RU2847339C1 - Gene therapy vector for eef1a2 and its application - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: recombinant adeno-associated virus (rAAV) virion is proposed for enhancing the expression of eEF1A2, containing a capsid and a vector genome, wherein the vector genome contains a polynucleotide sequence encoding the eEF1A2 protein, functionally linked to a promoter, wherein the promoter contains: a) the hSYN promoter containing a polynucleotide sequence that contains SEQ ID NO: 3; or b) the eSYN promoter. A method for treating and/or preventing a neurological disease or disorder in a subject in need thereof, comprising administering an rAAV virion to the subject; a method for ensuring expression of eEF1A2 in the brain of the subject in need thereof, comprising administering an rAAV virion to the subject; the use of a pharmaceutical composition containing the rAAV virion in the treatment of a subject who has or is suspected of having one or more mutations in the eEF1A2 gene; and a kit for enhancing eEF1A2 expression, comprising the rAAV virion and instructions for use.

EFFECT: providing therapy for diseases associated with EEF1A2.

55 cl, 12 dwg, 5 tbl, 5 ex

Description

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63/055,775, filed July 23, 2020, the contents of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the disclosure. The name of the text file containing the sequence listing is ROPA_019_02WO_ST25.txt. The text file, approximately 101 kilobytes in size, was generated on July 20, 2021, and is submitted electronically via EFS-Web.

LEVEL OF TECHNOLOGY

The EEF1A2 gene encodes eukaryotic elongation factor 1, alpha 2 (eEF1A2), a protein involved in protein synthesis, suppression of apoptosis, and regulation of actin function and cytoskeletal structure. Murine and human orthologs share identity at 462 of 463 amino acid positions. EEF1A2 is a potential oncogene because it is overexpressed in ovarian cancer. In ovarian cancer studies, a lentiviral vector encoding EEF1A2 was experimentally used to transduce immortalized ovarian surface epithelial (IOSE) cells, demonstrating that eEF1A2 promotes tumorigenesis in non-neoplastic progenitor cells. Sun et al. Int J Cancer . 123(8):1761–176 (2008).

EEF1A2 is expressed at high levels in the central nervous system (CNS), as well as in the heart and muscle. Complete loss of Eef1a2 in mice causes motor neuron degeneration, a phenotype termed "wasted," the genotype of which is called wst . Davies et al. Sci Rep. 7:46019 (2017). Point mutations in the human EEF1A2 gene have recently been shown to be various causes of epilepsy, intellectual disability, and/or autism. Cao et al. Human Molecular Genetics. 26(18):3545–3552 (2017); Lam et al. Mol Genet Genomic Med. 4(4):465–74 (2016); Nakajima et al. Clin Genet. 87(4):356–61 (2015).

Experiments using transgenic mice carrying wild-type Eef1a2 with a bacterial artificial chromosome (BAC) confirmed that wild-type Eefl1a2 , when present during development, complements the wst genotype. Newbury et al. J. Bio. Chem. 282:2891–50 (2007).

EEF1A2 -associated disease is rare. Only approximately 100 people worldwide have been identified as having an EEF1A2 mutation. The etiology of the disease remains poorly understood. Therefore, it is unclear whether reversal of the disease phenotype can be achieved through postnatal expression of wild-type EEF1A2 . Furthermore, delivery of gene therapy to the central nervous system is complex and unpredictable.

There is an unmet need for treatment of EEF1A2 -associated diseases. This paper presents a solution to this need using gene therapy.

ESSENCE OF THE INVENTION

The present invention generally relates to gene therapy for neurological diseases or disorders using adeno-associated virus (AAV)-based delivery of a polynucleotide encoding eEF1A2 or a functional variant thereof.

In one aspect, the present invention provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding the eEF1A2 protein or a functional variant thereof operably linked to a promoter. The promoter may be a neuronal-specific promoter, For example, the human synapsin 1 (hSYN) promoter. The capsid can be the AAV9 capsid or a functional variant. Other promoters or capsids can be used.

In another aspect, the present invention provides a method for treating and/or preventing a neurological disease or disorder in a subject in need thereof, comprising administering to the subject an rAAV virion according to the present invention or a pharmaceutical composition thereof. The rAAV virion can be administered by the intracerebral and/or intravenous route.

In another aspect, the present invention provides a method for expressing eEF1A2 in the brain of a subject in need thereof, comprising administering to the subject an rAAV virion according to the present invention or a pharmaceutical composition thereof. The rAAV virion can be administered by the intracerebral and/or intravenous route.

In further aspects, the present invention provides polynucleotides (e.g., vector genomes), pharmaceutical compositions, kits and other compositions and methods.

Various other aspects and embodiments are disclosed in the following detailed description. The present invention is limited solely by the appended claims.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows a domain diagram of eEF1A2 showing the point mutations associated with the disease.

Fig. 2 shows a vector diagram of a non-limiting example of a vector genome.

Fig. 3 shows a vector diagram of a non-limiting example of a vector genome.

Fig. 4 shows a vector diagram of a non-limiting example of a vector genome.

Fig. 5 shows a vector diagram of a non-limiting example of a vector genome.

Fig. 6 shows a vector diagram of a non-limiting example of a vector genome.

Figure 7 shows immunofluorescence microscopy of mice after neonatal injection, either intracerebral (IC) or intravenously (IV), of AAV9-hSyn-eEF1A2-2A-eGFP or control. Scale bar, 300 µm.

Figure 8A shows immunohistochemistry of mice after neonatal injection, either intracerebral (IC) or intravenous (IV), of AAV9-hSyn-eEF1A2-2A-eGFP or control. Figure 8B shows a magnified view of the same sections. Scale bar, 300 μm.

Figure 9A shows the survival of untreated wst / wst (null) mice compared to mice treated by the intracerebral (IC), intravenous (IV) route, or a combination of both (IC+IV).

Figure 9B shows weight loss in untreated wst / wst (null) mice compared to mice treated by the intracerebral (IC), intravenous (IV) route, or a combination of both (IC+IV).

Fig. 9C shows rotarod testing in untreated wst / wst (null) mice compared to mice treated by the intracerebral (IC), intravenous (IV) route, or a combination of both (IC+IV).

Fig. 9D shows inverted grid testing in untreated wst / wst (null) mice compared to mice treated by the intracerebral (IC), intravenous (IV) route, or a combination of both (IC+IV).

Figure 9E shows eEF1A2 expression in untreated wst / wst (null) mice compared to mice treated intracerebrally (IC), intravenously (IV), or a combination of both (IC+IV). Scale bar, 125 µm.

Fig. 9F shows eEF1A2 expression in untreated wst / wst (null) mice compared to mice treated by the intracerebral (IC), intravenous (IV) route, or a combination of both (IC+IV).

Figures 10A-10K show comparisons of AAV9 vectors containing the vector genomes shown in Figure 2 (“V1”), Figure 3 (“V2”), Figure 4 (“V3”), and Figure 6 (“V4”) administered at 2e10 ¹¹ vg/animal.

Figure 10A shows the Kaplan-Meier survival plot for wild type, FBS-treated, wst/wst , and intracerebroventricularly treated wst/wst animals with gene therapy treatment with V1, V2, V3, and V4.

Fig. 10B shows the weights of mice (mean ± SEM). Animals were weighed daily until postnatal age 35 and then weekly until sacrifice at P60 or a humane endpoint of 15% weight loss.

Fig. 10C shows the assessment of muscle strength by inverted grid testing on day P15.

Fig. 10D shows the assessment of muscle strength using rotarod testing on day P15.

Fig. 10E shows the assessment of muscle strength by inverted grid testing on day P23.

Fig. 10F shows the assessment of muscle strength using rotarod testing on day P23.

Fig. 10G shows representative immunostaining for eEF1A2 throughout the brain using free-floating immunohistochemistry for wild type with FBS (a buffered saline solution) as a physiological reference (n = 4–5 per group, scale bar 250 μm).

Fig. 10H shows representative immunohistofluorescence in cortical areas for neurons (NeuN marker) coexpressing eEF1A2 (n = 4.5 per group, 200 μm).

OnFig. 10IA representative immunoblot for eEF1A2 in the brain with quantification is shown, showing brain-wide eEF1A2 expression achieved with all gene therapy vectors with higher expression in the midbrain, cerebellum, and hindbrain regions compared with the V4 vector (mean ± SEM, two-way ANOVA).

Fig. 10J shows qPCR for human eEF1A2 transcript expression in the forebrain, showing the highest mRNA expression with the V1 vector (mean ± SEM, two-way ANOVA).

Figure 10K shows qPCR for human cortical eEF1A2 expression in the cerebral cortex, showing the highest mRNA expression with the V1 vector (mean ± SEM, two-way ANOVA).

Fig. 11A-11C.

Figure 11A shows the Kaplan-Meier survival plot of wild type, FBS-treated, FBS-treated D252H/+, knockout (D252H-/-), intracerebroventricularly treated with V3 gene therapy D252H-/- (wild type FBS, n = 12, D252H/+ FBS, n = 5, D252H-/- FBS, n = 4, and 2 x 10 ¹¹ vg/pup, V3-treated n = 5).

Figure 11B shows body weight over time (mean ± SEM, one-way ANOVA and Dunnett's multiple comparison).

OnFig. 11CAn assessment of motor skills using rotarod testing is presented (mean data ± SEM, two-way ANOVA and Dunnett's multiple comparison).

Figure 12A shows the Kaplan-Meier survival plot for Del22ex3 treated with high dose V3 (2 x 10 ¹¹ vg/pup, n=5), Del22ex3 treated with low dose V3 (2 x 10 ¹⁰ vg/pup, n=5), Del22ex3 controls treated with buffer solution (n=3), and wild-type controls treated with buffer solution (n=6).

Fig. 12B shows body weight over time (mean ± SEM).

Fig. 12C shows the assessment of motor function by grip strength manometry P22–25 (mean ± SEM)

OnFig. 12Dpresented P23 grip strength manometry(mean data ± S.E.M., two-way ANOVA and Tukey's multiple comparison).

Fig. 12E shows the motor assessment using rotarod testing P22–25 (mean ± SEM).

OnFig. 12FThe data from the rotarod test on P24 is presented (mean ± S.E.M., two-way ANOVA and Tukey's multiple comparison).

Fig. 12G shows neurological assessments from P21–25.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Definitions

Section headings are for organizational purposes only and should not be construed as limiting the described subject matter to particular aspects or embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practicing the present invention. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In the event of a dispute, the present description, including definitions, will control. Furthermore, the materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting.

All publications and patents mentioned in this document are hereby incorporated by reference in their entirety, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the event of a dispute, this application, including any definitions provided herein, will control. However, citation of any literature, article, publication, patent, patent publication, or patent application cited in this document does not constitute and should not be taken as an admission or suggestion in any way that they constitute valid prior art or are part of the general knowledge anywhere in the world.

In this specification, any concentration range, percentage range, ratio range, or whole number range shall be understood to include the value of any whole number within the stated range and, where appropriate, fractions thereof (e.g., one tenth and one hundredth of a whole number), unless otherwise stated. The term "about," when immediately preceding a number or figure, means that the number or figure is within a range of plus or minus 10%. As used herein, singular terms shall be understood to refer to "one or more" of the listed components, unless otherwise stated. The use of an alternative (e.g., "or" should be understood to mean either one, both, or any combination of alternatives. The term "and/or" should be understood to mean either one or both alternatives. As used in this document, the terms "include" and "contain" are used synonymously.

As used herein, the terms "identity" and "identical" refer, in part to a polypeptide or polynucleotide sequence, to the percentage of exactly matching residues in the results of an alignment of such a "query" sequence with a "subject" sequence, such as the results of an alignment generated by the BLAST algorithm. Identity is calculated, unless otherwise noted, over the entire length of the subject sequence. Thus, a query sequence "has at least x% identity" to a subject sequence if, in an alignment of the query sequence with the subject sequence, at least x% (rounded down) of the residues in the subject sequence align as an exact match with the corresponding residue in the query sequence. If the subject sequence has variable positions (e.g., residues marked with X), a match is considered to be an alignment with any residue in the query sequence.

As used herein, the term "AAV-based vector" or "rAAV-based vector" refers to a recombinant vector containing one or more polynucleotides (or transgenes) of interest flanked by AAV terminal repeat (ITR) sequences. Such AAV-based vectors can be replicated and packaged into infectious viral particles if they are present in a host cell that has been transfected with a plasmid encoding and expressing the rep and cap gene products. Alternatively, AAV-based vectors can be packaged into infectious particles using a host cell that has been stably engineered to express the rep and cap genes.

As used herein, the term "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle that consists of at least one AAV capsid protein and an encapsidated AAV-based polynucleotide vector. As used herein, if the particle contains a heterologous polynucleotide (i.e., When a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered into a mammalian cell, it is commonly referred to as an "AAV vector particle" or simply an "AAV-based vector." Thus, producing an AAV vector particle necessarily involves producing an AAV-based vector, since such a vector is contained within the AAV vector particle.

As used herein, the term "promoter" refers to a polynucleotide sequence capable of stimulating the initiation of transcription of RNA from a polynucleotide in a eukaryotic cell.

As used herein, the term “vector genome” refers to a polynucleotide sequence packaged by a vector (e.g., rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of the vector genome between the flanking ITR sequences. "Expression cassette" implies that the vector genome contains at least one gene encoding a gene product operably linked to an expression control element (e.g., promoter).

As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of developing or suffering from a disease, disorder, or pathological condition that is treatable or ameliorated by the recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may, for example, be a patient or subject diagnosed with a central nervous system disorder. The subject may have a mutation in the EEF1A2 gene or a deletion of all or part of the EEF1A2 gene or its regulatory sequences, resulting in aberrant expression of the eEF1A2 protein. The terms "subject" and "patient" are used interchangeably herein. A subject treated with the methods described herein may be an adult or a child. Subjects may vary in age.

As used herein, the term "variant" or "functional variant" refers interchangeably to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein that retains one or more required activities of the parent protein.

As used herein, the term "genetic disorder" refers to a partial or complete loss of function or aberrant activity of a gene. For example, a subject may have a genetic disorder of gene expression or function.EEF1A2, which reduces the expression of, or results in loss or aberrant function of, the eEF1A2 protein in at least some cells (e.g., neurons) of the subject.

As used herein, the term "treatment" refers to the alleviation of one or more symptoms of a disease or disorder. The term "prevention" refers to delaying or interrupting the onset of one or more symptoms of a disease or disorder or slowing the progression of an eEF1A2-associated neurological disease or disorder.

Protein or polynucleotide eEF1A2

The present invention provides compositions and methods of use related to the protein elongation factor 1-alpha 2 (eEF1A2). Various mutations in EEF1A2 , shown in Fig. 1 , are known to be associated with neurological disorders, including epilepsy, mental retardation, and/or autism. Both inherited and de novo mutations have been observed. In some cases, a heterozygous missense mutation is sufficient to cause disease.

The polypeptide sequence of eEF1A2 is as follows:

MGKEKTHINIVVIGHVDSGKSTTTGHLIYKCGGIDKRTIEKFEKEAAEMGKGSFKYAWVLDKLKAERERGITIDISLWKFETTKYYITIIDAPGHRDFIKNMITGTSQADCAVLI VAAGVGEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPAYSEKRYDEIVKEVSAYIKKIGYNPATVPFVPISGWHGDNMLEPSPNMPWFKGWKVERKEGNASGVSLLEA LDTILPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGILRPGMVVTFAPVNITTEVKSVEMHHEALSEALPGDNVGFNVKNVSVKDIRRGNVCGDSKSDPPQEAAQFTSQVIIL NHPGQISAGYSPVIDCHTAHIACKFAELKEKIDRRSGKKLEDNPKSLKSGDAAIVEMVPGKPMCVESFSQYPPLGRFAVRDMRQTVAVGVIKNVEKKSGGAGKVTKSAQKAQKAGK

(SEQ ID NO: 1).

In some embodiments, the eEF1A2 protein comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an eEF1A2 protein or a functional variant thereof operably linked to a promoter. In some embodiments, the present invention provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an eEF1A2 protein operably linked to a promoter. A polynucleotide encoding an eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTATGCCTG GGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTGATCGT GGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAGGAAG TCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGGCCC TGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGGTG AAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCTG AACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGTG CCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAG

(SEQ ID NO: 2).

The polynucleotide sequence encoding the eEF1A2 protein can be codon optimized.

A polynucleotide encoding the eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGTAAAGAAAAAACACATATTAATATAGTAGTAATCGGTCATGTTGACTCTGGAAAATCTACTACTACAGGACATTTGATTTATAAATGTGGAGGAATTGATAAAAGAACAATAGAAAAATTTGAAAAAGAAGCTGCTGAAATGGGTAAAGGTAGTTTTAAATATGCTTGG GTTTTGGATAAATTGAAAGCTGAAAGAGAAAGAGGAATTACAATTGATATTTCTTTGTGGAAATTTGAAACTACAAAATATTATATAACAATAATAGATGCTCCTGGACATAGAGATTTTATTAAAAATATGATTACAGGAACTTCTCAAGCAGATTGTGCTGTTTTGATAGTA GCAGCAGGAGTTGGTGAATTCGAAGCAGGCATTTCTAAAAATGGACAAACTAGAGAACATGCTTTGTTGGCTTATACATTGGGCGTAAAACAATTGATTGTAGGAGTTAATAAAATGGATTCTACTGAACCTGCATATTCTGAAAAAAGATATGATGAAATAGTAAAAGAAGTT TCTGCTTATATTAAAAAAATTGGTTATAATCCTGCTACAGTTCCATTTGTTCCTATTTCTGGATGGCATGGAGATAATATGTTGGAACCTAGTCCTAATATGCCTTGGTTTTAAAGGATGGAAAGTTGAAAGGAAAGAAGGAAATGCATCAGGAGTCTCCTTGTTGGAAGCTTTG GATACAATCTTGCCTCCAACAAGACCTACAGATAAACCTTTTGAGATTGCCTCTTCAAGATGTATATAAAATAGGAGGAATAGGAACAGTGCCAGTTGGAAGAGTAGAAACAGGTATATTGAGACCTGGAATGGTTGTAACATTTGCACCAGTTAATATAACTACTGAAGTAAAA TCTGTTGAAATGCATCATGAAGCTTTGTCTGAAGCTCTTCCTGGAGATAATGTAGGATTTAATGTTAAAAATGTAAGTGTAAAAGATATAAGAAGAGGAAATGTATGTGGTGATAGTAAATCAGATCCACCTCAAGAAGCAGCTCAATTTACATCACAAGTAATAATATTGAAT CATCCTGGACAAATTTCTGCAGGATATTCACCAGTAATAGATTGTCATACAGCACATATAGCTTGTAAATTTGCTGAATTGAAAGAAAAAATTGATAGAAGAAGTGGAAAAAAACTTGAAGATAATCCTAAATCATTGAAATCAGGAGATGCAGCTATTGTAGAAATGGTACCT GGAAAACCAATGTGTGTAGAATCTTTTTCTCAATATCCACCTCTCGGAAGATTTGCTGTTAGAGATATGAGACAAACAGTTGCAGTAGGAGTTATTAAAAATGTAGAAAAAAAAAGCGGAGGTGCAGGAAAGGTTACAAAATCCGCACAAAAAGCTCAAAAAGCTGGTAAATAA

(SEQ ID NO: 4).

A polynucleotide encoding the eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGCAAAGAAAAAACACATATAAACATTGTCGTTATCGGACACGTTGATTCTGGTAAAAGTACAACAACCGGTCACTTGATATACAAATGCGGGGGTATAGACAAACGCACTATTGAAAAGTTCGAGAAAGAAGCTGCGGAGATGGGCAAAGGCTCATTCAAGTACGCGTGG GTACTCGATAAGTTGAAAGCTGAACGCGAGAGGGGAATCACCATAGACATCTCACTTTGGAAATTCGAGACAACCAAGTATTACATAACTATTATAGATGCCCCAGGCCACAGGGATTTCATTAAAAATATGATAACCGGCACATCTCAAGCCGATTGCGCCGTACTCATCGTC GCCGCTGGTGTGGGTGAGTTCGAGGCAGGTATTTCTAAAAATGGCCAGACACGCGAACATGCTCTTCTGGCTTATACACTCGGGGTTAAACAGCTCATAGTAGGAGTGAATAAGATGGACTCCACTGAACCCGCCTATTCAGAAGCGCTATGACGAAATTGTAAAGGAGGTC TCAGCATATATTAAAAAAATTGGCTATAACCCAGCCACGGTGCCATTCGTCCCGATTAGTGGATGGCATGGTGACAATATGCTGGAACCAAGTCCCAATATGCCTTGGTTTAAGGGTTGGAAAGTAGAGCGGAAAGAGGGTAATGCTTCCGGCGTGTCATTGCTGGAGGCGCTT GACACGATACTCCCACCCACAAGGCCAACTGATAAGCCACTCCGATTGCCCTTGCAGGACGTGTACAAGATTGGGGAATTGGGACTGTGCCCGTCGGGCGCGTGGAGACGGGCATCCTCAGACCTGGGATGGTAGTCACTTTTGCCCCCGTCAACATAACGACTGAAGTTAAA TCAGTGGAAATGCATCACGAAGCTTTGAGTGAGGCGCTTCCCGGAGATAACGTTGGATTTAATGTCAAAAATGTCTCCGTTAAAGATATAAGAAGAGGAAACGTCTGCGGTGACTCAAAGTCAGACCCACCACAGGAGGCTGCTCAATTTACGAGTCAAGTAATAATTCTGAAT CACCCTGGGCAAATAAGTGCGGGATACTCTCCAGTCATCGATTGTCACACCGCCCATATTGCATGTAAGTTCGCAGAACTTAAGGAAAAGATCGACCGAAGAAGCGGAAAAAAATTGGAAGATAATCCGAAAAGTTTGAAAAGCGGTGACGCGGCGATTGTAGAGATGGTCCCT GGCAAACCGATGTGTGGAGTCTTTCAGTCAATATCCACCACTCGGTCGCTTTGCCGTGCGGGATATGCGACAGACCGTTGCTGTCGGCGTAATAAAAAACGTCGAAAAAAAGAGCGGTGGGGCTGGAAAAGTTACAAAATCCGCTCAAAAGGCACAGAAGGCGGGCAAGTGA

(SEQ ID NO: 5).

