtgtagg aagacagttc taattaggaa ggaggaatta 660

tactaggaag tagaggtaaa ggaagtgaga aggcttagta aatagaagaa atctgaactt 720

atcaaatagg ctagtgtata cccctggaga ttcttgatta agtgaattaa ctagatttta 780

gaaaggtatt gaggcagtta tttgcaagga acaattcaaa gtctggagac aagaagtcaa 840

gcaatacttt gagaagtggt tgagcttttt gggattggag agaattacac tattgagatt 900

tgttaaaaat attgaagaag gtttcacctt gtgaaattgt gtttctcaat tgaatctgag 960

aaacaattga gcagaggctg gagataattt tgtctaaaag acagtggggt acagggagca 1020

aaaaggccag ggagaaaggg aactgcagga attagtgctg agaagcagga gttagtggtt 1080

gaaggaggag atccaggccc agatacaggc aaataagtca attccctctc ccaagcttgg 1140

cagtcagccc tgcaggaacc aggataagag aaattgtttg gtaaaaacta acaaaccagg 1200

cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag tagccttgtt 1260

ctagtgtttt ttgtgttacc tctttttcct gaaaaaggaa ggtactgttg agttattgtt 1320

gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt gtttcccacc 1380

taaattgcac acttagaaac agttattggt aagccttata ttagatatat atagtgtctg 1440

gtagtcttac cctctctata attacacact ttttggcact gcccctttcc tgccttgcag 1500

agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt actcagtgct 1560

gtgtcccacc tgctgtgccc acagcctcaa acagccagtg acttcagagc caggaaacag 1620

ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg gccaagggga 1680

ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac caggactctt 1740

tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca ccagaggagc 1800

ccagagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg ggcttcctcc 1860

tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct tgtagttctt 1920

ggaagaggta caagaaaaca aagacctgac agaaggggtt gaggtggtga cagtgagaag 1980

caactggttg ttcagcactc tccttcttgt ccctctgtga agcctcttct accacaaagg 2040

gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag gtcagtcttg 2100

agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggagg gttcaggagg 2160

agaatttggg atttgcctgt gcccagctag cgtagataag tagcatggcg ggttaatcat 2220

taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 2280

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2340

gagcgagcga gcgcgcag 2358

<210> 15

<211> 2358

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_2945_psg11_v5_itr_to_itr

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_2945

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(2185)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2214)..(2358)

<223> 3' ITR

<400> 15

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat gtagtagaag gctttggctg agtattagtc cgtcagccca 420

agacttctaa tacttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttac caggtcagtt gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag 540

aaggtggaag ggatttcact ctgtttttag agataggatt ggaactaaga attttaagtc 600

tatttgggtg aaggggcagg gaagtgtagg aagacagttc taattaggaa ggaggaatta 660

tactaggaag tagaggtaaa ggaagtgaga aggcttagta aatagaagaa atctgaactt 720

atcaaatagg ctagtgtata cccctggaga ttcttgatta agtgaattaa ctagatttta 780

gaaaggtatt gaggcagtta tttgcaagga acaattcaaa gtctggagac aagaagtcaa 840

gcaatacttt gagaagtggt tgagcttttt gggattggag agaattacac tattgagatt 900

tgttaaaaat attgaagaag gtttcacctt gtgaaattgt gtttctcaat tgaatctgag 960

aaacaattga gcagaggctg gagataattt tgtctaaaag acagtggggt acagggagca 1020

aaaaggccag ggagaaaggg aactgcagga attagtgctg agaagcagga gttagtggtt 1080

gaaggaggag atccaggccc agatacaggc aaataagtca attccctctc ccaagcttgg 1140

cagtcagccc tgcaggaacc aggataagag aaattgtttg gtaaaaacta acaaaccagg 1200

cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag tagccttgtt 1260

ctagtgtttt ttgtgttacc tctttttcct gaaaaaggaa ggtactgttg agttattgtt 1320

gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt gtttcccacc 1380

taaattgcac acttagaaac agttattggt aagccttata ttagatatat atagtgtctg 1440

gtagtcttac cctctctata attacacact ttttggcact gcccctttcc tgccttgcag 1500

agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt actcagtgct 1560

gtgtcccacc tgctgtgccc acagcctcaa acagccagtg acttcagagc caggaaacag 1620

ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg gccaagggga 1680

ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac caggactctt 1740

tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca ccagaggagc 1800

ccagagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg ggcttcctcc 1860

tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct tgtagttctt 1920

ggaagaggta caagaaaaca aagacctgac agaaggggtt gaggtggtga cagtgagaag 1980

caactggttg ttcagcactc tccttcttgt ccctctgtga agcctcttct accacaaagg 2040

gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag gtcagtcttg 2100

agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggagg gttcaggagg 2160

agaatttggg atttgcctgt gcccagctag cgtagataag tagcatggcg ggttaatcat 2220

taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 2280

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2340

gagcgagcga gcgcgcag 2358

<210> 16

<211> 2358

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_3330_psg11_v5_itr_to_itr

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_3330

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(2185)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2214)..(2358)

<223> 3' ITR

<400> 16

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat atgctgagac tgataatgtg ggtattagtc cgccacatca 420

tccgtctcaa catttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttac caggtcagtt gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag 540

aaggtggaag ggatttcact ctgtttttag agataggatt ggaactaaga attttaagtc 600

tatttgggtg aaggggcagg gaagtgtagg aagacagttc taattaggaa ggaggaatta 660

tactaggaag tagaggtaaa ggaagtgaga aggcttagta aatagaagaa atctgaactt 720

atcaaatagg ctagtgtata cccctggaga ttcttgatta agtgaattaa ctagatttta 780

gaaaggtatt gaggcagtta tttgcaagga acaattcaaa gtctggagac aagaagtcaa 840

gcaatacttt gagaagtggt tgagcttttt gggattggag agaattacac tattgagatt 900

tgttaaaaat attgaagaag gtttcacctt gtgaaattgt gtttctcaat tgaatctgag 960

aaacaattga gcagaggctg gagataattt tgtctaaaag acagtggggt acagggagca 1020

aaaaggccag ggagaaaggg aactgcagga attagtgctg agaagcagga gttagtggtt 1080

gaaggaggag atccaggccc agatacaggc aaataagtca attccctctc ccaagcttgg 1140

cagtcagccc tgcaggaacc aggataagag aaattgtttg gtaaaaacta acaaaccagg 1200

cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag tagccttgtt 1260

ctagtgtttt ttgtgttacc tctttttcct gaaaaaggaa ggtactgttg agttattgtt 1320

gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt gtttcccacc 1380

taaattgcac acttagaaac agttattggt aagccttata ttagatatat atagtgtctg 1440

gtagtcttac cctctctata attacacact ttttggcact gcccctttcc tgccttgcag 1500

agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt actcagtgct 1560

gtgtcccacc tgctgtgccc acagcctcaa acagccagtg acttcagagc caggaaacag 1620

ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg gccaagggga 1680

ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac caggactctt 1740

tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca ccagaggagc 1800

ccagagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg ggcttcctcc 1860

tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct tgtagttctt 1920

ggaagaggta caagaaaaca aagacctgac agaaggggtt gaggtggtga cagtgagaag 1980

caactggttg ttcagcactc tccttcttgt ccctctgtga agcctcttct accacaaagg 2040

gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag gtcagtcttg 2100

agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggagg gttcaggagg 2160

agaatttggg atttgcctgt gcccagctag cgtagataag tagcatggcg ggttaatcat 2220

taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 2280

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 2340

gagcgagcga gcgcgcag 2358

<210> 17

<211> 2395

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 16-2-1755_AMELY V1_ITR _to_itr

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(335)

<223> miR16-2-1755

<220>

<221> misc_feature

<222> (336)..(341)

<223> terminator

<220>

<221> misc_feature

<222> (342)..(2222)

<223> AMELY_V1

<220>

<221> misc_feature

<222> (2251)..(2395)

<223> 3' ITR

<400> 17

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacttatgtt tggatgaact gacatacttg ttccactctc 240

gggttgaaat ctgaagtgtg tagtgaaata tatattaaac acacttcagg cttcaacccg 300

ttagtgtgac agggatacag caactatttt atcaattttt ttattatgca tatcacataa 360

ttttatttaa aaatcattta gattttgatt gcgtgaagta cacaaccagt attccatatc 420

ctaatttgtt ttgtaataca gtcttcaaaa gttaaatgca aagaaattgt catataaact 480

tcccttcatt tggttcaggt ttcagtgatt gtatatgttc atacattttt attttatcaa 540

cgattttaac tgatgtgtaa acttcatttt ttaaaaagat ttgacgtagt ataatttcag 600

ttttctctaa aaagaaatat agtccaaatg gtgaatttgt atgagtaaat gagcagttct 660

taccttattc aagaatactc attcataata tgttgtctca aaagtttcat tcaacacata 720

gcaaaaggaa caattaaata atttaattgt ttccagtgag taccatttac aatacatgta 780

ttcaaaccaa tgtacaacca cttaagttga actctataat ttccaaagtt tctttgttca 840

ttcgttgtca ttagatatta ggtgtccatg aagccttctt ctacctttgg aacacttaaa 900

gcctattatt ttataacaaa ataacaattc tttccataaa tagctttgtc aaggtgtgag 960

cacattataa atccacggca ttttctatgc acagagagaa catgtgcttt agactagaat 1020

agccaatgat agtcgtcaga ttacttggga ttcaatcaat caatcaattt tcaaaaattc 1080

tgccccatat cacaattatt aagccattta taacatatat tatgattttt taattcagaa 1140

gttatcacat ttatacagta gtatatcatc aatagcttgt ataaattgga attacaggtg 1200

gatacatata agtgatagat cacataatct tatatacgta gaaataagat tttatggatg 1260

tggataaagt ttttttattg attattccca ttcatttgta agtgcttctt gtaatgggag 1320

attcagtcat aaattgaggt aacttctttt taaaagctta atcatctcaa gttagactga 1380

aaactactga aaatgggaaa ccttcgtata ggttttttta aagagatttc ccatctacct 1440

ttcatagctt gttcaaatat atgcactcta aaaattaccc accccatctt atcttaagcc 1500

ttcactgcaa ttctagcctc ttcttgtgag tcaaatcttt gaaatgaaag aactgatatt 1560

cccttattgc tgttgccctt aaaattaaga gggtttaata atatattttt ctattaatgg 1620

gttgtcataa tatacagtct ggtagaaatt caataataat aaaatttaaa cctatcataa 1680

aaactgtgtt cttaattacc aagccattta gataaatttg tttataatgt aaagatacaa 1740

catattactt ggactaatag aatcatagat ctgtaactac tacaactcta gaccctgagt 1800

tgttggaccc cttagaactc tggggccacc tttgctgcac cattttcaca ggatttgcat 1860

tgtagtggct tcacagagag tgactccaac cagagaagca gcagtgttgt ctttgccatt 1920

atggccatag gaatattgta tttcaaaaat ttgtaactgt gtacacagac atgatttttt 1980

acaaactaaa tttcccatta gtgtctgtat gtggagtaca catggtattt tcttaaaaat 2040

gcctaatatt ttcagggaat tacagatata ttagaatcca tttgatttaa tttactttca 2100

tcccatgcag aaattcactg atgcggtagg aaataagaag aaggtaaaac tgtttatttc 2160

cataggtcct gcttttcatt gtaagagcaa agcaaacata aaattatgcc gcaataatgt 2220

gagctagcgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga 2280

tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 2340

tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcag 2395

<210> 18

<211> 2350

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 1-1_1784_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (488)..(2177)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2206)..(2350)

<223> 3' ITR

<400> 18

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacatgcag actgcctgct 360

tgggtacaga ccaaagagta gtcgaattat ggacctgcta agctaattaa ctactctttg 420

gtctgaactc aggccgggac ctctctcgcc gcactgaggg gcactccaca ccacgggggc 480

ctttttttat tttgcaaatc acaaaatttt atttaaaaat caattagatt ttgattgcct 540

gaagtacaca accagtattc caaatcctaa tttgttttgt aatacagtct tcaaaagtta 600

attgcaaaga aattgtcaaa taaacttccc ttcaattggt tcaggtttca gtgattgtat 660

ttgttcaaac aattttattt tatcaaggat tttaactgtt gtgtaaactt caatttttaa 720

aaagatttga cctagtataa tttcagtttt ctctaaaaag aaatatagtc caattggtga 780

atttgtttga gtaattgagc agttcttacc ttattcaaga atactcaatc aaaatttgtt 840

gtctcaaaag tttcaatcaa cacaaagcaa aaggaacaat taaataattt aattgtttcc 900

agtgagtacc aattacaata cttgtattca aaccagtgta caaccactta agttgaactc 960

tataatttcc aaagtttctt tgttcaatcc ttgtcaatag atattaggtg tccttgaagc 1020

cttcttctac ctttggaaca cttaaagcct attattttat aacaaaataa caattctttc 1080

caaaaatagc tttgtcaagg tgtgagcaca atataaatcc agggcaattt ctttgcacag 1140

agagaacttg tgctttagac tagaatagcc agtgatagtc ctcagattac ttgggattca 1200

atcaatcaat caattttcaa aaattctgcc ccaaatcaca attattaagc caattataac 1260

aaatattttg attttttaat tcagaagtta tcacaattat acagtagtat atcaacaata 1320

gcttgtataa attggaatta caggtggata caaataagtg atagatcaca aaatcttata 1380

tacctagaaa taagattttt tggttgtgga taaagttttt ttattgatta ttcccaatca 1440

attgtaagtg cttcttgtat tgggagattc agtcaaaaat tgaggtaact tctttttaaa 1500

agcttaatca actcaagtta gactgaaaac tactgaaatt gggaaacctt cctataggtt 1560

tttttaaaga gatttcccac ctacctttca aagcttgttc aaatatttgc actctaaaaa 1620

ttacccaccc caccttatct taagccttca ctgcaattct agcctcttct tgtgagtcaa 1680

atctttgaat tgaaagaact gatattccct tattgctgtt gcccttaaaa ttaagagggt 1740

ttaataatat atttttctat tattgggttg tcaaaatata cagtctggta gaaattcaat 1800

aataataaaa tttaaaccta tcaaaaaaac tgtgttctta attaccaagc caattagata 1860

aatttgttta tattgtaaag atacaacaaa ttacttggac taatagaatc aaagatctgt 1920

aactactaca actctagacc ctgagttgtt ggacccctta gaactctggg gccacctttg 1980

ctgcaccaat ttcacaggat ttgcagtgta gtggcttcac agagagtgac tccaaccaga 2040

gaagcagcag tgttgtcttt gccaatttgg ccaaaggaat attgtatttc aaaaatttgt 2100

aactgtgtac acagacttga ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg 2160

agtacacttg gtattttgct agcgtagata agtagcatgg cgggttaatc attaactaca 2220

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 2280

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 2340

gagcgcgcag 2350

<210> 19

<211> 2350

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 1-1_1784_psg11_v2 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(2177)

<223> PSG11_V2

<220>

<221> misc_feature

<222> (2206)..(2350)

<223> 3'ITR

<400> 19

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacatgcag actgcctgct 360

tgggtacaga ccaaagagta gtcgaattat ggacctgcta agctaattaa ctactctttg 420

gtctgaactc aggccgggac ctctctcgcc gcactgaggg gcactccaca ccacgggggc 480

ctttttttgt atacccctgg agattcttga ttaagtgaat taactagatt ttagaaaggt 540

attgaggcag ttatttgcaa ggaacaattc aaagtctgga gacaagaagt caagcaatac 600

tttgagaagt ggttgagctt tttgggattg gagagaatta cactattgag atttgttaaa 660

aatattgaag aaggtttcac cttgtgaaat tgtgtttctc aattgaatct gagaaacaat 720

tgagcagagg ctggagataa ttttgtctaa aagacagtgg ggtacaggga gcaaaaaggc 780

cagggagaaa gggaactgca ggaattagtg ctgagaagca ggagttagtg gttgaaggag 840

gagatccagg cccagataca ggcaaataag tcaattccct ctcccaagct tggcagtcag 900

ccctgcagga accaggataa gagaaaagaa gaaaagaaaa gaaggattga aggtgttgtt 960

attttacttg ggggagcgtc aggaacaagc ttgtaacttg agatactgta taattgtttc 1020

ttcaaggact acaatatttc tgttggaaaa ttgttgggag ttgattatat tcttgcagtt 1080

ttttttccct ctcaccttgt ttctcaggtg gtgatcagtc ctctgtcaat tctctactgc 1140

actcaggtat tttggaaggc tttcagacgt gagaaaggct gattgctatt ttctttgtca 1200

gtggaacttg tcacctttgc accttttctt ggttgcaact ttttctcagt gtctctgttt 1260

ggtaaaaact aacaaaccag gcttgattct ttgcagttag ctctttatct ttctctccca 1320

caagtagtca gtagccttgt tctagtgttt tttgtgttac ctctttttcc ggaaaaagga 1380

aggtactgtt gagttattgt tgttgatagt aatatagtag ctgacagtgg ttcctctgtt 1440

tgtggcaact tgtttcccac ctaaattgca cacttagaaa cagttattgg taagccttat 1500

attagatata tatagtgtct ggtagtctta ccctctctat aattacacac tttttggcac 1560

tgcccctttc ctgccttgca gagccccagg ggtgaatctt cctatttctc cagctctgca 1620

acagtcactg tactcagtgc tgtgtcccac gtgctgtgcc cacagcctca aacagccagt 1680

gacttcagag ccaggacgca gctcaggagt ctgccctgag gttgcttctc ttcttatttc 1740

cttgcagcct ggcccggggg aggcttggct tgaactggca gctcaattta gccaaattca 1800

ggacaggcca ccaggactct ttctccacac ttgctggtcc caccccaggt tgagtgaggc 1860

agggccagtc accagaggag cccggagcag agcaggaagc agagtctgag ctgctcctcc 1920

ctcacccaag gggcttcctc ctctcaattg ggggaaaagt gtgagcttgt ttcaaagcct 1980

cagttgttcc ttgtagttct tggaagaggt acaagaaaac aaagacgtga cagaaggggt 2040

tgaggtggtg acagtgagaa gcaactcgtt gttcagcact ctccttcttg tccctctgtg 2100

aagcctcttc taccacaaag ggctcagggc tgataaagcc ccctccctac ctttctcagg 2160

ccagacacaa ggtcagtgct agcgtagata agtagcatgg cgggttaatc attaactaca 2220

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 2280

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 2340

gagcgcgcag 2350

<210> 20

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 1-1_1784_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(710)

<223> SV40 termination

<220>

<221> misc_feature

<222> (711)..(2187)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 20

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacatgcag actgcctgct 360

tgggtacaga ccaaagagta gtcgaattat ggacctgcta agctaattaa ctactctttg 420

gtctgaactc aggccgggac ctctctcgcc gcactgaggg gcactccaca ccacgggggc 480

ctttttttca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 540

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 600

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 660

ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta attgtttcca 720

gtgagtacca attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct 780

ataatttcca aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc 840

ttcttctacc tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc 900

aaaaatagct ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga 960

gagaacttgt gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa 1020

tcaatcaatc aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca 1080

aatattttga ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag 1140

cttgtataaa ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat 1200

acctagaaat aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa 1260

ttgtaagtgc ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa 1320

gcttaatcaa ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt 1380

ttttaaagag atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat 1440

tacccacccc accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa 1500

tctttgaatt gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt 1560

taataatata tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata 1620

ataataaaat ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa 1680

atttgtttat attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta 1740

actactacaa ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc 1800

tgcaccaatt tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag 1860

aagcagcagt gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta 1920

actgtgtaca cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga 1980

gtacacttgg tattttctta aaattgccta atattttcag ggaattacag atatattaga 2040

atccaattga tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata 2100

agaagaaggt aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa 2160

acaaaaaatt ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 21

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 1-1_1784_sv40_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(710)

