JP2023154428A - Rational polyploid adeno-associated virus vectors and methods of making and using the same - Google Patents

Abstract

To provide isolated adeno-associated virus (AAV) virions, and to provide methods for producing and using the AAV virions.SOLUTION: The present invention provides a polyploid adeno-associated virus (AAV) capsid, the polyploid AAV capsid comprising: a capsid protein VP1 derived from one or two or more first AAV serotypes; a capsid protein VP2 derived from one or two or more first AAV serotypes; and a capsid protein VP3 derived from one or two or more second AAV serotypes, in any combination, at least one of the first AAV serotypes being different from at least one of the second AAV serotypes and different from at least one of the third AAV serotypes.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is filed under 35 U.S.C. No. 62/678,675, each of which is incorporated by reference in its entirety.

Statement of Government Support This invention was made with government support under Grant Numbers DK084033, AI117408, AI072176, CA016086, CA151652, HL125749 and HL112761 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

Description of electronic filing of sequence listing Filed under 37 CFR § 1.821, name 5470-786WO2_ST25.txt, size 111,597 bytes, created on July 31, 2018, filed by EFS-Web Sequence listings in ASCII text format will be provided in lieu of paper copies. This Sequence Listing is incorporated herein by reference for its disclosure.

TECHNICAL FIELD The present invention relates to methods for the production of streamlined polyploid virions with desired properties, virions, substantially homogeneous populations of such virions, methods for producing substantially homogeneous populations, and Target its use.

BACKGROUND OF THE INVENTION Adeno-associated virus (AAV) vectors have been used in over 100 clinical trials with promising results, particularly in the treatment of blindness and hemophilia B. AAV is non-pathogenic, has broad tissue tropism, and can infect dividing or non-dividing cells. More importantly, AAV vector transduction induced long-term therapeutic transgene expression in preclinical and clinical studies. To date, there are 12 serotypes of AAV isolated for gene delivery. Among them, AAV8 has been shown to be the best for mouse liver targeting. Extensive studies in preclinical animals with FIX deficiency and phase I/II clinical trials were conducted in patients with hemophilia B using AAV2 and AAV8. Although the results from these studies are very promising, the expression of FIX from patients treated with AAV/FIX is similar to that achieved in animal models, even using the same vector dose/kg. It was not proportional to that. When 1 × 10 ¹¹ AAV8 particles encoding FIX were administered systemically to FIX knockout mice, 160% of normal levels of FIX were detected in the blood. However, when 2×10 ¹¹ AAV8/FIX particles were administered, only 40% of FIX was achieved in primates and less than 1% of FIX was found in humans. The inconsistent FIX expression after AAV vector transduction among these species may be due to altered hepatocyte tropism in different species. Another interesting finding from the AAV FIX clinical trial is the capsid-specific cytotoxic T lymphocyte (CTL) response that wipes out AAV-transduced hepatic parenchymal cells and leads to treatment failure. This phenomenon was not found in animal models after AAV delivery, indicating yet another change between preclinical and clinical studies. FIX expression was detected in both clinical trials using either AAV2 or AAV8 when much higher doses of AAV/FIX vectors were used. However, blood FIX levels decreased 4 or 9 weeks after injection, respectively. Further studies suggested that AAV vector infection induced a capsid-specific CTL response, and this response likely eliminated AAV-transduced hepatic parenchymal cells. Therefore, results from these clinical trials highlight the need to explore effective approaches to enhance AAV transduction without increasing vector capsid burden. Any vector improvement that reduces AAV capsid antigen effects would impact the concern of burdensome vector production and would be a welcome addition to the development of viable gene therapy agents.

Adeno-associated virus (AAV), a non-pathogenic dependent parvovirus that requires a helper virus for efficient replication, is utilized as a viral vector for gene therapy because of its safety and simplicity. AAV has a wide range of hosts and cell type tropism that allows it to transform both dividing and non-dividing cells. To date, 12 AAV serotypes and over 100 variants have been identified. Different serotype capsids have different infectivity in tissues or cultured cells, depending on the main receptors and co-receptors on the cell surface or on the intracellular transport pathway itself. The main receptors for several serotypes of AAV have been determined, such as heparin sulfate proteoglycans (HSPGs) for AAV2 and AAV3, and N-linked sialic acid for AAV5, while the main receptors for AAV7 and AAV8 The body has not been identified. Interestingly, the transduction efficiency of AAV vectors in cultured cells does not always translate to transduction efficiency in animals. For example, AAV8 induces much higher transgene expression than other serotypes in mouse liver, but not in cultured cell lines.