A polynucleotide encoding the eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGTAAAGAAAAGACCCACATTAACATAGTAGTAATCGGTCATGTTGACTCTGGGAAAAGCACTACTACCGGACATTTGATCTATAAATGTGGGGGCATCGACAAAAGAACGATAGAGAAGTTTGAGAAGGAGGCGGCGGAGATGGGTAAAGGTAGTTTTAAGTACGCTTGG GTTTTGGACAAATTGAAAGCCGAGCGCGAGCGCGGCATTACCATTGACATTTCTCTCTGGAAATTCGAAACTACGAAGTATTATATAACAATAATAGACGCCCCCGGCCATCGGGACTTTATTAAAAACATGATTACAGGAACTAGCCAAGCAGATTGTGCTGTGCTGATAGTA GCGGCAGGGGTCGGGGAGTTCGAAGCAGGCATCTCTAAAATGGACAAACTCGAGAGCACGCCTTGTTGGCTTATACCTTGGGCGTAAAGCAGCTGATCGTAGGAGTTAATAAAATGGATTCCACTGAACCCGCATATAGCGAAAAGCGATATGACGAAATAGTAAAGGAAGTC TCAGCTTATATCAAGAAAATCGGTTACAATCCTGCGACGGTTCCATTCGTTCCTATCTCCGGGTGGCACGGCGATAATATGCTTGAGCCCAGTCCCAATATGCCCTGGTTCAAGGGGTGGAAGGTTGAGAGGAAGGAAGGCAATGCATCAGGCGTCAGCTTGTTGGAAGCTCTC GACACCATCCTGCCGCCCACGAGGCCCACAGACAAACCGTTGCGACTGCCTCTTCAAGATGTATACAAAATAGGCGGGATAGGAACCGTGCCGGTTGGACGAGTAGAGACGGGTATACTGCGGCCCGGAATGGTCGTGACGTTTGCACCCGTGAATATAACTACTGAGGTGAAG AGCGTCGAGATGCACCATGAAGCGCTGAGTGAAGCTCTCCCTGGCGATAACGTAGGGTTCAACGTGAAAAACGTAAGTGTAAAGGATATAAGGCGCGGAAATGTATGTGGTGACAGTAAAAGCGACCCGCCGCAAGAGGCGGCGCAATTCACATCACAGGTAATAATATTGAAT CACCCCGGCCAAATTTCCGCAGGCTACTCACCAGTCATAGATTGCCACACCGCCCACATAGCTTGTAAGTTCGCTGAGTTGAAAGAGAAGATTGATAGACGAAGTGGGAAGAAACTTGAAGACAATCCGAAGTCCCTGAAGTCCGGTGACGCAGCGATTGTAGAAATGGTACCG GGCAAGCCAATGTGTGTAGAGTCTTTCAGCCAGTACCCACCACTGGGGCGGTTCGCGGTGCGAGACATGAGGCAAACGGTTGCGGTCGGCGTCATTAAAAATGTCGAAAAAAAGAGTGGCGGTGCAGGTAAGGTCACAAAAAGCGCACAAAAGGCCCAGAAAGCCGGTAAGTGA

(SEQ ID NO: 6).

A polynucleotide encoding the eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGAAAGGAAAAAACTCACATAAACATTGTCGTCATCGGTCACGTAGACAGTGGCAAATCAACGACCACTGGACATCTCATCTATAAGTGTGGCGGTATTGACAAACGCACTATCGAGAAATTCGAAAAGGAGGCTGCTGAGATGGGCAAAGGCTCTTTCAAGTACGCATGG GTCCTGGATAAGCTGAAAGCGGAGCGAGAGAGAGGGATCACCATCGATATATCTCTGTGGAAATTTGAAACCACCAAGTACTACATCACAATTATTGATGCCCCAGGTCATAGGGATTTTATCAAGAACATGATCACCGGGACAAGCCAAGCCGACTGCGCAGTTCTCATAGTG GCGGCTGGAGTAGGGGAGTTTGAAGCAGGGATATCTAAGAATGGACAGACCCGCGAGCACGCCTTGCTGGCCTACACCCTGGGAGTGAAGCAGCTCATAGTTGGCGTCAATAAGATGGACAGCACCGAACCCGCCTACAGTGAGAAGAGGTATGACGAGATTGTGAAGGAGGTT TCTGCTTACATTAAAAAGATTGGCTATAACCCAGCTACTGTCCCATTCGTTCCAATCAGCGGCTGGCACGGTGATAACATGCTGGAGCCTAGTCCCAACATGCCGTGGTTCAAGGGGTGGAAGGTTGAACGCAAGGAGGGGAATGCCTCAGGCGTTTCCCTGCTGGAGGCCCTC GATACAATACTCCCCCCGACCCGGCCTACAGATAAACCGCTGCGACTGCCTCTTCAGGACGTGTATAAAATCGGGGGAATCGGCACAGTGCCCGTGGGCAGGGTAGAGACTGGCATCTTGCGGCCTGGAATGGTAGTCACCTTTGCCCCGGTTAATATCACAACGGAGGTGAAA TCTGTGGAGATGCATCACGAAGCACTGAGCGAGGCTCTGCCTGGTGACAACGTGGGATTTAACGTCAAAAACGTGTCAGTCAAGGACATCCGCCGCGGTAACGTTTGCGGAGATTCTAAGTCCGATCCCCCCCAGGAGGCAGCCCAATTTACCTCCCAAGTGATCATTCTGAAT CACCCAGGCCAAATTTCCGCCGGGTATTCCCCTGTGATTGACTGTCACACAGCACACATCGCATGCAAATTCGCCGAACTCAAGGAGAAAATTGATCGGAGAAGCGGTAAAAAACTGGAGGACAACCCAAAGTCCCTCAAGTCTGGGGATGCCGCCATCGTGGAGATGGTACCA GGCAAACCTATGTGCGTGGAAAGTTTTAGCCAGTACCCTCCACTGGGTCGCTTTGCTGTTCGGGATATGCGGCAGACAGTAGCGGTTGGGGTCATAAAAAACGTCGAGAAAAAGAGCGGAGGAGCTGGGAAAGTTACCAAATCCGCACAGAAGGCACAAAAAGCCGGAAAATGA

(SEQ ID NO: 7).

A polynucleotide encoding the eEF1A2 protein may comprise a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ATGGGCAAAGAGAAAACACATATTAACATTGTTGTTATCGGGCACGTTGATAGCGGCAAGTCCACTACCACTGGCCATCTGATTTACAAGTGCGGCGGAATCGATAAACGAACTATTGAAAAGTTCGAAAAAGAAGCCGCCGAGATGGGAAAGGGCTCCTTTAAATACGCTTGG GTCCTCGATAAACTCAAAGCAGAACGGGAGAGAGGAATCACCATCGATATATCCTTGTGGAAGTTCGAAACTACAAAATATTACATTACCATCATTGATGCGCCTGGGCACCGCGACTTCATTAAGAACATGATTACTGGCACCTCTCAAGCCGACTGCGCAGTGTTGATCGTA GCCGCAGGCGTCGGGGAGTTCGAAGCTGGGATCAGCAAGAACGGGCAGACTAGGGAACACGCTCTGCTCGCATATACTCTTGGCGTGAAACAGTTGATCGTTGGCGTGAACAAGATGGATTCAACTGAGCCTGCCTATTCTGAGAAACGATACGACGAGATTGTGAAAGAGGTT TCAGCTTACATCAAGAAAATTGGGTATAATCCCGCAACAGTTCCCTTCGTGCCCATCTCTGGGTGGCACGGCGACAACATGCTCGAACCATCCCCAAATATGCCATGGTTCAAGGGATGGAAGGTGGAGCGCAAAGAAGGCAACGCCTCCGGAGTGTCTCTGCTCGAGGCCCTG GACACCATTCTGCCCCCAACACGACCCACTGATAAGCCTCTGAGACTGCCACTGCAAGACGTTTACAAAATTGGGGGAATTGGAACCGTGCCTGTGGGTCGGGTGGAAACCGGAATCCTCAGACCCGGCATGGTGGTCACCTTCGCACCAGTGAATATAACGACAGAGGTCAAA TCTGTGGAGATGCACCATGAGGCATTGAGCGAGGCACTCCCAGGAGACAACGTGGGTTTCAACGTGAAAAATGTCTCAGTTAAGGACATCCGACGCGGCAACGTGTGCGGAGATAGCAAATCTGACCCCCCCCAGGAGGCCGCTCAATTCACAAGTCAGGTTATCATCCTTAAT CACCCTGGCCAAATATCTGCAGGCTACAGCCCCGTGATCGATTGTCACACAGCTCATATCGCCTGTAAATTTGCTGAACTCAAAGAAAAGATTGACCGCAGATCAGGAAAAAAGCTGGAGGACAACCCTAAAAGTCTGAAGTCCGGCGACGCTGCCATCGTGGAGATGGTCCCT GGGAAACCCATGTGCGTGGAGTCCTTTTCTCAGTACCCCCCTCTGGGACGATTCGCCGTGCGCGACATGAGACAGACTGTCGCCGTGGGCGTCATTAAAAATGTGGAAAAAAAATCAGGAGGTGCAGGGAAAGTGACAAAGAGTGCCCAGAAAGCACAGAAGGCTGGCAAGTGA

(SEQ ID NO: 8).

Optionally, the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 10). The Kozak sequence may overlap with the polynucleotide sequence encoding the eEF1A2 protein or a functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence (with the Kozak sequence underlined) that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

gccaccATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTAT GCCTGGGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTG ATCGTGGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAG GAAGTCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGG CCCTGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGG TGAAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCT GAACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGT GCCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAG

(SEQ ID NO: 9).

In some embodiments, the Kozak sequence is an alternative Kozak sequence comprising or consisting of any of:

(gcc)gccRccAUGG (SEQ ID NO: 11);

AGNNAUGN;

ANNAUGG;

ACCAUGG;

GACACCAUGG (SEQ ID NO: 12).

In some embodiments, the vector genome does not contain a Kozak sequence.

Vector genome

The AAV virions of the present invention comprise a vector genome. The vector genome may contain an expression cassette (or a polynucleotide cassette for gene editing purposes without the need for polynucleotide sequence expression). Any suitable inverted terminal repeats (ITRs) may be used. The ITRs may be of the same serotype as the capsid or of a different serotype (e.g., ITR from AAV2 can be used).

In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

(SEQ ID NO: 18).

In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to

GCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTA

(SEQ ID NO: 19).

In some embodiments, the 5' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTA

(SEQ ID NO: 20).

In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

(SEQ ID NO: 21).

In some embodiments, the 3' ITR comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to

TACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC

(SEQ ID NO: 63).

In some embodiments, the vector genome comprises one or more filler sequences that, for example, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

GCGGCAATTCAGTCGATAACTATAACGGTCCTAAGGTAGCGATTTAAATACGCGCTCTCTTAAGGTAGCCCCGGGACGCGTCAATTGACTACAAACCGAGTATCTGCAGAGGGCCCTGCGTATG (SEQ ID NO: 22);

CTTCTGAGGCGGAAAGAACCAGATCCTCTCTTAAGGTAGCATCGAGATTTAAATTAGGGATAACAGGGTAATGGCGCGGGCCGC (SEQ ID NO: 23) or

GTTACCCAGGCTGGAGTGCAGTGGCACATTTCTGCTCACTGCAACCTCCTCCTCCCTGGGTTC (SEQ ID NO: 24).

Promoters

In some embodiments, a polynucleotide sequence encoding an eEF1A2 protein or a functional variant thereof is operably linked to a promoter.

The present invention provides for the use of various promoters. Promoters useful in embodiments of the present invention include, but are not limited to, the cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PGK) gene promoter, or a promoter sequence consisting of the CMV enhancer and portions of the chicken beta-actin gene promoter and the rabbit beta-globin (CAG) gene promoter. In some cases, the promoter may be a synthetic promoter. Exemplary synthetic promoters are provided in Schlabach et al. PNAS USA . 107(6):2538–43 (2010). In some embodiments, the promoter comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical

ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT ATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTAATTTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCG GGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG

(SEQ ID NO: 14).

In some embodiments, a polynucleotide sequence encoding an eEF1A2 protein or a functional variant thereof is operably linked to an inducible promoter. The inducible promoter may be configured to cause transcriptional expression or not to cause transcriptional expression of the polynucleotide sequence in response to the addition or accumulation of an agent or in response to the removal, degradation, or dilution of the agent. The agent may be a drug. The agent may be tetracycline or one of its derivatives, including, but not limited to, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, a physically regulated promoter (i.e., A promoter that responds to the presence or absence of light or to low or high temperatures. Inducible promoters include promoters inducible by heavy metal ions (such as the murine mammary tumor virus (mMTV) promoter or various growth hormone gene promoters) and promoters from phage T7, which are active in the presence of T7 RNA polymerase. This list of inducible promoters is not exhaustive.

In some cases, the promoter is a tissue-specific promoter, such as a promoter capable of driving expression in a neuron to a greater extent than in a cell other than a neuronal cell. In some embodiments, the tissue-specific promoter is selected from any of a variety of neuron-specific promoters, including, but not limited to, hSYN1 (human synapsin), INA (alpha-internexin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (forkhead box A2), CaMKII (calmodulin-dependent protein kinase II), and NSE (neuron-specific enolase). In some cases, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is any of the CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, chicken beta-actin, and human beta-actin promoters.

In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs 3, 14, 16-17, and 25-30.

Table 3

PROMOTER SEQ ID NO: Human beta-actin (HuBa) 25 Chicken beta-actin (CBA) 26 Cytomegalovirus (CMV) 16 Cytomegalovirus (CMV) (second version) 17 CAG promoter 14 Human EF1-alpha (EF1-α) 27 Human synapsin1 (Syn), short version 28 Human synapsin1 (Syn) with 3′ extension 3 Human synapsin1 (Syn) with 5' extension 29 Human CamKIIa (CaMKIIa) 30 eSYN promoter 64

In a preferred embodiment, the vector genome comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3.

Additional illustrative examples of promoters include the SV40 late promoter from simian virus 40, the baculovirus polyhedron enhancer/promoter element, the herpes simplex virus thymidine kinase gene (HSV tk) promoter, the cytomegalovirus (CMV) immediate-early promoter, and various retroviral promoters, including LTR elements. A wide variety of other promoters are known and publicly available in the art, and the sequences of many such promoters are available in sequence databases such as GenBank.

Other regulatory elements

In some cases, the vectors of the present invention further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a sequence encoding peptide 2A, a WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) and an HPRE (hepatitis B post-transcriptional regulatory element).

In some embodiments, the vector comprises a CMV enhancer.

In certain embodiments, the vectors comprise one or more enhancers. In some embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin gene enhancer sequence, or an EF1-α enhancer sequence. The aforementioned sequences are known in the art. For example, the sequence of the immediate early (IE) enhancer of CMV is as follows:

ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA

(SEQ ID NO: 31).

In certain embodiments, the vectors comprise one or more introns. In some embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, or an EF1-α intron sequence.

In certain embodiments, the vectors comprise a polyA sequence. In certain embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the polyA signal may be a bovine growth hormone polyadenylation signal (bGHpA).

In certain embodiments, the vectors comprise one or more transcript stabilizing elements. In some embodiments, the transcript stabilizing element is a WPRE sequence, an HPRE sequence, a nuclear backbone attachment region, a 3'-UTR, or a 5'-UTR. In some embodiments, the vectors comprise both a 5'-UTR and a 3'-UTR.

In some embodiments, the vector comprises a 5' untranslated region (UTR) selected from Table 4. In some embodiments, the vector genome comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs 32-40.

Table 4

5 ' - UNTRANSLATED AREA SEQ ID NO: Human beta-actin exon/intron 32 Exon/intron of chicken beta actin + intron of rabbit globin 33 5'-UTR-Syn1 Hs 34 IE exon CMV 35 TPL-eMLP (adenoviral enhancer element) 36 Human EF1-α intron/exon 37 Human EF1-α intron A 38 5'-UTR of human CamKIIa 39 B-globin intron 40

In some embodiments, the vector comprises a 3' untranslated region selected from Table 5. In some embodiments, the vector genome comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs 41-49.

Table 5

3 ' - UNTRANSLATED AREA SEQ ID NO: WPRE(x) (woodchuck hepatitis mutant regulatory element - version 1) 41 WPRE(x) (woodchuck hepatitis mutant regulatory element - version 2) 42 WPRE(x) (woodchuck hepatitis mutant regulatory element - version 3) 43 CAAX 44 EES 45 HPRE 46 R2V17 (enhancer element derived from HepB) 47 3'-UTR (globin) 48 WPRE(r) 49

In some embodiments, the vector comprises a polyadenylation signal (polyA) selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs 50-54.

Table 6

POLYADENYLATION SITE SEQ ID NO: Rabbit globin (pAGlobin-Oc) 50 Bovine growth hormone (pAGH-Bt - version 1) 51 Bovine growth hormone (pAGH-Bt - version 2) 52 Bovine growth hormone (pAGH-Bt - version 3) 53 Human growth hormone (pAGH-Hs) 54

Exemplary vector genomes are depicted in Figs. 2-5 and are provided as SEQ ID NOs: 55-58 or 65-68. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NOs: 55-58 or 65-68, optionally with or without lowercase ITR sequences. The coding sequence is underlined. Alternative vector genome sequences are provided as SEQ ID NOs: 65-68.

V1 - Vector genome - 3144 bp (Fig. 2) (SEQ ID NO: 55)

cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctGCGGCAATTCAGTCGATAACTATAACGGTCCTAAGGTAGCGATTTAAATACGCGCTCTCTTAAGGTAGCCCCGGGACGCGTCAATTGACTACAAACCGAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTATGCCTGGGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTGATCGTGGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAGGAAGTCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGGCCCTGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGGTGAAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCTGAACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGTGCCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAGTGAAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGATCCTCTCTTAAGGTAGCATCGAGATTTAAATTAGGGATAACAGGGTAATGGCGCGGGCCGCaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

V2 - Vector genome - 3035 bp (Fig. 3) (SEQ ID NO: 56)

gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtaAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGGCCACCATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTATGCCTGGGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTGATCGTGGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAGGAAGTCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGGCCCTGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGGTGAAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCTGAACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGTGCCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAGTGATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCACTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCCCAAGTTGGGAAGAAACCTGTAGGGCCTGCGTTACCCAGGCTGGAGTGCAGTGGCACATTTCTGCTCACTGCAACCTCCTCCTCCCTGGGTTCtacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc

V3 - Vector genome - 3263 bp (Fig. 4) (SEQ ID NO: 57)

gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtaAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAAGTCGCAGCCACCATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTATGCCTGGGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTGATCGTGGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAGGAAGTCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGGCCCTGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGGTGAAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCTGAACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGTGCCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAGTGATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAATTCATTGCCTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCCCAAGTTGGGAAGAAACCTGTAGGGCCTGCGTTACCCAGGCTGGAGTGCAGTGGCACATTTCTGCTCACTGCAACCTCCTCCTCCCTGGGTTCtacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc

V4 - Vector genome - 4299 bp (Fig. 5) (SEQ ID NO: 58)

gcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagttaatgattaacccgccatgctacttatctacgtaCTCTGGAGACGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCGCCACCATGGGCAAGGAGAAGACCCACATCAACATCGTGGTCATCGGCCACGTGGACTCCGGAAAGTCCACCACCACGGGCCACCTCATCTACAAATGCGGAGGTATTGACAAAAGGACCATTGAGAAGTTCGAGAAGGAGGCGGCTGAGATGGGGAAGGGATCCTTCAAGTATGCCTGGGTGCTGGACAAGCTGAAGGCGGAGCGTGAGCGCGGCATCACCATCGACATCTCCCTCTGGAAGTTCGAGACCACCAAGTACTACATCACCATCATCGATGCCCCCGGCCACCGCGACTTCATCAAGAACATGATCACGGGTACATCCCAGGCGGACTGCGCAGTGCTGATCGTGGCGGCGGGCGTGGGCGAGTTCGAGGCGGGCATCTCCAAGAATGGGCAGACGCGGGAGCATGCCCTGCTGGCCTACACGCTGGGTGTGAAGCAGCTCATCGTGGGCGTGAACAAAATGGACTCCACAGAGCCGGCCTACAGCGAGAAGCGCTACGACGAGATCGTCAAGGAAGTCAGCGCCTACATCAAGAAGATCGGCTACAACCCGGCCACCGTGCCCTTTGTGCCCATCTCCGGCTGGCACGGTGACAACATGCTGGAGCCCTCCCCCAACATGCCGTGGTTCAAGGGCTGGAAGGTGGAGCGTAAGGAGGGCAACGCAAGCGGCGTGTCCCTGCTGGAGGCCCTGGACACCATCCTGCCCCCCACGCGCCCCACGGACAAGCCCCTGCGCCTGCCGCTGCAGGACGTGTACAAGATTGGCGGCATTGGCACGGTGCCCGTGGGCCGGGTGGAGACCGGCATCCTGCGGCCGGGCATGGTGGTGACCTTTGCGCCAGTGAACATCACCACTGAGGTGAAGTCAGTGGAGATGCACCACGAGGCTCTGAGCGAAGCTCTGCCCGGCGACAACGTCGGCTTCAATGTGAAGAACGTGTCGGTGAAGGACATCCGGCGGGGCAACGTGTGTGGGGACAGCAAGTCTGACCCGCCGCAGGAGGCTGCTCAGTTCACCTCCCAGGTCATCATCCTGAACCACCCGGGGCAGATTAGCGCCGGCTACTCCCCGGTCATCGACTGCCACACAGCCCACATCGCCTGCAAGTTTGCGGAGCTGAAGGAGAAGATTGACCGGCGCTCTGGCAAGAAGCTGGAGGACAACCCCAAGTCCCTGAAGTCTGGAGACGCGGCCATCGTGGAGATGGTGCCGGGAAAGCCCATGTGTGTGGAGAGCTTCTCCCAGTACCCGCCTCTCGGCCGCTTCGCCGTGCGCGACATGAGGCAGACGGTGGCCGTAGGCGTCATCAAGAACGTGGAGAAGAAGAGCGGCGGCGCCGGCAAGGTCACCAAGTCGGCGCAGAAGGCGCAGAAGGCGGGCAAGTGATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAGCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAATTCATTGCCTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCCCAAGTTGGGAAGAAACCTGTAGGGCCTGCGTTACCCAGGCTGGAGTGCAGTGGCACATTTCTGCTCACTGCAACCTCCTCCTCCCTGGGTTCtacgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc

In one embodiment, the expression cassette comprises, in order from 5' to 3', the HuBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(r), and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the Syn promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(r), 3'UTR (globin), and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the EF1α promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the HuBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the Syn promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), 3'UTR (globin), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(r), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(r), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the HuBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CMV promoter, a TPL/eMLP enhancer, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, 3'UTR (globin), and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the EF1α promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(r), and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the Syn promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the CaMKIIa promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), 3'UTR (globin), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the CBA promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, the 3'UTR (globin), and pAGlobin-Oc.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the CaMKIIa promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the EF1α promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, 3'UTR (globin), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CMV promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, R2V17, 3'UTR (globin), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CMV promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a Kozak sequence, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CAG promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CAG promoter, a Kozak sequence, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, WPRE(x), and pAGH-Bt.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', the hSYN promoter, a Kozak sequence, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CAG promoter, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

In one embodiment, the expression cassette comprises, in order from 5' to 3', a CAG promoter, a Kozak sequence, a polynucleotide sequence encoding eEF1A2 or a functional variant thereof, and pAGH-Hs.