<223> SV40 termination

<220>

<221> misc_feature

<222> (711)..(2187)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 21

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacatgcag actgcctgct 360

tgggtacaga ccaaagagta gtcgaattat ggacctgcta agctaattaa ctactctttg 420

gtctgaactc aggccgggac ctctctcgcc gcactgaggg gcactccaca ccacgggggc 480

ctttttttca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 540

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 600

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 660

ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta accaggtcag 720

ttgttgcttg tgaagaaggg tgtggttaag tctgcagtgc agaaggtgga agggatttca 780

ctctgttttt agagatagga ttggaactaa gaattttaag tctatttggg tgaaggggca 840

gggaagtgta ggaagacagt tctaattagg aaggaggaat tatactagga agtagaggta 900

aaggaagtga gaaggcttag taaatagaag aaatctgaac ttatcaaata ggctagtgta 960

tacccctgga gattcttgat taagtgaatt aactagattt tagaaaggta ttgaggcagt 1020

tatttgcaag gaacaattca aagtctggag acaagaagtc aagcaatact ttgagaagtg 1080

gttgagcttt ttgggattgg agagaattac actattgaga tttgttaaaa atattgaaga 1140

aggtttcacc ttgtgaaatt gtgtttctca attgaatctg agaaacaatt gagcagaggc 1200

tggagataat tttgtctaaa agacagtggg gtacagggag caaaaaggcc agggagaaag 1260

ggaactgcag gaattagtgc tgagaagcag gagttagtgg ttgaaggagg agatccaggc 1320

ccagatacag gcaaataagt caattccctc tcccaagctt ggcagtcagc cctgcaggaa 1380

ccaggataag agaaattgtt tggtaaaaac taacaaacca ggcttgattc tttgcagtta 1440

gctctttatc tttctctccc acaagtagtc agtagccttg ttctagtgtt ttttgtgtta 1500

cctctttttc ctgaaaaagg aaggtactgt tgagttattg ttgttgatag taatatagta 1560

gctgacagtg gttcctctgt ttgtggcaac ttgtttccca cctaaattgc acacttagaa 1620

acagttattg gtaagcctta tattagatat atatagtgtc tggtagtctt accctctcta 1680

taattacaca ctttttggca ctgccccttt cctgccttgc agagccccag gggtgaatct 1740

tcctatttct ccagctctgc aacagtcact gtactcagtg ctgtgtccca cctgctgtgc 1800

ccacagcctc aaacagccag tgacttcaga gccaggaaac agctcaggag tctgccctga 1860

ggttgcttct cttcttattt ccttgcagcc tggccaaggg gaggcttggc ttgaactggc 1920

agctcaattt agccaaattc aggacaggcc accaggactc tttctccaca cttgctggtc 1980

ccaccccagg ttgagtgagg cagggccagt caccagagga gcccagagca gagcaggaag 2040

cagagtctga gctgctcctc cctcacccaa ggggcttcct cctctcaatt gggggaaaag 2100

tgtgagcttg tttcaaagcc tcagttgttc cttgtagttc ttggaagagg tacaagaaaa 2160

caaagacctg acagaagggg ttgaggtgct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 22

<211> 2350

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_3330_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_3330

<220>

<221> misc_feature

<222> (482)..(487)

<223> terminator

<220>

<221> misc_feature

<222> (488)..(2177)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2206)..(2350)

<223> 3'ITR

<400> 22

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat atgctgagac tgataatgtg ggtattagtc cgccacatca 420

tccgtctcaa catttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttat tttgcaaatc acaaaatttt atttaaaaat caattagatt ttgattgcct 540

gaagtacaca accagtattc caaatcctaa tttgttttgt aatacagtct tcaaaagtta 600

attgcaaaga aattgtcaaa taaacttccc ttcaattggt tcaggtttca gtgattgtat 660

ttgttcaaac aattttattt tatcaaggat tttaactgtt gtgtaaactt caatttttaa 720

aaagatttga cctagtataa tttcagtttt ctctaaaaag aaatatagtc caattggtga 780

atttgtttga gtaattgagc agttcttacc ttattcaaga atactcaatc aaaatttgtt 840

gtctcaaaag tttcaatcaa cacaaagcaa aaggaacaat taaataattt aattgtttcc 900

agtgagtacc aattacaata cttgtattca aaccagtgta caaccactta agttgaactc 960

tataatttcc aaagtttctt tgttcaatcc ttgtcaatag atattaggtg tccttgaagc 1020

cttcttctac ctttggaaca cttaaagcct attattttat aacaaaataa caattctttc 1080

caaaaatagc tttgtcaagg tgtgagcaca atataaatcc agggcaattt ctttgcacag 1140

agagaacttg tgctttagac tagaatagcc agtgatagtc ctcagattac ttgggattca 1200

atcaatcaat caattttcaa aaattctgcc ccaaatcaca attattaagc caattataac 1260

aaatattttg attttttaat tcagaagtta tcacaattat acagtagtat atcaacaata 1320

gcttgtataa attggaatta caggtggata caaataagtg atagatcaca aaatcttata 1380

tacctagaaa taagattttt tggttgtgga taaagttttt ttattgatta ttcccaatca 1440

attgtaagtg cttcttgtat tgggagattc agtcaaaaat tgaggtaact tctttttaaa 1500

agcttaatca actcaagtta gactgaaaac tactgaaatt gggaaacctt cctataggtt 1560

tttttaaaga gatttcccac ctacctttca aagcttgttc aaatatttgc actctaaaaa 1620

ttacccaccc caccttatct taagccttca ctgcaattct agcctcttct tgtgagtcaa 1680

atctttgaat tgaaagaact gatattccct tattgctgtt gcccttaaaa ttaagagggt 1740

ttaataatat atttttctat tattgggttg tcaaaatata cagtctggta gaaattcaat 1800

aataataaaa tttaaaccta tcaaaaaaac tgtgttctta attaccaagc caattagata 1860

aatttgttta tattgtaaag atacaacaaa ttacttggac taatagaatc aaagatctgt 1920

aactactaca actctagacc ctgagttgtt ggacccctta gaactctggg gccacctttg 1980

ctgcaccaat ttcacaggat ttgcagtgta gtggcttcac agagagtgac tccaaccaga 2040

gaagcagcag tgttgtcttt gccaatttgg ccaaaggaat attgtatttc aaaaatttgt 2100

aactgtgtac acagacttga ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg 2160

agtacacttg gtattttgct agcgtagata agtagcatgg cgggttaatc attaactaca 2220

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 2280

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 2340

gagcgcgcag 2350

<210> 23

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_3330_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_3330

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(710)

<223> SV40 termination

<220>

<221> misc_feature

<222> (711)..(2187)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 23

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat atgctgagac tgataatgtg ggtattagtc cgccacatca 420

tccgtctcaa catttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 540

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 600

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 660

ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta attgtttcca 720

gtgagtacca attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct 780

ataatttcca aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc 840

ttcttctacc tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc 900

aaaaatagct ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga 960

gagaacttgt gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa 1020

tcaatcaatc aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca 1080

aatattttga ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag 1140

cttgtataaa ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat 1200

acctagaaat aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa 1260

ttgtaagtgc ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa 1320

gcttaatcaa ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt 1380

ttttaaagag atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat 1440

tacccacccc accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa 1500

tctttgaatt gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt 1560

taataatata tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata 1620

ataataaaat ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa 1680

atttgtttat attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta 1740

actactacaa ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc 1800

tgcaccaatt tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag 1860

aagcagcagt gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta 1920

actgtgtaca cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga 1980

gtacacttgg tattttctta aaattgccta atattttcag ggaattacag atatattaga 2040

atccaattga tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata 2100

agaagaaggt aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa 2160

acaaaaaatt ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 24

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_long_mir 100_3330_sv40_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(343)

<223> H1 long promoter

<220>

<221> misc_feature

<222> (344)..(481)

<223> miR100_3330

<220>

<221> misc_feature

<222> (482)..(488)

<223> terminator

<220>

<221> misc_feature

<222> (489)..(710)

<223> SV40 termination

<220>

<221> misc_feature

<222> (711)..(2187)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 24

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtggaattcg 120

aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 180

acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 240

gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 300

atttgggaat cttataagtt ctgtatgaga ccactctttc ccacccaaaa gagagaagat 360

attgaggcct gttgccacat atgctgagac tgataatgtg ggtattagtc cgccacatca 420

tccgtctcaa catttgtgtc tgttaggcaa tctcacggac ctggggcttt gcttatatgc 480

ctttttttca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 540

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 600

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 660

ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta accaggtcag 720

ttgttgcttg tgaagaaggg tgtggttaag tctgcagtgc agaaggtgga agggatttca 780

ctctgttttt agagatagga ttggaactaa gaattttaag tctatttggg tgaaggggca 840

gggaagtgta ggaagacagt tctaattagg aaggaggaat tatactagga agtagaggta 900

aaggaagtga gaaggcttag taaatagaag aaatctgaac ttatcaaata ggctagtgta 960

tacccctgga gattcttgat taagtgaatt aactagattt tagaaaggta ttgaggcagt 1020

tatttgcaag gaacaattca aagtctggag acaagaagtc aagcaatact ttgagaagtg 1080

gttgagcttt ttgggattgg agagaattac actattgaga tttgttaaaa atattgaaga 1140

aggtttcacc ttgtgaaatt gtgtttctca attgaatctg agaaacaatt gagcagaggc 1200

tggagataat tttgtctaaa agacagtggg gtacagggag caaaaaggcc agggagaaag 1260

ggaactgcag gaattagtgc tgagaagcag gagttagtgg ttgaaggagg agatccaggc 1320

ccagatacag gcaaataagt caattccctc tcccaagctt ggcagtcagc cctgcaggaa 1380

ccaggataag agaaattgtt tggtaaaaac taacaaacca ggcttgattc tttgcagtta 1440

gctctttatc tttctctccc acaagtagtc agtagccttg ttctagtgtt ttttgtgtta 1500

cctctttttc ctgaaaaagg aaggtactgt tgagttattg ttgttgatag taatatagta 1560

gctgacagtg gttcctctgt ttgtggcaac ttgtttccca cctaaattgc acacttagaa 1620

acagttattg gtaagcctta tattagatat atatagtgtc tggtagtctt accctctcta 1680

taattacaca ctttttggca ctgccccttt cctgccttgc agagccccag gggtgaatct 1740

tcctatttct ccagctctgc aacagtcact gtactcagtg ctgtgtccca cctgctgtgc 1800

ccacagcctc aaacagccag tgacttcaga gccaggaaac agctcaggag tctgccctga 1860

ggttgcttct cttcttattt ccttgcagcc tggccaaggg gaggcttggc ttgaactggc 1920

agctcaattt agccaaattc aggacaggcc accaggactc tttctccaca cttgctggtc 1980

ccaccccagg ttgagtgagg cagggccagt caccagagga gcccagagca gagcaggaag 2040

cagagtctga gctgctcctc cctcacccaa ggggcttcct cctctcaatt gggggaaaag 2100

tgtgagcttg tttcaaagcc tcagttgttc cttgtagttc ttggaagagg tacaagaaaa 2160

caaagacctg acagaagggg ttgaggtgct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 25

<211> 2305

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_AMELY_V1 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(245)

<223> multiple cloning site

<220>

<221> misc_feature

<222> (246)..(251)

<223> terminator

<220>

<221> misc_feature

<222> (252)..(2132)

<223> AMELY_V1

<220>

<221> misc_feature

<222> (2161)..(2305)

<223> 3'ITR

<400> 25

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacctacata agcaggtgat atgtaaattt cacctgctta 240

aatagttttt ttattatgca tatcacataa ttttatttaa aaatcattta gattttgatt 300

gcgtgaagta cacaaccagt attccatatc ctaatttgtt ttgtaataca gtcttcaaaa 360

gttaaatgca aagaaattgt catataaact tcccttcatt tggttcaggt ttcagtgatt 420

gtatatgttc atacattttt attttatcaa cgattttaac tgatgtgtaa acttcatttt 480

ttaaaaagat ttgacgtagt ataatttcag ttttctctaa aaagaaatat agtccaaatg 540

gtgaatttgt atgagtaaat gagcagttct taccttattc aagaatactc attcataata 600

tgttgtctca aaagtttcat tcaacacata gcaaaaggaa caattaaata atttaattgt 660

ttccagtgag taccatttac aatacatgta ttcaaaccaa tgtacaacca cttaagttga 720

actctataat ttccaaagtt tctttgttca ttcgttgtca ttagatatta ggtgtccatg 780

aagccttctt ctacctttgg aacacttaaa gcctattatt ttataacaaa ataacaattc 840

tttccataaa tagctttgtc aaggtgtgag cacattataa atccacggca ttttctatgc 900

acagagagaa catgtgcttt agactagaat agccaatgat agtcgtcaga ttacttggga 960

ttcaatcaat caatcaattt tcaaaaattc tgccccatat cacaattatt aagccattta 1020

taacatatat tatgattttt taattcagaa gttatcacat ttatacagta gtatatcatc 1080

aatagcttgt ataaattgga attacaggtg gatacatata agtgatagat cacataatct 1140

tatatacgta gaaataagat tttatggatg tggataaagt ttttttattg attattccca 1200

ttcatttgta agtgcttctt gtaatgggag attcagtcat aaattgaggt aacttctttt 1260

taaaagctta atcatctcaa gttagactga aaactactga aaatgggaaa ccttcgtata 1320

ggttttttta aagagatttc ccatctacct ttcatagctt gttcaaatat atgcactcta 1380

aaaattaccc accccatctt atcttaagcc ttcactgcaa ttctagcctc ttcttgtgag 1440

tcaaatcttt gaaatgaaag aactgatatt cccttattgc tgttgccctt aaaattaaga 1500

gggtttaata atatattttt ctattaatgg gttgtcataa tatacagtct ggtagaaatt 1560

caataataat aaaatttaaa cctatcataa aaactgtgtt cttaattacc aagccattta 1620

gataaatttg tttataatgt aaagatacaa catattactt ggactaatag aatcatagat 1680

ctgtaactac tacaactcta gaccctgagt tgttggaccc cttagaactc tggggccacc 1740

tttgctgcac cattttcaca ggatttgcat tgtagtggct tcacagagag tgactccaac 1800

cagagaagca gcagtgttgt ctttgccatt atggccatag gaatattgta tttcaaaaat 1860

ttgtaactgt gtacacagac atgatttttt acaaactaaa tttcccatta gtgtctgtat 1920

gtggagtaca catggtattt tcttaaaaat gcctaatatt ttcagggaat tacagatata 1980

ttagaatcca tttgatttaa tttactttca tcccatgcag aaattcactg atgcggtagg 2040

aaataagaag aaggtaaaac tgtttatttc cataggtcct gcttttcatt gtaagagcaa 2100

agcaaacata aaattatgcc gcaataatgt gagctagcgt agataagtag catggcgggt 2160

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 2220

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 2280

cctcagtgag cgagcgagcg cgcag 2305

<210> 26

<211> 2305

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_AMELY_V2 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(245)

<223> multiple cloning site

<220>

<221> misc_feature

<222> (246)..(251)

<223> terminator

<220>

<221> misc_feature

<222> (252)..(2132)

<223> AMELY_V2

<220>

<221> misc_feature

<222> (2161)..(2305)

<223> 3'ITR

<400> 26

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacctacata agcaggtgat atgtaaattt cacctgctta 240

aatagttttt ttattttgca aatcacaaaa ttttatttaa aaatcaatta gattttgatt 300

gcgtgaagta cacaaccagt attccaaatc ctaatttgtt ttgtaataca gtcttcaaaa 360

gttaattgca aagaaattgt caaataaact tcccttcaat tggttcaggt ttcagtgatt 420

gtatttgttc aaacaatttt attttatcaa cgattttaac tgttgtgtaa acttcaattt 480

ttaaaaagat ttgacgtagt ataatttcag ttttctctaa aaagaaatat agtccaattg 540

gtgaatttgt ttgagtaatt gagcagttct taccttattc aagaatactc aatcaaaatt 600

tgttgtctca aaagtttcaa tcaacacaaa gcaaaaggaa caattaaata atttaattgt 660

ttccagtgag taccaattac aatacttgta ttcaaaccag tgtacaacca cttaagttga 720

actctataat ttccaaagtt tctttgttca atcgttgtca atagatatta ggtgtccttg 780

aagccttctt ctacctttgg aacacttaaa gcctattatt ttataacaaa ataacaattc 840

tttccaaaaa tagctttgtc aaggtgtgag cacaatataa atccacggca atttctttgc 900

acagagagaa cttgtgcttt agactagaat agccagtgat agtcgtcaga ttacttggga 960

ttcaatcaat caatcaattt tcaaaaattc tgccccaaat cacaattatt aagccaatta 1020

taacaaatat tttgattttt taattcagaa gttatcacaa ttatacagta gtatatcaac 1080

aatagcttgt ataaattgga attacaggtg gatacaaata agtgatagat cacaaaatct 1140

tatatacgta gaaataagat tttttggttg tggataaagt ttttttattg attattccca 1200

atcaattgta agtgcttctt gtattgggag attcagtcaa aaattgaggt aacttctttt 1260

taaaagctta atcaactcaa gttagactga aaactactga aattgggaaa ccttcgtata 1320

ggttttttta aagagatttc ccacctacct ttcaaagctt gttcaaatat ttgcactcta 1380

aaaattaccc accccacctt atcttaagcc ttcactgcaa ttctagcctc ttcttgtgag 1440

tcaaatcttt gaattgaaag aactgatatt cccttattgc tgttgccctt aaaattaaga 1500

gggtttaata atatattttt ctattattgg gttgtcaaaa tatacagtct ggtagaaatt 1560

caataataat aaaatttaaa cctatcaaaa aaactgtgtt cttaattacc aagccaatta 1620

gataaatttg tttatattgt aaagatacaa caaattactt ggactaatag aatcaaagat 1680

ctgtaactac tacaactcta gaccctgagt tgttggaccc cttagaactc tggggccacc 1740

tttgctgcac caatttcaca ggatttgcag tgtagtggct tcacagagag tgactccaac 1800

cagagaagca gcagtgttgt ctttgccaat ttggccaaag gaatattgta tttcaaaaat 1860

ttgtaactgt gtacacagac ttgatttttt acaaactaaa tttcccaata gtgtctgttt 1920

gtggagtaca cttggtattt tcttaaaatt gcctaatatt ttcagggaat tacagatata 1980

ttagaatcca attgatttaa tttactttca acccttgcag aaattcactg ttgcggtagg 2040

aaataagaag aaggtaaaac tgtttatttc caaaggtcct gcttttcagt gtaagagcaa 2100

agcaaacaaa aaattttgcc gcaatattgt gagctagcgt agataagtag catggcgggt 2160

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 2220

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 2280

cctcagtgag cgagcgagcg cgcag 2305

<210> 27

<211> 2305

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_PSG11_V1 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(245)

<223> multiple cloning site

<220>

<221> misc_feature

<222> (246)..(251)

<223> terminator

<220>

<221> misc_feature

<222> (252)..(2132)

<223> PSG11_V1

<220>

<221> misc_feature

<222> (2161)..(2305)