Of the 12 serotypes listed above, several AAV serotypes and variants have been used in clinical trials. As the first characterized capsid, AAV2 is the most widely used for gene delivery such as RPE 65 for Leber congenital amaurosis and factor IX (FIX) for hemophilia B. Although the application of AAV vectors has proven to be safe and therapeutic efficacy has been achieved in these clinical trials, one of the main challenges of AAV vectors is the relatively large number of viral genomes. its low infectivity. The AAV8 vector is another vector that has been used in several clinical trials in hemophilia B patients. Results from AAV8/FIX liver-targeted delivery demonstrated clear species differences in transgene expression between mice, non-human primates, and humans. AAV8 10 ¹⁰ vg carrying the FIX gene was able to reach above physiological levels of FIX expression (>100%) in FIX knockout mice, but only high doses (2 × 10 ¹² vg/kg body weight) were detectable in humans. Possible FIX expression could be induced. Based on these results above, there is still a need to develop effective strategies to enhance AAV transduction.

Most people are naturally exposed to AAV. As a result, a large proportion of the population has developed neutralizing antibodies (Nab) against specific serotypes of AAV in their blood and other body fluids. The presence of Nab poses another major challenge for broader application of AAV in future clinical trials. To enhance AAV transduction or circumvent Nab activity, many approaches have been explored, particularly genetic modification of AAV capsids based on rational design and directed evolution methods. Although some AAV mutants have demonstrated high transduction with the ability to evade Nabs in vitro or in animal models, modification of capsid composition provides the ability to alter the cellular tropism of the parental AAV.

The present invention addresses the need in the art for AAV vectors that have a combination of desirable features.

Our previous studies showed that capsids from different AAV serotypes (AAV1-AAV5) were compatible to assemble AAV virions (the terms virion, capsid, virion, and particle , used interchangeably in this application), and have shown that most isolated AAV monoclonal antibodies recognized several sites located on different AAV subunits. In addition, studies with chimeric AAV capsids have demonstrated that higher transduction can be achieved by introducing domains for primary receptors from other serotypes or tissue-specific domains. Introduction of AAV9 glycan receptors into AAV2 capsids enhances AAV2 transduction. Substitution of the 100aa domain from AAV6 to AAV2 capsid increases muscle tropism. We have demonstrated that polyploid AAV vectors consisting of capsids from two or more AAV serotypes benefit from individual serotypes for higher transduction, but in certain embodiments, We discovered that there is a possibility that the tendency to Moreover, since the majority of Nabs recognize conformational epitopes and polyploid virions can have altered surface structures, these polyploid viruses have the ability to evade neutralization by Nabs. There is a possibility that the

One approach to generate rAAV with mixed or mosaic capsid shells was to add an AAV helper plasmid encoding capsid proteins (VP1, VP2, and VP3) from a mixture of AAV serotypes. This methodology is sometimes referred to as cross-dressing. In certain embodiments, cross-dressing can alter the antigenic pattern of certain virions. However, a wide variety of virions are produced. Moreover, the virions produced are mosaic with a mixture of serotypes. As a result, the population of virions produced retains some particles that will elicit an antigenic response. Therefore, it would be desirable to obtain a substantially homogeneous population of a given virion.

The inventors have now discovered a methodology that allows the rational design and production of such chimeric or shuffled virions. The resulting virions are sometimes called polyploids, haploids, or triploids to indicate that their capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes. Capsids were isolated for gene therapy in 12 AAV serotypes, other species of such genes, mutant serotypes, shuffled serotypes, e.g. AAV2 VP1.5 and AAV4 VP2, AAV4 VP3, or from any AAV serotype, including any other AAV serotype desired. This method allows for the production of the desired virion-only infectious virus, resulting in a substantially homogeneous population of virions.

AAV virions have T=1 icosahedral symmetry and consist of three structural viral proteins VP1, VP2, and VP3. Sixty copies of three viral proteins in a ratio of 1:1:8-10 (VP1:VP2:VP3, respectively) form the virion (Rayaprolu, V., et al., J. Virol. 87(24): 13150-13160 (2013).

In one embodiment, the AAV virion is an isolated virion that has at least one of the viral structural proteins VP1, VP2, and VP3 from a different serotype than other VPs, and each VP is from only one serotype. . For example, VP1 is derived only from AAV2, VP2 is derived only from AAV4, and VP3 is derived only from AAV8.

In an alternative embodiment, at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 necessary to form virion particles capable of encapsidating the AAV genome is combined with other viral proteins. A virion particle can be constructed that differs from at least one of the following. For each viral protein present (VP1, VP2, and/or VP3), the proteins are of the same type (eg, all AAV2 VP1). As an example, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, optimally, viral particles composed of chimeric AAV2/8-derived VP1/VP2 (N-terminus of AAV2 and C-terminus of AAV8) paired only with VP3 from AAV2; Chimeric VP1/VP2 28m-2P3 paired with VP3 only (N-terminus from AAV8 and C-terminus from AAV2, no VP3 start codon mutation). In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, optimal is chimeric VP1/VP2 28m-2P3 paired with VP3 derived only from AAV3. As another example, chimeras do not exist.