Adeno-associated virus-based vector

Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb, including two ~145-nt inverted terminal repeats (ITRs). There are several known AAV variants, which are also sometimes referred to as serotypes when classified based on antigenic epitopes. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is submitted to GenBank under accession number NC_002077; the complete genome of AAV-2 is submitted to GenBank under accession number NC_001401 and Srivastava et al., J. Virol., 45: 555–564 (1983); the complete genome of AAV-3 is submitted to GenBank under accession number NC_1829; The complete AAV-4 genome is submitted to GenBank under accession number NC_001829; the AAV-5 genome is submitted to GenBank under accession number AF085716; the complete AAV-6 genome is submitted to GenBank under accession number NC_00 1862; at least parts of the AAV-7 and AAV-8 genomes are submitted to GenBank under accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is submitted to Gao et al., J. Virol., 78: 6381–6388 (2004); the AAV-10 genome is submitted to Mol. Ther., 13(1): 67–76 (2006); and the AAV-11 genome is submitted to Virology, 330(2): 375–383 (2004). The AAVrh.74 genome sequence is provided in U.S. Patent 9,434,928, incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 for their relative locations on the map) drive the expression of two internal AAV open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), in combination with differential splicing of one AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that are ultimately responsible for viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and nonconsensus translation start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at position 95 of the AAV genome map. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97–129 (1992).

AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, such as for gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is asymptomatic. Moreover, AAV infects a wide range of mammalian cells, enabling targeting of a wide variety of tissues in vivo. Furthermore, AAV transduces slowly dividing and non-dividing cells and can persist for virtually the entire lifespan of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA into plasmids, enabling the construction of recombinant genomes. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITR of the AAV genome, some or all of the internal ∼4.3 kbp The genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA. To produce AAV-based vectors, the rep and cap proteins can be presented in trans orientation. Another important feature of AAV is that it is an extremely stable and active virus. It easily withstands the conditions used to inactivate adenoviruses (56° to 65°C for several hours), making cold storage of AAV less critical. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

The AAV DNA in the rAAV genomes may be from any AAV variant or serotype for which recombinant virus can be produced, including, but not limited to, the following AAV variants or serotypes: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAVrh10. The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692. Other types of rAAV variants are also provided, such as rAAV with mutations in the capsid. See, for example , Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

In some cases, rAAV contains a self-complementary genome. As defined herein, rAAV containing a "self-complementary" or "double-stranded" genome refers to rAAV that has been engineered such that the rAAV coding region is configured to form an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248–54 (2001). In some instances, the present invention contemplates the use of an rAAV containing a self-complementary genome because, upon infection (such as transduction), rather than awaiting cell-mediated synthesis of the second strand of the rAAV genome, the two complementary halves of the scAAV will associate to form a single unit of double-stranded DNA (dsDNA) ready for immediate replication and transcription. It will be appreciated that, instead of the full coding capacity of rAAV (4.7-6 kb), an rAAV containing a self-complementary genome may contain only approximately half that amount (≈2.4 kb).

In other cases, the rAAV-based vector contains a single-stranded genome. As defined herein, a "single standard" genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have single-stranded DNA genomes. There have been some indications that rAAVs should be scAAVs to achieve efficient cell transduction. However, the present invention provides rAAV-based vectors that may have single-stranded genomes rather than self-complementary genomes, with the understanding that other genetic modifications of the rAAV-based vector may be useful for achieving optimal gene transcription in target cells. In some cases, the present invention relates to single-stranded rAAV-based vectors capable of efficient gene transfer to the anterior segment of the mouse eye. See Wang et al. "Single-stranded adeno-associated virus achieves efficient gene transfer to the anterior segment in the mouse eye." PLoS ONE 12(8): e0182473 (2017).

In some cases, the rAAV-based vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692. Other types of rAAV variants are also provided, such as rAAV with mutations in the capsid. See, for example , Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV-based vector is of serotype AAV9. In some embodiments, the rAAV-based vector is of serotype AAV9 and comprises a single-stranded genome. In some embodiments, said rAAV-based vector is of the AAV9 serotype and comprises a self-complementary genome. In some embodiments, the rAAV-based vector comprises inverted terminal repeat (ITR) sequences from AAV2. In some embodiments, the rAAV-based vector comprises the AAV2 genome, such that the rAAV-based vector is an AAV-2/9-based vector, an AAV-2/6-based vector, or an AAV-2/8-based vector.

Full-length and capsid gene sequences for most known AAVs are provided in U.S. Patent No. 8,524,446, which is incorporated herein in its entirety.

AAV-based vectors may comprise a wild-type AAV sequence or may comprise one or more modifications to the wild-type AAV sequence. In certain embodiments, an AAV-based vector comprises one or more amino acid modifications, such as substitutions, deletions, or insertions, in the capsid protein, such as VP1, VP2, and/or VP3. In certain embodiments, the modification results in reduced immunogenicity upon administration of the AAV-based vector to a subject.

rAAV capsid proteins can be modified such that rAAV targets a specific target tissue of interest, such as neurons or, more specifically, a dopaminergic neuron.See, for example,,Albert et al. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models. Genes. 2017 Feb 8; see alsoU.S. Patent No. 6,180,613 and U.S. Patent Publication No. US20120082650A1, the disclosures of both of which are incorporated herein by reference. In some embodiments, rAAV is administered directly into the substantia nigra of a subject.

In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid may be an AAV2 capsid or a functional variant thereof. In some embodiments, the AAV2 capsid has at least 98%, 99%, or 100% identity to a reference AAV2 capsid, For example,

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSR LQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPP PQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

(SEQ ID NO: 59).

In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid may be an AAV9 capsid or a functional variant thereof. In some embodiments, the AAV9 capsid has at least 98%, 99%, or 100% identity to a reference AAV9 capsid, For example,

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLK YNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPP PQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

(SEQ ID NO: 15).

In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neutrophic variant thereof, such as, but not limited to, one of the variants described in International Patent Publication Nos. WO 2015/038958 A1 and WO 2017/100671 A1. For example, the AAV capsid may comprise at least 4 contiguous amino acids from the sequence TLAVPFK (SEQ ID NO:61) or KFPVALT (SEQ ID NO:62), e.g., inserted between the sequences encoding amino acids 588 and 589 of AAV9.

The capsid may be an AAV-PHP.B capsid or a functional variant thereof. In some embodiments, the AAV-PHP.B capsid has at least 98%, 99%, or 100% identity to a reference AAV-PHP.B capsid, For example,

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLK YNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDP QPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADV FMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK VMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHP PPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

(SEQ ID NO: 60).

Additional AAV capsids used in the rAAV virions of the present invention include those disclosed in Patent Publication Nos. WO 2009/012176 A2 and WO 2015/168666 A2.

Pharmaceutical compositions and kits

In one aspect, the present invention provides pharmaceutical compositions comprising an rAAV virion of the present invention and one or more pharmaceutically acceptable carriers, diluents, or excipients.

For administration, such as by injection, various solutions, such as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered if necessary, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as Pluronic™ F-68, at a concentration of 0.001% or 0.01%. rAAV dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media used can be readily prepared by standard techniques well known to those skilled in the art.

Pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Microbial activity can be prevented using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and others. In many cases, the inclusion of isotonic agents, such as sugars or sodium chloride, is preferable. Prolonged absorption of injectable formulations can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating rAAV in the required amount in an appropriate solvent with various other ingredients listed above, if necessary, followed by filter sterilization. Typically, dispersions are prepared by incorporating the sterilized active ingredient into a sterile carrier medium containing the basic dispersion medium and the required other ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods are vacuum drying and lyophilization, which yield a powder of the active ingredient plus any additional required ingredient from a previously filter-sterilized solution.

In another aspect, the present invention provides a kit comprising a rAAV virion of the present invention and instructions for use.

Methods of application

In one aspect, the present invention provides a method for increasing eEF1A2 activity in a cell, comprising contacting the cell with an rAAV of the present invention. In another aspect, the present invention provides a method for increasing eEF1A2 activity in a subject, comprising administering an rAAV of the present invention. In some embodiments, the cell and/or subject are deficient in eEF1A2 expression levels and/or activity and/or contain a loss-of-function mutation in eEF1A2. The cell can be a neuron, such as a dopaminergic neuron.

In some embodiments, the method promotes the survival of neurons in cell culture and/or in vivo .

Treatment methods

In another aspect, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an rAAV virion of the present invention. In some embodiments, the disease or disorder is a neurological disease or disorder. In some embodiments, the subject has a genetic disorder of eEF1A2 expression or function. In some embodiments, the disease or disorder is eEF1A2 deficiency and/or an eEF1A2-associated neurological disorder (OMIM No. 617309, 616393, 616409) phenotypic spectrum disorders such as intellectual disability, developmental delay, epileptic encephalopathy, and autism spectrum disorder.

AAV-mediated delivery of eEF1A2 protein to the CNS can extend lifespan, prevent neuronal degeneration, prevent or attenuate neurobehavioral disorders, degenerative epilepto-dysketic encephalopathy, epilepsy, and dystonia.

The present invention also provides various forms of combination therapy. Specifically, combinations of the methods of the present invention with standard treatments (e.g., corticosteroids or topical antihypertensive agents), as well as combinations with novel therapies, are contemplated. In some cases, a subject may be treated with steroids to prevent or reduce the immune response to administration of the rAAV described herein.

A therapeutically effective amount of an rAAV-based vector, such as for intraventricular (ICV) injection or intracerebellopontine cistern (ICM) injection, is a rAAV dose in the range of about 1e12 vg/kg to about 5e12 vg/kg, or about 1e13 vg/kg to about 5e13 vg/kg, or about 1e14 vg/kg to about 5e14 vg/kg, or about 1e15 vg/kg to about 5e15 vg/kg based on brain weight. For intravenous administration, doses range from 1213-1e14 vg/kg body weight. The present invention also provides compositions containing these ranges of rAAV vector.

For example, in particular embodiments, the therapeutically effective amount of the rAAV vector is a dose of about 1e10 vg, about 2e10 vg, about 3e10 vg, about 4e10 vg, about 5e10 vg, about 6e10 vg, about 7e10 vg, about 8e10 vg, about 9e10 vg, about 1e12 vg, about 2e12 vg, about 3e12 vg, about 4e12 vg, or about 5e12 vg. The present invention also provides compositions comprising these doses of the rAAV vector.

In some embodiments, for example, where an ICV injection is performed, the therapeutically effective amount of the rAAV vector is a dose in the range of 1e10 vg/hemisphere to 1e13 vg/hemisphere, or about 1e10 vg/hemisphere, about 1e11 vg/hemisphere, about 1e12 vg/hemisphere, or about 1e13 vg/hemisphere. In some embodiments, for example, where an ICM injection is performed, the therapeutically effective amount of the rAAV vector is a dose in the range of 1e10 vg in total to 1e14 vg in total, or about 1e10 vg in total, about 1e11 vg in total, about 1e12 vg in total, about 1e13 vg in total, or about 1e14 vg in total.

In some embodiments, the therapeutic composition comprises more than about 1e9, 1e10, or 1e11 rAAV-based vector genomes per volume of the therapeutic composition injected. In embodiments, the therapeutic composition comprises more than about 1e10, 1e11, 1e12, or 1e13 rAAV-based vector genomes per ml. In certain embodiments, the therapeutic composition comprises less than about 1e14, 1e13, or 1e12 rAAV-based vector genomes per ml.

Evidence of functional improvement, clinical benefit, or efficacy in patients can be assessed by analyzing surrogate markers of seizure frequency reduction (myoclonic and generalized tonic-clonic seizures), brain growth, and body growth using the UK-WHO pediatric head circumference, height, and weight charts. Measurements of cognitive, motor, speech, and language functions using standard disease assessment scales, such as the Childhood Seizure History and Medication Log. Cognitive and developmental assessments, including the Peabody Motor Development Scales, the 2nd edition (PDMS-2), and the Bayley Infant Development Scales, 3rd Editions were modified, if necessary, depending on the child's level of disability. Gross Motor Function Measure (GFMF-88), Pediatric Evaluation of Disability (PEDI). Improvements in disease components can be demonstrated by these or similar scales, as well as patient-reported quality-of-life outcomes, such as the Caregiver Global Impression of Change in Seizure Duration (CGICSD) on a 3-point scale (decrease, no change, or increase in mean duration), the Pediatric Quality of Life Questionnaire (PedsQL™), and the Vineland Adaptive Behavior Scales 2nd version. Based on baseline and magnetic resonance imaging of the brain, improvements in myelination and brain volume may be seen after treatment. Cardiac defects have been observed in patients with the autosomal dominant neurodevelopmental disorder EEF1A2, including cardiomyopathy, aortic defects, and ventricular septal defect (Kaneko et al., 2021; Carvill et al., 2020; McLachlan et al., 2019). A homozygous variant of EEF1A2 has been identified in isolated relatives with global developmental delay, epilepsy, growth retardation, dilated cardiomyopathy, and premature death (Cao et al., 2017). Cardiac parameters can be monitored with a baseline electrocardiogram and echocardiogram.

Clinical benefits may include increased lifespan, achievement of normal neurodevelopmental milestones, reduction in the frequency or severity of seizures (including myoclonic, clonic, generalized tonic-clonic, and/or epileptic spasms), improvement or absence of hypotonia, or movement disorders such as choreoathetosis, dystonia, and/or ataxia. Evidence of neuroprotective and/or neurorestorative effects may be evident on magnetic resonance imaging (MRI) by characterizing the degree of myelination during development, the thickness of the corpus callosum, and the degree of cortical and/or cerebellar atrophy. Favorable changes in electroencephalogram (EEG) activity will be evident by a reduction in multifocal discharges and/or generalized spiking activity.

In some embodiments, for example, when administered intravenously, the therapeutically effective amount of the rAAV vector is a dose in the range of about 1e12 vg/kg to 1e14 vg/kg of the subject's total body weight. For example, in particular embodiments, the therapeutically effective amount of the rAAV vector is a dose of about 1e12 vg/kg, about 2e12 vg/kg, about 3e12 vg/kg, about 4e12 vg/kg, about 5e12 vg/kg, about 6e12 vg/kg, about 7e12 vg/kg, about 8e12 vg/kg, about 9e12 vg/kg, about 1e13 vg/kg, about 2e13 vg/kg, about 3e13 vg/kg, about 4e13 vg/kg, about 5e13 vg/kg, about 6e13 vg/kg, about 7e13 vg/kg, about 8e13 vg/kg, about 9e13 vg/kg, or about 1e14 vg. Evidence of cardiac benefit may include stable cardiac function on echocardiogram.

Introduction of compositions

Administration of an effective dose of the compositions can be carried out by routes standard in the art, including, but not limited to, systemic, local, direct injection, intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputaminal, intrahippocampal, intrastriatal (putamen and/or caudate), intracortical, or intracerebroventricular administration. In some cases, administration involves intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputaminal, intrahippocampal, intrastriatal (putamen and/or caudate), or intracerebroventricular injection. Administration can be carried out by intrathecal injection with or without Trendelenburg tilt.

In some embodiments, the present invention provides for local and systemic administration of an effective dose of rAAV and the compositions of the present invention. For example, systemic administration can be administered into the circulatory system so that the effect is throughout the body. Systemic administration includes parenteral administration via injection, infusion, or implantation.

In particular, rAAV administration according to the present invention can be accomplished by any physical method that allows the transport of the recombinant rAAV-based vector into the target tissue of the animal. Administration includes, but is not limited to, injection into the central nervous system (CNS) or cerebrospinal fluid (CSF) and/or directly into the brain.

In some embodiments, the methods of the present invention involve intracerebroventricular, intracisternal, intrathecal, or intraparenchymal delivery. Infusion can be performed using a dedicated cannula, catheter, syringe/needle, or an infusion pump. Optionally, injection site targeting can be accomplished using MRI-guided imaging. Administration can involve delivering an effective amount of rAAV virion or a pharmaceutical composition comprising rAAV virion to the CNS. This can be accomplished, for example, by unilateral intraventricular injection, bilateral intraventricular injection, infusion into the pontocerebellar cistern with a Trendelenburg tilt procedure or infusion into the pontocerebellar cistern without a Trendelenburg tilt procedure, intrathecal infusion with a Trendelenburg tilt procedure or intrathecal infusion without a Trendelenburg tilt procedure. The compositions of the present invention can additionally be administered intravenously.

Direct CNS delivery may involve targeted delivery to the intraventricular space, either unilaterally or bilaterally, of specific neuronal regions or more general brain areas containing neural targets. Selection of the intraventricular space, brain region, and/or neural target(s) for an individual patient and subsequent intraoperative delivery of AAV can be accomplished using a variety of imaging modalities (MRI, CT, CT in combination with co-MRI) and using any of a number of planning software tools (e.g., Stealth System, Clearpoint Neuronavigation System, Brainlab, Neuroinspire, etc.). Targeting and delivery to the intraventricular space or brain region can involve the use of standard stereotactic frames (Leksell, CRW) or frameless approaches with or without intraoperative MRI. Actual delivery of the AAV can be accomplished by injection through a needle or cannulas with or without an internal lumen coated with a material to prevent AAV vector adsorption (e.g., Smartflow cannulas, MRI Interventions cannulas). The delivery device interfaces with syringes and automated infusion or microinfusion pumps with preprogrammed infusion rates and volumes. The syringe/needle combination or the needle alone can be connected directly to the stereotactic frame. Infusion can involve a constant flow rate or variable flow rate with convection-enhanced delivery.

EXAMPLES

Example 1. Selecting a Promoter

Biodistribution studies in neonatal wild-type mice were conducted to select a promoter to restore eEF1A2 expression in neurons. The human synapsin (hSYN) promoter demonstrated superior neuronal selectivity and strong neuronal expression compared to all other candidate promoters shown in Table 1. Surprisingly, the hSYN promoter demonstrated greater neuronal selectivity than eSYN and the other promoters tested.

Table 6

Promoter Brain cell tropism of the eEF1A2 transgene CMV Neuronal and glial EF1a Strong neuronal Enhanced synapsin (eSYN) Neuronal and glial Human synapsin (hSYN) Strong neuronal

Example 2. Gene therapy rescue with AAV9 for an EEF1A2 knockout mouse model

The present inventors have developed a new approach to treating subjects (e.g., children) suffering from mutations in the geneEEF1A2Eukaryotic translation elongation factor 1 alpha 2 (eEF1A2) is required for the delivery of aminoacyl transfer RNA to the ribosome for protein synthesis. Mutations in the geneEEF1A2are associated with severe mental retardation, autism, and epilepsy. There are currently no effective treatments. A knockout mouse modelEEF1A2(emaciated mice) has been well characterized. Exhausted (wst/wst) mice exhibit gait disturbances and tremors after weaning, followed by paralysis and motor neuron degeneration by 23 days of age. Using this mouse model, we tested whether protein function could be restored using gene therapy. We engineered adeno-associated virus 9 (AAV9) using the pan-neuronal promoter, human synapsin, to drive cDNA expression.EEF1A2human (hSyn-eEF1A2). The eGFP marker gene was inserted to monitor the expression of the construct.in vivo. Immunofluorescence (Fig. 7) revealed neuronal targeting following neonatal IC or IV injection of AAV9-hSyn-eEF1A2-T2A-eGFP. Immunohistochemical staining (Fig. 8) confirmed widespread transgene expression in the CNS after both routes of administration from a single rAAV injection (for the eEF1A2-2A-eGFP marker or the eGFP marker alone).