<223> 3'ITR

<400> 27

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacctacata agcaggtgat atgtaaattt cacctgctta 240

aatagttttt tacgaggtca gatgttgctt gtgatgaagg gtgtggttaa gtctgcagtg 300

cagatggtgg aagggatttc actctgtttt tagacatagc attggaacta agaattttat 360

gtctatatgg gtgaaggggc agggatgtgt aggaagacag ttctaattag gatggaggaa 420

ttatactagg aagtagaggt aaaggaagtg agaaggctta gtaaatagaa gaaatgtgaa 480

cttatcaaat aggctagtgt atacccctgg agattcttga ttaagtgaat taactagatt 540

ttagaaaggt aatgaggcag ttatatgcaa ggaacatttc aaagtctgga gacaagaagt 600

caagcaatac tttgagaagt ggttgagctt tatgggaatg gagagaatta cactattgag 660

atatgttaaa aataatgaag aaggtttcac catgtgaaat tgtgtttctc atttgaatct 720

gagaaacaaa tgagcagagg ctggagataa ttatgtctaa aagacagtgg ggtacaggga 780

gcataaaggc cagggagaaa gggaactgca ggaattagtg ctgagaagca ggagttagtg 840

gatgaaggag gagatccagg cccagataca ggcaaataag tcatttccct ctcccaagca 900

tggcagtcag ccctgcagga accaggataa gagaaaaatg gttggtaaaa actaacatac 960

caggcttgat tctttgcagt tagctcttta tctttctctc ccacaagtag tcagtagcct 1020

tgttctagtg ttttatgtgt tacctctttt tccggaaaaa ggaaggtact gttgagttaa 1080

tgatgatgat agtaatatag tagctgacat tggttcctct gtatgtggca tcttgtttcc 1140

catctaaatt gcacacttag aaacagttaa tggtaagcct tatattagat atatatagtg 1200

tctggtagtc ttaccctctc tataattaca cactttttgg cactgcccct ttcctgccat 1260

gcagagcccc aggggtgaat cttcctattt ctccagctct gcatcagtca ctgtactcag 1320

tgctgtgtcc cacgtgctgt gcccacagcc tcatacagcc agtgacttca gagccaggac 1380

gcagctcagg agtctgccct gaggttgctt ctcttcttat ttccttgcag cctggcccgg 1440

gggaggcttg gcttgaactg gcagctcaat ttagccaaat tcaggacagg ccaccaggac 1500

tctttctcca cacatgctgg tcccacccca ggttgagtga ggcagggcca gtcaccagag 1560

gagcccggag cagagcagga agcagagtct gagctgctcc tccctcaccc aaggggcttc 1620

ctcctctcat ttgggggaaa agtgtgagct tgtttcaaag cctcagatgt tccttgtagt 1680

tcatggaaga ggtacaagaa aacaaagacg tgacagaagg ggatgaggtg gtgacagtga 1740

gaagcaactc gatgttcagc actctccttc ttgtccctct gtgaagcctc ttctaccaca 1800

tagggctcag ggctgataaa gccccctccc tacctttctc aggccagaca caaggtcagt 1860

catgagaaaa cagaaaaaaa aggagaagag agtctgtaga gacaaattgg gagggttcag 1920

gaggagaatt tgggatttgc ctgtgcccat gggacacagg ctgggaataa aaatgttttc 1980

ctgactcttc tctgaaagct agatagactc cacctaaaac cctattgcca aggatgctgg 2040

gcccacagtt gatgtcatct gagggaaggg gaagctcctt gtctcttaaa gggacacagt 2100

gaccctctga gccaagacac accctcaagt ccgctagcgt agataagtag catggcgggt 2160

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 2220

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 2280

cctcagtgag cgagcgagcg cgcag 2305

<210> 28

<211> 2305

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_PSG11_V2 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(245)

<223> multiple cloning site

<220>

<221> misc_feature

<222> (246)..(251)

<223> terminator

<220>

<221> misc_feature

<222> (252)..(2132)

<223> PSG11_V2

<220>

<221> misc_feature

<222> (2161)..(2305)

<223> 3'ITR

<400> 28

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacctacata agcaggtgat atgtaaattt cacctgctta 240

aatagttttt tgtatacccc tggagattct tgattaagtg aattaactag attttagaaa 300

ggtattgagg cagttatttg caaggaacaa ttcaaagtct ggagacaaga agtcaagcaa 360

tactttgaga agtggttgag ctttttggga ttggagagaa ttacactatt gagatttgtt 420

aaaaatattg aagaaggttt caccttgtga aattgtgttt ctcaattgaa tctgagaaac 480

aattgagcag aggctggaga taattttgtc taaaagacag tggggtacag ggagcaaaaa 540

ggccagggag aaagggaact gcaggaatta gtgctgagaa gcaggagtta gtggttgaag 600

gaggagatcc aggcccagat acaggcaaat aagtcaattc cctctcccaa gcttggcagt 660

cagccctgca ggaaccagga taagagaaaa gaagaaaaga aaagaaggat tgaaggtgtt 720

gttattttac ttgggggagc gtcaggaaca agcttgtaac ttgagatact gtataattgt 780

ttcttcaagg actacaatat ttctgttgga aaattgttgg gagttgatta tattcttgca 840

gttttttttc cctctcacct tgtttctcag gtggtgatca gtcctctgtc aattctctac 900

tgcactcagg tattttggaa ggctttcaga cgtgagaaag gctgattgct attttctttg 960

tcagtggaac ttgtcacctt tgcacctttt cttggttgca actttttctc agtgtctctg 1020

tttggtaaaa actaacaaac caggcttgat tctttgcagt tagctcttta tctttctctc 1080

ccacaagtag tcagtagcct tgttctagtg ttttttgtgt tacctctttt tccggaaaaa 1140

ggaaggtact gttgagttat tgttgttgat agtaatatag tagctgacag tggttcctct 1200

gtttgtggca acttgtttcc cacctaaatt gcacacttag aaacagttat tggtaagcct 1260

tatattagat atatatagtg tctggtagtc ttaccctctc tataattaca cactttttgg 1320

cactgcccct ttcctgcctt gcagagcccc aggggtgaat cttcctattt ctccagctct 1380

gcaacagtca ctgtactcag tgctgtgtcc cacgtgctgt gcccacagcc tcaaacagcc 1440

agtgacttca gagccaggac gcagctcagg agtctgccct gaggttgctt ctcttcttat 1500

ttccttgcag cctggcccgg gggaggcttg gcttgaactg gcagctcaat ttagccaaat 1560

tcaggacagg ccaccaggac tctttctcca cacttgctgg tcccacccca ggttgagtga 1620

ggcagggcca gtcaccagag gagcccggag cagagcagga agcagagtct gagctgctcc 1680

tccctcaccc aaggggcttc ctcctctcaa ttgggggaaa agtgtgagct tgtttcaaag 1740

cctcagttgt tccttgtagt tcttggaaga ggtacaagaa aacaaagacg tgacagaagg 1800

ggttgaggtg gtgacagtga gaagcaactc gttgttcagc actctccttc ttgtccctct 1860

gtgaagcctc ttctaccaca aagggctcag ggctgataaa gccccctccc tacctttctc 1920

aggccagaca caaggtcagt cttgagaaaa cagaaaaaaa aggagaagag agtctgtaga 1980

gacaaattgg gcaaattgtt ttcctgactc ttctctgaaa gctagataga ctccacctaa 2040

aaccctattg ccaaggttgc tggggagaat aaagccacag ttgttgtcaa ctgagggaag 2100

gggaagctcc ttgtctctta aagggacaca gtgctagcgt agataagtag catggcgggt 2160

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 2220

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 2280

cctcagtgag cgagcgagcg cgcag 2305

<210> 29

<211> 2410

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 1-1_1784_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (351)..(356)

<223> terminator

<220>

<221> misc_feature

<222> (357)..(2237)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2266)..(2410)

<223> 3'ITR

<400> 29

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacatgcaga ctgcctgctt gggtacagac 240

caaagagtag tcgaattatg gacctgctaa gctaattaac tactctttgg tctgaactca 300

ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc tttttttatt 360

ttgcaaatca caaaatttta tttaaaaatc aattagattt tgattgcctg aagtacacaa 420

ccagtattcc aaatcctaat ttgttttgta atacagtctt caaaagttaa ttgcaaagaa 480

attgtcaaat aaacttccct tcaattggtt caggtttcag tgattgtatt tgttcaaaca 540

attttatttt atcaaggatt ttaactgttg tgtaaacttc aatttttaaa aagatttgac 600

ctagtataat ttcagttttc tctaaaaaga aatatagtcc aattggtgaa tttgtttgag 660

taattgagca gttcttacct tattcaagaa tactcaatca aaatttgttg tctcaaaagt 720

ttcaatcaac acaaagcaaa aggaacaatt aaataattta attgtttcca gtgagtacca 780

attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct ataatttcca 840

aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc ttcttctacc 900

tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc aaaaatagct 960

ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga gagaacttgt 1020

gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa tcaatcaatc 1080

aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca aatattttga 1140

ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag cttgtataaa 1200

ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat acctagaaat 1260

aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa ttgtaagtgc 1320

ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa gcttaatcaa 1380

ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt ttttaaagag 1440

atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat tacccacccc 1500

accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa tctttgaatt 1560

gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt taataatata 1620

tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata ataataaaat 1680

ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa atttgtttat 1740

attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta actactacaa 1800

ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc tgcaccaatt 1860

tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag aagcagcagt 1920

gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta actgtgtaca 1980

cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga gtacacttgg 2040

tattttctta aaattgccta atattttcag ggaattacag atatattaga atccaattga 2100

tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata agaagaaggt 2160

aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa acaaaaaatt 2220

ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc attaactaca 2280

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 2340

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 2400

gagcgcgcag 2410

<210> 30

<211> 2368

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 1-1_1784_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(2195)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2224)..(2368)

<223> 3'ITR

<400> 30

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacatgcaga ctgcctgctt gggtacagac 240

caaagagtag tcgaattatg gacctgctaa gctaattaac tactctttgg tctgaactca 300

ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc tttttttacc 360

aggtcagttg ttgcttgtga agaagggtgt ggttaagtct gcagtgcaga aggtggaagg 420

gatttcactc tgtttttaga gataggattg gaactaagaa ttttaagtct atttgggtga 480

aggggcaggg aagtgtagga agacagttct aattaggaag gaggaattat actaggaagt 540

agaggtaaag gaagtgagaa ggcttagtaa atagaagaaa tctgaactta tcaaataggc 600

tagtgtatac ccctggagat tcttgattaa gtgaattaac tagattttag aaaggtattg 660

aggcagttat ttgcaaggaa caattcaaag tctggagaca agaagtcaag caatactttg 720

agaagtggtt gagctttttg ggattggaga gaattacact attgagattt gttaaaaata 780

ttgaagaagg tttcaccttg tgaaattgtg tttctcaatt gaatctgaga aacaattgag 840

cagaggctgg agataatttt gtctaaaaga cagtggggta cagggagcaa aaaggccagg 900

gagaaaggga actgcaggaa ttagtgctga gaagcaggag ttagtggttg aaggaggaga 960

tccaggccca gatacaggca aataagtcaa ttccctctcc caagcttggc agtcagccct 1020

gcaggaacca ggataagaga aattgtttgg taaaaactaa caaaccaggc ttgattcttt 1080

gcagttagct ctttatcttt ctctcccaca agtagtcagt agccttgttc tagtgttttt 1140

tgtgttacct ctttttcctg aaaaaggaag gtactgttga gttattgttg ttgatagtaa 1200

tatagtagct gacagtggtt cctctgtttg tggcaacttg tttcccacct aaattgcaca 1260

cttagaaaca gttattggta agccttatat tagatatata tagtgtctgg tagtcttacc 1320

ctctctataa ttacacactt tttggcactg cccctttcct gccttgcaga gccccagggg 1380

tgaatcttcc tatttctcca gctctgcaac agtcactgta ctcagtgctg tgtcccacct 1440

gctgtgccca cagcctcaaa cagccagtga cttcagagcc aggaaacagc tcaggagtct 1500

gccctgaggt tgcttctctt cttatttcct tgcagcctgg ccaaggggag gcttggcttg 1560

aactggcagc tcaatttagc caaattcagg acaggccacc aggactcttt ctccacactt 1620

gctggtccca ccccaggttg agtgaggcag ggccagtcac cagaggagcc cagagcagag 1680

caggaagcag agtctgagct gctcctccct cacccaaggg gcttcctcct ctcaattggg 1740

ggaaaagtgt gagcttgttt caaagcctca gttgttcctt gtagttcttg gaagaggtac 1800

aagaaaacaa agacctgaca gaaggggttg aggtggtgac agtgagaagc aactggttgt 1860

tcagcactct ccttcttgtc cctctgtgaa gcctcttcta cccaaagggc tcagggctga 1920

taaagccccc tccctacctt tctcaggcca gacacaaggt cagtcttgag aaaacagaaa 1980

aaaaaggaga agagagtctg tagagacaaa ttgggagggt tcaggaggag aatttgggat 2040

ttgcctgtgc ccaagggaca caggctggga atcaaattgt tttcctgact cttctctgaa 2100

agctagatag actccaccta aaaccctatt gccaaggttg ctgggccaca gttgttgtca 2160

actgagggaa ggggaagctc cttgtctctt aaagggctag cgtagataag tagcatggcg 2220

ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg 2280

ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 2340

cggcctcagt gagcgagcga gcgcgcag 2368

<210> 31

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 1-1_1784_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(579)

<223> SV40 termination

<220>

<221> misc_feature

<222> (580)..(2187)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 31

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacatgcaga ctgcctgctt gggtacagac 240

caaagagtag tcgaattatg gacctgctaa gctaattaac tactctttgg tctgaactca 300

ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc tttttttcag 360

acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 420

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 480

aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg 540

aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatatagtcc aattggtgaa 600

tttgtttgag taattgagca gttcttacct tattcaagaa tactcaatca aaatttgttg 660

tctcaaaagt ttcaatcaac acaaagcaaa aggaacaatt aaataattta attgtttcca 720

gtgagtacca attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct 780

ataatttcca aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc 840

ttcttctacc tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc 900

aaaaatagct ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga 960

gagaacttgt gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa 1020

tcaatcaatc aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca 1080

aatattttga ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag 1140

cttgtataaa ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat 1200

acctagaaat aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa 1260

ttgtaagtgc ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa 1320

gcttaatcaa ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt 1380

ttttaaagag atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat 1440

tacccacccc accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa 1500

tctttgaatt gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt 1560

taataatata tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata 1620

ataataaaat ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa 1680

atttgtttat attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta 1740

actactacaa ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc 1800

tgcaccaatt tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag 1860

aagcagcagt gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta 1920

actgtgtaca cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga 1980

gtacacttgg tattttctta aaattgccta atattttcag ggaattacag atatattaga 2040

atccaattga tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata 2100

agaagaaggt aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa 2160

acaaaaaatt ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 32

<211> 2369

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 1-1_1784_sv40_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(579)

<223> SV40 termination

<220>

<221> misc_feature

<222> (580)..(2196)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2225)..(2369)

<223> 3'ITR

<400> 32

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacatgcaga ctgcctgctt gggtacagac 240

caaagagtag tcgaattatg gacctgctaa gctaattaac tactctttgg tctgaactca 300

ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc tttttttcag 360

acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 420

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 480

aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg 540

aggtttttta aagcaagtaa aacctctaca aatgtggtaa ccaggtcagt tgttgcttgt 600

gaagaagggt gtggttaagt ctgcagtgca gaaggtggaa gggatttcac tctgttttta 660

gagataggat tggaactaag aattttaagt ctatttgggt gaaggggcag ggaagtgtag 720

gaagacagtt ctaattagga aggaggaatt atactaggaa gtagaggtaa aggaagtgag 780

aaggcttagt aaatagaaga aatctgaact tatcaaatag gctagtgtat acccctggag 840

attcttgatt aagtgaatta actagatttt agaaaggtat tgaggcagtt atttgcaagg 900

aacaattcaa agtctggaga caagaagtca agcaatactt tgagaagtgg ttgagctttt 960

tgggattgga gagaattaca ctattgagat ttgttaaaaa tattgaagaa ggtttcacct 1020

tgtgaaattg tgtttctcaa ttgaatctga gaaacaattg agcagaggct ggagataatt 1080

ttgtctaaaa gacagtgggg tacagggagc aaaaaggcca gggagaaagg gaactgcagg 1140

aattagtgct gagaagcagg agttagtggt tgaaggagga gatccaggcc cagatacagg 1200

caaataagtc aattccctct cccaagcttg gcagtcagcc ctgcaggaac caggataaga 1260

gaaattgttt ggtaaaaact aacaaaccag gcttgattct ttgcagttag ctctttatct 1320

ttctctccca caagtagtca gtagccttgt tctagtgttt tttgtgttac ctctttttcc 1380

tgaaaaagga aggtactgtt gagttattgt tgttgatagt aatatagtag ctgacagtgg 1440

ttcctctgtt tgtggcaact tgtttcccac ctaaattgca cacttagaaa cagttattgg 1500

taagccttat attagatata tatagtgtct ggtagtctta ccctctctat aattacacac 1560

tttttggcac tgcccctttc ctgccttgca gagccccagg ggtgaatctt cctatttctc 1620

cagctctgca acagtcactg tactcagtgc tgtgtcccac ctgctgtgcc cacagcctca 1680

aacagccagt gacttcagag ccaggaaaca gctcaggagt ctgccctgag gttgcttctc 1740

ttcttatttc cttgcagcct ggccaagggg aggcttggct tgaactggca gctcaattta 1800

gccaaattca ggacaggcca ccaggactct ttctccacac ttgctggtcc caccccaggt 1860

tgagtgaggc agggccagtc accagaggag cccagagcag agcaggaagc agagtctgag 1920

ctgctcctcc ctcacccaag gggcttcctc ctctcaattg ggggaaaagt gtgagcttgt 1980

ttcaaagcct cagttgttcc ttgtagttct tggaagaggt acaagaaaac aaagacctga 2040

cagaaggggt tgaggtggtg acagtgagaa gcaactggtt gttcagcact ctccttcttg 2100

tccctctgtg aagcctcttc taccacaaag ggctcagggc tgataaagcc ccctccctac 2160

ctttctcagg ccagacacaa ggtcagtctt gagaaagcta gcgtagataa gtagcatggc 2220

gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc 2280

gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 2340

gcggcctcag tgagcgagcg agcgcgcag 2369

<210> 33

<211> 2410

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 100_3330_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR100_3330

<220>

<221> misc_feature

<222> (351)..(356)

<223> terminator

<220>

<221> misc_feature

<222> (357)..(2237)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2266)..(2410)

<223> 3'ITR

<400> 33

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacccaaaag agagaagata ttgaggcctg 240

ttgccacata tgctgagact gataatgtgg gtattagtcc gccacatcat ccgtctcaac 300

atttgtgtct gttaggcaat ctcacggacc tggggctttg cttatatgcc tttttttatt 360

ttgcaaatca caaaatttta tttaaaaatc aattagattt tgattgcctg aagtacacaa 420

ccagtattcc aaatcctaat ttgttttgta atacagtctt caaaagttaa ttgcaaagaa 480

attgtcaaat aaacttccct tcaattggtt caggtttcag tgattgtatt tgttcaaaca 540

attttatttt atcaaggatt ttaactgttg tgtaaacttc aatttttaaa aagatttgac 600

ctagtataat ttcagttttc tctaaaaaga aatatagtcc aattggtgaa tttgtttgag 660

taattgagca gttcttacct tattcaagaa tactcaatca aaatttgttg tctcaaaagt 720

ttcaatcaac acaaagcaaa aggaacaatt aaataattta attgtttcca gtgagtacca 780

attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct ataatttcca 840

aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc ttcttctacc 900

tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc aaaaatagct 960

ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga gagaacttgt 1020

gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa tcaatcaatc 1080

aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca aatattttga 1140

ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag cttgtataaa 1200

ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat acctagaaat 1260

aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa ttgtaagtgc 1320

ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa gcttaatcaa 1380

ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt ttttaaagag 1440

atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat tacccacccc 1500

accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa tctttgaatt 1560

gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt taataatata 1620

tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata ataataaaat 1680

ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa atttgtttat 1740

attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta actactacaa 1800

ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc tgcaccaatt 1860

tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag aagcagcagt 1920

gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta actgtgtaca 1980

cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga gtacacttgg 2040

tattttctta aaattgccta atattttcag ggaattacag atatattaga atccaattga 2100

tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata agaagaaggt 2160

aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa acaaaaaatt 2220

ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc attaactaca 2280

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 2340

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 2400

gagcgcgcag 2410

<210> 34

<211> 2368

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 100_3330_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR100_3330