In one embodiment, AAV virions encapsidating the AAV genome (including the heterologous genes between the two AAV ITRs) can be formed with only two viral structural proteins, VP1 and VP3. In one embodiment, the virion is conformationally correct, ie, has T=1 icosahedral symmetry. In one embodiment, the virion is infectious.

The population consists of at least 10 ¹ virions, at least 10 ² virions, at least 10 ³ virions, at least 10 ⁴ virions, at least 10 ⁵ virions, ...at least 10 ¹⁰ virions, at least 10 ¹¹ virions, at least 10 ¹² virions, at least 10 ¹⁵ virions, at least 10 ¹⁷ virions. In one embodiment, the population is at least 100 virus particles. In one embodiment, the population is between 10 ⁹ and 10 ¹² virions.

In one embodiment, the population comprises at least 1×10 ⁴ viral genomes (vg)/ml, at least 1×10 ⁵ viral genomes (vg)/ml, at least 1×10 ⁶ viral genomes (vg)/ml, at least 1 x 10 ⁷ viral genomes (vg)/ml, at least 1 x 10 ⁸ viral genomes (vg)/ml, at least 1 x 10 ⁹ viral genomes (vg)/ml, at least 1 x 10 ¹⁰ per vg/ml , at least per 1×10 ¹¹ vg/ml, at least per 1×10 ¹² vg/ml. In one embodiment, the population ranges from about 1×10 ⁵ vg/ml to about 1×10 ¹³ vg/ml.

A substantially homogeneous population is at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9% of the desired virions. In one embodiment, the population is completely homogeneous.

AAV2 and AAV8 have been used for clinical applications. In one embodiment, we first characterized haploid AAV viruses derived from AAV2 and AAV8 for transduction efficiency in vitro and in vivo, as well as for immune responses such as Nab evasion capacity, i.e., antigenic responses. Ta. In that study, we found that the virus yield of haploid vectors was not compromised and that the heparin binding profile was associated with the incorporation of AAV2 capsid subunit proteins. Haploid vector AAV2/8 initiated higher transduction in muscle and liver of mice. When applied to the FIX-deficient mouse model, higher FIX expression and improved bleeding phenotype correction were observed in haploid vector-treated mice compared to the AAV8 group. Importantly, haploid virus AAV2/8 had low binding affinity for A20 and was able to evade neutralization from anti-AAV2 sera. The following polyploid viruses AAV2/8/9 were generated from capsids of three serotypes (AAV2, 8 and 9). It was demonstrated that the ability of haploid AAV2/8/9 to evade neutralizing antibodies was significantly improved relative to sera immunized with the parental serotype.

Thus, in one aspect, the invention provides capsid protein VP1 derived from one or more first AAV serotypes and capsid protein VP3 derived from one or more second AAV serotypes. and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes. . Preferably such a population is substantially homogeneous. In some embodiments, a capsid of the invention comprises capsid protein VP2 in any combination, wherein the capsid protein VP2 is derived from one or more third AAV serotypes, wherein one or At least one of the two or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype.

In some embodiments, AAV virions can be formed from the three typical viral structural proteins VP1, VP2, and VP3 (e.g., Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids also fall within the scope of this invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion encapsidating an AAV genome, wherein at least one of the viral capsid structural proteins is different from other viral capsid structural proteins, and , isolated AAV virions in which each viral capsid structural protein is exclusively of the same type. In a further embodiment, the isolated AAV virion has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3, wherein the two viral proteins are derived from the AAV genome. is sufficient to form AAV virions that encapsidate the virus, at least one of the viral structural proteins present is derived from a different serotype than the other viral structural proteins, and VP1 is derived from only one serotype; VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype. For example, VP1.5 can be derived from AAV serotype 2 and VP3 can be derived from AAV serotype 8.

In some embodiments, a capsid of the invention comprises capsid protein VP1.5 from one or more fourth AAV serotypes in any combination, wherein one or more At least one of the fourth AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsid proteins described herein can include capsid protein VP2.