A gene therapy vector has proven effective in treating malnourished (wst/wst) mice. Eef1a2 knockout mice^−/−(wst/wst) mostly survived (3/4) with IC administration, and all survived with both IC and IV administration(Fig. 9A). Untreated mice died at P23. IC or IC/IV mice also did not show weight loss compared to wild-type mice, whereas untreated control mice showed weight loss leading to death at P23 (Fig. 9B). Analysis using rotarod and inverted mesh did not demonstrate a decrease in the effectiveness of the treated (Fig. 9C And Fig. 9D). The results were significant for both the two-way ANOVA and Dunnett's multiple comparison tests.

eEF1A2 expression was observed throughout the brain in wild-type, IC, and combination-treated mice ( Fig. 9E and Fig. 9F ). eEF1A2 expression was present in spinal cord tissue in the wild-type, IC, and combination-treated groups. However, expression was absent in the untreated depleted group and in the IV-treated groups (F).

Example 3. Gene therapy rescue with AAV9 for mouse models of EEF1A2 ^D252H , or EEF1A2 ^G70S , or EEF1A2 ^E122K

The efficiency of the vector design is shown in Figs. 2–5 and Fig. 6 , and various codon optimizations are compared to identify vectors with higher efficiency. Experiments are conducted in mouse models that reproduce three mutations found in humans (D252H, G70S, and/or E122K) and/or in a mouse model with a severe neurodegenerative phenotype (Del.22.ex3). Experiments are conducted in both neonatal mice and at later stages of development to adulthood to confirm that AAV vectors encoding eEF1A2 can restore survival, avoid weight loss, and restore behavioral phenotypes. Positive effects on neurobehavioral test results include performance on a rotating cylinder (rotarod), the ability to grasp a suspended wire surface (wire test), the foot-deflection test, behavior on an inverted grid, grip strength, and open-field behavior, including observations of normal exploratory activity or abnormal behavior (e.g., "twitching behavior"). To assess neurobehavioral function in mice injected with AAV9-eEF1A2 compared to untreated controls, a general neurological assessment can be obtained from a battery of tests that includes hindlimb contraction, gait, kyphosis, and ledge walking. Changes in seizure frequency and electroencephalogram (EEG) in Del.22.ex3 mice (or other mouse strains with abnormal EEG) with extended lifespan reveal positive effects in mitigating abnormal electrical activity in the central nervous system. Biochemical and histological analyses confirm the superior performance of the vector constructs in terms of eEF1A2 expression levels and distribution in the CNS. Tissue analysis includes determination of mRNA, DNA, vector copy number, and transgene protein expression in fresh tissues using Western blotting and ELISA, as well as in fixed CNS sections using immunolabeling.

Example 4. Effects of AAV9-mediated delivery of human eEF1A2 protein to the central nervous system in the wst/wst mouse model of eEF1A2 deficiency

The leading model of eEF1A2-associated disorders is the wasted mouse model, in which spontaneous deletion of the first exon and all promoter elements of the EEf1A2 gene results in eEF1A2 null mice ( wst / wst ). In untreated animals, 31% of wst / wst mice die between P20–22, and surviving wst / wst mice exhibit weakened weight gain, tremors, followed by weight loss, with the remainder all dying by day 24. Untreated animals can also exhibit reduced grip strength and impaired performance in the rotarod test, with animals experiencing the longest progression of tremors, progressive paralysis, and weight loss. Animals in our wst / wst colony appear to have a more severe degree of disease, with more acute deterioration and earlier death. We observed weakened grip strength in the inverted grid fall delay test at P23. With wst / wst animals surviving only to P24, we did not observe a decrease in performance in the rotarod test to significantly distinguish wst/wst from wild-type animals.

Materials and methods

Caring for animals

All animal experiments were carried out in accordance with the UK Home Office and Animals (Scientific Procedures) Act 1986 and in accordance with the guidelines of the University College London Ethics Committee. Null mouse modelwst/wsteEF1A2 used in this study has been described previously (Chambers D et al.PNAS95:4463-8 (1998). Heterozygous mice were time-crossed to produce litters with mixed genotypes. Pups were genotyped at P0 using primers (primers EEF1A2, mutated, F 5' ACCAGTGGTTTCACCTGCTC 3', EEF1A2, common, R 5' CACTGTGGGGGCTCTGGTTT 3', EEF1A2 WT F 5' CAGAGCTTCACTCAGTCTG 3').

Administration of AAV9-eEF1A2 vectors or control samples was performed by bilateral intracerebroventricular injection to homozygous neonates.wst/wstpups or WT pups from a single litter at P0, and animals were monitored until a humane endpoint (weight loss ≥15%) or sacrifice at P60. Intracerebroventricular injections were directed into the lateral ventricle of P0-1 mice as previously described (Newbery HJ, et al.J Neuropathol Exp Neurol64:295–303 (2005)). A 33-gauge needle (Hamilton) was inserted perpendicular to the injection site to a depth of 3 mm, and 5 μl of vector was injected into the lateral ventricle over 5 seconds. The pup was quickly returned to its mother. Group sizes were 6 wst/wst mice treated with gene therapy with 14–16 control mice from one litter of 7.

Behavioral research

Mice were weighed regularly and assessed for changes in general well-being and achievement of a humane endpoint. Behavioral tests using a rotarod and the inverted grid test were performed on P23. All behavioral tests were performed by investigators blinded to the experimental group of animals. Mice were placed on a rotarod (Harvard Apparatus®) at a constant acceleration of 4 to 40 rpm for a maximum of 5 minutes. The time it took for mice to fall from the rod was recorded in 3 trials (fall latency) for each animal on each testing day. In the inverted grid test, mice were placed on a stainless steel grid (41 x 25 cm), which was placed on a 30-cm elevated plastic transparent box. The latency to fall from the inverted grid was recorded, for a maximum of 5 minutes. The inverted grid test was repeated 3 times for each mouse on each testing day.

Histological and immunohistochemical analyses of mouse tissues

Mice were culled by terminal transcardial perfusion using PBS. The collected tissues (brain and internal organs) were divided in half to allow for different processing techniques. Brains used for immunohistochemistry were postfixed in 4% PFA for 48 hours and transferred to 30% sucrose solution for cryoprotection at 4°C.^oC prior to sectioning. Brains were fixed on a freezing microtome (ThermoFisher® HM430) at 40 μm thickness in any of the coronal planes. Free-floating immunohistochemistry-based analyses were performed using brain sections collected at 240 μm intervals for whole-brain immunohistochemistry. Briefly, free-floating sections were blocked in 15% normal goat serum (Vector Laboratories®)-Tris-buffered saline with 0.1% Triton-X (TBS-T) (Sigma®) for 1 hour at room temperature and incubated in rabbit eEF1A2 primary antibody (Proteintech®) in 10% normal goat serum-TBS-T overnight at 4^oC.^.The following day, sections were incubated with appropriate species-specific secondary antibodies (Vector Laboratories®) for 1 hour at room temperature, washed in TBS, and then incubated with Vectastain avidin-biotin solution (Vector Laboratories®). The reaction was visualized with 3,3'-diaminobenzidine (DAB) (Sigma®). The DAB reaction was stopped with ice-cold 1x TBS, and sections were washed before mounting on double-coated gelatinized glass slides. Fixed sections were air-dried and dehydrated in 100% ethanol for 10 min and in dehydrating solution (Histoclear™, National Diagnostics®) for 30 min before being mounted with fixative medium (DPX, VWR International®) and coverslipped.

For immunofluorescence, brain sections were blocked in 15% goat serum for 30 min and then incubated with primary antibodies (rabbit eEF1A2 1:1000 Proteintech® and mouse NeuN 1:1000 Milipore) diluted in 10% normal goat serum TBS-T 0.3% overnight at 4°C. Sections were washed in 1xTBS and incubated for 2 h with the appropriate species-specific secondary antibodies labeled with Alexa 488 and Alexa 594 (all from Invitrogen®) diluted in 10% normal goat serum at room temperature. NV4ei was stained with DAPI (Sigma Aldrich®) for 2 min. Brain sections were mounted on double-coated glass slides and coverslipped using Fluromount G™ (Thermofisher Scientific®).

Light microscopy and fluorescence imaging were performed using a Leica DM 4000 coupled to a Leica DFC420 camera. Confocal images were acquired using a Leica TCS SP5 AOBS confocal microscope. Images were analyzed using Image J software (National Institutes of Health).

Immunoblotting

Proteins were isolated from mouse brain tissue in ice-cold 0.32 M sucrose supplemented with protease inhibitor (Roche®) using a Qiagen® tissue lyser and centrifuged at 4°C for 15 minutes. Protein concentration was measured using a Pierce BCA protein assay kit (Thermo Scientific®): 10 μg of protein was denatured with Laemmli buffer (Bio-Rad Laboratories®) containing dithiothreitol (DTT). Proteins were separated using Mini-PROTEAN TGX™ Stain Free Gels (Bio-Rad Laboratories®) and transferred to a Trans-Blot Turbo Transfer membrane (Bio-Rad Laboratories®). After blocking in Biorad® blocking buffer for 1 hour at room temperature, the membranes were incubated with primary antibodies rabbit eEF1A2 (Proteintech®, 1:1000) and mouse GAPDH (Ab Cam®, 1:10,000) at 4°C overnight. The membranes were then incubated with secondary goat anti-rabbit IgG StarBright™ Blue 520 (1:3000) and goat anti-mouse IgG StarBright™ Blue 700 (1:3000). Immunoreactive proteins were visualized using Chemidoc MP (Bio-Rad Laboratories®). Loaded samples had n = 4–5 biological replicates.

qRT-PCR analysis of mRNA transcript expression

RNA was isolated from brain homogenates (forebrain, cerebral cortex, n = 4–5 biological replicates per group) using the RNeasy™ Mini Kit (Qiagen®) according to the manufacturer’s instructions and quantified on an Omega Fluostar™. Contaminating DNA was removed from total RNA (1 μg) using a DNase I Cleanup Kit (NEB®) before reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Applied Bioscience®). Then, 10 ng of DNA or synthesized cDNA were used to perform multiplex RT-qPCR of hEEF1A2 and mGAPDH (eEF1A2 forward: ATCGTGGGCGTGAACAAA, eEF1A2 reverse: GGTTGTAGCCGATCTTCTTGAT, eEF1A2 probe: ATCGTCAAGGAAGTCAGCGCCTAC and mouse GAPDH forward: ACGGCAAATTCAACGGCAC, reverse: TAGTGGGGTCTCGCTCCTGG, probe: TTGTCATCAACGGGAAGCCCATCA with Luna Taqman™ mastermix (NEB®) in Quantstudio™ Real-Time PCR System (Applied Biosystems®). GAPDH was used as an endogenous control, and relative fold changes were calculated.

Statistical analysis

Statistical analysis, tailored to each experiment, was performed using GraphPad Prism™ version 8. The in vivo experimental design and sample sizes were developed using NC3R guidelines and power calculations. For most animal experiment analyses, one- or two-way ANOVA with Bonferroni or Tukey's multiple comparisons were performed.

Results

Figures 10A-10K show comparisons of AAV9 vectors containing the vector genomes shown in Figure 2 (“V1”; SEQ ID NO: 55), Figure 3 (“V2”; SEQ ID NO: 56), Figure 4 (“V3”; SEQ ID NO: 57), and Figure 6 (“V4”; SEQ ID NO: 58) that were administered 2e10 ¹¹ vg/animal.

OnFig. 10AThe Kaplan-Meier survival plot for wild type treated FBS is shown,wst/wst, intracerebroventricularly treated animalswst/wstusing gene therapy with V1, V2, V3 and V4 (2 x 10¹¹wg/pup, all gene therapy treated groups had n = 6, wild type FBS, n = 14, wst/wst FBS = 17. All gene therapy treated animals showed a significant increase in lifespan (mean lifespan V1 = 4.4 days, p = 0.001, V2 = 3.8 days p = 0.0014, V3 = 10.7 days p < 0.0001, V4 = 4.4 days p = 0.001 Mantel-Cox log-rank test).Fig. 10BThe mice weights are shown (mean ± S.E.M.), with animals weighed daily until postnatal age 35 and then weekly until sacrifice at P60 or a humane endpoint of 15% weight loss.Fig. 10C,Fig. 10D,Fig. 10E And Fig. 10FMuscle strength assessment using inverted grid testing and rotarod testing is presented. (n = 4-7 per group, each animal was tested three times at 15 and 23 days of age). In these tests, no significant difference was observed betweenwst/wstand FBS wild-type controls when tested using a rotarod. A significant decrease in grip strength was observed in animalswst/wstFBS were observed at P23 compared to WT, and no effect of gene therapy vectors was observed on these behavioral outcomes (means ± S.E.M., two-way ANOVA).Fig. 10GRepresentative immunostaining for eEF1A2 throughout the brain using free-floating immunohistochemistry for wild type with FBS as a physiological reference (n = 4–5 per group, scale bar 250 μm) is shown. All animalswst/wstV1-4-injected mice express eEF1A2 throughout the brain with the darkest staining and highest expression observed with V3.Fig. 10HRepresentative immunohistofluorescence in cortical areas for neurons (NeuN marker) coexpressing eEF1A2 (n = 4.5 per group, 200 μm) is shown. All demonstrate a neuronal expression profile with higher expression compared to wild type. The highest expression, as observed by fluorescence intensity, was in V3, demonstrating expression in all cortical layers. All images were captured with constant imaging settings. More discrete expression in cortical layers 4 and 5 was observed with V2 and V4.Fig. 10IA representative immunoblot for eEF1A2 in the brain is shown with quantification showing brain-wide eEF1A2 expression achieved with all gene therapy vectors with higher expression in the midbrain, cerebellum, and hindbrain regions compared with the V4 vector (mean ± S.E.M, two-way ANOVA).Fig. 10JqPCR for human eEF1A2 transcript expression in the forebrain is presented, showing the highest mRNA expression with the V1 vector (mean ± S.E.M, two-way ANOVA).Fig. 10KqPCR for human cortical eEF1A2 expression in the forebrain is presented, showing the highest mRNA expression with the V1 vector (mean ± S.E.M, two-way ANOVA).

Conclusions

These experiments demonstrate that all four vectors are able to restore eEF1A2 expression in a mouse model harboring homozygous null mutations in the EEF1A2 gene (designated wst in mice). Surprisingly, vectors V1, V2, and V3 are able to efficiently and significantly extend the survival of wst/wst mice beyond P23 ( Fig. 10A ). Moreover, the experiment unexpectedly demonstrates that eEF1A2 transgene expression in the brain, particularly in the forebrain ( Fig. 10J ) and cortex ( Fig. 10K ), is higher in V3 than in V1, V2, or V4. Without being bound by theory, it appears that, contrary to expectations, inclusion of the 5′-UTR (SEQ ID NO: 34) and/or 3′-UTR (SEQ ID NO: 48) therapeutically increases gene expression, prolonging survival.

Example 5. Effects of AAV9-mediated delivery of human eEF1A2 protein to the central nervous system in the D252H mouse model of eEF1A2 disease

Heterozygous de novo mutations in EEF1A2 , encoding the tissue-specific translation elongation factor eEF1A2, have been shown to cause neurodevelopmental disorders, including frequent severe epilepsy and intellectual disability. Approximately 50 different missense mutations have been identified, but no obvious loss-of-function mutations have been identified, although large heterozygous deletions are known to be compatible with life. An eEF1A2 knockin mouse model carrying the disease causing the D252H missense mutation is more severely affected than null homozygotes on the same genetic basis, demonstrating decreased weight, increased neurological scores, and death by P23. Mice heterozygous for the missense mutation do not exhibit behavioral abnormalities but have sex-specific deficits in body weight and motor function with a transient impairment of grip strength. Phenotyping of this new D252H mouse, along with the del22ex3 null mouse model, confirms that the D252H mutation results in gain-of-function (Davies, Faith CJ, et al. Human Molecular Genetics (2020)). This example describes gene therapy studies in both heterozygous and homozygous D252H mice.

Materials and methods

The survival and weight of treated and untreated mice were monitored throughout development. Behavioral tests were also performed at 18–24 days of age.

Caring for animals

All animal experiments were conducted in accordance with the UK Home Office and Animals (Scientific Procedures) Act 1986 and in accordance with the guidelines of the University College London Ethics Committee. Heterozygous eEF1A2 D252H knockin mice were time-crossed to produce litters with mixed genotypes. Pups were genotyped at P0 using primers 5'-3' AGGCTACCCCTTAGGCAGGT, TGAACAAATGGTAGGTGGGAGG. Following PCR amplification, samples were restriction digested with Hin1II (Thermo Fisher).

Introduction of test and control samples

Administration of 5 μl of V3 vector or FB formulation was performed by unilateral intracerebroventricular injection at a dose of 1.8 x 10 ¹¹ vg/pup into neonatal homozygous knockin mice (D252H-/-). Test or FB controls were injected into wild type and heterozygous D252H mice via a 33-gauge Hamilton needle (Fisher Scientific®, Loughborough, UK) using the injection site coordinates reported by Kim et al. Kim, JY et al. J Vis Exp 91:51863 (2014). In newborn pups, the lambdoid suture was identified, and the intended injection site was located two-fifths of the way from the lambdoid suture to the eye, located approximately 0.8–1 mm lateral to the sagittal suture, midway between lambda and bregma. After injections, the pups were returned to their mothers.

Body weight

Individual animal body weight data were collected from P1–P32 and then weekly thereafter. A weight loss of ≥15% will meet the humane endpoint criteria for euthanasia.

Behavioral testing

All behavioral testing analyses were performed by investigators blinded to the experimental animal group. Rotarod Test. Rotarod training/testing was conducted on P18–24. Mice were placed on a rotarod (Harvard Apparatus) at constant acceleration from 4 to 40 rpm for a maximum of 2 minutes. The time it took for mice to fall from the rod was recorded with 3 trials (fall latency) for each animal on each testing day. Inverted Grid Test. For the inverted grid test, mice were placed on a stainless steel grid (41 x 25 cm), which was placed on a 30-cm elevated plastic transparent box. The latency to fall from the inverted grid was recorded, with a maximum of 2 minutes. The inverted grid test was repeated 3 times for each mouse on each testing day.

Results

OnFig. 11A-11CThe phenotype of untreated D252H-/- mice is presented.Fig. 11A.Kaplan-Meier survival plot of wild-type, FBS-treated, knockout (D252H-/-), intracerebroventricularly treated with V3 gene therapy D252H-/- (2 x 10¹¹vg/pup, treated with V3 = 2, treated with V4 = 1, wild-type FBS n = 5, D252H-/- FBS = 3. OnFig. 11BThe weight of mice is shown (mean ± S.E.M.).Fig. 11CAn assessment of motor skills is presented by testing using (mean ± S.E.M., two-way ANOVA and Dunnett's multiple comparison).

Conclusions

These experiments demonstrate that AAV9-mediated eEF1A2 expression in V3-treated D252H-/- mice can provide increased survival compared to untreated D252H-/- controls. Furthermore, although rotarod performance is consistently low in homozygous D252H-/- mice, V3-treated D252H-/- mice show a consistent improvement over time. In a prespecified long-term safety/tolerability study, eEF1A2 overexpression following intracerebroventricular V3 administration in heterozygous D252H/+ mice did not appear to have a detrimental effect on survival or functional outcome measures. Ongoing analyses in both homozygous and heterozygous D252H mice will further characterize the long-term effects of AAV9-mediated eEF1A2 overexpression in this eEF1A2 deficiency model.

Example 6. Effects of AAV9-mediated delivery of human eEF1A2 protein to the central nervous system in the Del22ex3 mouse model of eEF1A2 disease

The CRISPR/Cas9-generated Del.22.ex.3 eEF1A2 mouse model was generated to knock out eEF1A2 expression (Davies, Faith CJ, et al.Human Molecular Genetics(2020). A 22-base pair deletion in exon 3 of Eef1a2, resulting from CRISPR/Cas9 mutagenesis, resulted in a null mutation. These Del22ex3 mice exhibit a severe phenotype, such that the mice do not survive much beyond disease onset (~21-25 days), suffering from early motor neuron degeneration with paralysis and additional clinically similar symptoms of fatal epileptic seizures. This example describes gene therapy studies in Del22ex3 eEF1A2 homozygous null mice.

Materials and methods

The survival and body weight of treated and untreated mice were monitored throughout development. Behavioral tests were also performed during the critical age window of P21–25 days.

Caring for animals

All animal experiments were conducted in accordance with the UK Home Office and Animals (Scientific Procedures) Act 1986 and in accordance with the guidelines of the University College London Ethics Committee. Heterozygous Del22ex3 mice were time-crossed to produce litters with mixed genotypes. Pups were genotyped at P0 using primers 5'-3'5'-TGAGTTGTGCCTCTACCCTT-3' and 5' - TACAGGCACATCCCAGGTGT-3'.

Introduction of test and control samples

Intracerebroventricular injections of 10 μl (5 μl per hemisphere, bilaterally) of V3 vector or FBS were administered to neonatal homozygous Del22ex3 (Del22ex3) mice at a dose of 2x1011 ^vg /pup (high dose V3) or ^2x1010 vg/pup (low dose V3). FBS (5 μl bilaterally) as a control was administered to wild-type and homozygous Del22ex3 mice via a 33-gauge Hamilton needle (Fisher Scientific®, Loughborough, UK) using the injection site coordinates reported by Kim et al. Kim, JY et al. J Vis Exp 91:51863 (2014). In newborn pups, the lambdoid suture was identified, and the intended injection site was located two-fifths of the way from the lambdoid suture to the eye, located approximately 0.8–1 mm lateral to the sagittal suture, midway between lambda and bregma. After injections, the pups were returned to their mothers.

Body weight

Individual animal body weight data were collected daily from P1–P30, and then weekly. A weight loss of ≥15% was used as the humane endpoint for euthanasia.