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(2195)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2224)..(2368)

<223> 3'ITR

<400> 34

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacccaaaag agagaagata ttgaggcctg 240

ttgccacata tgctgagact gataatgtgg gtattagtcc gccacatcat ccgtctcaac 300

atttgtgtct gttaggcaat ctcacggacc tggggctttg cttatatgcc tttttttacc 360

aggtcagttg ttgcttgtga agaagggtgt ggttaagtct gcagtgcaga aggtggaagg 420

gatttcactc tgtttttaga gataggattg gaactaagaa ttttaagtct atttgggtga 480

aggggcaggg aagtgtagga agacagttct aattaggaag gaggaattat actaggaagt 540

agaggtaaag gaagtgagaa ggcttagtaa atagaagaaa tctgaactta tcaaataggc 600

tagtgtatac ccctggagat tcttgattaa gtgaattaac tagattttag aaaggtattg 660

aggcagttat ttgcaaggaa caattcaaag tctggagaca agaagtcaag caatactttg 720

agaagtggtt gagctttttg ggattggaga gaattacact attgagattt gttaaaaata 780

ttgaagaagg tttcaccttg tgaaattgtg tttctcaatt gaatctgaga aacaattgag 840

cagaggctgg agataatttt gtctaaaaga cagtggggta cagggagcaa aaaggccagg 900

gagaaaggga actgcaggaa ttagtgctga gaagcaggag ttagtggttg aaggaggaga 960

tccaggccca gatacaggca aataagtcaa ttccctctcc caagcttggc agtcagccct 1020

gcaggaacca ggataagaga aattgtttgg taaaaactaa caaaccaggc ttgattcttt 1080

gcagttagct ctttatcttt ctctcccaca agtagtcagt agccttgttc tagtgttttt 1140

tgtgttacct ctttttcctg aaaaaggaag gtactgttga gttattgttg ttgatagtaa 1200

tatagtagct gacagtggtt cctctgtttg tggcaacttg tttcccacct aaattgcaca 1260

cttagaaaca gttattggta agccttatat tagatatata tagtgtctgg tagtcttacc 1320

ctctctataa ttacacactt tttggcactg cccctttcct gccttgcaga gccccagggg 1380

tgaatcttcc tatttctcca gctctgcaac agtcactgta ctcagtgctg tgtcccacct 1440

gctgtgccca cagcctcaaa cagccagtga cttcagagcc aggaaacagc tcaggagtct 1500

gccctgaggt tgcttctctt cttatttcct tgcagcctgg ccaaggggag gcttggcttg 1560

aactggcagc tcaatttagc caaattcagg acaggccacc aggactcttt ctccacactt 1620

gctggtccca ccccaggttg agtgaggcag ggccagtcac cagaggagcc cagagcagag 1680

caggaagcag agtctgagct gctcctccct cacccaaggg gcttcctcct ctcaattggg 1740

ggaaaagtgt gagcttgttt caaagcctca gttgttcctt gtagttcttg gaagaggtac 1800

aagaaaacaa agacctgaca gaaggggttg aggtggtgac agtgagaagc aactggttgt 1860

tcagcactct ccttcttgtc cctctgtgaa gcctcttcta cccaaagggc tcagggctga 1920

taaagccccc tccctacctt tctcaggcca gacacaaggt cagtcttgag aaaacagaaa 1980

aaaaaggaga agagagtctg tagagacaaa ttgggagggt tcaggaggag aatttgggat 2040

ttgcctgtgc ccaagggaca caggctggga atcaaattgt tttcctgact cttctctgaa 2100

agctagatag actccaccta aaaccctatt gccaaggttg ctgggccaca gttgttgtca 2160

actgagggaa ggggaagctc cttgtctctt aaagggctag cgtagataag tagcatggcg 2220

ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg 2280

ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg 2340

cggcctcagt gagcgagcga gcgcgcag 2368

<210> 35

<211> 2360

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 100_3330_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR100_3330

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(579)

<223> SV40 termination

<220>

<221> misc_feature

<222> (580)..(2187)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2216)..(2360)

<223> 3'ITR

<400> 35

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacccaaaag agagaagata ttgaggcctg 240

ttgccacata tgctgagact gataatgtgg gtattagtcc gccacatcat ccgtctcaac 300

atttgtgtct gttaggcaat ctcacggacc tggggctttg cttatatgcc tttttttcag 360

acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 420

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 480

aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg 540

aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatatagtcc aattggtgaa 600

tttgtttgag taattgagca gttcttacct tattcaagaa tactcaatca aaatttgttg 660

tctcaaaagt ttcaatcaac acaaagcaaa aggaacaatt aaataattta attgtttcca 720

gtgagtacca attacaatac ttgtattcaa accagtgtac aaccacttaa gttgaactct 780

ataatttcca aagtttcttt gttcaatcct tgtcaataga tattaggtgt ccttgaagcc 840

ttcttctacc tttggaacac ttaaagccta ttattttata acaaaataac aattctttcc 900

aaaaatagct ttgtcaaggt gtgagcacaa tataaatcca gggcaatttc tttgcacaga 960

gagaacttgt gctttagact agaatagcca gtgatagtcc tcagattact tgggattcaa 1020

tcaatcaatc aattttcaaa aattctgccc caaatcacaa ttattaagcc aattataaca 1080

aatattttga ttttttaatt cagaagttat cacaattata cagtagtata tcaacaatag 1140

cttgtataaa ttggaattac aggtggatac aaataagtga tagatcacaa aatcttatat 1200

acctagaaat aagatttttt ggttgtggat aaagtttttt tattgattat tcccaatcaa 1260

ttgtaagtgc ttcttgtatt gggagattca gtcaaaaatt gaggtaactt ctttttaaaa 1320

gcttaatcaa ctcaagttag actgaaaact actgaaattg ggaaaccttc ctataggttt 1380

ttttaaagag atttcccacc tacctttcaa agcttgttca aatatttgca ctctaaaaat 1440

tacccacccc accttatctt aagccttcac tgcaattcta gcctcttctt gtgagtcaaa 1500

tctttgaatt gaaagaactg atattccctt attgctgttg cccttaaaat taagagggtt 1560

taataatata tttttctatt attgggttgt caaaatatac agtctggtag aaattcaata 1620

ataataaaat ttaaacctat caaaaaaact gtgttcttaa ttaccaagcc aattagataa 1680

atttgtttat attgtaaaga tacaacaaat tacttggact aatagaatca aagatctgta 1740

actactacaa ctctagaccc tgagttgttg gaccccttag aactctgggg ccacctttgc 1800

tgcaccaatt tcacaggatt tgcagtgtag tggcttcaca gagagtgact ccaaccagag 1860

aagcagcagt gttgtctttg ccaatttggc caaaggaata ttgtatttca aaaatttgta 1920

actgtgtaca cagacttgat tttttacaaa ctaaatttcc caatagtgtc tgtttgtgga 1980

gtacacttgg tattttctta aaattgccta atattttcag ggaattacag atatattaga 2040

atccaattga tttaatttac tttcaaccct tgcagaaatt cactgttggg gtaggaaata 2100

agaagaaggt aaaactgttt atttccaaag gtcctgcttt tcagtgtaag agcaaagcaa 2160

acaaaaaatt ttgccccaat attgtgagct agcgtagata agtagcatgg cgggttaatc 2220

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 2280

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 2340

gtgagcgagc gagcgcgcag 2360

<210> 36

<211> 2369

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_natural_mir 100_3330_sv40_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(350)

<223> miR100_3330

<220>

<221> misc_feature

<222> (351)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(579)

<223> SV40 termination

<220>

<221> misc_feature

<222> (580)..(2196)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2225)..(2369)

<223> 3'ITR

<400> 36

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cacccaaaag agagaagata ttgaggcctg 240

ttgccacata tgctgagact gataatgtgg gtattagtcc gccacatcat ccgtctcaac 300

atttgtgtct gttaggcaat ctcacggacc tggggctttg cttatatgcc tttttttcag 360

acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 420

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 480

aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg 540

aggtttttta aagcaagtaa aacctctaca aatgtggtaa ccaggtcagt tgttgcttgt 600

gaagaagggt gtggttaagt ctgcagtgca gaaggtggaa gggatttcac tctgttttta 660

gagataggat tggaactaag aattttaagt ctatttgggt gaaggggcag ggaagtgtag 720

gaagacagtt ctaattagga aggaggaatt atactaggaa gtagaggtaa aggaagtgag 780

aaggcttagt aaatagaaga aatctgaact tatcaaatag gctagtgtat acccctggag 840

attcttgatt aagtgaatta actagatttt agaaaggtat tgaggcagtt atttgcaagg 900

aacaattcaa agtctggaga caagaagtca agcaatactt tgagaagtgg ttgagctttt 960

tgggattgga gagaattaca ctattgagat ttgttaaaaa tattgaagaa ggtttcacct 1020

tgtgaaattg tgtttctcaa ttgaatctga gaaacaattg agcagaggct ggagataatt 1080

ttgtctaaaa gacagtgggg tacagggagc aaaaaggcca gggagaaagg gaactgcagg 1140

aattagtgct gagaagcagg agttagtggt tgaaggagga gatccaggcc cagatacagg 1200

caaataagtc aattccctct cccaagcttg gcagtcagcc ctgcaggaac caggataaga 1260

gaaattgttt ggtaaaaact aacaaaccag gcttgattct ttgcagttag ctctttatct 1320

ttctctccca caagtagtca gtagccttgt tctagtgttt tttgtgttac ctctttttcc 1380

tgaaaaagga aggtactgtt gagttattgt tgttgatagt aatatagtag ctgacagtgg 1440

ttcctctgtt tgtggcaact tgtttcccac ctaaattgca cacttagaaa cagttattgg 1500

taagccttat attagatata tatagtgtct ggtagtctta ccctctctat aattacacac 1560

tttttggcac tgcccctttc ctgccttgca gagccccagg ggtgaatctt cctatttctc 1620

cagctctgca acagtcactg tactcagtgc tgtgtcccac ctgctgtgcc cacagcctca 1680

aacagccagt gacttcagag ccaggaaaca gctcaggagt ctgccctgag gttgcttctc 1740

ttcttatttc cttgcagcct ggccaagggg aggcttggct tgaactggca gctcaattta 1800

gccaaattca ggacaggcca ccaggactct ttctccacac ttgctggtcc caccccaggt 1860

tgagtgaggc agggccagtc accagaggag cccagagcag agcaggaagc agagtctgag 1920

ctgctcctcc ctcacccaag gggcttcctc ctctcaattg ggggaaaagt gtgagcttgt 1980

ttcaaagcct cagttgttcc ttgtagttct tggaagaggt acaagaaaac aaagacctga 2040

cagaaggggt tgaggtggtg acagtgagaa gcaactggtt gttcagcact ctccttcttg 2100

tccctctgtg aagcctcttc taccacaaag ggctcagggc tgataaagcc ccctccctac 2160

ctttctcagg ccagacacaa ggtcagtctt gagaaagcta gcgtagataa gtagcatggc 2220

gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc 2280

gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg 2340

gcggcctcag tgagcgagcg agcgcgcag 2369

<210> 37

<211> 2401

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 1-1_1784_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (342)..(347)

<223> terminator

<220>

<221> misc_feature

<222> (348)..(2228)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2257)..(2401)

<223> 3' ITR

<400> 37

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc ctttttttat tttgcaaatc 360

acaaaatttt atttaaaaat caattagatt ttgattgcct gaagtacaca accagtattc 420

caaatcctaa tttgttttgt aatacagtct tcaaaagtta attgcaaaga aattgtcaaa 480

taaacttccc ttcaattggt tcaggtttca gtgattgtat ttgttcaaac aattttattt 540

tatcaaggat tttaactgtt gtgtaaactt caatttttaa aaagatttga cctagtataa 600

tttcagtttt ctctaaaaag aaatatagtc caattggtga atttgtttga gtaattgagc 660

agttcttacc ttattcaaga atactcaatc aaaatttgtt gtctcaaaag tttcaatcaa 720

cacaaagcaa aaggaacaat taaataattt aattgtttcc agtgagtacc aattacaata 780

cttgtattca aaccagtgta caaccactta agttgaactc tataatttcc aaagtttctt 840

tgttcaatcc ttgtcaatag atattaggtg tccttgaagc cttcttctac ctttggaaca 900

cttaaagcct attattttat aacaaaataa caattctttc caaaaatagc tttgtcaagg 960

tgtgagcaca atataaatcc agggcaattt ctttgcacag agagaacttg tgctttagac 1020

tagaatagcc agtgatagtc ctcagattac ttgggattca atcaatcaat caattttcaa 1080

aaattctgcc ccaaatcaca attattaagc caattataac aaatattttg attttttaat 1140

tcagaagtta tcacaattat acagtagtat atcaacaata gcttgtataa attggaatta 1200

caggtggata caaataagtg atagatcaca aaatcttata tacctagaaa taagattttt 1260

tggttgtgga taaagttttt ttattgatta ttcccaatca attgtaagtg cttcttgtat 1320

tgggagattc agtcaaaaat tgaggtaact tctttttaaa agcttaatca actcaagtta 1380

gactgaaaac tactgaaatt gggaaacctt cctataggtt tttttaaaga gatttcccac 1440

ctacctttca aagcttgttc aaatatttgc actctaaaaa ttacccaccc caccttatct 1500

taagccttca ctgcaattct agcctcttct tgtgagtcaa atctttgaat tgaaagaact 1560

gatattccct tattgctgtt gcccttaaaa ttaagagggt ttaataatat atttttctat 1620

tattgggttg tcaaaatata cagtctggta gaaattcaat aataataaaa tttaaaccta 1680

tcaaaaaaac tgtgttctta attaccaagc caattagata aatttgttta tattgtaaag 1740

atacaacaaa ttacttggac taatagaatc aaagatctgt aactactaca actctagacc 1800

ctgagttgtt ggacccctta gaactctggg gccacctttg ctgcaccaat ttcacaggat 1860

ttgcagtgta gtggcttcac agagagtgac tccaaccaga gaagcagcag tgttgtcttt 1920

gccaatttgg ccaaaggaat attgtatttc aaaaatttgt aactgtgtac acagacttga 1980

ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg agtacacttg gtattttctt 2040

aaaattgcct aatattttca gggaattaca gatatattag aatccaattg atttaattta 2100

ctttcaaccc ttgcagaaat tcactgttgg ggtaggaaat aagaagaagg taaaactgtt 2160

tatttccaaa ggtcctgctt ttcagtgtaa gagcaaagca aacaaaaaat tttgccccaa 2220

tattgtgagc tagcgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc 2280

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 2340

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 2400

g 2401

<210> 38

<211> 2402

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 1-1_1784_psg11_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR-1-1 1784

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(2229)

<223> PSG11_V3

<220>

<221> misc_feature

<222> (2258)..(2402)

<223> 3'ITR

<400> 38

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc ctttttttgt atacccctgg 360

agattcttga ttaagtgaat taactagatt ttagaaaggt attgaggcag ttatttgcaa 420

ggaacaattc aaagtctgga gacaagaagt caagcaatac tttgagaagt ggttgagctt 480

tttgggattg gagagaatta cactattgag atttgttaaa aatattgaag aaggtttcac 540

cttgtgaaat tgtgtttctc aattgaatct gagaaacaat tgagcagagg ctggagataa 600

ttttgtctaa aagacagtgg ggtacaggga gcaaaaaggc cagggagaaa gggaactgca 660

ggaattagtg ctgagaagca ggagttagtg gttgaaggag gagatccagg cccagataca 720

ggcaaataag tcaattccct ctcccaagct tggcagtcag ccctgcagga accaggataa 780

gagaaaagaa gaaaagaaaa gaaggattga aggtgttgtt attttacttg ggggagcctc 840

aggaacaagc ttgtaacttg agatactgta taattgtttc ttcaaggact acaatatttc 900

tgttggaaaa ttgttgggag ttgattatat tcttgcagtt ttttttccct ctcaccttgt 960

ttctcaggtg gtgatcagtc ctctgtcaat tctctactgc actcaggtat tttggaaggc 1020

tttcagaggt gagaaaggct gattgctatt ttctttgtca gtggaacttg tcacctttgc 1080

accttttctt ggttgcaact ttttctcagt gtctctgttt ggtaaaaact aacaaaccag 1140

gcttgattct ttgcagttag ctctttatct ttctctccca caagtagtca gtagccttgt 1200

tctagtgttt tttgtgttac ctctttttcc tgaaaaagga aggtactgtt gagttattgt 1260

tgttgatagt aatatagtag ctgacagtgg ttcctctgtt tgtggcaact tgtttcccac 1320

ctaaattgca cacttagaaa cagttattgg taagccttat attagatata tatagtgtct 1380

ggtagtctta ccctctctat aattacacac tttttggcac tgcccctttc ctgccttgca 1440

gagccccagg ggtgaatctt cctatttctc cagctctgca acagtcactg tactcagtgc 1500

tgtgtcccac ctgctgtgcc cacagcctca aacagccagt gacttcagag ccaggaccca 1560

gctcaggagt ctgccctgag gttgcttctc ttcttatttc cttgcagcct ggccctgggg 1620

aggcttggct tgaactggca gctcaattta gccaaattca ggacaggcca ccaggactct 1680

ttctccacac ttgctggtcc caccccaggt tgagtgaggc agggccagtc accagaggag 1740

ccctgagcag agcaggaagc agagtctgag ctgctcctcc ctcacccaag gggcttcctc 1800

ctctcaattg ggggaaaagt gtgagcttgt ttcaaagcct cagttgttcc ttgtagttct 1860

tggaagaggt acaagaaaac aaagacctga cagaaggggt tgaggtggtg acagtgagaa 1920

gcaactggtt gttcagcact ctccttcttg tccctctgtg aagcctcttc taccacaaag 1980

ggctcagggc tgataaagcc ccctccctac ctttctcagg ccagacacaa ggtcagtctt 2040

gagaaaacag aaaaaaaagg agaagagagt ctgtagagac aaattgggca aattgttttc 2100

ctgactcttc tctgaaagct agatagactc cacctaaaac cctattgcca aggttgctgg 2160

ggagaataaa gccacagttg ttgtcaactg agggaagggg aagctccttg tctcttaaag 2220

ggacacagtg ctagcgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 2280

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 2340

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 2400

ag 2402

<210> 39

<211> 2359

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 1-1_1784_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(2186)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2215)..(2359)