Thus, in certain embodiments, at least one of the viral structural proteins can be a chimeric viral structural protein, ie, contain segments from more than one protein. In one embodiment, the chimeric viral structural proteins are all from the same serotype. In another embodiment, the chimeric viral structural protein is composed of components from two or more serotypes, but the serotypes differ from at least one other serotype. In one embodiment, the viral structural proteins are not chimeric. In one embodiment, the chimeric AAV structural protein does not include structural amino acids derived from canine parvovirus. In one embodiment, the chimeric AAV structural protein does not include structural amino acids from b19 parvovirus. In one embodiment, the chimeric AAV structural protein does not include structural amino acids derived from canine parvovirus or b19 parvovirus. In one embodiment, the chimeric AAV structural protein contains only structural amino acids derived from AAV.

In some embodiments, only virions containing at least one viral protein that is different from other viral proteins are produced. For example, optimally VP1 and VP2 are derived from the same serotype and VP3 is derived from an alternate serotype. In other embodiments, optimally VP1 is derived from one serotype and VP2 and VP3 are derived from another serotype. In another embodiment, only particles are produced in which VP1 is derived from one serotype, VP2 is derived from a second serotype, and VP3 is derived from yet another serotype.

This can be done, for example, by changing splice donor sites, splice acceptor sites, initiation codons, and combinations thereof, by site-specific deletions and/or additions.

Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Deletion of the start codon, typically by substitution of M11 and M138, renders expression of VP1 and VP2 inoperable, while a similar deletion of the VP3 start codon is not sufficient. This is because the viral capsid ORF contains multiple ATG codons with varying strengths as initiation codons. Therefore, care must be taken when designing constructs that do not express VP3 to ensure that alternative VP3 species are not produced. For VP3, deletion of M138 is required, or if VP2 but not VP3 is desired, deletion of M203 plus M211 and 235 is typically the best approach (Warrington, K. H. Jr., et al., J. of Virol. 78(12): 6595-6609 (June 2004)). This can be done by mutation such as substitution or other means known in the art. Corresponding start codons in other serotypes can also be readily determined, and it can also be determined whether additional ATG sequences in eg VP3 can function as alternative start codons.

This allows a method for producing a population of substantially homogeneous polyploid virion populations, such as haploid or triploid virus particles.

The invention also provides an AAV capsid comprising capsid protein VP1 and capsid protein VP2 in any combination, wherein capsid protein VP1 is from one or more first AAV serotypes, and capsid protein VP2 is derived from one or more second AAV serotypes, and at least one of the first AAV serotypes is different from at least one of the second AAV serotypes. provide.

In some embodiments, the capsid comprises capsid protein VP3 in any combination, and the capsid protein VP3 is from one or more third AAV serotypes, and the capsid protein VP3 is from one or more third AAV serotypes. At least one of the third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsids described herein can include capsid protein VP1.5.

The present invention provides that an adeno-associated virus (AAV) capsid comprises capsid protein VP1 and capsid protein VP1.5 in any combination, wherein capsid protein VP1 is derived from one or more than one first AAV serotype. and the capsid protein VP1.5 is from one or more second AAV serotypes, and at least one of the first AAV serotypes is derived from at least one of the second AAV serotypes. Further providing an AAV capsid that is different from one.

In a further aspect, the invention provides a viral vector comprising (a) an AAV capsid of the invention and (b) a nucleic acid comprising at least one terminal repeat sequence and enveloped by the AAV capsid. The viral vector can be an AAV particle, and the capsid protein, capsid, viral vector, and/or AAV particle of the invention can be present in a composition further comprising a pharmaceutically acceptable carrier.

A method of producing an AAV particle comprising an AAV capsid according to any preceding claim, comprising: (a) one or more plasmids that together provide all the functions and genes necessary for assembling the AAV particle; transfecting a host cell; (b) introducing one or more nucleic acid constructs into a packaging or producer cell line to provide together all the functions and genes necessary to assemble AAV particles; (c) introducing into a host cell one or more recombinant baculovirus vectors that together provide all the functions and genes necessary to assemble the AAV particles; and/or (d) AAV particles. Further provided herein are methods comprising introducing into a host cell one or more recombinant herpesvirus vectors that together provide all the functions and genes necessary to assemble a vector.

In a further aspect, the invention provides a method of administering a nucleic acid to a cell, the method comprising contacting the cell with a viral vector of the invention and/or a composition of the invention.

Also provided herein are methods of delivering a nucleic acid to a subject, the method comprising administering to the subject a viral vector and/or composition of the invention.

Additionally, provided herein are capsid proteins, capsids, viral vectors, AAV particles, and/or compositions of the invention for use as medicaments in the beneficial treatment of disorders or diseases.

These and other aspects of the invention are addressed in more detail in the description of the invention below.