Behavioral testing

Training and testing using the rotarod were conducted on days P21–25 (P21 is considered the training day). All behavioral analyses were performed by a researcher blinded to the experimental group of animals. Mice were placed on a rotarod (Harvard Apparatus) at a constant acceleration of 4 to 40 rpm for a maximum of 2 minutes. The time it took for mice to fall from the rod was recorded over three trials (fall latency) for each animal on each testing day.

Limb muscle strength was measured from P21 to P25 using a grip strength meter (Bioseb®) (P21 is considered the training day). Grip strength of all four limbs or forelimbs was measured three times, with a 1-minute break between each test to allow the mouse to rest. In each test, the mouse was held by the base of the tail and lowered onto the net until it grasped either with its forelimbs or with all four paws.

Neurological assessment

Ledge test . Each mouse was placed on a ledge of an empty cage and allowed to explore freely. Mice were observed walking along the edge of the cage and landing on it and were scored accordingly: 0 = confident gait and good landing; 1 = stumbles and wobbles while walking; 2 = stumbles and wobbles, slips off the ledge but recovers; 3 = unable to walk along the ledge.

Hindlimb compression test . The mouse was grasped by the base of the tail and suspended in midair for 10 seconds. Hindlimb position was observed and scored as follows: 0 = hindlimb consistently pointing outward away from the abdomen, 1 = hindlimb slightly retracted toward the body for more than 50% of the suspension time, 2 = hindlimb pointing down toward the abdomen for more than 50% of the suspension time, 3 = hindlimb fully retracted touching the abdomen for more than 50% of the suspension time.

Gait test . The animal was placed on a flat surface with its head facing away from the researcher, then observed from behind while walking, and its behavior was scored as follows: 0 = the mouse moves normally, body weight is supported on all limbs, the abdomen does not touch the ground, and both hind limbs engage equally; 1 = slight tremors, a slightly raised pelvis, or a slight waddling motion; 2 = severe tremors, a raised pelvis, or a pronounced waddling motion; 3 = incoherent, stuttering movements with a raised pelvis and a strong waddling motion. The mouse may not move at all.

Kyphosis test . The mouse was placed on a flat surface and observed from the side while it walked, scoring it as follows: 0 = easily straightens the spine when walking, 1 = mild kyphosis (curvature of the spine) but mostly able to straighten the spine when walking, 2 = unable to fully straighten the spine and maintains moderate but persistent kyphosis, 3 = maintains marked kyphosis when walking or sitting.

Grip strength manometry. Limb muscle strength was measured at P21-25 ages and will be measured again at P30 and P60 using a grip strength meter (Bioseb®). Forelimb grip strength was measured three times, with a 1-minute break between each test to allow the mouse to rest. In each test, the mouse was held by the base of the tail and lowered onto the net until it grasped the net with its forepaws.

Results

Figure 12A shows the Kaplan-Meier survival plot for Del22ex3 mice treated with high dose V3 (2 x 10 ¹¹ vg/pup, n=5), Del22ex3 mice treated with low dose V3 (2 x 10 ¹⁰ vg/pup, n=5), Del22ex3 controls treated with buffer (n=3), and wild-type controls treated with buffer (n=6). Figure 12B shows the weight of mice over time (mean ± SEM). Figure 12C shows the motor assessment by grip strength manometry at P22-25 (mean ± SEM).

Figure 12D shows grip strength manometry at P23 ( mean ± SEM, two-way ANOVA and Tukey's multiple comparison). Figure 12E shows motility assessment by rotarod testing at P22–25 (mean ± SEM).

OnFig. 12FThe data from the rotarod test on P24 is presented (mean ± S.E.M., two-way ANOVA and Tukey's multiple comparison).

Fig. 12G shows neurological assessments from P21–25.

Conclusions

This experiment demonstrates a dose-dependent benefit of AAV9-mediated eEF1A2 expression in V3-positive Del22ex3 mice. Therapeutic efficacy is demonstrated by increased survival to postnatal day 36 (the most recent time point assessed to date), increased body weight, muscle strength, and motor behavior measured by grip strength and rotarod testing, and attenuation of normal neurological decline. Extended survival, up to postnatal day 36, is observed in animals treated with high doses of V3 compared to untreated Del22ex3 homozygous controls, which survive only to postnatal day 25. Survival duration is also increased compared to controls up to postnatal day 28 in the low-dose V3 group. Increased body weight gain comparable to single-litter wild-type controls is observed in the high-dose V3 group. Grip strength manometry demonstrates a decrease in muscle strength in untreated controls, with an increase in grip strength with age in wild-type animals. High-dose V3-treated animals exhibit increased grip strength, which is maintained from P22-25 compared to untreated controls. At postnatal day 23, high-dose V3-treated mice exhibit significantly stronger grip strength manometry (p = 0.0046), which is questionable for single-litter wild-type mice. No effect is observed with the low dose of V3. Fall latency in the rotarod test demonstrates sustained efficacy with high doses of V3 from P22-25 compared to untreated and low-dose V3-treated mice, which show a trend to decrease with age. At postnatal day 24, high-dose V3-treated animals demonstrate significantly higher performance scores in the rotarod test compared to untreated animals (p=0.0016) and are comparable to the wild-type littermate. No effect is observed in the low-dose V3 group. Further evidence of the benefit of AAV9-eEF1A2 is found in the neurological scores of V3-treated animals. Neurological scores in untreated Del22.ex3 controls increase with age from P21-25, consistent with the decline in neurobehavioral scores. This is not observed in high-dose V3-treated animals, which are comparable to the wild-type littermate from P21-25. Consistently lower neurological scores are also observed in the low-dose V3 group compared to untreated Del22.ex3 at least through P24.

--->

SEQUENCE LIST

<110> UCL Business, Ltd

<120> GENE THERAPY VECTOR FOR eEF1A2 AND ITS APPLICATION

<130>ROPA-019/02WO 329592-2245

<150> 63/055,775

<151> 2020-07-23

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 463

<212> Protein

<213> Homo sapiens

<400> 1

Met Gly Lys Glu Lys Thr His Ile Asn Ile Val Val Ile Gly His Val

1 5 10 15

Asp Ser Gly Lys Ser Thr Thr Thr Gly His Leu Ile Tyr Lys Cys Gly

20 25 30

Gly Ile Asp Lys Arg Thr Ile Glu Lys Phe Glu Lys Glu Ala Ala Glu

35 40 45

Met Gly Lys Gly Ser Phe Lys Tyr Ala Trp Val Leu Asp Lys Leu Lys

50 55 60

Ala Glu Arg Glu Arg Gly Ile Thr Ile Asp Ile Ser Leu Trp Lys Phe

65 70 75 80

Glu Thr Thr Lys Tyr Tyr Ile Thr Ile Ile Asp Ala Pro Gly His Arg

85 90 95

Asp Phe Ile Lys Asn Met Ile Thr Gly Thr Ser Gln Ala Asp Cys Ala

100 105 110

Val Leu Ile Val Ala Ala Gly Val Gly Glu Phe Glu Ala Gly Ile Ser

115 120 125

Lys Asn Gly Gln Thr Arg Glu His Ala Leu Leu Ala Tyr Thr Leu Gly

130 135 140

Val Lys Gln Leu Ile Val Gly Val Asn Lys Met Asp Ser Thr Glu Pro

145 150 155 160

Ala Tyr Ser Glu Lys Arg Tyr Asp Glu Ile Val Lys Glu Val Ser Ala

165 170 175

Tyr Ile Lys Lys Ile Gly Tyr Asn Pro Ala Thr Val Pro Phe Val Pro

180 185 190

Ile Ser Gly Trp His Gly Asp Asn Met Leu Glu Pro Ser Pro Asn Met

195 200 205

Pro Trp Phe Lys Gly Trp Lys Val Glu Arg Lys Glu Gly Asn Ala Ser

210 215 220

Gly Val Ser Leu Leu Glu Ala Leu Asp Thr Ile Leu Pro Pro Thr Arg

225 230 235 240

Pro Thr Asp Lys Pro Leu Arg Leu Pro Leu Gln Asp Val Tyr Lys Ile

245 250 255

Gly Gly Ile Gly Thr Val Pro Val Gly Arg Val Glu Thr Gly Ile Leu

260 265 270

Arg Pro Gly Met Val Val Thr Phe Ala Pro Val Asn Ile Thr Thr Glu

275 280 285

Val Lys Ser Val Glu Met His His Glu Ala Leu Ser Glu Ala Leu Pro

290 295 300

Gly Asp Asn Val Gly Phe Asn Val Lys Asn Val Ser Val Lys Asp Ile

305 310 315 320

Arg Arg Gly Asn Val Cys Gly Asp Ser Lys Ser Asp Pro Pro Gln Glu

325 330 335

Ala Ala Gln Phe Thr Ser Gln Val Ile Ile Leu Asn His Pro Gly Gln

340 345 350

Ile Ser Ala Gly Tyr Ser Pro Val Ile Asp Cys His Thr Ala His Ile

355 360 365

Ala Cys Lys Phe Ala Glu Leu Lys Glu Lys Ile Asp Arg Arg Ser Gly

370 375 380

Lys Lys Leu Glu Asp Asn Pro Lys Ser Leu Lys Ser Gly Asp Ala Ala

385 390 395 400

Ile Val Glu Met Val Pro Gly Lys Pro Met Cys Val Glu Ser Phe Ser

405 410 415

Gln Tyr Pro Pro Leu Gly Arg Phe Ala Val Arg Asp Met Arg Gln Thr

420 425 430

Val Ala Val Gly Val Ile Lys Asn Val Glu Lys Lys Ser Gly Gly Ala

435 440 445

Gly Lys Val Thr Lys Ser Ala Gln Lys Ala Gln Lys Ala Gly Lys

450 455 460

<210> 2

<211> 1389

<212> DNA

<213> Homo sapiens

<400> 2

atgggcaagg agaagaccca catcaacatc gtggtcatcg gccacgtgga ctccggaaag 60

tccaccacca cgggccacct catctacaaa tgcggaggta ttgacaaaag gaccattgag 120

aagttcgaga aggaggcggc tgagatgggg aagggatcct tcaagtatgc ctgggtgctg 180

gacaagctga aggcggagcg tgagcgcggc atcaccatcg acatctccct ctggaagttc 240

gagaccacca agtactacat caccatcatc gatgcccccg gccaccgcga cttcatcaag 300

aacatgatca cgggtacatc ccaggcggac tgcgcagtgc tgatcgtggc ggcgggcgtg 360

ggcgagttcg aggcgggcat ctccaagaat gggcagacgc gggagcatgc cctgctggcc 420

tacacgctgg gtgtgaagca gctcatcgtg ggcgtgaaca aaatggactc cacagagccg 480

gcctacagcg agaagcgcta cgacgagatc gtcaaggaag tcagcgccta catcaagaag 540

atcggctaca acccggccac cgtgcccttt gtgcccatct ccggctggca cggtgacaac 600

atgctggagc cctcccccaa catgccgtgg ttcaagggct ggaaggtgga gcgtaaggag 660

ggcaacgcaa gcggcgtgtc cctgctggag gccctggaca ccatcctgcc ccccacgcgc 720

cccacggaca agcccctgcg cctgccgctg caggacgtgt acaagattgg cggcattggc 780

acggtgcccg tgggccgggt ggagaccggc atcctgcggc cgggcatggt ggtgaccttt 840

gcgccagtga acatcaccac tgaggtgaag tcagtggaga tgcaccacga ggctctgagc 900

gaagctctgc ccggcgacaa cgtcggcttc aatgtgaaga acgtgtcggt gaaggacatc 960

cggcggggca acgtgtgtgg ggacagcaag tctgacccgc cgcaggaggc tgctcagttc 1020

acctcccagg tcatcatcct gaaccacccg gggcagatta gcgccggcta ctccccggtc 1080

atcgactgcc acacagccca catcgcctgc aagtttgcgg agctgaagga gaagattgac 1140

cggcgctctg gcaagaagct ggaggacaac cccaagtccc tgaagtctgg agacgcggcc 1200

atcgtggaga tggtgccggg aaagccatg tgtgtggaga gcttctccca gtacccgcct 1260

ctcggccgct tcgccgtgcg cgacatgagg cagacggtgg ccgtaggcgt catcaagaac 1320

gtggagaaga agagcggcgg cgccggcaag gtcaccaagt cggcgcagaa ggcgcagaag 1380

gcggggcaag 1389

<210> 3

<211> 448

<212> DNA

<213> Homo sapiens

<400> 3

agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 60

ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 120

gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 180

gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 240

gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 300

cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 360

cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg 420

aggagtcgtg tcgtgcctga gagcgcag 448

<210> 4

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized polynucleotide variant of eEF1A2

<400> 4

atgggtaaag aaaaaacaca tattaatata gtagtaatcg gtcatgttga ctctggaaaa 60

tctactacta caggacattt gatttataaa tgtggaggaa ttgataaaag aacaatagaa 120

aaatttgaaa aagaagctgc tgaaatgggt aaaggtagtt ttaaatatgc ttgggttttg 180

gataaattga aagctgaaag agaaagagga attacaattg atatttcttt gtggaaattt 240

gaaactacaa aatattatat aacaataata gatgctcctg gacatagaga ttttattaaa 300

aatatgatta caggaacttc tcaagcagat tgtgctgttt tgatagtagc agcaggagtt 360

ggtgaattcg aagcaggcat ttctaaaaat ggacaaacta gagaacatgc tttgttggct 420

tatacattgg gcgtaaaaca attgattgta ggagttaata aaatggattc tactgaacct 480

gcatattctg aaaaaagata tgatgaaata gtaaaagaag tttctgctta tattaaaaaa 540

attggttata atcctgctac agttccatt gttcctattt ctggatggca tggagataat 600

atgttggaac ctagtcctaa tatgccttgg tttaaaggat ggaaagttga aaggaaagaa 660

ggaaatgcat caggagtctc cttgttggaa gctttggata caatcttgcc tccaacaaga 720

cctacagata aacctttgag attgcctctt caagatgtat ataaaatagg aggaatagga 780

acagtgccag ttggaagagt agaaacaggt atattgagac ctggaatggt tgtaacattt 840

gcaccagtta atataactac tgaagtaaaa tctgttgaaa tgcatcatga agctttgtct 900

gaagctcttc ctggagataa tgtaggattt aatgttaaaa atgtaagtgt aaaagatata 960

agaagaggaa atgtatgtgg tgatagtaaa tcagatccac ctcaagaagc agctcaattt 1020

acatcacaag taataatatt gaatcatcct ggacaaattt ctgcaggata ttcaccagta 1080

atagattgtc atacagcaca tatagcttgt aaatttgctg aattgaaaga aaaaattgat 1140

agaagaagtg gaaaaaaact tgaagataat cctaaatcat tgaaatcagg agatgcagct 1200

attgtagaaa tggtacctgg aaaaccaatg tgtgtagaat ctttttctca atatccacct 1260

ctcggaagat ttgctgttag agatatgaga caaacagttg cagtaggagt tattaaaaat 1320

gtagaaaaaa aaagcggagg tgcaggaaag gttacaaaat ccgcacaaaa agctcaaaaa 1380

gctggtaaat aa 1392

<210> 5

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized polynucleotide variant of eEF1A2

<400> 5

atgggcaaag aaaaaacaca tataaacatt gtcgttatcg gacacgttga ttctggtaaa 60

agtacaacaa ccggtcactt gatatacaaa tgcgggggta tagacaaacg cactattgaa 120

aagttcgaga aagaagctgc ggagatgggc aaaggctcat tcaagtacgc gtgggtactc 180

gataagttga aagctgaacg cgagagggga atcaccatag acatctcact ttggaaattc 240

gagacaacca agtattacat aactattata gatgccccag gccacaggga tttcattaaa 300

aatatgataa ccggcacatc tcaagccgat tgcgccgtac tcatcgtcgc cgctggtgtg 360

ggtgagttcg aggcaggtat ttctaaaaat ggccagacac gcgaacatgc tcttctggct 420

tatacactcg gggttaaaca gctcatagta ggagtgaata agatggactc cactgaaccc 480

gcctattcag agaagcgcta tgacgaaatt gtaaaggagg tctcagcata tattaaaaaa 540

attggctata acccagccac ggtgccattc gtcccgatta gtggatggca tggtgacaat 600

atgctggaac caagtcccaa tatgccttgg tttaagggtt ggaaagtaga gcggaaagag 660

ggtaatgctt ccggcgtgtc attgctggag gcgcttgaca cgatactccc acccacaagg 720

ccaactgata agccactccg attgcccttg caggacgtgt acaagattgg gggaattggg 780

actgtgcccg tcgggcgcgt ggagacgggc atcctcagac ctgggatggt agtcactttt 840

gcccccgtca acataacgac tgaagttaaa tcagtggaaa tgcatcacga agctttgagt 900

gaggcgcttc ccggagataa cgttggattt aatgtcaaaa atgtctccgt taaagatata 960

agaagaggaa acgtctgcgg tgactcaaag tcagacccac cacaggaggc tgctcaattt 1020

acgagtcaag taataattct gaatcaccct gggcaaataa gtgcgggata ctctccagtc 1080

atcgattgtc acaccgccca tattgcatgt aagttcgcag aacttaagga aaagatcgac 1140

cgaagaagcg gaaaaaaatt ggaagataat ccgaaaagtt tgaaaagcgg tgacgcggcg 1200

attgtagaga tggtccctgg caaaccgatg tgtgtggagt ctttcagtca atatccacca 1260

ctcggtcgct ttgccgtgcg ggatatgcga cagaccgttg ctgtcggcgt aataaaaaac 1320

gtcgaaaaaa agagcggtgg ggctggaaaa gttacaaaat ccgctcaaaa ggcacagaag 1380

gcggggcaagtga 1392

<210> 6

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized polynucleotide variant of eEF1A2

<400> 6

atgggtaaag aaaagaccca cattaacata gtagtaatcg gtcatgttga ctctgggaaa 60

agcactacta ccggacattt gatctataaa tgtggggggca tcgacaaaag aacgatagag 120

aagtttgaga aggaggcggc ggagatgggt aaaggtagtt ttaagtacgc ttgggttttg 180

gacaaattga aagccgagcg cgagcgcggc attaccattg acatttctct ctggaaattc 240

gaaactacga agtattatat aacaataata gacgcccccg gccatcggga ctttattaaa 300

aacatgatta caggaactag ccaagcagat tgtgctgtgc tgatagtagc ggcaggggtc 360

ggggagttcg aagcaggcat ctctaaaaat ggacaaactc gagagcacgc cttgttggct 420

tataccttgg gcgtaaagca gctgatcgta ggagttaata aaatggattc cactgaaccc 480

gcatatagcg aaaagcgata tgacgaaata gtaaaggaag tctcagctta tatcaagaaa 540

atcggttaca atcctgcgac ggttccattc gttcctatct ccgggtggca cggcgataat 600

atgcttgagc ccagtcccaa tatgccctgg ttcaaggggt ggaaggttga gaggaaggaa 660

ggcaatgcat caggcgtcag cttgttggaa gctctcgaca ccatcctgcc gcccacgagg 720

cccacagaca aaccgttgcg actgcctctt caagatgtat acaaaatagg cgggatagga 780

accgtgccgg ttggacgagt agagacgggt atactgcggc ccggaatggt cgtgacgttt 840

gcacccgtga atataactac tgaggtgaag agcgtcgaga tgcaccatga agcgctgagt 900

gaagctctcc ctggcgataa cgtagggttc aacgtgaaaa acgtaagtgt aaaggatata 960

aggcgcggaa atgtatgtgg tgacagtaaa agcgacccgc cgcaagaggc ggcgcaattc 1020

acatcacagg taataatatt gaatcacccc ggccaaattt ccgcaggcta ctcaccagtc 1080

atagattgcc acaccgccca catagcttgt aagttcgctg agttgaaaga gaagattgat 1140

agacgaagtg ggaagaaact tgaagacaat ccgaagtccc tgaagtccgg tgacgcagcg 1200

attgtagaaa tggtaccggg caagccaatg tgtgtagagt ctttcagcca gtacccacca 1260

ctggggcggt tcgcggtgcg agacatgagg caaacggttg cggtcggcgt cattaaaaat 1320

gtcgaaaaaa agagtggcgg tgcaggtaag gtcacaaaaa gcgcacaaaa ggcccagaaa 1380

gccggtaagtga 1392

<210> 7

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized polynucleotide variant of eEF1A2

<400> 7

atgggaaagg aaaaaactca cataaacatt gtcgtcatcg gtcacgtaga cagtggcaaa 60

tcaacgacca ctggacatct catctataag tgtggcggta ttgacaaacg cactatcgag 120

aaattcgaaa aggaggctgc tgagatgggc aaaggctctt tcaagtacgc atgggtcctg 180

gataagctga aagcggagcg agagagagg atcaccatcg atatatctct gtggaaattt 240

gaaaccacca agtactacat cacaattatt gatgccccag gtcataggga ttttatcaag 300

aacatgatca ccgggacaag ccaagccgac tgcgcagttc tcatagtggc ggctggagta 360

ggggagtttg aagcagggat atctaagaat ggacagaccc gcgagcacgc cttgctggcc 420

tacaccctgg gagtgaagca gctcatagtt ggcgtcaata agatggacag caccgaaccc 480

gcctacagtg agaagaggta tgacgagatt gtgaaggagg tttctgctta cattaaaaag 540

attggctata acccagctac tgtcccattc gttccaatca gcggctggca cggtgataac 600

atgctggagc ctagtcccaa catgccgtgg ttcaaggggt ggaaggttga acgcaaggag 660

gggaatgcct caggcgtttc cctgctggag gccctcgata caatactccc cccgacccgg 720

cctacagata aaccgctgcg actgcctctt caggacgtgt ataaaatcgg gggaatcggc 780

acagtgcccg tgggcagggt agagactggc atcttgcggc ctggaatggt agtcaccttt 840

gccccggtta atatcacaac ggaggtgaaa tctgtggaga tgcatcacga agcactgagc 900

gaggctctgc ctggtgacaa cgtgggattt aacgtcaaaa acgtgtcagt caaggacatc 960

cgccgcggta acgtttgcgg agattctaag tccgatcccc cccaggaggc agcccaattt 1020

acctcccaag tgatcattct gaatcaccca ggccaaattt ccgccgggta ttcccctgtg 1080

attgactgtc acacagcaca catcgcatgc aaattcgccg aactcaagga gaaaattgat 1140

cggagaagcg gtaaaaaact ggaggacaac ccaaagtccc tcaagtctgg ggatgccgcc 1200

atcgtggaga tggtaccagg caaacctatg tgcgtggaaa gttttagcca gtaccctcca 1260

ctgggtcgct ttgctgttcg ggatatgcgg cagacagtag cggttggggt cataaaaaac 1320

gtcgagaaaa agagcggagg agctgggaaa gttaccaaat ccgcacagaa ggcacaaaaa 1380

gccggaaaatga 1392

<210> 8

<211> 1392

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized polynucleotide variant of eEF1A2