<223> 3'ITR

<400> 39

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc ctttttttac caggtcagtt 360

gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag aaggtggaag ggatttcact 420

ctgtttttag agataggatt ggaactaaga attttaagtc tatttgggtg aaggggcagg 480

gaagtgtagg aagacagttc taattaggaa ggaggaatta tactaggaag tagaggtaaa 540

ggaagtgaga aggcttagta aatagaagaa atctgaactt atcaaatagg ctagtgtata 600

cccctggaga ttcttgatta agtgaattaa ctagatttta gaaaggtatt gaggcagtta 660

tttgcaagga acaattcaaa gtctggagac aagaagtcaa gcaatacttt gagaagtggt 720

tgagcttttt gggattggag agaattacac tattgagatt tgttaaaaat attgaagaag 780

gtttcacctt gtgaaattgt gtttctcaat tgaatctgag aaacaattga gcagaggctg 840

gagataattt tgtctaaaag acagtggggt acagggagca aaaaggccag ggagaaaggg 900

aactgcagga attagtgctg agaagcagga gttagtggtt gaaggaggag atccaggccc 960

agatacaggc aaataagtca attccctctc ccaagcttgg cagtcagccc tgcaggaacc 1020

aggataagag aaattgtttg gtaaaaacta acaaaccagg cttgattctt tgcagttagc 1080

tctttatctt tctctcccac aagtagtcag tagccttgtt ctagtgtttt ttgtgttacc 1140

tctttttcct gaaaaaggaa ggtactgttg agttattgtt gttgatagta atatagtagc 1200

tgacagtggt tcctctgttt gtggcaactt gtttcccacc taaattgcac acttagaaac 1260

agttattggt aagccttata ttagatatat atagtgtctg gtagtcttac cctctctata 1320

attacacact ttttggcact gcccctttcc tgccttgcag agccccaggg gtgaatcttc 1380

ctatttctcc agctctgcaa cagtcactgt actcagtgct gtgtcccacc tgctgtgccc 1440

acagcctcaa acagccagtg acttcagagc caggaaacag ctcaggagtc tgccctgagg 1500

ttgcttctct tcttatttcc ttgcagcctg gccaagggga ggcttggctt gaactggcag 1560

ctcaatttag ccaaattcag gacaggccac caggactctt tctccacact tgctggtccc 1620

accccaggtt gagtgaggca gggccagtca ccagaggagc ccagagcaga gcaggaagca 1680

gagtctgagc tgctcctccc tcacccaagg ggcttcctcc tctcaattgg gggaaaagtg 1740

tgagcttgtt tcaaagcctc agttgttcct tgtagttctt ggaagaggta caagaaaaca 1800

aagacctgac agaaggggtt gaggtggtga cagtgagaag caactggttg ttcagcactc 1860

tccttcttgt ccctctgtga agcctcttct acccaaaggg ctcagggctg ataaagcccc 1920

ctccctacct ttctcaggcc agacacaagg tcagtcttga gaaaacagaa aaaaaaggag 1980

aagagagtct gtagagacaa attgggaggg ttcaggagga gaatttggga tttgcctgtg 2040

cccaagggac acaggctggg aatcaaattg ttttcctgac tcttctctga aagctagata 2100

gactccacct aaaaccctat tgccaaggtt gctgggccac agttgttgtc aactgaggga 2160

aggggaagct ccttgtctct taaagggcta gcgtagataa gtagcatggc gggttaatca 2220

ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 2280

tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 2340

tgagcgagcg agcgcgcag 2359

<210> 40

<211> 2351

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 1-1_1784_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(570)

<223> SV40 termination

<220>

<221> misc_feature

<222> (571)..(2178)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2207)..(2351)

<223> 3' ITR

<400> 40

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc ctttttttca gacatgataa 360

gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 420

gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 480

acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt 540

aaagcaagta aaacctctac aaatgtggta aaatatagtc caattggtga atttgtttga 600

gtaattgagc agttcttacc ttattcaaga atactcaatc aaaatttgtt gtctcaaaag 660

tttcaatcaa cacaaagcaa aaggaacaat taaataattt aattgtttcc agtgagtacc 720

aattacaata cttgtattca aaccagtgta caaccactta agttgaactc tataatttcc 780

aaagtttctt tgttcaatcc ttgtcaatag atattaggtg tccttgaagc cttcttctac 840

ctttggaaca cttaaagcct attattttat aacaaaataa caattctttc caaaaatagc 900

tttgtcaagg tgtgagcaca atataaatcc agggcaattt ctttgcacag agagaacttg 960

tgctttagac tagaatagcc agtgatagtc ctcagattac ttgggattca atcaatcaat 1020

caattttcaa aaattctgcc ccaaatcaca attattaagc caattataac aaatattttg 1080

attttttaat tcagaagtta tcacaattat acagtagtat atcaacaata gcttgtataa 1140

attggaatta caggtggata caaataagtg atagatcaca aaatcttata tacctagaaa 1200

taagattttt tggttgtgga taaagttttt ttattgatta ttcccaatca attgtaagtg 1260

cttcttgtat tgggagattc agtcaaaaat tgaggtaact tctttttaaa agcttaatca 1320

actcaagtta gactgaaaac tactgaaatt gggaaacctt cctataggtt tttttaaaga 1380

gatttcccac ctacctttca aagcttgttc aaatatttgc actctaaaaa ttacccaccc 1440

caccttatct taagccttca ctgcaattct agcctcttct tgtgagtcaa atctttgaat 1500

tgaaagaact gatattccct tattgctgtt gcccttaaaa ttaagagggt ttaataatat 1560

atttttctat tattgggttg tcaaaatata cagtctggta gaaattcaat aataataaaa 1620

tttaaaccta tcaaaaaaac tgtgttctta attaccaagc caattagata aatttgttta 1680

tattgtaaag atacaacaaa ttacttggac taatagaatc aaagatctgt aactactaca 1740

actctagacc ctgagttgtt ggacccctta gaactctggg gccacctttg ctgcaccaat 1800

ttcacaggat ttgcagtgta gtggcttcac agagagtgac tccaaccaga gaagcagcag 1860

tgttgtcttt gccaatttgg ccaaaggaat attgtatttc aaaaatttgt aactgtgtac 1920

acagacttga ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg agtacacttg 1980

gtattttctt aaaattgcct aatattttca gggaattaca gatatattag aatccaattg 2040

atttaattta ctttcaaccc ttgcagaaat tcactgttgg ggtaggaaat aagaagaagg 2100

taaaactgtt tatttccaaa ggtcctgctt ttcagtgtaa gagcaaagca aacaaaaaat 2160

tttgccccaa tattgtgagc tagcgtagat aagtagcatg gcgggttaat cattaactac 2220

aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 2280

gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 2340

cgagcgcgca g 2351

<210> 41

<211> 2351

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 1-1_1784_sv40_psg11_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR1-1_1784

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(570)

<223> SV40 termination

<220>

<221> misc_feature

<222> (571)..(2178)

<223> PSG11_V3

<220>

<221> misc_feature

<222> (2207)..(2351)

<223> 3' ITR

<400> 41

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc ctttttttca gacatgataa 360

gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 420

gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 480

acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt 540

aaagcaagta aaacctctac aaatgtggta gtacagggag caaaaaggcc agggagaaag 600

ggaactgcag gaattagtgc tgagaagcag gagttagtgg ttgaaggagg agatccaggc 660

ccagatacag gcaaataagt caattccctc tcccaagctt ggcagtcagc cctgcaggaa 720

ccaggataag agaaaagaag aaaagaaaag aaggattgaa ggtgttgtta ttttacttgg 780

gggagcctca ggaacaagct tgtaacttga gatactgtat aattgtttct tcaaggacta 840

caatatttct gttggaaaat tgttgggagt tgattatatt cttgcagttt tttttccctc 900

tcaccttgtt tctcaggtgg tgatcagtcc tctgtcaatt ctctactgca ctcaggtatt 960

ttggaaggct ttcagaggtg agaaaggctg attgctattt tctttgtcag tggaacttgt 1020

cacctttgca ccttttcttg gttgcaactt tttctcagtg tctctgtttg gtaaaaacta 1080

acaaaccagg cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag 1140

tagccttgtt ctagtgtttt ttgtgttacc tctttttcct gaaaaaggaa ggtactgttg 1200

agttattgtt gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt 1260

gtttcccacc taaattgcac acttagaaac agttattggt aagccttata ttagatatat 1320

atagtgtctg gtagtcttac cctctctata attacacact ttttggcact gcccctttcc 1380

tgccttgcag agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt 1440

actcagtgct gtgtcccacc tgctgtgccc acagcctcaa acagccagtg acttcagagc 1500

caggacccag ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg 1560

gccctgggga ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac 1620

caggactctt tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca 1680

ccagaggagc cctgagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg 1740

ggcttcctcc tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct 1800

tgtagttctt ggaagaggta caagaaaaca aagacctgac agaaggggtt gaggtggtga 1860

cagtgagaag caactggttg ttcagcactc tccttcttgt ccctctgtga agcctcttct 1920

accacaaagg gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag 1980

gtcagtcttg agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggcaa 2040

attgttttcc tgactcttct ctgaaagcta gatagactcc acctaaaacc ctattgccaa 2100

ggttgctggg gagaataaag ccacagttgt tgtcaactga gggaagggga agctccttgt 2160

ctcttaaagg gacacagtgc tagcgtagat aagtagcatg gcgggttaat cattaactac 2220

aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 2280

gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 2340

cgagcgcgca g 2351

<210> 42

<211> 2401

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 100_3330_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR100_3330

<220>

<221> misc_feature

<222> (342)..(347)

<223> terminator

<220>

<221> misc_feature

<222> (348)..(2228)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2257)..(2401)

<223> 3'ITR

<400> 42

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccccaaaa gagagaagat attgaggcct gttgccacat 240

atgctgagac tgataatgtg ggtattagtc cgccacatca tccgtctcaa catttgtgtc 300

tgttaggcaa tctcacggac ctggggcttt gcttatatgc ctttttttat tttgcaaatc 360

acaaaatttt atttaaaaat caattagatt ttgattgcct gaagtacaca accagtattc 420

caaatcctaa tttgttttgt aatacagtct tcaaaagtta attgcaaaga aattgtcaaa 480

taaacttccc ttcaattggt tcaggtttca gtgattgtat ttgttcaaac aattttattt 540

tatcaaggat tttaactgtt gtgtaaactt caatttttaa aaagatttga cctagtataa 600

tttcagtttt ctctaaaaag aaatatagtc caattggtga atttgtttga gtaattgagc 660

agttcttacc ttattcaaga atactcaatc aaaatttgtt gtctcaaaag tttcaatcaa 720

cacaaagcaa aaggaacaat taaataattt aattgtttcc agtgagtacc aattacaata 780

cttgtattca aaccagtgta caaccactta agttgaactc tataatttcc aaagtttctt 840

tgttcaatcc ttgtcaatag atattaggtg tccttgaagc cttcttctac ctttggaaca 900

cttaaagcct attattttat aacaaaataa caattctttc caaaaatagc tttgtcaagg 960

tgtgagcaca atataaatcc agggcaattt ctttgcacag agagaacttg tgctttagac 1020

tagaatagcc agtgatagtc ctcagattac ttgggattca atcaatcaat caattttcaa 1080

aaattctgcc ccaaatcaca attattaagc caattataac aaatattttg attttttaat 1140

tcagaagtta tcacaattat acagtagtat atcaacaata gcttgtataa attggaatta 1200

caggtggata caaataagtg atagatcaca aaatcttata tacctagaaa taagattttt 1260

tggttgtgga taaagttttt ttattgatta ttcccaatca attgtaagtg cttcttgtat 1320

tgggagattc agtcaaaaat tgaggtaact tctttttaaa agcttaatca actcaagtta 1380

gactgaaaac tactgaaatt gggaaacctt cctataggtt tttttaaaga gatttcccac 1440

ctacctttca aagcttgttc aaatatttgc actctaaaaa ttacccaccc caccttatct 1500

taagccttca ctgcaattct agcctcttct tgtgagtcaa atctttgaat tgaaagaact 1560

gatattccct tattgctgtt gcccttaaaa ttaagagggt ttaataatat atttttctat 1620

tattgggttg tcaaaatata cagtctggta gaaattcaat aataataaaa tttaaaccta 1680

tcaaaaaaac tgtgttctta attaccaagc caattagata aatttgttta tattgtaaag 1740

atacaacaaa ttacttggac taatagaatc aaagatctgt aactactaca actctagacc 1800

ctgagttgtt ggacccctta gaactctggg gccacctttg ctgcaccaat ttcacaggat 1860

ttgcagtgta gtggcttcac agagagtgac tccaaccaga gaagcagcag tgttgtcttt 1920

gccaatttgg ccaaaggaat attgtatttc aaaaatttgt aactgtgtac acagacttga 1980

ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg agtacacttg gtattttctt 2040

aaaattgcct aatattttca gggaattaca gatatattag aatccaattg atttaattta 2100

ctttcaaccc ttgcagaaat tcactgttgg ggtaggaaat aagaagaagg taaaactgtt 2160

tatttccaaa ggtcctgctt ttcagtgtaa gagcaaagca aacaaaaaat tttgccccaa 2220

tattgtgagc tagcgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc 2280

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 2340

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 2400

g 2401

<210> 43

<211> 2359

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 100_3330_psg11_v5 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR100_3330

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(2186)

<223> PSG11_V5

<220>

<221> misc_feature

<222> (2215)..(2359)

<223> 3'ITR

<400> 43

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccccaaaa gagagaagat attgaggcct gttgccacat 240

atgctgagac tgataatgtg ggtattagtc cgccacatca tccgtctcaa catttgtgtc 300

tgttaggcaa tctcacggac ctggggcttt gcttatatgc ctttttttac caggtcagtt 360

gttgcttgtg aagaagggtg tggttaagtc tgcagtgcag aaggtggaag ggatttcact 420

ctgtttttag agataggatt ggaactaaga attttaagtc tatttgggtg aaggggcagg 480

gaagtgtagg aagacagttc taattaggaa ggaggaatta tactaggaag tagaggtaaa 540

ggaagtgaga aggcttagta aatagaagaa atctgaactt atcaaatagg ctagtgtata 600

cccctggaga ttcttgatta agtgaattaa ctagatttta gaaaggtatt gaggcagtta 660

tttgcaagga acaattcaaa gtctggagac aagaagtcaa gcaatacttt gagaagtggt 720

tgagcttttt gggattggag agaattacac tattgagatt tgttaaaaat attgaagaag 780

gtttcacctt gtgaaattgt gtttctcaat tgaatctgag aaacaattga gcagaggctg 840

gagataattt tgtctaaaag acagtggggt acagggagca aaaaggccag ggagaaaggg 900

aactgcagga attagtgctg agaagcagga gttagtggtt gaaggaggag atccaggccc 960

agatacaggc aaataagtca attccctctc ccaagcttgg cagtcagccc tgcaggaacc 1020

aggataagag aaattgtttg gtaaaaacta acaaaccagg cttgattctt tgcagttagc 1080

tctttatctt tctctcccac aagtagtcag tagccttgtt ctagtgtttt ttgtgttacc 1140

tctttttcct gaaaaaggaa ggtactgttg agttattgtt gttgatagta atatagtagc 1200

tgacagtggt tcctctgttt gtggcaactt gtttcccacc taaattgcac acttagaaac 1260

agttattggt aagccttata ttagatatat atagtgtctg gtagtcttac cctctctata 1320

attacacact ttttggcact gcccctttcc tgccttgcag agccccaggg gtgaatcttc 1380

ctatttctcc agctctgcaa cagtcactgt actcagtgct gtgtcccacc tgctgtgccc 1440

acagcctcaa acagccagtg acttcagagc caggaaacag ctcaggagtc tgccctgagg 1500

ttgcttctct tcttatttcc ttgcagcctg gccaagggga ggcttggctt gaactggcag 1560

ctcaatttag ccaaattcag gacaggccac caggactctt tctccacact tgctggtccc 1620

accccaggtt gagtgaggca gggccagtca ccagaggagc ccagagcaga gcaggaagca 1680

gagtctgagc tgctcctccc tcacccaagg ggcttcctcc tctcaattgg gggaaaagtg 1740

tgagcttgtt tcaaagcctc agttgttcct tgtagttctt ggaagaggta caagaaaaca 1800

aagacctgac agaaggggtt gaggtggtga cagtgagaag caactggttg ttcagcactc 1860

tccttcttgt ccctctgtga agcctcttct acccaaaggg ctcagggctg ataaagcccc 1920

ctccctacct ttctcaggcc agacacaagg tcagtcttga gaaaacagaa aaaaaaggag 1980

aagagagtct gtagagacaa attgggaggg ttcaggagga gaatttggga tttgcctgtg 2040

cccaagggac acaggctggg aatcaaattg ttttcctgac tcttctctga aagctagata 2100

gactccacct aaaaccctat tgccaaggtt gctgggccac agttgttgtc aactgaggga 2160

aggggaagct ccttgtctct taaagggcta gcgtagataa gtagcatggc gggttaatca 2220

ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc 2280

tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag 2340

tgagcgagcg agcgcgcag 2359

<210> 44

<211> 2351

<212> DNA

<213> artificial sequence

<220>

<223> scaav_h1_short_mir 100_3330_sv40_amely_v3 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(341)

<223> miR100_3330

<220>

<221> misc_feature

<222> (342)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(570)

<223> SV40 termination

<220>

<221> misc_feature

<222> (571)..(2178)

<223> AMELY_V3

<220>

<221> misc_feature

<222> (2207)..(2351)

<223> 3'ITR

<400> 44

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccccaaaa gagagaagat attgaggcct gttgccacat 240