In vitro haploid virus transduction profiles. Haploid or parental virus was added to Huh7 or C2C12 cells at 10 ⁴ vg/cell. Cells were lysed 48 hours after transduction for luciferase assay. Data represent the average of three separate infections, and standard deviation is indicated by error bars. Haploid virus transduction in mouse muscle. 1×10 ¹⁰ vg of haploid virus, parental virus or virus mixed with AAV2 and AAV8 was injected via direct intramuscular injection into C57BL/6 mice. Four mice were included in each group. (Panel A) After one week, luciferase gene expression was imaged by IVIS imaging system. (Panel B) Photon signals were measured and calculated. Data represent the average of luciferase gene expression values for 4 injected mice in each group, and standard deviations are indicated by error bars. Supine: left leg - AAV8 or haploid or mixed virus, right leg - AAV2. Haploid virus transduction in mouse liver. 3×10 ¹⁰ vg of haploid virus was administered via intravenous injection. One week after injection, luciferase expression was imaged by IVIS imaging system (panel A) and photon signal was measured and calculated (panel B). Two weeks after injection, mice were euthanized, livers were removed for DNA extraction, AAV genome copies in the liver were measured by qPCR (panel C), and relative luciferase expression per number of AAV genome copies was calculated. (Panel D). Data represent the mean and standard deviation from 4 mice. Therapeutic levels of fix via haploid virus delivery. FIX knockout mice were injected with 1×10 ¹⁰ vg of each vector via the tail vein. Blood samples were collected 1, 2 and 4 weeks after injection. (Panel A) hFIX protein levels were tested by enzyme-linked immunosorbent assay. (Panel B) hFIX function was tested by hFIX-specific coagulation one-step method. Six weeks after injection, blood loss was determined by measuring the absorbance at A575 of hemoglobin content in saline solution (panel C). Data represent the mean and standard deviation from 5 mice (knockout and normal mice, no AAV treatment, as control) or 8 mice (AAV8 FIX or AAV2/8 1:3/FIX treatment groups). Transduction of haploid AAV82 derived from AAV2 and AAV8. Panel A. Composition of AAV capsid subunits. Panel B. Western blot for haploid viruses. Panel C. Representative imaging and quantification of liver transduction. Panel D. Representative imaging and quantification of muscle transduction. Liver transduction with triploid virus AAV2/8/9. 3×10 ¹⁰ vg of haploid virus was injected via the retroorbital vein. One week after injection, luciferase gene expression was imaged by the IVIS imaging system (panel A) and photon signals were measured and calculated (panel B). Data represent the mean and standard deviation from 5 mice. AAV stability against heating. Haploid design by mutating the start codon of capsid protein VP1. Haploid design by mutating splice acceptor site A2. Haploid design by mutating splice acceptor site A1. Haploid design by mutating the start codon and splice acceptor site A2 of the capsid protein for VP2/VP3. Haploid design by mutating the initiation codon and splice acceptor site A1 of capsid protein VP1. Production of haploid vectors using two plasmids. Production of haploid vectors using three plasmids. Production of haploid vectors using four plasmids. Schematic diagram showing the use of DNA shuffling to obtain virions with desired characteristics. A plasmid containing the DNA sequence for the AAV2 capsid protein (SEQ ID NO:139) in which the start codons for VP1 and VP2 have been mutated. A plasmid containing the DNA sequence for the AAV2 capsid protein (SEQ ID NO:140) in which the start codon for VP1 has been mutated. A plasmid containing the DNA sequence for the AAV2 capsid protein (SEQ ID NO:141) in which the start codons for VP2 and VP3 have been mutated. A plasmid containing the DNA sequence for the AAV2 capsid protein (SEQ ID NO:142) in which the start codon for VP2 has been mutated. Single or multiple subunits are substituted to generate novel polyploid AAV capsids. Figures 22A-C: Liver transduction of haploid vector H-AAV82. (22A) Composition of AAV capsid subunits. Haploid AAV virus was produced from cotransfection of two plasmids, one encoding VP1 and VP2 and another encoding VP3. (22B) 3 × 10 ¹⁰ AAV vector particles were injected into C57BL mice via the retroorbital vein. Imaging was performed one week later. (22C) Quantification of liver transduction. Data represent the mean and standard deviation of 5 mice. Figure 23A-B: Muscle transduction of haploid vector H-AAV82. 1×10 ⁹ AAV/luc particles were injected into the hind limb muscles of mice. Three weeks after injection, images were taken for 3 minutes. Supine: left leg - haploid AAV, right leg - AAV2. (23A) Representative imaging. (23B) Data from four mice after intramuscular injection. Fold increase in transduction was calculated by transduction from haploid AAV to AAV2. Figures 24A-C: Liver transduction of haploid vector H-AAV92. (24A) Composition of AAV capsid subunits. Haploid AAV virus was produced from co-transfection of two plasmids, one encoding AAV9 VP1 and VP2 and another encoding AAV2 VP3. (24B) 3 × ¹⁰ AAV vector particles were injected into C57BL mice via the retroorbital vein. Imaging was performed one week later. (24C) Quantification of liver transduction. Data represent the mean and standard deviation of 5 mice. Figures 25A-C: Liver transduction of haploid vector H-AAV82G9. (25A) Composition of AAV capsid subunits. Haploid AAV virus was produced from co-transfection of two plasmids, one encoding AAV8 VP1 and VP2 and another encoding AAV2G9 VP3. (25B) 3 × 10 ¹⁰ AAV vector particles were injected into C57BL mice via the retroorbital vein. Imaging was performed 1 week after AAV administration. (25C) Quantification of liver transduction. Data represent the mean and standard deviation of 5 mice. Figures 26A-D: Liver transduction of haploids AAV83, AAV93 and AAVrh10-3. (26A) Composition of AAV capsid subunits. (26B) Representative imaging. (26C) Quantification of liver transduction. (26D) Quantification of the viral genome in the indicated organs when compared to mouse lamin (internal control for expression levels). See description of Figure 26-1. Figures 27A-D: Transduction of haploid AAV82 from AAV2 and AAV8. (27A) Composition of AAV capsid subunits. (27B) Western blot for haploid viruses. (27C) Representative imaging and quantification of liver transduction. (27D) Representative imaging and quantification of muscle transduction. Analysis of haploid capabilities for binding and trafficking. AAV stability against heating. Detection of N-terminal exposure under different pH.