<400> 8

atgggcaaag agaaaacaca tattaacatt gttgttatcg ggcacgttga tagcggcaag 60

tccactacca ctggccatct gatttacaag tgcggcggaa tcgataaacg aactattgaa 120

aagttcgaaa aagaagccgc cgagatggga aagggctcct ttaaatacgc ttgggtcctc 180

gataaactca aagcagaacg ggagagagga atcaccatcg atatatcctt gtggaagttc 240

gaaactacaa aatattacat taccatcatt gatgcgcctg ggcaccgcga cttcattaag 300

aacatgatta ctggcacctc tcaagccgac tgcgcagtgt tgatcgtagc cgcaggcgtc 360

ggggagttcg aagctgggat cagcaagaac gggcagacta gggaacacgc tctgctcgca 420

tatactcttg gcgtgaaaca gttgatcgtt ggcgtgaaca agatggattc aactgagcct 480

gcctattctg agaaacgata cgacgagatt gtgaaagagg tttcagctta catcaagaaa 540

attgggtata atcccgcaac agttcccttc gtgcccatct ctgggtggca cggcgacaac 600

atgctcgaac catccccaaa tatgccatgg ttcaagggat ggaaggtgga gcgcaaagaa 660

ggcaacgcct ccggagtgtc tctgctcgag gccctggaca ccattctgcc cccaacacga 720

cccactgata agcctctgag actgccactg caagacgttt acaaaattgg gggaattgga 780

accgtgcctg tgggtcgggt ggaaaccgga atcctcagac ccggcatggt ggtcaccttc 840

gcaccagtga atataacgac agaggtcaaa tctgtggaga tgcaccatga ggcattgagc 900

gaggcactcc caggagacaa cgtgggtttc aacgtgaaaa atgtctcagt taaggacatc 960

cgacgcggca acgtgtgcgg agatagcaaa tctgaccccc cccaggaggc cgctcaattc 1020

acaagtcagg ttatcatcct taatcaccct ggccaaatat ctgcaggcta cagccccgtg 1080

atcgattgtc acacagctca tatcgcctgt aaatttgctg aactcaaaga aaagattgac 1140

cgcagatcag gaaaaaagct ggaggacaac cctaaaagtc tgaagtccgg cgacgctgcc 1200

atcgtggaga tggtccctgg gaaacccatg tgcgtggagt ccttttctca gtacccccct 1260

ctgggacgat tcgccgtgcg cgacatgaga cagactgtcg ccgtgggcgt cattaaaaat 1320

gtggaaaaaa aatcaggagg tgcagggaaa gtgacaaaga gtgcccagaa agcacagaag 1380

gctggcaagtga 1392

<210> 9

<211> 1395

<212> DNA

<213> Artificial sequence

<220>

<223> Human eEF1A2 with Kozak sequence

<400> 9

gccaccatgg gcaaggagaa gacccacatc aacatcgtgg tcatcggcca cgtggactcc 60

ggaaagtcca ccaccacggg ccacctcatc tacaaatgcg gaggtattga caaaaggacc 120

attgagaagt tcgagaagga ggcggctgag atggggaagg gatccttcaa gtatgcctgg 180

gtgctggaca agctgaaggc ggagcgtgag cgcggcatca ccatcgacat ctccctctgg 240

aagttcgaga ccaccaagta ctacatcacc atcatcgatg cccccggcca ccgcgacttc 300

atcaagaaca tgatcacggg tacatcccag gcggactgcg cagtgctgat cgtggcggcg 360

ggcgtgggcg agttcgaggc gggcatctcc aagaatgggc agacgcggga gcatgccctg 420

ctggcctaca cgctgggtgt gaagcagctc atcgtgggcg tgaacaaaat ggactccaca 480

gagccggcct acagcgagaa gcgctacgac gagatcgtca aggaagtcag cgcctacatc 540

aagaagatcg gctacaaccc ggccaccgtg ccctttgtgc ccatctccgg ctggcacggt 600

gacaacatgc tggagccctc ccccaacatg ccgtggttca agggctggaa ggtggagcgt 660

aaggaggca acgcaagcgg cgtgtccctg ctggaggccc tggacaccat cctgcccccc 720

acgcgcccca cggacaagcc cctgcgcctg ccgctgcagg acgtgtacaa gattggcggc 780

attggcacgg tgcccgtggg ccgggtggag accggcatcc tgcggccggg catggtggtg 840

acctttgcgc cagtgaacat caccactgag gtgaagtcag tggagatgca ccacgaggct 900

ctgagcgaag ctctgcccgg cgacaacgtc ggcttcaatg tgaagaacgt gtcggtgaag 960

gacatccggc ggggcaacgt gtgtggggac agcaagtctg acccgccgca ggaggctgct 1020

cagttcacct cccaggtcat catcctgaac cacccggggc agattagcgc cggctactcc 1080

ccggtcatcg actgccacac agcccacatc gcctgcaagt ttgcggagct gaaggagaag 1140

attgaccggc gctctggcaa gaagctggag gacaacccca agtccctgaa gtctggagac 1200

gcggccatcg tggagatggt gccgggaaag cccatgtgtg tggagagctt ctcccagtac 1260

ccgcctctcg gccgcttcgc cgtgcgcgac atgaggcaga cggtggccgt aggcgtcatc 1320

aagaacgtgg agaagaagag cggcggcgcc ggcaaggtca ccaagtcggc gcagaaggcg 1380

cagaaggcgg gcaag 1395

<210> 10

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Kozak sequence motif

<400> 10

gccaccatgg 10

<210> 11

<211> 13

<212> RNA

<213> Artificial sequence

<220>

<223> alternative Kozak sequence motif

<400> 11

gccgccrcca ugg 13

<210> 12

<211> 10

<212> RNA

<213> Artificial sequence

<220>

<223> Alternative Kozak sequence motif

<400> 12

gacaccaugg 10

<210> 13

<211> 168

<212> DNA

<213> Adeno-associated virus

<400> 13

tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 60

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 120

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgc 168

<210> 14

<211> 573

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-engineered CAG promoter from human betaherpavirus 5

<400> 14

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 60

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 120

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 180

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 240

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga 300

ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt 360

gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggcgcg 420

cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 480

cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 540

ggccctataa aaagcgaagc gcgcggcggg cgg 573

<210> 15

<211> 736

<212> Protein

<213> Adeno-associated virus 9

<400> 15

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 16

<211> 220

<212> DNA

<213> Human betaherpesvirus 5

<400> 16

tggtgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac tcacggggat 60

ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg 120

actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt aggcgtgtac 180

ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 220

<210> 17

<211> 583

<212> DNA

<213> Human betaherpesvirus 5

<400> 17

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgt 583

<210> 18

<211> 141

<212> DNA

<213> Adeno-associated virus

<400> 18

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc t 141

<210> 19

<211> 168

<212> DNA

<213> Adeno-associated virus

<400> 19

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgta 168

<210> 20

<211> 170

<212> DNA

<213> Adeno-associated virus

<400> 20

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta 170

<210> 21

<211> 141

<212> DNA

<213> Adeno-associated virus

<400> 21

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgcag g 141

<210> 22

<211> 124

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector filler sequence

<400> 22

gcggcaattc agtcgataac tataacggtc ctaaggtagc gatttaaata cgcgctctct 60

taaggtagcc ccgggacgcg tcaattgact acaaaccgag tatctgcaga gggccctgcg 120

tag 124

<210> 23

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector filler sequence

<400> 23

cttctgaggc ggaaagaacc agatcctctc ttaaggtagc atcgagattt aaattaggga 60

taacagggta atggcgcggg ccgc 84

<210> 24

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector filler sequence

<400> 24

gttacccagg ctggagtgca gtggcacatt tctgctcact gcaacctcct cctccctggg 60

ttc 63

<210> 25

<211> 253

<212> DNA

<213> Homo sapiens

<400> 25

gcccagcacc ccaaggcggc caacgccaaa actctccctc ctcctcttcc tcaatctcgc 60

tctcgctctt tttttttttc gcaaaaggag gggagagggg gtaaaaaaat gctgcactgt 120

gcggcgaagc cggtgagtga gcggcgcggg gccaatcagc gtgcgccgtt ccgaaagttg 180

ccttttatgg ctcgagcggc cgcggcggcg ccctataaaa cccagcggcg cgacgcgcca 240

ccaccgccga gtc 253

<210> 26

<211> 281

<212> DNA

<213> Gallus gallus

<400> 26

ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc 60

aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg 120

ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 180

cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 240

ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg a 281

<210> 27

<211> 455

<212> DNA

<213> Homo sapiens

<400> 27

caacctttgg agctaagcca gcaatggtag agggaagatt ctgcacgtcc cttccaggcg 60

gcctccccgt caccaccccc cccaacccgc cccgaccgga gctgagagta attcatacaa 120

aaggactcgc ccctgccttg gggaatccca gggaccgtcg ttaaactccc actaacgtag 180

aacccagaga tcgctgcgtt cccgccccct cacccgcccg ctctcgtcat cactgaggtg 240

gagaatagca tgcgtgaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 300

tccccgagaa gttgggggga ggggtcggca attgaacggg tgcctagaga aggtggcgcg 360

gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 420

aaccgtatat aagtgcagta gtcgccgtga acgtt 455

<210> 28

<211> 401

<212> DNA

<213> Homo sapiens

<400> 28

agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga 60

ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga 120

gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg 180

gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc 240

gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg 300

cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg 360

cgaccatctg cgctgcggcg ccggcgactc agcgctgcct c 401

<210> 29

<211> 422

<212> DNA

<213> Homo sapiens

<400> 29

ctgcagaggg ccctgcgtat gagtgcaagt gggttttagg accaggatga ggcggggtgg 60

gggtgcctac ctgacgaccg accccgaccc actggacaag cacccaaccc ccattcccca 120

aattgcgcat cccctatcag agagggggag gggaaacagg atgcggcgag gcgcgtgcgc 180

actgccagct tcagcaccgc ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg 240

ccgcctcagc actgaaggcg cgctgacgtc actcgccggt cccccgcaaa ctccccttcc 300

cggccacctt ggtcgcgtcc gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg 360

cgagataggg gggcacgggc gcgaccatct gcgctgcggc gccggcgact cagcgctgcc 420

tc 422

<210> 30

<211> 281

<212> DNA

<213> Homo sapiens

<400> 30

acttgtggac aaagtttgct ctattccacc tcctccaggc cctccttggg tccatcaccc 60

caggggtgct gggtccatcc cacccccagg cccacacagg cttgcagtat tgtgtgcggt 120

atggtcaggg cgtccgagag caggtttcgc agtggaaggc aggcaggtgt tggggaggca 180

gttaccgggg caacgggaac agggcgtttt ggaggtggtt gccatgggga cctggatgct 240

gacgaaggct cgcgaggctg tgagcagcca cagtgccctg c 281

<210> 31

<211> 293

<212> DNA

<213> Human betaherpesvirus 5

<400> 31

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 60

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 120

gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 180

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 240

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta cca 293

<210> 32

<211> 953

<212> DNA

<213> Homo sapiens

<400> 32

cgcgtccgcc cgcgagcaca gagcctcgcc tttgccgatc cgccgcccgt ccacacccgc 60

cgccaggtaa gcccggccag ccgaccgggg catgcggccg cggcccttcg cccgtgcaga 120

gccgccgtct gggccgcagc ggggggcgca tggggcggaa ccggaccgcc gtggggggcg 180

cggggagaagc ccctgggcct ccggagatgg gggacacccc acgccagttc gcaggcgcga 240

ggccgcgctc gggcgggcgc gctccggggg tgccgctctc ggggcggggg caaccggcgg 300

ggtctttgtc tgagccgggc tcttgccaat ggggatcgca cggtgggcgc ggcgtagccc 360

ccgtcaggcc cggtgggggc tggggcgcca tgcgcgtgcg cgctggtcct ttgggcgcta 420

actgcgtgcg cgctgggaat tggcgctaat tgcgcgtgcg cgctgggact caatggcgct 480

aatcgcgcgt gcgttctggg gcccgggcgc ttgcgccact tcctgcccga gccgctggcg 540

cccgaggtg tggccgctgc gtgcgcgcgc gcgacccggt cgctgtttga accgggcgga 600

ggcggggctg gcgcccggtt gggagggggt tggggcctgg cttcctgccg cgcgccgcgg 660

ggacgcctcc gaccagtgtt tgccttttat ggtaataacg cggccggccc ggcttccttt 720

gtccccaatc tgggcgcgcg ccggcgcccc ctggcggcct aaggactcgg cgcgccggaa 780

gtggccaggg cggcagcggc tgctcttggc ggccccgagg tgactatagc cttcttttgt 840

gtcttgatag ttcgccagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc 900

tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttc 953

<210> 33

<211> 1068

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-Made - Chicken Beta-Actin Exon/Intron with Intron

rabbit globin

<400> 33

gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc 60

cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg 120

ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc 180

cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg 240

tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc 300

gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc 360

ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc 420

gtggggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc 480

cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc 540

gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 600

ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc 660

tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 720

ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc 780

tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc 840

cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg 900

ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc 960

ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg 1020

ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattc 1068

<210> 34

<211> 126

<212> DNA

<213> Homo sapiens

<400> 34

agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagctg tgctcctggg 60

caccgcgcag tccgcccccg cggctcctgg ccagaccacc cctaggaccc cctgccccaa 120

gtcgca 126

<210> 35

<211> 121

<212> DNA

<213> Human betaherpesvirus 5

<400> 35

tcagatcgcc tggagaggcc atccacgctg ttttgacctc catagtggac accgggaccg 60

atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga 120

c 121

<210> 36

<211> 512

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-Created - Enhancer Element for Adenovirus Delivery

<400> 36

ctcactctct tccgcatcgc tgtctgcgag ggccagctgt tgggctcgcg gttgaggaca 60

aactcttcgc ggtctttcca gtactcttgg atcggaaacc cgtcggcctc cgaacggtac 120

tccgccaccg agggacctga gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa 180

ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc gtggcgggcg gcagcgggtg 240

gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg taattaaagt aggcggtctt 300

gagacggcgg atggtcgagg tgaggtgtgg caggcttgag atccagctgt tggggtgagt 360

actccctctc aaaagcgggc attacttctg cgctaagatt gtcagtttcc aaaaacgagg 420

aggatttgat attcacctgg cccgatctgg ccatacactt gagtgacaat gacatccact 480

ttgcctttct ctccacaggt gtccactccc ag 512

<210> 37

<211> 956

<212> DNA

<213> Homo sapiens

<400> 37

ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg 60

cctggcctct ttacggggtta tggcccttgc gtgccttgaa ttacttccac ctggctccag 120

tacgtgattc ttgatcccga gctggagcca ggggcggggcc ttgcgcttta ggagcccctt 180

cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 240

ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 300

gacgtgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caggatctgc 360

acactggtat ttcggttttt gggcccgcgg ccggcgacgg ggcccgtgcg tcccagcgca 420

catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 480

aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 540

cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 600

ctgctccagg gggctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 660

ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 720

accgggcgcc gtccaggcac ctcgattagt tctggagctt ttggagtacg tcgtctttag 780

gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 840

ttaggccagc ttggcacttg atgtaattct ccttggaatt tggccttttt gagtttggat 900

cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcag 956

<210> 38

<211> 939

<212> DNA

<213> Homo sapiens

<400> 38

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 60

ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 120

agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 180

gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 240

ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 300

tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 360

tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 420

ggcctgcgag cgcggccacc gagaatcgga cggggggtagt ctcaagctgg ccggcctgct 480

ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 540

tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 600

aaatggagga cgcggcgctc ggggagagcgg gcgggtgagt cacccacaca aaggaaaagg 660

gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 720

cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 780

tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 840

ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 900

cctcagacag tggttcaaag tttttttctt ccatttcag 939

<210> 39

<211> 83

<212> DNA

<213> Homo sapiens

<400> 39

tcagaagccc cgggctcgtc agtcaaaccg gttctctgtt tgcactcggc agcacgggca 60

ggcaagtggt ccctaggttc ggg 83

<210> 40

<211> 476

<212> DNA

<213> Homo sapiens

<400> 40

gtgagtctat gggacccttg atgttttctt tccccttctt ttctatggtt aagttcatgt 60

cataggaagg ggagaagtaa cagggtacac atattgacca aatcagggta attttgcatt 120

tgtaatttta aaaaatgctt tcttctttta atatactttt ttgtttatct tatttctaat 180

actttcccta atctctttct ttcagggcaa taatgataca atgtatcatg cctctttgca 240

ccattctaaa gaataacagt gataatttct gggttaaggc aatagcaata tttctgcata 300

taaatatttc tgcatataaa ttgtaactga tgtaagaggt ttcatattgc taatagcagc 360

tacaatccag ctaccattct gcttttattt tatggttggg ataaggctgg attattctga 420

gtccaagcta ggcccttttg ctaatcatgt tcatacctct tatcttcctc ccacag 476

<210> 41

<211> 589

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created, mutable regulatory element of marmot hepatitis

<400> 41

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 42

<211> 588

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created, mutable regulatory element of marmot hepatitis

<400> 42

tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 60

ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 120

ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 180

gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 240

ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 300

tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacaggg ctcggctgtt 360

gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 420

ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 480

tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 540

ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgca 588

<210> 43

<211> 755

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created, mutable regulatory element of marmot hepatitis

<400> 43

ttcctgttaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact 60

atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg 120

cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg 180

aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa 240

cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc 300

ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacaggg 360

ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccgc 420

ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 480

cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 540

cgcctcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgccca 600

tgtatctttt tcacctgtgc cttgtttttg cctgtgttcc gcgtcctact tttcaagcct 660

ccaagctgtg ccttgggcgg ctttggggca tggacataga tccctataaa gaatttggtt 720

catcttatca gttgttgaat tttcttcctt tggac 755

<210> 44

<211> 12

<212> DNA

<213> Artificial sequence

<220>

<223> CAAX Motive

<400> 44

tgtgtgataa tg 12

<210> 45

<211> 810

<212> DNA

<213> Homo sapiens

<400> 45

ctgttctcat cacatcatat caaggttata taccatcaat attgccacag atgttactta 60

gccttttaat atttctctaa tttagtgtat atgcaatgat agttctctga tttctgagat 120

tgagtttctc atgtgtaatg attatttaga gtttctcttt catctgttca aatttttgtc 180

tagttttatt ttttactgat ttgtaagact tctttttata atctgcatat tacaattctc 240

tttactgggg tgttgcaaat attttctgtc attctatggc ctgacttttc ttaatggttt 300

tttaatttta aaaataagtc ttaatattca tgcaatctaa ttaacaatct tttctttgtg 360

gttaggactt tgagtcataa gaaatttttc tctacactga agtcatgatg gcatgcttct 420

atattatttt ctaaaagatt taaagttttg ccttctccat ttagacttat aattcactgg 480

aatttttttg tgtgtatggt atgacatatg ggttcccttt tattttttac atataaatat 540

atttccctgt ttttctaaaa aagaaaaaga tcatcatttt cccattgtaa aatgccatat 600

ttttttcata ggtcacttac atatatcaat gggtctgttt ctgagctcta ctctatttta 660

tcagcctcac tgtctatccc cacacatctc atgctttgct ctaaatcttg atatttagtg 720

gaacattctt tcccattttg ttctacaaga atatttttgt tattgtcttt gggctttcta 780

tatacatttt gaaatgaggt tgacaagtta 810

<210> 46

<211> 726

<212> DNA

<213> Hepatitis B virus

<400> 46

ataacaggcc tattgattgg aaagtttgtc aacgaattgt gggtcttttg gggtttgctg 60

ccccttttac gcaatgtgga tatcctgctt taatgccttt atatgcatgt atacaagcaa 120

aacaggcttt tactttctcg ccaacttaca aggcctttct cagtaaacag tatatgaccc 180

tttaccccgt tgctcggcaa cggcctggtc tgtgccaagt gtttgctgac gcaaccccca 240

ctggttgggg cttggccata ggccatcagc gcatgcgtgg aacctttgtg tctcctctgc 300

cgatccatac tgcggaactc ctagccgctt gttttgctcg cagcaggtct ggagcaaacc 360

tcatcgggac cgacaattct gtcgtactct cccgcaagta tacatcgttt ccatggctgc 420

taggctgtgc tgccaactgg atcctgcgcg ggacgtcctt tgtttacgtc ccgtcggcgc 480

tgaatcccgc ggacgacccc tcccggggcc gcttggggct ctaccgcccg cttctccgtc 540

tgccgtaccg tccgaccacg gggcgcacct ctctttacgc ggactccccg tctgtgcctt 600

ctcatctgcc ggaccgtgtg cacttcgctt cacctctgca cgtcgcatgg aggccaccgt 660

gaacgcccac cggaacctgc ccaaggtctt gcataagagg actcttggac tttcagcaat 720

gtcatc 726

<210> 47

<211> 755

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-engineered enhancer element for HepB delivery