atgctgagac tgataatgtg ggtattagtc cgccacatca tccgtctcaa catttgtgtc 300

tgttaggcaa tctcacggac ctggggcttt gcttatatgc ctttttttca gacatgataa 360

gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 420

gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 480

acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt 540

aaagcaagta aaacctctac aaatgtggta aaatatagtc caattggtga atttgtttga 600

gtaattgagc agttcttacc ttattcaaga atactcaatc aaaatttgtt gtctcaaaag 660

tttcaatcaa cacaaagcaa aaggaacaat taaataattt aattgtttcc agtgagtacc 720

aattacaata cttgtattca aaccagtgta caaccactta agttgaactc tataatttcc 780

aaagtttctt tgttcaatcc ttgtcaatag atattaggtg tccttgaagc cttcttctac 840

ctttggaaca cttaaagcct attattttat aacaaaataa caattctttc caaaaatagc 900

tttgtcaagg tgtgagcaca atataaatcc agggcaattt ctttgcacag agagaacttg 960

tgctttagac tagaatagcc agtgatagtc ctcagattac ttgggattca atcaatcaat 1020

caattttcaa aaattctgcc ccaaatcaca attattaagc caattataac aaatattttg 1080

attttttaat tcagaagtta tcacaattat acagtagtat atcaacaata gcttgtataa 1140

attggaatta caggtggata caaataagtg atagatcaca aaatcttata tacctagaaa 1200

taagattttt tggttgtgga taaagttttt ttattgatta ttcccaatca attgtaagtg 1260

cttcttgtat tgggagattc agtcaaaaat tgaggtaact tctttttaaa agcttaatca 1320

actcaagtta gactgaaaac tactgaaatt gggaaacctt cctataggtt tttttaaaga 1380

gatttcccac ctacctttca aagcttgttc aaatatttgc actctaaaaa ttacccaccc 1440

caccttatct taagccttca ctgcaattct agcctcttct tgtgagtcaa atctttgaat 1500

tgaaagaact gatattccct tattgctgtt gcccttaaaa ttaagagggt ttaataatat 1560

atttttctat tattgggttg tcaaaatata cagtctggta gaaattcaat aataataaaa 1620

tttaaaccta tcaaaaaaac tgtgttctta attaccaagc caattagata aatttgttta 1680

tattgtaaag atacaacaaa ttacttggac taatagaatc aaagatctgt aactactaca 1740

actctagacc ctgagttgtt ggacccctta gaactctggg gccacctttg ctgcaccaat 1800

ttcacaggat ttgcagtgta gtggcttcac agagagtgac tccaaccaga gaagcagcag 1860

tgttgtcttt gccaatttgg ccaaaggaat attgtatttc aaaaatttgt aactgtgtac 1920

acagacttga ttttttacaa actaaatttc ccaatagtgt ctgtttgtgg agtacacttg 1980

gtattttctt aaaattgcct aatattttca gggaattaca gatatattag aatccaattg 2040

atttaattta ctttcaaccc ttgcagaaat tcactgttgg ggtaggaaat aagaagaagg 2100

taaaactgtt tatttccaaa ggtcctgctt ttcagtgtaa gagcaaagca aacaaaaaat 2160

tttgccccaa tattgtgagc tagcgtagat aagtagcatg gcgggttaat cattaactac 2220

aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 2280

gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 2340

cgagcgcgca g 2351

<210> 45

<211> 1881

<212> DNA

<213> artificial sequence

<220>

<223> PSG11_V1 stuffer sequence

<400> 45

acgaggtcag atgttgcttg tgatgaaggg tgtggttaag tctgcagtgc agatggtgga 60

agggatttca ctctgttttt agacatagca ttggaactaa gaattttatg tctatatggg 120

tgaaggggca gggatgtgta ggaagacagt tctaattagg atggaggaat tatactagga 180

agtagaggta aaggaagtga gaaggcttag taaatagaag aaatgtgaac ttatcaaata 240

ggctagtgta tacccctgga gattcttgat taagtgaatt aactagattt tagaaaggta 300

atgaggcagt tatatgcaag gaacatttca aagtctggag acaagaagtc aagcaatact 360

ttgagaagtg gttgagcttt atgggaatgg agagaattac actattgaga tatgttaaaa 420

ataatgaaga aggtttcacc atgtgaaatt gtgtttctca tttgaatctg agaaacaaat 480

gagcagaggc tggagataat tatgtctaaa agacagtggg gtacagggag cataaaggcc 540

agggagaaag ggaactgcag gaattagtgc tgagaagcag gagttagtgg atgaaggagg 600

agatccaggc ccagatacag gcaaataagt catttccctc tcccaagcat ggcagtcagc 660

cctgcaggaa ccaggataag agaaaaatgg ttggtaaaaa ctaacatacc aggcttgatt 720

ctttgcagtt agctctttat ctttctctcc cacaagtagt cagtagcctt gttctagtgt 780

tttatgtgtt acctcttttt ccggaaaaag gaaggtactg ttgagttaat gatgatgata 840

gtaatatagt agctgacatt ggttcctctg tatgtggcat cttgtttccc atctaaattg 900

cacacttaga aacagttaat ggtaagcctt atattagata tatatagtgt ctggtagtct 960

taccctctct ataattacac actttttggc actgcccctt tcctgccatg cagagcccca 1020

ggggtgaatc ttcctatttc tccagctctg catcagtcac tgtactcagt gctgtgtccc 1080

acgtgctgtg cccacagcct catacagcca gtgacttcag agccaggacg cagctcagga 1140

gtctgccctg aggttgcttc tcttcttatt tccttgcagc ctggcccggg ggaggcttgg 1200

cttgaactgg cagctcaatt tagccaaatt caggacaggc caccaggact ctttctccac 1260

acatgctggt cccaccccag gttgagtgag gcagggccag tcaccagagg agcccggagc 1320

agagcaggaa gcagagtctg agctgctcct ccctcaccca aggggcttcc tcctctcatt 1380

tgggggaaaa gtgtgagctt gtttcaaagc ctcagatgtt ccttgtagtt catggaagag 1440

gtacaagaaa acaaagacgt gacagaaggg gatgaggtgg tgacagtgag aagcaactcg 1500

atgttcagca ctctccttct tgtccctctg tgaagcctct tctaccacat agggctcagg 1560

gctgataaag ccccctccct acctttctca ggccagacac aaggtcagtc atgagaaaac 1620

agaaaaaaaa ggagaagaga gtctgtagag acaaattggg agggttcagg aggagaattt 1680

gggatttgcc tgtgcccatg ggacacaggc tgggaataaa aatgttttcc tgactcttct 1740

ctgaaagcta gatagactcc acctaaaacc ctattgccaa ggatgctggg cccacagttg 1800

atgtcatctg agggaagggg aagctccttg tctcttaaag ggacacagtg accctctgag 1860

ccaagacaca ccctcaagtc c 1881

<210> 46

<211> 1881

<212> DNA

<213> artificial sequence

<220>

<223> PSG11_V2 stuffer sequence

<400> 46

gtatacccct ggagattctt gattaagtga attaactaga ttttagaaag gtattgaggc 60

agttatttgc aaggaacaat tcaaagtctg gagacaagaa gtcaagcaat actttgagaa 120

gtggttgagc tttttgggat tggagagaat tacactattg agatttgtta aaaatattga 180

agaaggtttc accttgtgaa attgtgtttc tcaattgaat ctgagaaaca attgagcaga 240

ggctggagat aattttgtct aaaagacagt ggggtacagg gagcaaaaag gccagggaga 300

aagggaactg caggaattag tgctgagaag caggagttag tggttgaagg aggagatcca 360

ggcccagata caggcaaata agtcaattcc ctctcccaag cttggcagtc agccctgcag 420

gaaccaggat aagagaaaag aagaaaagaa aagaaggatt gaaggtgttg ttattttact 480

tgggggagcg tcaggaacaa gcttgtaact tgagatactg tataattgtt tcttcaagga 540

ctacaatatt tctgttggaa aattgttggg agttgattat attcttgcag ttttttttcc 600

ctctcacctt gtttctcagg tggtgatcag tcctctgtca attctctact gcactcaggt 660

attttggaag gctttcagac gtgagaaagg ctgattgcta ttttctttgt cagtggaact 720

tgtcaccttt gcaccttttc ttggttgcaa ctttttctca gtgtctctgt ttggtaaaaa 780

ctaacaaacc aggcttgatt ctttgcagtt agctctttat ctttctctcc cacaagtagt 840

cagtagcctt gttctagtgt tttttgtgtt acctcttttt ccggaaaaag gaaggtactg 900

ttgagttatt gttgttgata gtaatatagt agctgacagt ggttcctctg tttgtggcaa 960

cttgtttccc acctaaattg cacacttaga aacagttatt ggtaagcctt atattagata 1020

tatatagtgt ctggtagtct taccctctct ataattacac actttttggc actgcccctt 1080

tcctgccttg cagagcccca ggggtgaatc ttcctatttc tccagctctg caacagtcac 1140

tgtactcagt gctgtgtccc acgtgctgtg cccacagcct caaacagcca gtgacttcag 1200

agccaggacg cagctcagga gtctgccctg aggttgcttc tcttcttatt tccttgcagc 1260

ctggcccggg ggaggcttgg cttgaactgg cagctcaatt tagccaaatt caggacaggc 1320

caccaggact ctttctccac acttgctggt cccaccccag gttgagtgag gcagggccag 1380

tcaccagagg agcccggagc agagcaggaa gcagagtctg agctgctcct ccctcaccca 1440

aggggcttcc tcctctcaat tgggggaaaa gtgtgagctt gtttcaaagc ctcagttgtt 1500

ccttgtagtt cttggaagag gtacaagaaa acaaagacgt gacagaaggg gttgaggtgg 1560

tgacagtgag aagcaactcg ttgttcagca ctctccttct tgtccctctg tgaagcctct 1620

tctaccacaa agggctcagg gctgataaag ccccctccct acctttctca ggccagacac 1680

aaggtcagtc ttgagaaaac agaaaaaaaa ggagaagaga gtctgtagag acaaattggg 1740

caaattgttt tcctgactct tctctgaaag ctagatagac tccacctaaa accctattgc 1800

caaggttgct ggggagaata aagccacagt tgttgtcaac tgagggaagg ggaagctcct 1860

tgtctcttaa agggacacag t 1881

<210> 47

<211> 1881

<212> DNA

<213> artificial sequence

<220>

<223> PSG11_V3 stuffer sequence

<400> 47

gtatacccct ggagattctt gattaagtga attaactaga ttttagaaag gtattgaggc 60

agttatttgc aaggaacaat tcaaagtctg gagacaagaa gtcaagcaat actttgagaa 120

gtggttgagc tttttgggat tggagagaat tacactattg agatttgtta aaaatattga 180

agaaggtttc accttgtgaa attgtgtttc tcaattgaat ctgagaaaca attgagcaga 240

ggctggagat aattttgtct aaaagacagt ggggtacagg gagcaaaaag gccagggaga 300

aagggaactg caggaattag tgctgagaag caggagttag tggttgaagg aggagatcca 360

ggcccagata caggcaaata agtcaattcc ctctcccaag cttggcagtc agccctgcag 420

gaaccaggat aagagaaaag aagaaaagaa aagaaggatt gaaggtgttg ttattttact 480

tgggggagcc tcaggaacaa gcttgtaact tgagatactg tataattgtt tcttcaagga 540

ctacaatatt tctgttggaa aattgttggg agttgattat attcttgcag ttttttttcc 600

ctctcacctt gtttctcagg tggtgatcag tcctctgtca attctctact gcactcaggt 660

attttggaag gctttcagag gtgagaaagg ctgattgcta ttttctttgt cagtggaact 720

tgtcaccttt gcaccttttc ttggttgcaa ctttttctca gtgtctctgt ttggtaaaaa 780

ctaacaaacc aggcttgatt ctttgcagtt agctctttat ctttctctcc cacaagtagt 840

cagtagcctt gttctagtgt tttttgtgtt acctcttttt cctgaaaaag gaaggtactg 900

ttgagttatt gttgttgata gtaatatagt agctgacagt ggttcctctg tttgtggcaa 960

cttgtttccc acctaaattg cacacttaga aacagttatt ggtaagcctt atattagata 1020

tatatagtgt ctggtagtct taccctctct ataattacac actttttggc actgcccctt 1080

tcctgccttg cagagcccca ggggtgaatc ttcctatttc tccagctctg caacagtcac 1140

tgtactcagt gctgtgtccc acctgctgtg cccacagcct caaacagcca gtgacttcag 1200

agccaggacc cagctcagga gtctgccctg aggttgcttc tcttcttatt tccttgcagc 1260

ctggccctgg ggaggcttgg cttgaactgg cagctcaatt tagccaaatt caggacaggc 1320

caccaggact ctttctccac acttgctggt cccaccccag gttgagtgag gcagggccag 1380

tcaccagagg agccctgagc agagcaggaa gcagagtctg agctgctcct ccctcaccca 1440

aggggcttcc tcctctcaat tgggggaaaa gtgtgagctt gtttcaaagc ctcagttgtt 1500

ccttgtagtt cttggaagag gtacaagaaa acaaagacct gacagaaggg gttgaggtgg 1560

tgacagtgag aagcaactgg ttgttcagca ctctccttct tgtccctctg tgaagcctct 1620

tctaccacaa agggctcagg gctgataaag ccccctccct acctttctca ggccagacac 1680

aaggtcagtc ttgagaaaac agaaaaaaaa ggagaagaga gtctgtagag acaaattggg 1740

caaattgttt tcctgactct tctctgaaag ctagatagac tccacctaaa accctattgc 1800

caaggttgct ggggagaata aagccacagt tgttgtcaac tgagggaagg ggaagctcct 1860

tgtctcttaa agggacacag t 1881

<210> 48

<211> 1697

<212> DNA

<213> artificial sequence

<220>

<223> PSG11_V5 stuffer sequence

<400> 48

accaggtcag ttgttgcttg tgaagaaggg tgtggttaag tctgcagtgc agaaggtgga 60

agggatttca ctctgttttt agagatagga ttggaactaa gaattttaag tctatttggg 120

tgaaggggca gggaagtgta ggaagacagt tctaattagg aaggaggaat tatactagga 180

agtagaggta aaggaagtga gaaggcttag taaatagaag aaatctgaac ttatcaaata 240

ggctagtgta tacccctgga gattcttgat taagtgaatt aactagattt tagaaaggta 300

ttgaggcagt tatttgcaag gaacaattca aagtctggag acaagaagtc aagcaatact 360

ttgagaagtg gttgagcttt ttgggattgg agagaattac actattgaga tttgttaaaa 420

atattgaaga aggtttcacc ttgtgaaatt gtgtttctca attgaatctg agaaacaatt 480

gagcagaggc tggagataat tttgtctaaa agacagtggg gtacagggag caaaaaggcc 540

agggagaaag ggaactgcag gaattagtgc tgagaagcag gagttagtgg ttgaaggagg 600

agatccaggc ccagatacag gcaaataagt caattccctc tcccaagctt ggcagtcagc 660

cctgcaggaa ccaggataag agaaattgtt tggtaaaaac taacaaacca ggcttgattc 720

tttgcagtta gctctttatc tttctctccc acaagtagtc agtagccttg ttctagtgtt 780

ttttgtgtta cctctttttc ctgaaaaagg aaggtactgt tgagttattg ttgttgatag 840

taatatagta gctgacagtg gttcctctgt ttgtggcaac ttgtttccca cctaaattgc 900

acacttagaa acagttattg gtaagcctta tattagatat atatagtgtc tggtagtctt 960

accctctcta taattacaca ctttttggca ctgccccttt cctgccttgc agagccccag 1020

gggtgaatct tcctatttct ccagctctgc aacagtcact gtactcagtg ctgtgtccca 1080

cctgctgtgc ccacagcctc aaacagccag tgacttcaga gccaggaaac agctcaggag 1140

tctgccctga ggttgcttct cttcttattt ccttgcagcc tggccaaggg gaggcttggc 1200

ttgaactggc agctcaattt agccaaattc aggacaggcc accaggactc tttctccaca 1260

cttgctggtc ccaccccagg ttgagtgagg cagggccagt caccagagga gcccagagca 1320

gagcaggaag cagagtctga gctgctcctc cctcacccaa ggggcttcct cctctcaatt 1380

gggggaaaag tgtgagcttg tttcaaagcc tcagttgttc cttgtagttc ttggaagagg 1440

tacaagaaaa caaagacctg acagaagggg ttgaggtggt gacagtgaga agcaactggt 1500

tgttcagcac tctccttctt gtccctctgt gaagcctctt ctaccacaaa gggctcaggg 1560

ctgataaagc cccctcccta cctttctcag gccagacaca aggtcagtct tgagaaaaca 1620

gaaaaaaaag gagaagagag tctgtagaga caaattggga gggttcagga ggagaatttg 1680

ggatttgcct gtgccca 1697

<210> 49

<211> 1881

<212> DNA

<213> artificial sequence

<220>

<223> AMELY_V1 stuffer sequence

<400> 49

tattatgcat atcacataat tttatttaaa aatcatttag attttgattg cgtgaagtac 60

acaaccagta ttccatatcc taatttgttt tgtaatacag tcttcaaaag ttaaatgcaa 120

agaaattgtc atataaactt cccttcattt ggttcaggtt tcagtgattg tatatgttca 180

tacattttta ttttatcaac gattttaact gatgtgtaaa cttcattttt taaaaagatt 240

tgacgtagta taatttcagt tttctctaaa aagaaatata gtccaaatgg tgaatttgta 300

tgagtaaatg agcagttctt accttattca agaatactca ttcataatat gttgtctcaa 360

aagtttcatt caacacatag caaaaggaac aattaaataa tttaattgtt tccagtgagt 420

accatttaca atacatgtat tcaaaccaat gtacaaccac ttaagttgaa ctctataatt 480

tccaaagttt ctttgttcat tcgttgtcat tagatattag gtgtccatga agccttcttc 540

tacctttgga acacttaaag cctattattt tataacaaaa taacaattct ttccataaat 600

agctttgtca aggtgtgagc acattataaa tccacggcat tttctatgca cagagagaac 660

atgtgcttta gactagaata gccaatgata gtcgtcagat tacttgggat tcaatcaatc 720

aatcaatttt caaaaattct gccccatatc acaattatta agccatttat aacatatatt 780

atgatttttt aattcagaag ttatcacatt tatacagtag tatatcatca atagcttgta 840

taaattggaa ttacaggtgg atacatataa gtgatagatc acataatctt atatacgtag 900

aaataagatt ttatggatgt ggataaagtt tttttattga ttattcccat tcatttgtaa 960

gtgcttcttg taatgggaga ttcagtcata aattgaggta acttcttttt aaaagcttaa 1020

tcatctcaag ttagactgaa aactactgaa aatgggaaac cttcgtatag gtttttttaa 1080

agagatttcc catctacctt tcatagcttg ttcaaatata tgcactctaa aaattaccca 1140

ccccatctta tcttaagcct tcactgcaat tctagcctct tcttgtgagt caaatctttg 1200

aaatgaaaga actgatattc ccttattgct gttgccctta aaattaagag ggtttaataa 1260

tatatttttc tattaatggg ttgtcataat atacagtctg gtagaaattc aataataata 1320

aaatttaaac ctatcataaa aactgtgttc ttaattacca agccatttag ataaatttgt 1380

ttataatgta aagatacaac atattacttg gactaataga atcatagatc tgtaactact 1440

acaactctag accctgagtt gttggacccc ttagaactct ggggccacct ttgctgcacc 1500

attttcacag gatttgcatt gtagtggctt cacagagagt gactccaacc agagaagcag 1560

cagtgttgtc tttgccatta tggccatagg aatattgtat ttcaaaaatt tgtaactgtg 1620

tacacagaca tgatttttta caaactaaat ttcccattag tgtctgtatg tggagtacac 1680

atggtatttt cttaaaaatg cctaatattt tcagggaatt acagatatat tagaatccat 1740

ttgatttaat ttactttcat cccatgcaga aattcactga tgcggtagga aataagaaga 1800

aggtaaaact gtttatttcc ataggtcctg cttttcattg taagagcaaa gcaaacataa 1860

aattatgccg caataatgtg a 1881

<210> 50

<211> 1881

<212> DNA

<213> artificial sequence

<220>

<223> AMELY_V2 stuffer sequence

<400> 50

tattttgcaa atcacaaaat tttatttaaa aatcaattag attttgattg cgtgaagtac 60

acaaccagta ttccaaatcc taatttgttt tgtaatacag tcttcaaaag ttaattgcaa 120

agaaattgtc aaataaactt cccttcaatt ggttcaggtt tcagtgattg tatttgttca 180

aacaatttta ttttatcaac gattttaact gttgtgtaaa cttcaatttt taaaaagatt 240

tgacgtagta taatttcagt tttctctaaa aagaaatata gtccaattgg tgaatttgtt 300

tgagtaattg agcagttctt accttattca agaatactca atcaaaattt gttgtctcaa 360

aagtttcaat caacacaaag caaaaggaac aattaaataa tttaattgtt tccagtgagt 420

accaattaca atacttgtat tcaaaccagt gtacaaccac ttaagttgaa ctctataatt 480

tccaaagttt ctttgttcaa tcgttgtcaa tagatattag gtgtccttga agccttcttc 540

tacctttgga acacttaaag cctattattt tataacaaaa taacaattct ttccaaaaat 600

agctttgtca aggtgtgagc acaatataaa tccacggcaa tttctttgca cagagagaac 660

ttgtgcttta gactagaata gccagtgata gtcgtcagat tacttgggat tcaatcaatc 720

aatcaatttt caaaaattct gccccaaatc acaattatta agccaattat aacaaatatt 780

ttgatttttt aattcagaag ttatcacaat tatacagtag tatatcaaca atagcttgta 840

taaattggaa ttacaggtgg atacaaataa gtgatagatc acaaaatctt atatacgtag 900

aaataagatt ttttggttgt ggataaagtt tttttattga ttattcccaa tcaattgtaa 960

gtgcttcttg tattgggaga ttcagtcaaa aattgaggta acttcttttt aaaagcttaa 1020

tcaactcaag ttagactgaa aactactgaa attgggaaac cttcgtatag gtttttttaa 1080

agagatttcc cacctacctt tcaaagcttg ttcaaatatt tgcactctaa aaattaccca 1140

ccccacctta tcttaagcct tcactgcaat tctagcctct tcttgtgagt caaatctttg 1200

aattgaaaga actgatattc ccttattgct gttgccctta aaattaagag ggtttaataa 1260

tatatttttc tattattggg ttgtcaaaat atacagtctg gtagaaattc aataataata 1320

aaatttaaac ctatcaaaaa aactgtgttc ttaattacca agccaattag ataaatttgt 1380

ttatattgta aagatacaac aaattacttg gactaataga atcaaagatc tgtaactact 1440

acaactctag accctgagtt gttggacccc ttagaactct ggggccacct ttgctgcacc 1500

aatttcacag gatttgcagt gtagtggctt cacagagagt gactccaacc agagaagcag 1560

cagtgttgtc tttgccaatt tggccaaagg aatattgtat ttcaaaaatt tgtaactgtg 1620

tacacagact tgatttttta caaactaaat ttcccaatag tgtctgtttg tggagtacac 1680

ttggtatttt cttaaaattg cctaatattt tcagggaatt acagatatat tagaatccaa 1740

ttgatttaat ttactttcaa cccttgcaga aattcactgt tgcggtagga aataagaaga 1800

aggtaaaact gtttatttcc aaaggtcctg cttttcagtg taagagcaaa gcaaacaaaa 1860

aattttgccg caatattgtg a 1881

<210> 51

<211> 1690

<212> DNA

<213> artificial sequence

<220>

<223> AMELY_V3 stuffer sequence

<400> 51

tattttgcaa atcacaaaat tttatttaaa aatcaattag attttgattg cctgaagtac 60

acaaccagta ttccaaatcc taatttgttt tgtaatacag tcttcaaaag ttaattgcaa 120