DETAILED DESCRIPTION OF THE INVENTION The invention will now be described with reference to the accompanying drawings in which representative aspects of the invention are shown. However, this invention may be embodied in different forms and should not be considered limited to the embodiments set forth herein. Rather, these aspects are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Unless defined otherwise, 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 belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, accession numbers, and other references mentioned herein are incorporated by reference in their entirety.

All amino acid position designations of AAV capsid virus structural proteins in the present specification and appended claims refer to the VP1 capsid subunit numbering (native AAV2 VP1 capsid protein: GenBank accession number AAC03780 or YP680426). ). It will be appreciated by those skilled in the art that the modifications described herein, if inserted into the AAV cap gene, may result in modifications to the structural viral proteins VP1, VP2, and/or VP3 that make up the capsid subunit. There will be. Alternatively, the capsid subunits may be expressed independently to achieve modification in only one or two of the capsid subunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3). I can do it.

Definitions The following terms are used in this specification and the appended claims.

Unless the context clearly dictates otherwise, the singular forms (a, an, the) are intended to include the plural forms.

Additionally, as used herein, the term "about" when referring to a measurable value such as a length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, refers to a specific amount. It is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of.

In addition, "and/or" as used herein is interpreted as an alternative ("or") in addition to any and all possible combinations of one or more of the related items. If a combination is used, it refers to the absence of and includes the combination.

As used herein, the transitional phrase "consisting essentially of" means that the claim is limited to the specific materials or steps recited in the claim as well as the "essential and novel features of the claimed invention." This means that it should be construed as including "anything that does not substantially affect the See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in original), and see also MPEP §2111.03. Accordingly, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be construed as equivalent to "comprising." It is specifically intended that the various features of the invention described herein can be used in any combination, unless the context dictates otherwise.

Moreover, the invention also contemplates that any feature or combination of features set forth herein may be excluded or omitted in some aspects of the invention.

To further illustrate, for example, where the specification indicates that certain amino acids can be selected from A, G, I, L, and/or V, the term also refers to any respective subset of these amino acids, such as A, G . Moreover, such language also indicates that one or more of the particular amino acids can be discarded (eg, by negative condition). For example, in certain embodiments, an amino acid is not A, G, or I; not A; not G or V, etc., as if each such possible disclaimer was expressly set forth herein. It is.

As used herein, the terms "reduce", "reduces", "reduction" and similar terms mean at least about 25%, 35%, 50%, 75%, 80% , 85%, 90%, 95%, 97% or greater reduction.

As used herein, the terms "enhance," "enhances," "augmentation" and similar terms mean at least about 25%, 50%, 75%, 100%, 150% , 200%, 300%, 400%, 500% or greater increase.

The term "parvovirus" as used herein encompasses the family Parvoviridae, which includes autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, mouse minute virus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy Includes parvovirus, B19 virus, and any other autonomous parvoviruses now known or later discovered. Other autonomous parvoviruses are known to those skilled in the art. See, eg, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV types AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6 Types AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, eg, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Several relatively new AAV serotypes and clades have been identified (e.g., Gao et al., (2004) J. Virology 78:6381-6388; Morris et al., (2004) Virology 33-:375- 383; and see Table 3).