<400> 47

ttcctgtaaa caggcctatt gattggaaag tttgtcaacg aattgtgggt cttttggggt 60

ttgctgcccc ttttacgcaa tgtggatatc ctgctttaat gcctttatat gcatgtatac 120

aagcaaaaca ggcttttact ttctcgccaa cttacaaggc ctttctcagt aaacagtata 180

tgacccttta ccccgttgct cggcaacggc ctggtctgtg ccaagtgttt gctgacgcaa 240

cccccactgg ttggggcttg gccataggcc atcagcgcat gcgtggaacc tttgtgtctc 300

ctctgccgat ccatactgcg gaactcctag ccgcttgttt tgctcgcagc tggactggag 360

caaacctcat cgggaccgac aattctgtcg tactctcccg caagcactca ccgtttccgc 420

ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt 480

cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc 540

cgcctcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgccca 600

tgtatctttt tcacctgtgc cttgtttttg cctgtgttcc gcgtcctact tttcaagcct 660

ccaagctgtg ccttgggcgg ctttggggca tggacataga tccctataaa gaatttggtt 720

catcttatca gttgttgaat tttcttcctt tggac 755

<210> 48

<211> 94

<212> DNA

<213> Homo sapiens

<400> 48

gctggagcct cggtagccgt tcctcctgcc cgctgggcct cccaacgggc cctcctcccc 60

tccttgcacc ggcccttcct ggtctttgaa taaa 94

<210> 49

<211> 596

<212> DNA

<213> Marmot hepatitis virus

<400> 49

attcgagcat cttaccgcca tttattccca tatttgttct gtttttcttg atttgggtat 60

acatttaaat gttaataaaa caaaatggtg gggcaatcat ttacattttt agggatatgt 120

aattactagt tcaggtgtat tgccacaaga caaacatgtt aagaaacttt cccgttattt 180

acgctctgtt cctgttaatc aacctctgga ttacaaaatt tgtgaaagat tgactgatat 240

tcttaactat gttgctcctt ttacgctgtg tggatatgct gctttaatgc ctctgtatca 300

tgctattgct tcccgtacgg ctttcgtttt ctcctccttg tataaatcct ggttgctgtc 360

tctttatgag gagttgtggc ccgttgtccg tcaacgtggc gtggtgtgct ctgtgtttgc 420

tgacgcaacc cccactggct ggggcattgc caccacctgt caactccttt ctgggacttt 480

cgctttcccc ctcccgatcg ccacggcaga actcatcgcc gcctgccttg cccgctgctg 540

gacaggggct aggttgctgg gcactgataa ttccgtggtg ttgtcgggga agggcc 596

<210> 50

<211> 387

<212> DNA

<213> Oryctolagus cuniculus

<400> 50

tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc 60

actcggaaga acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta 120

gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg 180

tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac 240

ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa 300

attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat 360

agctgtccct cttctcttat ggagatc 387

<210> 51

<211> 251

<212> DNA

<213> Bos taurus

<400> 51

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 60

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 120

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aatacaatag 180

caggcatgct ggggatgcgg tgggctctat gggtacccag gtgctgaaga attgacccgg 240

ttcctcctgg g 251

<210> 52

<211> 251

<212> DNA

<213> Bos taurus

<400> 52

ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 60

tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 120

ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 180

caggcatgct ggggatgcgg tgggctctat gggtacccag gtgctgaaga attgacccgg 240

ttcctcctgg g 251

<210> 53

<211> 225

<212> DNA

<213> Bos taurus

<400> 53

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225

<210> 54

<211> 202

<212> DNA

<213> Homo sapiens

<400> 54

ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 60

actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 120

gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcccaagttg 180

ggaagaaacc tgtagggcct gc 202

<210> 55

<211> 3144

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector genome construct

<400> 55

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggcaatt cagtcgataa ctataacggt cctaaggtag 180

cgatttaaat acgcgctctc ttaaggtagc cccgggacgc gtcaattgac tacaaaccga 240

gtatctgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg 300

gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca acccccattc 360

cccaaattgc gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt 420

gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc 480

accgccgcct cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc 540

ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga 600

ggcgcgagat aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc 660

tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagatgggca 720

aggagaagac ccacatcaac atcgtggtca tcggccacgt ggactccgga aagtccacca 780

ccacgggcca cctcatctac aaatgcggag gtattgacaa aaggaccatt gagaagttcg 840

agaaggaggc ggctgagatg gggaagggat ccttcaagta tgcctgggtg ctggacaagc 900

tgaaggcgga gcgtgagcgc ggcatcacca tcgacatctc cctctggaag ttcgagacca 960

ccaagtacta catcaccatc atcgatgccc ccggccaccg cgacttcatc aagaacatga 1020

tcacgggtac atcccaggcg gactgcgcag tgctgatcgt ggcggcgggc gtgggcgagt 1080

tcgaggcggg catctccaag aatgggcaga cgcgggagca tgccctgctg gcctacacgc 1140

tgggtgtgaa gcagctcatc gtgggcgtga acaaaatgga ctccacagag ccggcctaca 1200

gcgagaagcg ctacgacgag atcgtcaagg aagtcagcgc ctacatcaag aagatcggct 1260

acaacccggc caccgtgccc tttgtgccca tctccggctg gcacggtgac aacatgctgg 1320

agccctcccc caacatgccg tggttcaagg gctggaaggt ggagcgtaag gagggcaacg 1380

caagcggcgt gtccctgctg gaggccctgg acaccatcct gccccccacg cgccccacgg 1440

acaagcccct gcgcctgccg ctgcaggacg tgtacaagat tggcggcatt ggcacggtgc 1500

ccgtgggccg ggtggagacc ggcatcctgc ggccgggcat ggtggtgacc tttgcgccag 1560

tgaacatcac cactgaggtg aagtcagtgg agatgcacca cgaggctctg agcgaagctc 1620

tgcccggcga caacgtcggc ttcaatgtga agaacgtgtc ggtgaaggac atccggcggg 1680

gcaacgtgtg tggggacagc aagtctgacc cgccgcagga ggctgctcag ttcacctccc 1740

aggtcatcat cctgaaccac ccggggcaga ttagcgccgg ctactccccg gtcatcgact 1800

gccacacagc ccacatcgcc tgcaagtttg cggagctgaa ggagaagatt gaccggcgct 1860

ctggcaagaa gctggaggac aaccccaagt ccctgaagtc tggagacgcg gccatcgtgg 1920

agatggtgcc gggaaagccc atgtgtgtgg agagcttctc ccagtacccg cctctcggcc 1980

gcttcgccgt gcgcgacatg aggcagacgg tggccgtagg cgtcatcaag aacgtggaga 2040

agaagagcgg cggcgccggc aaggtcacca agtcggcgca gaaggcgcag aaggcgggca 2100

agtgaaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 2160

ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 2220

cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 2280

agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 2340

ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 2400

tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 2460

ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc 2520

tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 2580

ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 2640

gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctgtgc 2700

cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag 2760

gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta 2820

ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag 2880

acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca 2940

gatcctctct taaggtagca tcgagattta aattagggat aacagggtaa tggcgcgggc 3000

cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg 3060

aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg 3120

agcgagcgcg cagctgcctg cagg 3144

<210> 56

<211> 3035

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector genome construct

<400> 56

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtaag tgcaagtggg 180

ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact 240

ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg 300

aaacaggatg cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc 360

gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc tgacgtcact 420

cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg 480

cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg accatctgcg 540

ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc 600

gtgcctgaga gcgcaggcca ccatgggcaa ggagaagacc cacatcaaca tcgtggtcat 660

cggccacgtg gactccggaa agtccaccac cacggggccac ctcatctaca aatgcggagg 720

tattgacaaa aggaccattg agaagttcga gaaggaggcg gctgagatgg ggaagggatc 780

cttcaagtat gcctgggtgc tggacaagct gaaggcggag cgtgagcgcg gcatcaccat 840

cgacatctcc ctctggaagt tcgagaccac caagtactac atcaccatca tcgatgcccc 900

cggccaccgc gacttcatca agaacatgat cacgggtaca tcccaggcgg actgcgcagt 960

gctgatcgtg gcggcgggcg tgggcgagtt cgaggcgggc atctccaaga atgggcagac 1020

gcgggagcat gccctgctgg cctacacgct gggtgtgaag cagctcatcg tgggcgtgaa 1080

caaaatggac tccacagagc cggcctacag cgagaagcgc tacgacgaga tcgtcaagga 1140

agtcagcgcc tacatcaaga agatcggcta caacccggcc accgtgccct ttgtgcccat 1200

ctccggctgg cacggtgaca acatgctgga gccctccccc aacatgccgt ggttcaaggg 1260

ctggaaggtg gagcgtaagg agggcaacgc aagcggcgtg tccctgctgg aggccctgga 1320

caccatcctg ccccccacgc gccccacgga caagcccctg cgcctgccgc tgcaggacgt 1380

gtacaagatt ggcggcattg gcacggtgcc cgtgggccgg gtggagaccg gcatcctgcg 1440

gccgggcatg gtggtgacct ttgcgccagt gaacatcacc actgaggtga agtcagtgga 1500

gatgcaccac gaggctctga gcgaagctct gcccggcgac aacgtcggct tcaatgtgaa 1560

gaacgtgtcg gtgaaggaca tccggcgggg caacgtgtgt ggggacagca agtctgaccc 1620

gccgcaggag gctgctcagt tcacctccca ggtcatcatc ctgaaccacc cggggcagat 1680

tagcgccggc tactccccgg tcatcgactg ccacacagcc cacatcgcct gcaagtttgc 1740

ggagctgaag gagaagattg accggcgctc tggcaagaag ctggaggaca accccaagtc 1800

cctgaagtct ggagacgcgg ccatcgtgga gatggtgccg ggaaagccca tgtgtgtgga 1860

gagcttctcc cagtacccgc ctctcggccg cttcgccgtg cgcgacatga ggcagacggt 1920

ggccgtaggc gtcatcaaga acgtggagaa gaagagcggc ggcgccggca aggtcaccaa 1980

gtcggcgcag aaggcgcaga aggcgggcaa gtgatcaacc tctggattac aaaatttgtg 2040

aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 2100

taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 2160

aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 2220

tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2280

tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2340

gccttgcccg ctgctggaca ggggctcggc tgttggggcac tgacaattcc gtggtgttgt 2400

cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg 2460

ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2520

tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 2580

ccctttgggc cgcctccccg cactgcccgg gtggcatccc tgtgacccct ccccagtgcc 2640

tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag 2700

ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt 2760

atggagcaag gggcccaagt tgggaagaaa cctgtagggc ctgcgttacc caggctggag 2820

tgcagtggca catttctgct cactgcaacc tcctcctccc tgggttctac gtagataagt 2880

agcatggcgg gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct 2940

ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 3000

ttgcccgggc ggcctcagtg agcgagcgag cgcgc 3035

<210> 57

<211> 3263

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector genome construct

<400> 57

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtaag tgcaagtggg 180

ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact 240

ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg 300

aaacaggatg cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc 360

gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc tgacgtcact 420

cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg 480

cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg accatctgcg 540

ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc 600

gtgcctgaga gcgcagagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg 660

cagctgtgct cctgggcacc gcgcagtccg cccccgcggc tcctggccag accaccccta 720

ggaccccctg ccccaagtcg cagccaccat gggcaaggag aagacccaca tcaacatcgt 780

ggtcatcggc cacgtggact ccggaaagtc caccaccacg ggccacctca tctacaaatg 840

cggaggtatt gacaaaagga ccattgagaa gttcgagaag gaggcggctg agatggggaa 900

gggatccttc aagtatgcct gggtgctgga caagctgaag gcggagcgtg agcgcggcat 960

caccatcgac atctccctct ggaagttcga gaccaccaag tactacatca ccatcatcga 1020

tgcccccggc caccgcgact tcatcaagaa catgatcacg ggtacatccc aggcggactg 1080

cgcagtgctg atcgtggcgg cgggcgtggg cgagttcgag gcgggcatct ccaagaatgg 1140

gcagacgcgg gagcatgccc tgctggccta cacgctgggt gtgaagcagc tcatcgtggg 1200

cgtgaacaaa atggactcca cagagccggc ctacagcgag aagcgctacg acgagatcgt 1260

caaggaagtc agcgcctaca tcaagaagat cggctacaac ccggccaccg tgccctttgt 1320

gcccatctcc ggctggcacg gtgacaacat gctggagccc tcccccaaca tgccgtggtt 1380

caagggctgg aaggtggagc gtaagggaggg caacgcaagc ggcgtgtccc tgctggaggc 1440

cctggacacc atcctgcccc ccacgcgccc cacggacaag cccctgcgcc tgccgctgca 1500

ggacgtgtac aagattggcg gcattggcac ggtgcccgtg ggccgggtgg agaccggcat 1560

cctgcggccg ggcatggtgg tgacctttgc gccagtgaac atcaccactg aggtgaagtc 1620

agtggagatg caccacgagg ctctgagcga agctctgccc ggcgacaacg tcggcttcaa 1680

tgtgaagaac gtgtcggtga aggacatccg gcggggcaac gtgtgtgggg acagcaagtc 1740

tgacccgccg caggaggctg ctcagttcac ctcccaggtc atcatcctga accacccggg 1800

gcagattagc gccggctact ccccggtcat cgactgccac acagcccaca tcgcctgcaa 1860

gtttgcggag ctgaaggaga agattgaccg gcgctctggc aagaagctgg aggacaaccc 1920

caagtccctg aagtctggag acgcggccat cgtggagatg gtgccgggaa agcccatgtg 1980

tgtggagagc ttctcccagt acccgcctct cggccgcttc gccgtgcgcg acatgaggca 2040

gacggtggcc gtaggcgtca tcaagaacgt ggagaagaag agcggcggcg ccggcaaggt 2100

caccaagtcg gcgcagaagg cgcagaaggc gggcaagtga tcaacctctg gattacaaaa 2160

tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 2220

ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2280

tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2340

gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2400

gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2460

ccgcctgcct tgcccgctgc tggacaggg ctcggctgtt gggcactgac aattccgtgg 2520

tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc 2580

tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2640

gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 2700

ggatctccct ttgggccgcc tccccgcagc tggagcctcg gtagccgttc ctcctgcccg 2760

ctgggcctcc caacgggccc tcctcccctc cttgcaccgg cccttcctgg tctttgaata 2820

aattcattgc ctgcccgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct 2880

ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt 2940

gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg 3000

gcccaagttg ggaagaaacc tgtagggcct gcgttaccca ggctggagtg cagtggcaca 3060

tttctgctca ctgcaacctc ctcctccctg ggttctacgt agataagtag catggcgggt 3120

taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 3180

gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 3240

cctcagtgag cgagcgagcg cgc 3263

<210> 58

<211> 4299

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-created vector genome construct

<400> 58

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtact ctggagacgc 180

gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 240

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 300

tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 360

agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 420

atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 480

atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc 540

ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg 600

ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga ggcggagagg 660

tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg 720

gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct 780

tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 840

gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt 900

ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga 960

gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag 1020

cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct 1080

ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg 1140

ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg 1200

gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga 1260

gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc 1320

gggcgggggg tggcggcagg tggggggtgcc gggcggggcg gggccgcctc gggccgggga 1380

gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag 1440

ccgcagccat tgccttttat ggtaatcgtg cgagagggcg caggacttc ctttgtccca 1500

aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc 1560

gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 1620

ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg 1680

ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc 1740

tgctaaccat gttcatgcct tcttcttttt cctacagcgc caccatgggc aaggagaaga 1800

cccacatcaa catcgtggtc atcggccacg tggactccgg aaagtccacc accacgggcc 1860

acctcatcta caaatgcgga ggtattgaca aaaggaccat tgagaagttc gagaaggagg 1920

cggctgagat ggggaaggga tccttcaagt atgcctgggt gctggacaag ctgaaggcgg 1980

agcgtgagcg cggcatcacc atcgacatct ccctctggaa gttcgagacc accaagtact 2040

acatcaccat catcgatgcc cccggccacc gcgacttcat caagaacatg atcacgggta 2100

catcccaggc ggactgcgca gtgctgatcg tggcggcggg cgtgggcgag ttcgaggcgg 2160

gcatctccaa gaatgggcag acgcgggagc atgccctgct ggcctacacg ctgggtgtga 2220

agcagctcat cgtgggcgtg aacaaaatgg actccacaga gccggcctac agcgagaagc 2280

gctacgacga gatcgtcaag gaagtcagcg cctacatcaa gaagatcggc tacaacccgg 2340

ccaccgtgcc ctttgtgccc atctccggct ggcacggtga caacatgctg gagccctccc 2400

ccaacatgcc gtggttcaag ggctggaagg tggagcgtaa ggagggcaac gcaagcggcg 2460

tgtccctgct ggaggccctg gacaccatcc tgccccccac gcgccccacg gacaagcccc 2520

tgcgcctgcc gctgcaggac gtgtacaaga ttggcggcat tggcacggtg cccgtgggcc 2580

gggtggagac cggcatcctg cggccggggca tggtggtgac ctttgcgcca gtgaacatca 2640

ccactgaggt gaagtcagtg gagatgcacc acgaggctct gagcgaagct ctgcccggcg 2700

acaacgtcgg cttcaatgtg aagaacgtgt cggtgaagga catccggcgg ggcaacgtgt 2760

gtggggacag caagtctgac ccgccgcagg aggctgctca gttcacctcc caggtcatca 2820

tcctgaacca cccggggcag attagcgccg gctactcccc ggtcatcgac tgccacacag 2880

cccacatcgc ctgcaagttt gcggagctga aggagaagat tgaccggcgc tctggcaaga 2940

agctggagga caaccccaag tccctgaagt ctggagacgc ggccatcgtg gagatggtgc 3000

cgggaaagcc catgtgtgtg gagagcttct cccagtaccc gcctctcggc cgcttcgccg 3060

tgcgcgacat gaggcagacg gtggccgtag gcgtcatcaa gaacgtggag aagaagagcg 3120

gcggcgccgg caaggtcacc aagtcggcgc agaaggcgca gaaggcgggc aagtgatcaa 3180

cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 3240

acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 3300

ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 3360

gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 3420

ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 3480

acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 3540

actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 3600

gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 3660

gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 3720

cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcagctgga gcctcggtag 3780

ccgttcctcc tgcccgctgg gcctcccaac gggccctcct cccctccttg caccggccct 3840

tcctggtctt tgaataaatt cattgcctgc ccgggtggca tccctgtgac ccctccccag 3900

tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat 3960

taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg 4020

tggtatggag caaggggccc aagttgggaa gaaacctgta gggcctgcgt tacccaggct 4080

ggagtgcagt ggcacatttc tgctcactgc aacctcctcc tccctgggtt ctacgtagat 4140

aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact 4200

ccctctctgc gcgctcgctc gctcactgag gccggggcgac caaaggtcgc ccgacgcccg 4260

ggctttgccc gggcggcctc agtgagcgag cgagcgcgc 4299

<210> 59

<211> 735

<212> Protein

<213> Adeno-associated virus 2

<400> 59

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 60

<211> 743

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construct - AAV9 variant

<400> 60

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Arg Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Thr Leu Ala Val