agaaattgtc aaataaactt cccttcaatt ggttcaggtt tcagtgattg tatttgttca 180

aacaatttta ttttatcaag gattttaact gttgtgtaaa cttcaatttt taaaaagatt 240

tgacctagta taatttcagt tttctctaaa aagaaatata gtccaattgg tgaatttgtt 300

tgagtaattg agcagttctt accttattca agaatactca atcaaaattt gttgtctcaa 360

aagtttcaat caacacaaag caaaaggaac aattaaataa tttaattgtt tccagtgagt 420

accaattaca atacttgtat tcaaaccagt gtacaaccac ttaagttgaa ctctataatt 480

tccaaagttt ctttgttcaa tccttgtcaa tagatattag gtgtccttga agccttcttc 540

tacctttgga acacttaaag cctattattt tataacaaaa taacaattct ttccaaaaat 600

agctttgtca aggtgtgagc acaatataaa tccagggcaa tttctttgca cagagagaac 660

ttgtgcttta gactagaata gccagtgata gtcctcagat tacttgggat tcaatcaatc 720

aatcaatttt caaaaattct gccccaaatc acaattatta agccaattat aacaaatatt 780

ttgatttttt aattcagaag ttatcacaat tatacagtag tatatcaaca atagcttgta 840

taaattggaa ttacaggtgg atacaaataa gtgatagatc acaaaatctt atatacctag 900

aaataagatt ttttggttgt ggataaagtt tttttattga ttattcccaa tcaattgtaa 960

gtgcttcttg tattgggaga ttcagtcaaa aattgaggta acttcttttt aaaagcttaa 1020

tcaactcaag ttagactgaa aactactgaa attgggaaac cttcctatag gtttttttaa 1080

agagatttcc cacctacctt tcaaagcttg ttcaaatatt tgcactctaa aaattaccca 1140

ccccacctta tcttaagcct tcactgcaat tctagcctct tcttgtgagt caaatctttg 1200

aattgaaaga actgatattc ccttattgct gttgccctta aaattaagag ggtttaataa 1260

tatatttttc tattattggg ttgtcaaaat atacagtctg gtagaaattc aataataata 1320

aaatttaaac ctatcaaaaa aactgtgttc ttaattacca agccaattag ataaatttgt 1380

ttatattgta aagatacaac aaattacttg gactaataga atcaaagatc tgtaactact 1440

acaactctag accctgagtt gttggacccc ttagaactct ggggccacct ttgctgcacc 1500

aatttcacag gatttgcagt gtagtggctt cacagagagt gactccaacc agagaagcag 1560

cagtgttgtc tttgccaatt tggccaaagg aatattgtat ttcaaaaatt tgtaactgtg 1620

tacacagact tgatttttta caaactaaat ttcccaatag tgtctgtttg tggagtacac 1680

ttggtatttt 1690

<210> 52

<211> 231

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)..(231)

<223> H1 long promoter

<400> 52

ggaattcgaa cgctgacgtc atcaacccgc tccaaggaat cgcgggccca gtgtcactag 60

gcgggaacac ccagcgcgcg tgcgccctgg caggaagatg gctgtgaggg acaggggagt 120

ggcgccctgc aatatttgca tgtcgctatg tgttctggga aatcaccata aacgtgaaat 180

gtctttggat ttgggaatct tataagttct gtatgagacc actctttccc a 231

<210> 53

<211> 100

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)..(100)

<223> H1 native promoter

<400> 53

atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatg tctttggatt 60

tgggaatctt ataagttctg tatgagacca ctctttccca 100

<210> 54

<211> 91

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)..(91)

<223> H1 short promoter

<400> 54

atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatg tctttggatt 60

tgggaatctt ataagttctg tatgagacca c 91

<210> 55

<211> 200

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_PSG11_V1

<400> 55

acgaggtcag atgttgcttg tgatgaaggg tgtggttaag tctgcagtgc agatggtgga 60

agggatttca ctctgttttt agacatagca ttggaactaa gaattttatg tctatatggg 120

tgaaggggca gggatgtgta ggaagacagt tctaattagg atggaggaat tatactagga 180

agtagaggta aaggaagtga 200

<210> 56

<211> 200

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_MCS_PSG11_V2

<400> 56

gtatacccct ggagattctt gattaagtga attaactaga ttttagaaag gtattgaggc 60

agttatttgc aaggaacaat tcaaagtctg gagacaagaa gtcaagcaat actttgagaa 120

gtggttgagc tttttgggat tggagagaat tacactattg agatttgtta aaaatattga 180

agaaggtttc accttgtgaa 200

<210> 57

<211> 106

<212> DNA

<213> artificial sequence

<220>

<223> TRS-absent 5' ITR

<400> 57

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgg 106

<210> 58

<211> 145

<212> DNA

<213> artificial sequence

<220>

<223> 3' ITR

<400> 58

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 60

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 120

cctcagtgag cgagcgagcg cgcag 145

<210> 59

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 1755 guide (DNA)

<400> 59

tcgggttgaa atctgaagtg tg 22

<210> 60

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 1755 passenger (DNA)

<400> 60

cacactccag ctttcaaacc gt 22

<210> 61

<211> 138

<212> DNA

<213> artificial sequence

<220>

<223> miR100_1755 (DNA)

<400> 61

cccaaaagag agaagatatt gaggcctgtt gccacatcgg gttgaaatct gaagtgtggt 60

attagtccgc acactccagc tttcaaaccg ttgtgtctgt taggcaatct cacggacctg 120

gggctttgct tatatgcc 138

<210> 62

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 2945 guide (DNA)

<400> 62

tgtagtagaa ggctttggct ga 22

<210> 63

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 2945 passenger (DNA)

<400> 63

tcagcccaag acttctaata ct 22

<210> 64

<211> 138

<212> DNA

<213> artificial sequence

<220>

<223> miR100_2945 (DNA)

<400> 64

cccaaaagag agaagatatt gaggcctgtt gccacatgta gtagaaggct ttggctgagt 60

attagtccgt cagcccaaga cttctaatac ttgtgtctgt taggcaatct cacggacctg 120

gggctttgct tatatgcc 138

<210> 65

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 2586 guide (DNA)

<400> 65

tagattcaga agtagaactt gg 22

<210> 66

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 2586 passenger (DNA)

<400> 66

ccaagtccta attctgactc tt 22

<210> 67

<211> 138

<212> DNA

<213> artificial sequence

<220>

<223> miR100_2586 (DNA)

<400> 67

cccaaaagag agaagatatt gaggcctgtt gccacataga ttcagaagta gaacttgggt 60

attagtccgc caagtcctaa ttctgactct ttgtgtctgt taggcaatct cacggacctg 120

gggctttgct tatatgcc 138

<210> 68

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 3330 guide (DNA)

<400> 68

tatgctgaga ctgataatgt gg 22

<210> 69

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 3330 passenger (DNA)

<400> 69

ccacatcatc cgtctcaaca tt 22

<210> 70

<211> 138

<212> DNA

<213> artificial sequence

<220>

<223> miR100_3330 (DNA)

<400> 70

cccaaaagag agaagatatt gaggcctgtt gccacatatg ctgagactga taatgtgggt 60

attagtccgc cacatcatcc gtctcaacat ttgtgtctgt taggcaatct cacggacctg 120

gggctttgct tatatgcc 138

<210> 71

<211> 22

<212> RNA

<213> artificial sequence

<220>

<223> 1784 (also 14792) guide (RNA)

<400> 71

auuaacuacu cuuuggucug aa 22

<210> 72

<211> 22

<212> RNA

<213> artificial sequence

<220>

<223> 1784 (also 14792) passenger (RNA)

<400> 72

uccagaccaa auauuaguua au 22

<210> 73

<211> 138

<212> RNA

<213> artificial sequence

<220>

<223> mir1.1_1784 (or mir1.1_ 14792) (RNA)

<400> 73

caugcagacu gccugcuugg guacagacca aagaguaguc gaauuaugga ccugcuaagc 60

uaauuaacua cucuuugguc ugaacucagg ccgggaccuc ucucgccgca cugaggggca 120

cuccacacca cgggggcc 138

<210> 74

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 1784 (also 14792) guide (DNA)

<400> 74

attaactact ctttggtctg aa 22

<210> 75

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> 1784 (also 14792) passenger (DNA)

<400> 75

tccagaccaa atattagtta at 22

<210> 76

<211> 138

<212> DNA

<213> artificial sequence

<220>

<223> mir1.1_1784 (or mir1.1_ 14792) (DNA)

<400> 76

catgcagact gcctgcttgg gtacagacca aagagtagtc gaattatgga cctgctaagc 60

taattaacta ctctttggtc tgaactcagg ccgggacctc tctcgccgca ctgaggggca 120

ctccacacca cgggggcc 138

<210> 77

<211> 222

<212> DNA

<213> artificial sequence

<220>

<223> SV40 transcription termination Signal

<400> 77

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 60

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 120

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt 180

gggaggtttt ttaaagcaag taaaacctct acaaatgtgg ta 222

<210> 78

<211> 2402

<212> DNA

<213> artificial sequence

<220>

<223> scAAV9_H1_miR-1-1-XD-14792_PSG11_V1 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(342)

<223> miR-1-1_XD-14792

<220>

<221> misc_feature

<222> (343)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(2229)

<223> PSG11_V1

<220>

<221> misc_feature

<222> (2258)..(2402)

<223> 3' ITR

<400> 78

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc cgttttttac gaggtcagat 360

gttgcttgtg atgaagggtg tggttaagtc tgcagtgcag atggtggaag ggatttcact 420

ctgtttttag acatagcatt ggaactaaga attttatgtc tatatgggtg aaggggcagg 480

gatgtgtagg aagacagttc taattaggat ggaggaatta tactaggaag tagaggtaaa 540

ggaagtgaga aggcttagta aatagaagaa atgtgaactt atcaaatagg ctagtgtata 600

cccctggaga ttcttgatta agtgaattaa ctagatttta gaaaggtaat gaggcagtta 660

tatgcaagga acatttcaaa gtctggagac aagaagtcaa gcaatacttt gagaagtggt 720

tgagctttat gggaatggag agaattacac tattgagata tgttaaaaat aatgaagaag 780

gtttcaccat gtgaaattgt gtttctcatt tgaatctgag aaacaaatga gcagaggctg 840

gagataatta tgtctaaaag acagtggggt acagggagca taaaggccag ggagaaaggg 900

aactgcagga attagtgctg agaagcagga gttagtggat gaaggaggag atccaggccc 960

agatacaggc aaataagtca tttccctctc ccaagcatgg cagtcagccc tgcaggaacc 1020

aggataagag aaaaatggtt ggtaaaaact aacataccag gcttgattct ttgcagttag 1080

ctctttatct ttctctccca caagtagtca gtagccttgt tctagtgttt tatgtgttac 1140

ctctttttcc ggaaaaagga aggtactgtt gagttaatga tgatgatagt aatatagtag 1200

ctgacattgg ttcctctgta tgtggcatct tgtttcccat ctaaattgca cacttagaaa 1260

cagttaatgg taagccttat attagatata tatagtgtct ggtagtctta ccctctctat 1320

aattacacac tttttggcac tgcccctttc ctgccatgca gagccccagg ggtgaatctt 1380

cctatttctc cagctctgca tcagtcactg tactcagtgc tgtgtcccac gtgctgtgcc 1440

cacagcctca tacagccagt gacttcagag ccaggacgca gctcaggagt ctgccctgag 1500

gttgcttctc ttcttatttc cttgcagcct ggcccggggg aggcttggct tgaactggca 1560

gctcaattta gccaaattca ggacaggcca ccaggactct ttctccacac atgctggtcc 1620

caccccaggt tgagtgaggc agggccagtc accagaggag cccggagcag agcaggaagc 1680

agagtctgag ctgctcctcc ctcacccaag gggcttcctc ctctcatttg ggggaaaagt 1740

gtgagcttgt ttcaaagcct cagatgttcc ttgtagttca tggaagaggt acaagaaaac 1800

aaagacgtga cagaagggga tgaggtggtg acagtgagaa gcaactcgat gttcagcact 1860

ctccttcttg tccctctgtg aagcctcttc taccacatag ggctcagggc tgataaagcc 1920

ccctccctac ctttctcagg ccagacacaa ggtcagtcat gagaaaacag aaaaaaaagg 1980

agaagagagt ctgtagagac aaattgggag ggttcaggag gagaatttgg gatttgcctg 2040

tgcccatggg acacaggctg ggaataaaaa tgttttcctg actcttctct gaaagctaga 2100

tagactccac ctaaaaccct attgccaagg atgctgggcc cacagttgat gtcatctgag 2160

ggaaggggaa gctccttgtc tcttaaaggg acacagtgac cctctgagcc aagacacacc 2220

ctcaagtccg ctagcgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc 2280

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 2340

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 2400

ag 2402

<210> 79

<211> 2411

<212> DNA

<213> artificial sequence

<220>

<223> scAAV 9_h1_natural_mir-1-XD-14792_psg11_v2 (5 'itr..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(212)

<223> H1 native promoter

<220>

<221> misc_feature

<222> (213)..(351)

<223> miR1-1-XD-14792

<220>

<221> misc_feature

<222> (352)..(357)

<223> terminator

<220>

<221> misc_feature

<222> (358)..(2238)

<223> PSG11_V2

<220>

<221> misc_feature

<222> (2267)..(2411)

<223> 3' ITR

<400> 79

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cactctttcc cgcatgcaga ctgcctgctt gggtacagac 240

caaagagtag tcgaattatg gacctgctaa gctaattaac tactctttgg tctgaactca 300

ggccgggacc tctctcgccg cactgagggg cactccacac cacgggggcc gttttttgta 360

tacccctgga gattcttgat taagtgaatt aactagattt tagaaaggta ttgaggcagt 420

tatttgcaag gaacaattca aagtctggag acaagaagtc aagcaatact ttgagaagtg 480

gttgagcttt ttgggattgg agagaattac actattgaga tttgttaaaa atattgaaga 540

aggtttcacc ttgtgaaatt gtgtttctca attgaatctg agaaacaatt gagcagaggc 600

tggagataat tttgtctaaa agacagtggg gtacagggag caaaaaggcc agggagaaag 660

ggaactgcag gaattagtgc tgagaagcag gagttagtgg ttgaaggagg agatccaggc 720

ccagatacag gcaaataagt caattccctc tcccaagctt ggcagtcagc cctgcaggaa 780

ccaggataag agaaaagaag aaaagaaaag aaggattgaa ggtgttgtta ttttacttgg 840

gggagcgtca ggaacaagct tgtaacttga gatactgtat aattgtttct tcaaggacta 900

caatatttct gttggaaaat tgttgggagt tgattatatt cttgcagttt tttttccctc 960

tcaccttgtt tctcaggtgg tgatcagtcc tctgtcaatt ctctactgca ctcaggtatt 1020

ttggaaggct ttcagacgtg agaaaggctg attgctattt tctttgtcag tggaacttgt 1080

cacctttgca ccttttcttg gttgcaactt tttctcagtg tctctgtttg gtaaaaacta 1140

acaaaccagg cttgattctt tgcagttagc tctttatctt tctctcccac aagtagtcag 1200

tagccttgtt ctagtgtttt ttgtgttacc tctttttccg gaaaaaggaa ggtactgttg 1260

agttattgtt gttgatagta atatagtagc tgacagtggt tcctctgttt gtggcaactt 1320

gtttcccacc taaattgcac acttagaaac agttattggt aagccttata ttagatatat 1380

atagtgtctg gtagtcttac cctctctata attacacact ttttggcact gcccctttcc 1440

tgccttgcag agccccaggg gtgaatcttc ctatttctcc agctctgcaa cagtcactgt 1500

actcagtgct gtgtcccacg tgctgtgccc acagcctcaa acagccagtg acttcagagc 1560

caggacgcag ctcaggagtc tgccctgagg ttgcttctct tcttatttcc ttgcagcctg 1620

gcccggggga ggcttggctt gaactggcag ctcaatttag ccaaattcag gacaggccac 1680

caggactctt tctccacact tgctggtccc accccaggtt gagtgaggca gggccagtca 1740

ccagaggagc ccggagcaga gcaggaagca gagtctgagc tgctcctccc tcacccaagg 1800

ggcttcctcc tctcaattgg gggaaaagtg tgagcttgtt tcaaagcctc agttgttcct 1860

tgtagttctt ggaagaggta caagaaaaca aagacgtgac agaaggggtt gaggtggtga 1920

cagtgagaag caactcgttg ttcagcactc tccttcttgt ccctctgtga agcctcttct 1980

accacaaagg gctcagggct gataaagccc cctccctacc tttctcaggc cagacacaag 2040

gtcagtcttg agaaaacaga aaaaaaagga gaagagagtc tgtagagaca aattgggcaa 2100

attgttttcc tgactcttct ctgaaagcta gatagactcc acctaaaacc ctattgccaa 2160

ggttgctggg gagaataaag ccacagttgt tgtcaactga gggaagggga agctccttgt 2220

ctcttaaagg gacacagtgc tagcgtagat aagtagcatg gcgggttaat cattaactac 2280

aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 2340

gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 2400

cgagcgcgca g 2411

<210> 80

<211> 2395

<212> DNA

<213> artificial sequence

<220>

<223> H1_short_miR 16-2-3330_AMELY_V1 (5 'ITR..3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(335)

<223> miR16-2-3330

<220>

<221> misc_feature

<222> (336)..(341)

<223> terminator

<220>

<221> misc_feature

<222> (342)..(2222)

<223> AMELY_V1

<220>

<221> misc_feature

<222> (2251)..(2395)

<223> 3' ITR

<400> 80

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacttatgtt tggatgaact gacatacttg ttccactcta 240

tgctgagact gataatgtgg tagtgaaata tatattaaac cacattatcg ttctcagcat 300

ttagtgtgac agggatacag caactatttt atcaattttt ttattatgca tatcacataa 360

ttttatttaa aaatcattta gattttgatt gcgtgaagta cacaaccagt attccatatc 420

ctaatttgtt ttgtaataca gtcttcaaaa gttaaatgca aagaaattgt catataaact 480

tcccttcatt tggttcaggt ttcagtgatt gtatatgttc atacattttt attttatcaa 540

cgattttaac tgatgtgtaa acttcatttt ttaaaaagat ttgacgtagt ataatttcag 600

ttttctctaa aaagaaatat agtccaaatg gtgaatttgt atgagtaaat gagcagttct 660

taccttattc aagaatactc attcataata tgttgtctca aaagtttcat tcaacacata 720

gcaaaaggaa caattaaata atttaattgt ttccagtgag taccatttac aatacatgta 780

ttcaaaccaa tgtacaacca cttaagttga actctataat ttccaaagtt tctttgttca 840

ttcgttgtca ttagatatta ggtgtccatg aagccttctt ctacctttgg aacacttaaa 900

gcctattatt ttataacaaa ataacaattc tttccataaa tagctttgtc aaggtgtgag 960

cacattataa atccacggca ttttctatgc acagagagaa catgtgcttt agactagaat 1020

agccaatgat agtcgtcaga ttacttggga ttcaatcaat caatcaattt tcaaaaattc 1080

tgccccatat cacaattatt aagccattta taacatatat tatgattttt taattcagaa 1140

gttatcacat ttatacagta gtatatcatc aatagcttgt ataaattgga attacaggtg 1200

gatacatata agtgatagat cacataatct tatatacgta gaaataagat tttatggatg 1260

tggataaagt ttttttattg attattccca ttcatttgta agtgcttctt gtaatgggag 1320

attcagtcat aaattgaggt aacttctttt taaaagctta atcatctcaa gttagactga 1380

aaactactga aaatgggaaa ccttcgtata ggttttttta aagagatttc ccatctacct 1440

ttcatagctt gttcaaatat atgcactcta aaaattaccc accccatctt atcttaagcc 1500

ttcactgcaa ttctagcctc ttcttgtgag tcaaatcttt gaaatgaaag aactgatatt 1560

cccttattgc tgttgccctt aaaattaaga gggtttaata atatattttt ctattaatgg 1620

gttgtcataa tatacagtct ggtagaaatt caataataat aaaatttaaa cctatcataa 1680

aaactgtgtt cttaattacc aagccattta gataaatttg tttataatgt aaagatacaa 1740

catattactt ggactaatag aatcatagat ctgtaactac tacaactcta gaccctgagt 1800

tgttggaccc cttagaactc tggggccacc tttgctgcac cattttcaca ggatttgcat 1860

tgtagtggct tcacagagag tgactccaac cagagaagca gcagtgttgt ctttgccatt 1920

atggccatag gaatattgta tttcaaaaat ttgtaactgt gtacacagac atgatttttt 1980

acaaactaaa tttcccatta gtgtctgtat gtggagtaca catggtattt tcttaaaaat 2040

gcctaatatt ttcagggaat tacagatata ttagaatcca tttgatttaa tttactttca 2100

tcccatgcag aaattcactg atgcggtagg aaataagaag aaggtaaaac tgtttatttc 2160

cataggtcct gcttttcatt gtaagagcaa agcaaacata aaattatgcc gcaataatgt 2220

gagctagcgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga 2280

tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 2340

tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcag 2395

<210> 81

<211> 2395

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_miR16-2-1479_AMELY_V1 (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 short promoter