The genomic sequences and native terminal repeat (TR), Rep protein, and capsid subunit sequences of various serotypes of AAV and autonomous parvoviruses are known in the art. Such sequences can be found in the literature or public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF 043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, See AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; the disclosures of which are incorporated herein by reference to teach parvovirus and AAV nucleic acid and amino acid sequences. For example, also Srivistava et al., (1983) J. Virology 45:555; Chiarini et al., (1998) J. Virology 71:6823; Chiarini et al., (1999) J. Virology 73:1309; Bantel -Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., ( 1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; Morris et al., (2004) Virology 33-:375- 383; International Publication See also WO 00/28061, WO 99/61601, WO 98/11244; and US Pat. No. 6,156,303; these disclosures contain is incorporated herein by reference to teach. See also Table 1.

The capsid structure of autonomous parvoviruses and AAV is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99:10405-10), AAV4 (Padron et al., (2005) J. Virol. 79: 5047-58), AAV5 (Walters et al. al., (2004) J. Virol. 78: 3361-71) and CPV (Xie et al., (1996) J. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251: 1456-64) for the description of the crystal structure.

The term "tropism" as used herein refers to preferential entry of a virus into certain cells or tissues, optionally followed by expression in the cell of sequences carried by the viral genome. (e.g. transcription and optionally translation), followed by expression of the heterologous nucleic acid of interest, e.g. for recombinant viruses.

As used herein, “systemic tropism” and “systemic transduction” (and equivalent terms) mean that the viral capsid or viral vector of the invention tropism for and/or transduction of the heart, liver, kidney and/or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (eg, brain, nerve cells, etc.) is observed. In other embodiments, systemic transduction of myocardial tissue is achieved.

"Selective tropism" or "specific tropism" as used herein refers to the delivery and/or specific transduction of a viral vector to certain target cells and/or certain tissues. .

Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined with reference to appropriate controls (e.g., control transduction or tropism, respectively). at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or greater). In certain embodiments, the viral vector efficiently transduces or has an efficient tropism for neuronal cells and cardiomyocytes. Appropriate controls will depend on a variety of factors, including the desired tropism and/or transduction profile.

Similarly, whether a virus "does not efficiently transduce" or "do not have efficient tropism" or similar terms to a target tissue can be determined with reference to appropriate controls. In certain embodiments, the viral vector does not efficiently transduce (ie, does not have efficient tropism) liver, kidney, gonads, and/or germ cells. In certain embodiments, the transduction (e.g., undesired transduction) of a tissue (e.g., liver) is lower than the level of transduction of the desired target tissue (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and/or cells of the central nervous system). 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less.

In some embodiments of the invention, AAV particles comprising capsids of the invention exhibit efficient transduction of certain tissues/cells and very low transduction of certain tissues/cells where transduction is undesirable. Multiple phenotypes can be exhibited, such as levels of transduction (eg, reduced transduction).

The term "polypeptide" as used herein includes both peptides and proteins, unless otherwise specified.

A "polynucleotide" is a sequence of nucleotide bases, which can be RNA, DNA, or a DNA-RNA hybrid sequence (including both natural and non-natural nucleotides), but in typical embodiments is single-stranded or double-stranded. Any stranded DNA sequence.

As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") refers to eg, cellular or viral structural components or other polypeptides or nucleic acids commonly found in association with the polynucleotide. In typical embodiments, "isolated" nucleotides are enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more compared to the starting material.

Similarly, an "isolated" polypeptide is one that is commonly found in association with at least some other component of a naturally occurring organism or virus, such as a structural component or polypeptide of a cell or virus. refers to a polypeptide that is at least partially separated from a polypeptide or nucleic acid. In typical embodiments, an "isolated" polypeptide is enriched by at least about 10-fold, 100-fold, 1000-fold, 1000-fold, 10,000-fold or more compared to the starting material.

"Isolated cell" refers to a cell that has been separated from other components with which it is normally associated in its natural state. For example, isolated cells can be cells in culture and/or cells in a pharmaceutically acceptable carrier of the invention. Thus, isolated cells can be delivered and/or introduced into a subject. In some embodiments, isolated cells can be cells that have been removed from a subject, manipulated ex vivo as described herein, and then returned to the subject.

Populations of virions can be generated by any of the methods described herein. In one embodiment, the population is at least 10 ¹ virions. In one embodiment, the population comprises at least 10 ² virions, at least 10 ³ virions, at least 10 ⁴ virions, at least 10 ⁵ virions, at least 10 ⁶ virions, at least 10 ⁷ virions, at least 10 ⁸ virions, at least 10 ⁹ virions, at least 10 10 virions, at least 10 ¹¹ virions, at least 10 ¹² virions, at least 10 ¹³ virions, at least ^{10 14 virions} , at least 10 ¹⁵ virions, at least 10 ¹⁶ virions, or at least 10 ¹⁷ virions. A population of virions can be heterogeneous or homogeneous (eg, substantially homogeneous or completely homogeneous).