580 585 590

Pro Phe Lys Ala Gln Ala Gln Thr Gly Trp Val Gln Asn Gln Gly Ile

595 600 605

Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro

610 615 620

Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His Pro Ser Pro

625 630 635 640

Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln Ile Leu Ile

645 650 655

Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe Asn Lys Asp

660 665 670

Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val

675 680 685

Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro

690 695 700

Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn Val Glu Phe

705 710 715 720

Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr

725 730 735

Arg Tyr Leu Thr Arg Asn Leu

740

<210> 61

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide insert

<400> 61

Thr Leu Ala Val Pro Phe Lys

1 5

<210> 62

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide insert

<400> 62

Lys Phe Pro Val Ala Leu Thr

1 5

<210> 63

<211> 168

<212> DNA

<213> Adeno-associated virus

<400> 63

tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag 60

ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 120

cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgc 168

<210> 64

<211> 851

<212> DNA

<213> Artificial sequence

<220>

<223> Lab-Made - eSYN Promoter Polynucleotide

<400> 64

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccatgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccatg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gctgcagagg gccctgcgta tgagtgcaag tgggttttag 420

gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa 480

gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag 540

gatgcggcga ggcgcgtcgc gactgccagc ttcagcaccg cggacagtgc cttcgccccc 600

gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg 660

tcccccgcaa actcccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc 720

cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg 780

cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggagagtcg tgtcgtgcct 840

gagagcgcag g 851

<210> 65

<211> 3014

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial vector genome

<400> 65

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggcaatt cagtcgataa ctataacggt cctaaggacg 180

cgtagtgcaa gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac 240

cgaccccgac ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc 300

agagagggg aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc 360

gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg 420

cgcgctgacg tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt 480

ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg 540

gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag 600

cggagggagtc gtgtcgtgcc tgagagcgca gatgggcaag gagaagaccc acatcaacat 660

cgtggtcatc ggccacgtgg actccggaaa gtccaccacc acgggccacc tcatctacaa 720

atgcggaggt attgacaaaa ggaccattga gaagttcgag aaggaggcgg ctgagatggg 780

gaagggatcc ttcaagtatg cctgggtgct ggacaagctg aaggcggagc gtgagcgcgg 840

catcaccatc gacatctccc tctggaagtt cgagaccacc aagtactaca tcaccatcat 900

cgatgccccc ggccaccgcg acttcatcaa gaacatgatc acgggtacat cccaggcgga 960

ctgcgcagtg ctgatcgtgg cggcgggcgt gggcgagttc gaggcgggca tctccaagaa 1020

tgggcagacg cgggagcatg ccctgctggc ctacacgctg ggtgtgaagc agctcatcgt 1080

gggcgtgaac aaaatggact ccacagagcc ggcctacagc gagaagcgct acgacgagat 1140

cgtcaaggaa gtcagcgcct acatcaagaa gatcggctac aacccggcca ccgtgccctt 1200

tgtgcccatc tccggctggc acggtgacaa catgctggag ccctccccca acatgccgtg 1260

gttcaagggc tggaaggtgg agcgtaagga gggcaacgca agcggcgtgt ccctgctgga 1320

ggccctggac accatcctgc cccccacgcg cccccacggac aagcccctgc gcctgccgct 1380

gcaggacgtg tacaagattg gcggcattgg cacggtgccc gtgggccggg tggagaccgg 1440

catcctgcgg ccgggcatgg tggtgacctt tgcgccagtg aacatcacca ctgaggtgaa 1500

gtcagtggag atgcaccacg aggctctgag cgaagctctg cccggcgaca acgtcggctt 1560

caatgtgaag aacgtgtcgg tgaaggacat ccggcggggc aacgtgtgtg gggacagcaa 1620

gtctgacccg ccgcaggagg ctgctcagtt cacctcccag gtcatcatcc tgaaccaccc 1680

ggggcagatt agcgccggct actccccggt catcgactgc cacacagccc acatcgcctg 1740

caagtttgcg gagctgaagg agaagattga ccggcgctct ggcaagaagc tggaggacaa 1800

ccccaagtcc ctgaagtctg gagacgcggc catcgtggag atggtgccgg gaaagcccat 1860

gtgtgtggag agcttctccc agtacccgcc tctcggccgc ttcgccgtgc gcgacatgag 1920

gcagacggtg gccgtaggcg tcatcaagaa cgtggagaag aagagcggcg gcgccggcaa 1980

ggtcaccaag tcggcgcaga aggcgcagaa ggcgggcaag tgatcaacct ctggattaca 2040

aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 2100

acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 2160

ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac 2220

gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 2280

cctgtcagct cctttccgggg actttcgctt tccccctccc tattgccacg gcggaactca 2340

tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 2400

tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga 2460

ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 2520

cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 2580

gtcggatctc cctttgggcc gcctccccgc ctgtgccttc tagttgccag ccatctgttg 2640

tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 2700

aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 2760

gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 2820

cggtgggctc tatggattta aattagggat aacagggtaa tggcgcgggc cgcaggaacc 2880

cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 2940

accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 3000

cagctgcctg cagg 3014

<210> 66

<211> 3049

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial vector genome

<400> 66

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtact ctggagacgc 180

gtagtgcaag tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc 240

gaccccgacc cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca 300

gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg 360

cggacagtgc cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc 420

gcgctgacgt cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc 480

cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg 540

cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc 600

ggagggagtcg tgtcgtgcct gagagcgcag gccaccatgg gcaaggagaa gacccacatc 660

aacatcgtgg tcatcggcca cgtggactcc ggaaagtcca ccaccacgggg ccacctcatc 720

tacaaatgcg gaggtattga caaaaggacc attgagaagt tcgagaagga ggcggctgag 780

atggggaagg gatccttcaa gtatgcctgg gtgctggaca agctgaaggc ggagcgtgag 840

cgcggcatca ccatcgacat ctccctctgg aagttcgaga ccaccaagta ctacatcacc 900

atcatcgatg cccccggcca ccgcgacttc atcaagaaca tgatcacggg tacatcccag 960

gcggactgcg cagtgctgat cgtggcggcg ggcgtgggcg agttcgaggc gggcatctcc 1020

aagaatgggc agacgcggga gcatgccctg ctggcctaca cgctgggtgt gaagcagctc 1080

atcgtgggcg tgaacaaaat ggactccaca gagccggcct acagcgagaa gcgctacgac 1140

gagatcgtca aggaagtcag cgcctacatc aagaagatcg gctacaaccc ggccaccgtg 1200

ccctttgtgc ccatctccgg ctggcacggt gacaacatgc tggagccctc ccccaacatg 1260

ccgtggttca agggctggaa ggtggagcgt aaggaggca acgcaagcgg cgtgtccctg 1320

ctggaggccc tggacaccat cctgcccccc acgcgcccca cggacaagcc cctgcgcctg 1380

ccgctgcagg acgtgtacaa gattggcggc attggcacgg tgcccgtggg ccgggtggag 1440

accggcatcc tgcggccggg catggtggtg acctttgcgc cagtgaacat caccactgag 1500

gtgaagtcag tggagatgca ccacgaggct ctgagcgaag ctctgcccgg cgacaacgtc 1560

ggcttcaatg tgaagaacgt gtcggtgaag gacatccggc ggggcaacgt gtgtggggac 1620

agcaagtctg acccgccgca ggaggctgct cagttcacct cccaggtcat catcctgaac 1680

cacccggggc agattagcgc cggctactcc ccggtcatcg actgccacac agcccacatc 1740

gcctgcaagt ttgcggagct gaaggagaag attgaccggc gctctggcaa gaagctggag 1800

gacaacccca agtccctgaa gtctggagac gcggccatcg tggagatggt gccgggaaag 1860

cccatgtgtg tggagagctt ctcccagtac ccgcctctcg gccgcttcgc cgtgcgcgac 1920

atgaggcaga cggtggccgt aggcgtcatc aagaacgtgg agaagaagag cggcggcgcc 1980

ggcaaggtca ccaagtcggc gcagaaggcg cagaaggcgg gcaagtgatc aacctctgga 2040

ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 2100

tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 2160

ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 2220

gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 2280

caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 2340

actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 2400

ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 2460

ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 2520

tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 2580

gacgagtcgg atctcccttt gggccgcctc cccgcactgc ccgggtggca tccctgtgac 2640

ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc 2700

ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg 2760

tggagggggg tggtatggag caaggggccc aagttgggaa gaaacctgta gggcctgcgt 2820

tacccaggct ggagtgcagt ggcacatttc tgctcactgc aacctcctcc tccctgggtt 2880

ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga 2940

gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 3000

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgc 3049

<210> 67

<211> 3277

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial vector genome

<400> 67

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtact ctggagacgc 180

gtagtgcaag tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc 240

gaccccgacc cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca 300

gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg 360

cggacagtgc cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc 420

gcgctgacgt cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc 480

cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg 540

cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc 600

ggagggagtcg tgtcgtgcct gagagcgcag agtctgcggt gggcagcgga ggagtcgtgt 660

cgtgcctgag agcgcagctg tgctcctggg caccgcgcag tccgcccccg cggctcctgg 720

ccagaccacc cctaggaccc cctgccccaa gtcgcagcca ccatgggcaa ggagaagacc 780

cacatcaaca tcgtggtcat cggccacgtg gactccggaa agtccaccac cacgggccac 840

ctcatctaca aatgcggagg tattgacaaa aggaccattg agaagttcga gaaggaggcg 900

gctgagatgg ggaagggatc cttcaagtat gcctgggtgc tggacaagct gaaggcggag 960

cgtgagcgcg gcatcaccat cgacatctcc ctctggaagt tcgagaccac caagtactac 1020

atcaccatca tcgatgcccc cggccaccgc gacttcatca agaacatgat cacgggtaca 1080

tcccaggcgg actgcgcagt gctgatcgtg gcggcgggcg tgggcgagtt cgaggcgggc 1140

atctccaaga atgggcagac gcgggagcat gccctgctgg cctacacgct gggtgtgaag 1200

cagctcatcg tgggcgtgaa caaaatggac tccacagagc cggcctacag cgagaagcgc 1260

tacgacgaga tcgtcaagga agtcagcgcc tacatcaaga agatcggcta caacccggcc 1320

accgtgccct ttgtgcccat ctccggctgg cacggtgaca acatgctgga gccctccccc 1380

aacatgccgt ggttcaaggg ctggaaggtg gagcgtaagg agggcaacgc aagcggcgtg 1440

tccctgctgg aggccctgga caccatcctg ccccccacgc gccccacgga caagcccctg 1500

cgcctgccgc tgcaggacgt gtacaagatt ggcggcattg gcacggtgcc cgtgggccgg 1560

gtggagaaccg gcatcctgcg gccgggcatg gtggtgacct ttgcgccagt gaacatcacc 1620

actgaggtga agtcagtgga gatgcaccac gaggctctga gcgaagctct gcccggcgac 1680

aacgtcggct tcaatgtgaa gaacgtgtcg gtgaaggaca tccggcgggg caacgtgtgt 1740

ggggacagca agtctgaccc gccgcaggag gctgctcagt tcacctccca ggtcatcatc 1800

ctgaaccacc cggggcagat tagcgccggc tactccccgg tcatcgactg ccacacagcc 1860

cacatcgcct gcaagtttgc ggagctgaag gagaagattg accggcgctc tggcaagaag 1920

ctggaggaca accccaagtc cctgaagtct ggagacgcgg ccatcgtgga gatggtgccg 1980

ggaaagccca tgtgtgtgga gagcttctcc cagtacccgc ctctcggccg cttcgccgtg 2040

cgcgacatga ggcagacggt ggccgtaggc gtcatcaaga acgtggagaa gaagagcggc 2100

ggcgccggca aggtcaccaa gtcggcgcag aaggcgcaga aggcgggcaa gtgatcaacc 2160

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 2220

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 2280

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 2340

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 2400

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 2460

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 2520

tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 2580

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 2640

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 2700

ccctcagacg agtcggatct ccctttgggc cgcctccccg cagctggagc ctcggtagcc 2760

gttcctcctg cccgctgggc ctcccaacgg gccctcctcc cctccttgca ccggcccttc 2820

ctggtctttg aataaattca ttgcctgccc gggtggcatc cctgtgaccc ctccccagtg 2880

cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta 2940

agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg 3000

gtatggagca aggggcccaa gttgggaaga aacctgtagg gcctgcgtta cccaggctgg 3060

agtgcagtgg cacatttctg ctcactgcaa cctcctcctc cctgggttct acgtagataa 3120

gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 3180

ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 3240

ctttgcccgg gcggcctcag tgagcgagcg agcgcgc 3277

<210> 68

<211> 3498

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial vector genome

<400> 68

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtact ctggagacgc 180

gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 240

acgtcaatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg 300

taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac 360

gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt acatgacctt atgggacttt 420

cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga ggtgagcccc 480

acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt gtatttattt 540

attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg cgcgccaggc 600

ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat 660

cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat 720

aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc 780

tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 840

agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt 900

aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc 960

actttttttc aggttgggcc accatgggca aggagaagac ccacatcaac atcgtggtca 1020

tcggccacgt ggactccgga aagtccacca ccacggggcca cctcatctac aaatgcggag 1080

gtattgacaa aaggaccatt gagaagttcg agaaggaggc ggctgagatg gggaagggat 1140

ccttcaagta tgcctgggtg ctggacaagc tgaaggcgga gcgtgagcgc ggcatcacca 1200

tcgacatctc cctctggaag ttcgagacca ccaagtacta catcaccatc atcgatgccc 1260

ccggccaccg cgacttcatc aagaacatga tcacgggtac atcccaggcg gactgcgcag 1320

tgctgatcgt ggcggcgggc gtgggcgagt tcgaggcggg catctccaag aatgggcaga 1380

cgcgggagca tgccctgctg gcctacacgc tgggtgtgaa gcagctcatc gtgggcgtga 1440

acaaaatgga ctccacagag ccggcctaca gcgagaagcg ctacgacgag atcgtcaagg 1500

aagtcagcgc ctacatcaag aagatcggct acaacccggc caccgtgccc tttgtgccca 1560

tctccggctg gcacggtgac aacatgctgg agccctcccc caacatgccg tggttcaagg 1620

gctggaaggt ggagcgtaag gagggcaacg caagcggcgt gtccctgctg gaggccctgg 1680

acaccatcct gccccccacg cgccccacgg acaagcccct gcgcctgccg ctgcaggacg 1740

tgtacaagat tggcggcatt ggcacggtgc ccgtgggccg ggtggagacc ggcatcctgc 1800

ggccgggcat ggtggtgacc tttgcgccag tgaacatcac cactgaggtg aagtcagtgg 1860

agatgcacca cgaggctctg agcgaagctc tgcccggcga caacgtcggc ttcaatgtga 1920

agaacgtgtc ggtgaaggac atccggcggg gcaacgtgtg tggggacagc aagtctgacc 1980

cgccgcagga ggctgctcag ttcacctccc aggtcatcat cctgaaccac ccggggcaga 2040

ttagcgccgg ctactccccg gtcatcgact gccacacagc ccacatcgcc tgcaagtttg 2100

cggagctgaa ggagaagatt gaccggcgct ctggcaagaa gctggaggac aaccccaagt 2160

ccctgaagtc tggagacgcg gccatcgtgg agatggtgcc gggaaagccc atgtgtgtgg 2220

agagcttctc ccagtacccg cctctcggcc gcttcgccgt gcgcgacatg aggcagacgg 2280

tggccgtagg cgtcatcaag aacgtggaga agaagagcgg cggcgccggc aaggtcacca 2340

agtcggcgca gaaggcgcag aaggcgggca agtgatcaac ctctggatta caaaatttgt 2400

gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 2460

ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 2520

aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 2580

gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 2640

ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 2700

tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 2760

tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc 2820

gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 2880

ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc 2940

tccctttggg ccgcctcccc gcagctggag cctcggtagc cgttcctcct gcccgctggg 3000

cctcccaacg ggccctcctc ccctccttgc accggccctt cctggtcttt gaataaattc 3060

attgcctgcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag 3120

ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg 3180

actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggccca 3240

agttgggaag aaacctgtag ggcctgcgtt acccaggctg gagtgcagtg gcacatttct 3300

gctcactgca acctcctcct ccctgggttc tacgtagata agtagcatgg cgggttaatc 3360

attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 3420

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 3480

gtgagcgagc gagcgcgc 3498

<---

Claims (57)

1. A recombinant adeno-associated virus (rAAV) virion for enhancing eEF1A2 expression, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding the eEF1A2 protein operably linked to a promoter, wherein the promoter comprises: a) an hSYN promoter comprising a polynucleotide sequence that comprises SEQ ID NO: 3; or b) eSYN promoter. 2. The rAAV virion of claim 1, wherein the eSYN promoter comprises a polynucleotide sequence that comprises SEQ ID NO: 64. 3. The rAAV virion of claim 1 or 2, wherein the polynucleotide sequence encoding the eEF1A2 protein is characterized by at least 70%, at least 80%, or at least 90% identity with SEQ ID NO: 2. 4. The rAAV virion of any one of claims 1-3, wherein the polynucleotide sequence encoding the eEF1A2 protein is characterized by at least 95% or at least 99% identity with SEQ ID NO: 2. 5. The rAAV virion of claim 4, wherein the polynucleotide sequence encoding the eEF1A2 protein comprises the polynucleotide sequence of SEQ ID NO: 2. 6. The rAAV virion of any one of claims 1-5, wherein the eEF1A2 protein is characterized by at least 70%, at least 80%, or at least 90% identity to SEQ ID NO: 1. 7. The rAAV virion of claim 6, wherein the eEF1A2 protein is characterized by at least 95% or at least 99% identity to SEQ ID NO: 1. 8. The rAAV virion of claim 7, wherein the eEF1A2 protein comprises the polynucleotide sequence of SEQ ID NO: 1. 9. The rAAV virion of any one of claims 1-8, wherein the vector genome comprises a polyadenylation (polyA) site. 10. The rAAV virion of claim 9, wherein the polyA sequence is a bGH polyadenylation site. 11. The rAAV virion of claim 10, wherein the bGH polyadenylation site is characterized by at least 95% identity to SEQ ID NO: 53. 12. The rAAV virion of claim 9, wherein the polyA sequence is an hGH polyadenylation site. 13. The rAAV virion of claim 12, wherein the hGH polyadenylation site is characterized by at least 95% identity to SEQ ID NO: 54. 14. The rAAV virion of any one of claims 1-13, wherein the vector genome comprises a WPRE(x) element. 15. The rAAV virion of claim 14, wherein the WPRE(x) element is characterized by at least 95% identity to SEQ ID NO: 42. 16. The rAAV virion of claim 14, wherein the WPRE(x) element is characterized by at least 95% identity with SEQ ID NO: 41 or SEQ ID NO: 43. 17. The rAAV virion of any one of claims 1-16, wherein the vector genome comprises a Kozak sequence. 18. The rAAV virion of claim 17, wherein the Kozak sequence is SEQ ID NO: 10. 19. The rAAV virion of any one of claims 1-18, wherein the vector genome comprises a 5' untranslated region (UTR) that is characterized by at least 95% identity to one or more of SEQ ID NOs: 32-40. 20. The rAAV virion of any one of claims 1-19, wherein the vector genome comprises a 3' untranslated region (UTR) that is characterized by at least 95% identity to one or more of SEQ ID NOs: 41-49. 21. The rAAV virion of any one of claims 1-20, wherein the vector genome comprises a 5'-inverted terminal repeat (ITR) having a sequence that is at least 95% identical to SEQ ID NO: 19 or SEQ ID NO: 20. 22. The rAAV virion of any one of claims 1-21, wherein the vector genome comprises a 3'-inverted terminal repeat (ITR) having a sequence at least 95% identical to SEQ ID NO: 21 or SEQ ID NO: 63. 23. The rAAV virion of any one of claims 1-22, wherein the capsid is an AAV9 capsid. 24. The rAAV virion of any one of claims 1-23, wherein the capsid is characterized by at least 98%, 99% or 100% identity to SEQ ID NO: 15. 25. The rAAV virion of any one of claims 1-24, wherein the vector genome comprises a polynucleotide sequence that is characterized by at least 90%, 95%, 99% or 100% identity with any one of SEQ ID NOs: 55-58 or 65-68. 26. A method for treating and/or preventing a neurological disease or disorder in a subject in need thereof, comprising administering to the subject an rAAV virion according to any one of claims 1-25. 27. The method of claim 26, wherein the subject has or is suspected of having one or more mutations in the eEF1A2 gene. 28. The method according to claim 26 or 27, wherein the neurological disease or disorder comprises epilepsy. 29. The method according to any one of paragraphs 26-28, wherein the neurological disease or disorder involves mental retardation. 30. The method according to any one of paragraphs 26-29, wherein the neurological disease or disorder comprises autism. 31. The method according to any one of claims 26-30, wherein the step of administering comprises intracerebroventricular administration. 32. The method according to any one of paragraphs 26-31, wherein the step of administering comprises intravenous administration. 33. The method according to any one of claims 26-32, wherein the step of administering includes, simultaneously or sequentially, both intracerebroventricular administration and intravenous administration. 34. The method according to any one of paragraphs 26-33, wherein the subject is a mammal. 35. The method of any one of claims 26-34, wherein the method provides for prevention of deterioration in neurological performance in the subject compared to an untreated control subject. 36. The method according to any one of claims 26-35, wherein the method provides for an improvement in muscle strength and/or motor skills, wherein the muscle strength and/or motor skills are optionally assessed using an inverted grid test or a rotarod test. 37. A method for providing expression of eEF1A2 in the brain of a subject in need thereof, comprising administering to the subject an rAAV virion according to any one of claims 1-25. 38. The method of claim 37, wherein the method provides higher expression in the forebrain, optionally comparing the expression to a reference vector comprising a vector genome comprising an hSYN promoter, a Kozak sequence, an eEF1A2 transgene and/or a human globulin polyadenylation sequence (hGH). 39. The method of claim 37, wherein the method provides higher expression in the cerebral cortex, optionally comparing the expression to a reference vector comprising a vector genome comprising an hSYN promoter, a Kozak sequence, an eEF1A2 transgene and/or a human globulin polyadenylation sequence (hGH). 40. The method according to any one of claims 37-39, wherein the rAAV virion vector genome does not contain a Kozak sequence. 41. The method according to any one of claims 37-40, wherein the rAAV virion vector genome does not contain a human globulin polyadenylation sequence (hGH). 42. The method according to any one of claims 37-41, wherein the rAAV virion vector genome comprises a bovine globulin polyadenylation sequence (bGH). 43. The method according to any one of claims 37-42, wherein the subject has or is suspected of having one or more mutations in the eEF1A2 gene. 44. The method of any one of claims 37-43, wherein the subject suffers from or is at risk of a neurological disease or disorder. 45. The method of claim 44, wherein the neurological disease or disorder comprises epilepsy. 46. The method according to claim 44 or 45, wherein the neurological disease or disorder involves mental retardation. 47. The method according to any one of paragraphs 44-46, wherein the neurological disease or disorder comprises autism. 48. The method according to any one of claims 37-47, wherein the step of administering comprises intracerebroventricular administration. 49. The method according to any one of claims 37-47, wherein the step of administering comprises intravenous administration. 50. The method according to any one of claims 37-47, wherein the step of administering includes, simultaneously or sequentially, both intracerebroventricular administration and intravenous administration. 51. The method according to any one of paragraphs 37-50, wherein the subject is a mammal. 52. The method of any one of claims 37-51, wherein the method provides for prevention of deterioration in neurological performance in the subject compared to an untreated control subject. 53. The method according to any one of claims 37-52, wherein the method provides for an improvement in muscle strength and/or motor skills, wherein the muscle strength and/or motor skills are optionally assessed using an inverted grid test or a rotarod test. 54. Use of a pharmaceutical composition comprising an rAAV virion according to any one of claims 1-25 in the treatment of a subject who has or is suspected of having one or more mutations in the eEF1A2 gene. 55. A kit for increasing the expression of eEF1A2, containing the rAAV virion according to any one of claims 1-25 and instructions for use.