<220>

<221> misc_feature

<222> (204)..(335)

<223> miR16-2-1479

<220>

<221> misc_feature

<222> (336)..(341)

<223> terminator

<220>

<221> misc_feature

<222> (342)..(2222)

<223> AMELY_V1

<220>

<221> misc_feature

<222> (2251)..(2395)

<223> 3' ITR

<400> 81

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggacgc gtatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac cacttatgtt tggatgaact gacatacttg ttccactctt 240

gctaactggt ttgcccttgc tagtgaaata tatattaaag caagggcaag acagttagca 300

ttagtgtgac agggatacag caactatttt atcaattttt ttattatgca tatcacataa 360

ttttatttaa aaatcattta gattttgatt gcgtgaagta cacaaccagt attccatatc 420

ctaatttgtt ttgtaataca gtcttcaaaa gttaaatgca aagaaattgt catataaact 480

tcccttcatt tggttcaggt ttcagtgatt gtatatgttc atacattttt attttatcaa 540

cgattttaac tgatgtgtaa acttcatttt ttaaaaagat ttgacgtagt ataatttcag 600

ttttctctaa aaagaaatat agtccaaatg gtgaatttgt atgagtaaat gagcagttct 660

taccttattc aagaatactc attcataata tgttgtctca aaagtttcat tcaacacata 720

gcaaaaggaa caattaaata atttaattgt ttccagtgag taccatttac aatacatgta 780

ttcaaaccaa tgtacaacca cttaagttga actctataat ttccaaagtt tctttgttca 840

ttcgttgtca ttagatatta ggtgtccatg aagccttctt ctacctttgg aacacttaaa 900

gcctattatt ttataacaaa ataacaattc tttccataaa tagctttgtc aaggtgtgag 960

cacattataa atccacggca ttttctatgc acagagagaa catgtgcttt agactagaat 1020

agccaatgat agtcgtcaga ttacttggga ttcaatcaat caatcaattt tcaaaaattc 1080

tgccccatat cacaattatt aagccattta taacatatat tatgattttt taattcagaa 1140

gttatcacat ttatacagta gtatatcatc aatagcttgt ataaattgga attacaggtg 1200

gatacatata agtgatagat cacataatct tatatacgta gaaataagat tttatggatg 1260

tggataaagt ttttttattg attattccca ttcatttgta agtgcttctt gtaatgggag 1320

attcagtcat aaattgaggt aacttctttt taaaagctta atcatctcaa gttagactga 1380

aaactactga aaatgggaaa ccttcgtata ggttttttta aagagatttc ccatctacct 1440

ttcatagctt gttcaaatat atgcactcta aaaattaccc accccatctt atcttaagcc 1500

ttcactgcaa ttctagcctc ttcttgtgag tcaaatcttt gaaatgaaag aactgatatt 1560

cccttattgc tgttgccctt aaaattaaga gggtttaata atatattttt ctattaatgg 1620

gttgtcataa tatacagtct ggtagaaatt caataataat aaaatttaaa cctatcataa 1680

aaactgtgtt cttaattacc aagccattta gataaatttg tttataatgt aaagatacaa 1740

catattactt ggactaatag aatcatagat ctgtaactac tacaactcta gaccctgagt 1800

tgttggaccc cttagaactc tggggccacc tttgctgcac cattttcaca ggatttgcat 1860

tgtagtggct tcacagagag tgactccaac cagagaagca gcagtgttgt ctttgccatt 1920

atggccatag gaatattgta tttcaaaaat ttgtaactgt gtacacagac atgatttttt 1980

acaaactaaa tttcccatta gtgtctgtat gtggagtaca catggtattt tcttaaaaat 2040

gcctaatatt ttcagggaat tacagatata ttagaatcca tttgatttaa tttactttca 2100

tcccatgcag aaattcactg atgcggtagg aaataagaag aaggtaaaac tgtttatttc 2160

cataggtcct gcttttcatt gtaagagcaa agcaaacata aaattatgcc gcaataatgt 2220

gagctagcgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga 2280

tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 2340

tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcag 2395

<210> 82

<211> 2331

<212> DNA

<213> artificial sequence

<220>

<223> scAAV_H1_miR1-1-XD-14792_CBh_GFP_SV40p (5' ITR .. 3' ITR)

<220>

<221> misc_feature

<222> (1)..(106)

<223> 5' ITR

<220>

<221> misc_feature

<222> (113)..(203)

<223> H1 promoter

<220>

<221> misc_feature

<222> (204)..(342)

<223> miR1-1-XD-14792

<220>

<221> misc_feature

<222> (343)..(348)

<223> terminator

<220>

<221> misc_feature

<222> (349)..(634)

<223> CMV early enhancer contains an 18-bp deletion relative to the standard CMV enhancer

<220>

<221> misc_feature

<222> (636)..(913)

<223> chicken beta actin promoter

<220>

<221> misc_feature

<222> (983)..(1079)

<223> SV40 intron with splice donor and acceptor sites

<220>

<221> misc_feature

<222> (1153)..(1869)

<223> GFP, mammalian codon optimization

<220>

<221> misc_feature

<222> (1870)..(1899)

<223> Myc epitope tag

<220>

<221> misc_feature

<222> (1939)..(2161)

<223> SV40 termination

<220>

<221> misc_feature

<222> (2187)..(2331)

<223> 3' ITR

<400> 82

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggctcg agatatttgc 120

atgtcgctat gtgttctggg aaatcaccat aaacgtgaaa tgtctttgga tttgggaatc 180

ttataagttc tgtatgagac caccatgcag actgcctgct tgggtacaga ccaaagagta 240

gtcgaattat ggacctgcta agctaattaa ctactctttg gtctgaactc aggccgggac 300

ctctctcgcc gcactgaggg gcactccaca ccacgggggc cgttttttcg ttacataact 360

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaatagt 420

aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca 480

cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 540

taaatggccc gcctggcatt gtgcccagta catgacctta tgggactttc ctacttggca 600

gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt 660

cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 720

attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg 780

gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc 840

gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag 900

cgcgcggcgg gcgggagcgg gatcagccac cgcggtggcg gcctagagtc gacgaggaac 960

tgaaaaacca gaaagttaac tggtaagttt agtctttttg tcttttattt caggtcccgg 1020

atccggtggt ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt tacttctagg 1080

cctgtacgga agtgttactt ctgctctaaa agctgcggaa ttgtacccgc ggccgatcca 1140

ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 1200

gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 1260

gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 1320

ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 1380

gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 1440

cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 1500

ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1560

atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1620

aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1680

gttcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1740

cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1800

gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1860

ctgtacaagg agcagaagct gatcagcgag gaggacctgt agcctgcagg ggccctcgac 1920

ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa 1980

ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 2040

taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 2100

aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta 2160

accggtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga 2220

gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 2280

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca g 2331

Claims

1. A vector stuffer sequence comprising a nucleic acid having a length of about 1300 to about 2300 nucleotides and at least 75% identity to any one of: SEQ ID NO. 45-51; 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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.

2. The vector stuffer sequence according to claim 1, wherein said nucleic acid is about 1500-2000 nucleotides in length.

3. The vector stuffer sequence according to claim 1 or 2, wherein said nucleic acid is about 1600 to 1900 nucleotides in length.

4. A vector stuffer sequence according to any one of claims 1 to 3, wherein said nucleic acid has at least 80% identity to any one of: SEQ ID NO. 45-51; 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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.

5. The vector stuffer sequence according to any one of claims 1 to 4, wherein said nucleic acid has at least 85% identity to any one of: SEQ ID NO. 45-51; 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 to 2187 of SEQ ID NO. 20; nucleotides 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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.

6. The vector stuffer sequence according to any one of claims 1 to 5, wherein said nucleic acid has at least 90% identity to any one of: SEQ ID NO. 45-51; 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 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; and 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.

7. The vector stuffer sequence according to any one of claims 1 to 6, wherein said nucleic acid has at least 95% identity to any one of: SEQ ID NO. 45-51; 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 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; and nucleotides 342-2222 of SEQ ID NO. 80; nucleotides 342-2222 of SEQ ID NO. 81.

8. The vector stuffer sequence according to any one of claims 1 to 7, wherein said nucleic acid has at least 97% identity to any one of: SEQ ID NO. 45-51; 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 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; and 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.

9. The vector stuffer sequence according to any one of claims 1 to 8, wherein said nucleic acid comprises or consists of any one of: SEQ ID NO. 45-51; 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 711 to 2187 of SEQ ID NO. 21; nucleotides 488-2177 of SEQ ID NO. 22; nucleotides 711 to 2187 of SEQ ID NO. 23; nucleotides 711 to 2187 of SEQ ID NO. 24; nucleotides 252-2132 of SEQ ID NO. 25; nucleotide 252-2132 of SEQ ID NO. 26; nucleotide 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; nucleotide 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; and 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.

10. The vector stuffer sequence according to any one of claims 1 to 9, wherein said vector is an adeno-associated virus (AAV) vector, optionally wherein said AAV vector is self-complementing.

11. The vector stuffer sequence according to any one of claims 1 to 10, wherein said vector stuffer sequence is adjacent to an expression construct comprising a heterologous nucleic acid sequence.

12. The vector stuffer sequence according to claim 11, wherein said heterologous nucleic acid sequence encodes a therapeutic agent.

13. The vector stuffer sequence according to claim 12, wherein said therapeutic agent comprises a nucleic acid encoding an inhibitory nucleic acid.

14. The vector stuffer sequence according to claim 13, wherein said inhibitory nucleic acid comprises siRNA, miRNA, shRNA or dsRNA.

15. The vector stuffer sequence according to claim 14, wherein said inhibitory nucleic acid comprises a miRNA targeting a gene associated with a neurodegenerative disease, optionally wherein said neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, age-related edge TDP-43 encephalopathy, chronic traumatic encephalopathy, lewy body dementia, corticobasal degeneration, progressive Supranuclear Palsy (PSP), dementia with guam parkinsonism (G-PDC), pick's disease, hippocampal sclerosis, huntington's disease, parkinson's disease or alzheimer's disease.

16. The vector stuffer sequence according to claim 15, wherein said neurodegenerative disease is a polyglutamine repeat disease.

17. The vector stuffer sequence according to any one of claims 13 to 16, wherein said inhibitory nucleic acid targets ATXN2.

18. The vector stuffer sequence according to any one of claims 13 to 17, wherein said heterologous nucleic acid encodes an artificial miRNA comprising:

(a) A guide sequence selected from SEQ ID NOS 1-4 and 71;

(b) The guide sequence provided in SEQ ID NO. 1 and the passenger sequence provided by SEQ ID NO. 5; the guide sequence provided in SEQ ID NO. 2 and the passenger sequence provided by SEQ ID NO. 6; a guide sequence provided in SEQ ID NO. 3 and a passenger sequence provided by SEQ ID NO. 7; a guide sequence provided by SEQ ID NO. 4 and a passenger sequence provided by SEQ ID NO. 8; or a guide sequence provided by SEQ ID NO. 71 and a passenger sequence provided by SEQ ID NO. 72; or alternatively

(c) The sequence provided by any one of SEQ ID NOS 9-12 and 73.

19. The vector stuffer sequence according to any one of claims 11 to 18, wherein said expression construct comprises a promoter, polyadenylation signal, termination signal, or any combination thereof.

20. The vector stuffer sequence according to claim 19, wherein said promoter is an H1 promoter, optionally wherein:

(a) The H1 promoter is an H1 long promoter comprising SEQ ID NO. 52; nucleotides 113-343 of any one of SEQ ID NOs 13-16 and 18-24;

(b) The H1 promoter is an H1 promoter comprising SEQ ID NO. 53; or alternatively

(c) The H1 promoter is an H1 short promoter comprising nucleotide 113-203 of SEQ ID NO. 54 or any of SEQ ID NO. 17, 25-28 and 37-44.

21. The vector stuffer sequence according to claim 19 or 20, wherein said termination signal is an SV40 termination signal, optionally wherein said SV40 termination signal comprises the sequence provided by SEQ ID No. 77.

22. A recombinant AAV vector comprising the vector stuffer sequence according to any one of claims 1 to 21.

23. The recombinant AAV vector of claim 22, wherein the AAV vector is self-complementing.

24. The recombinant AAV vector of claim 22 or 23, wherein the AAV vector comprises 5 'Inverted Terminal Repeats (ITRs) and 3' ITRs flanking the expression construct and the vector stuffer sequence.

25. The recombinant AAV vector of claim 24, wherein the 5'itr and the 3' itr 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.

26. The recombinant AAV vector of claim 24 or 25, wherein one of the 5'itr and the 3' itr lacks a functional terminal dissociation site.

27. The recombinant AAV vector of claim 26, wherein the 5' itr lacks a functional terminal dissociation site.

28. The recombinant AAV vector of any one of claims 24-27, wherein:

(a) The 5' ITR comprises SEQ ID NO. 57; or nucleotides 1 to 106 of any one of SEQ ID NOS 13 to 24, 29 to 44 and 78 to 82; and/or

(b) The 3' ITR comprises SEQ ID NO. 58; nucleotides 2192-2358 of any one of SEQ ID NOs 13-16; nucleotides 2229-2395 of SEQ ID NO. 17; nucleotides 2184-2350 of SEQ ID NO. 18; nucleotides 2214-2358 of any one of SEQ ID NOs 13-16; nucleotides 2251-2395 of SEQ ID NO. 17; 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 2161-2305 of SEQ ID NO. 26; nucleotides 2161-2305 of SEQ ID NO. 27; nucleotides 2161-2305 of SEQ ID NO. 28; nucleotides 2266 to 2410 of SEQ ID NO. 29; nucleotides 2224-2368 of SEQ ID NO. 30; nucleotides 2216-2360 of SEQ ID NO. 31; nucleotides 2225-2369 of SEQ ID NO. 32; nucleotides 2266 to 2410 of SEQ ID NO. 33; nucleotides 2224-2368 of SEQ ID NO. 34; nucleotides 2216-2360 of SEQ ID NO. 35; nucleotides 2225-2369 of SEQ ID NO. 36; nucleotides 2257-2401 of SEQ ID NO. 37; nucleotides 2258-2402 of SEQ ID NO. 38; nucleotides 2215-2359 of SEQ ID NO. 39; nucleotides 2207-2351 of SEQ ID NO. 40; nucleotides 2207-2351 of SEQ ID NO. 41; nucleotides 2257-2401 of SEQ ID NO. 42; nucleotides 2215-2359 of SEQ ID NO. 43; nucleotides 2207-2351 of SEQ ID NO. 44; nucleotides 2258-2402 of SEQ ID NO. 78; nucleotides 2267-2411 of SEQ ID NO. 79; nucleotides 2251-2395 of SEQ ID NO. 80; nucleotides 2251-2395 of SEQ ID NO. 81; or nucleotides 2187-2331 of SEQ ID NO. 82.

29. The recombinant AAV vector of any one of claims 22-28, comprising:

(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, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(b) The nucleotide sequence of SEQ ID NO. 14, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(c) The nucleotide sequence of SEQ ID NO. 15, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(d) The nucleotide sequence of SEQ ID NO. 16, wherein nucleotides 344-481 are replaced by 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 344-481 are replaced by a sequence encoding a miRNA of interest;

(g) The nucleotide sequence of SEQ ID NO. 19, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(h) The nucleotide sequence of SEQ ID NO. 20, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(i) The nucleotide sequence of SEQ ID NO. 21, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(j) The nucleotide sequence of SEQ ID NO. 22, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(k) The nucleotide sequence of SEQ ID NO. 23, wherein nucleotides 344-481 are replaced by a sequence encoding a miRNA of interest;

(l) The nucleotide sequence of SEQ ID NO. 24, wherein nucleotides 344-481 are replaced by 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, wherein 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 204-341 are replaced by a sequence encoding a miRNA of interest;

(v) The nucleotide sequence of SEQ ID NO. 38, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(w) the nucleotide sequence of SEQ ID NO. 39, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(x) The nucleotide sequence of SEQ ID NO. 40, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(y) the nucleotide sequence of SEQ ID NO. 41, wherein nucleotides 204-341 are replaced with a sequence encoding a miRNA of interest;

(z) the nucleotide sequence of SEQ ID NO. 42, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(aa) the nucleotide sequence of SEQ ID NO. 43, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(bb) the nucleotide sequence of SEQ ID NO. 44, wherein nucleotides 204-341 are replaced by a sequence encoding a miRNA of interest;

(cc) the nucleotide sequence of SEQ ID NO. 78, wherein nucleotides 204-342 are replaced with a sequence encoding an artificial miRNA of interest;

(dd) the nucleotide sequence of SEQ ID NO. 79, wherein nucleotides 213-351 are replaced by a sequence encoding an artificial miRNA of interest;

(ee) the nucleotide sequence shown in SEQ ID NO. 80, wherein nucleotides 204-335 are replaced with a sequence encoding an artificial miRNA of interest; or alternatively

(ff) the nucleotide sequence shown in SEQ ID NO. 81, wherein nucleotides 204-335 are replaced with a sequence encoding an artificial miRNA of interest.

30. A rAAV particle comprising the rAAV vector of any one of claims 22-29.

31. The rAAV particle of claim 30, wherein the rAAV particle comprises a capsid protein.

32. The rAAV particle of claim 31, wherein the capsid protein is capable of crossing the blood brain barrier.

33. The rAAV particle of claim 31 or 32, wherein the capsid protein is an AAV9 capsid protein.

34. A method of delivering a therapeutic agent to a subject, the method comprising administering the rAAV particle of any one of claims 28-32 to the subject.

35. The method of claim 34, wherein the subject has or is at risk of developing a neurodegenerative disease.

36. The method of claim 34 or 35, wherein the administering comprises direct injection into the CNS of the subject.

37. The method of claim 36, wherein the direct injection is an intra-brain injection, an intra-brain parenchymal injection, an intrathecal injection, an intrastriatal injection, an subpial injection, or any combination thereof.

38. The method of claim 36, wherein the direct injection is direct injection into cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracisternal injection, intraventricular injection, and/or lumbar intra-injection.

39. The method of any one of claims 35 to 38, wherein the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, primary lateral sclerosis, progressive muscular atrophy, edge-dominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, lewy body dementia, corticobasal degeneration, progressive Supranuclear Palsy (PSP), dementia-island-type parkinsonism ALS syndrome (G-PDC), pick's disease, hippocampus sclerosis, huntington's disease, parkinson's disease, or alzheimer's disease.