A "substantially homogeneous population" as that term is used herein refers to a population of nearly identical virions within which there are few contaminant virions (those that are not identical). A substantially homogeneous population is at least 90% identical virions (e.g., desired virions), at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, at least 99%, at least 99.5%, at least 99.9% identical virions.

A population of virions that is completely homogeneous contains only identical virions.

As used herein, by "isolating" or "purifying" (or grammatical equivalents) a viral vector or virus particle or population of virus particles; is meant to be at least partially separated from at least some of the other components in the starting material. In typical embodiments, the "isolated" or "purified" viral vector or virus particle or population of virus particles is at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or It is more concentrated than that.

"Therapeutic polypeptide" means a polypeptide that is capable of alleviating, reducing, preventing, delaying and/or stabilizing symptoms caused by an absence or defect in a protein in a cell or a subject, and/or otherwise in a subject. A polypeptide that confers a benefit, for example, anticancer activity or improved graft viability or induces an immune response.

The term "treat," "treating," or "treatment of" (and grammatical variations thereof) reduces the severity of, at least partially ameliorates or stabilizes the condition in question; and/or some alleviation, reduction, reduction or stabilization in at least one clinical symptom is achieved and/or it is meant that there is a delay in the progression of the disease or disorder.

The terms "prevent", "preventing" and "prophylaxis" (and grammatical variations thereof) refer to the prevention and/or delay of the onset of a disease, disorder and/or clinical symptom in a subject and/or of the present invention. Refers to a reduction in the severity of the onset of a disease, disorder and/or clinical symptom compared to the severity that would occur in the absence of the method. Prevention can be complete, eg, the complete absence of disease, disorder and/or clinical symptoms. Prevention is also partial, such that the severity of the appearance and/or development of the disease, disorder and/or clinical symptoms in the subject is substantially less than the severity that would occur in the absence of the invention. You can also do that.

As used herein, a "therapeutically effective" amount is an amount sufficient to provide some improvement or benefit to the subject. Stated otherwise, a "therapeutically effective" amount is an amount that provides some alleviation, alleviation, reduction or stabilization of at least one clinical symptom in a subject. Those skilled in the art will recognize that the therapeutic effect need not be complete or curative, so long as some benefit is provided to the subject.

As used herein, a "prophylactically effective" amount prevents and/or delays the onset of a disease, disorder, and/or clinical symptom in a subject as compared to what would occur in the absence of a method of the invention. and/or to reduce and/or delay the severity of the disease, disorder and/or onset of clinical symptoms in the subject. Those skilled in the art will recognize that the level of prophylaxis need not be complete, so long as some prophylactic benefit is provided to the subject.

The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein to refer to a nucleic acid sequence that is not naturally occurring in a virus. Generally, a heterologous nucleic acid molecule or nucleotide sequence includes an open reading frame encoding a polypeptide and/or untranslated RNA of interest (eg, for delivery to a cell and/or subject).

As used herein, the term "viral vector," "vector," or "gene delivery vector" refers to a vector genome (e.g., viral DNA [vDNA]) that functions as a nucleic acid delivery vehicle and is packaged within a virion. ) refers to virus (e.g. AAV) particles containing Alternatively, in some situations, the term "vector" may be used to refer to the vector genome/vDNA alone.

An "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that includes one or more heterologous nucleic acid sequences. rAAV vectors generally require only the cis terminal repeats (TR) to generate virus. All other viral sequences are not required and can be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, the rAAV vector genome retains only one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Coding sequences for structural and nonstructural proteins can be provided in trans (eg, from vectors such as plasmids or by stably incorporating the sequences into packaging cells). In aspects of the invention, the rAAV vector genome includes at least one TR sequence (e.g., AAV TR sequence), optionally containing two TRs (e.g., two AAV TRs). TRs can be the same or different from each other.

The term "terminal repeat" or "TR" refers to a terminal repeat that forms a hairpin structure and functions as a terminal inverted repeat (i.e., performs a desired function such as replication, viral packaging, integration and/or proviral rescue, etc.). mediator) any viral terminal repeats or synthetic sequences. A TR can be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences such as the TR sequences of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable viral sequences (e.g. , the SV40 hairpin that serves as the SV40 origin of replication) can be used as a TR, which can be further modified by truncations, substitutions, deletions, insertions and/or additions. Additionally, the TR can be partially synthetic or fully synthetic, such as a "double D sequence" as described in Samulski et al., US Pat. No. 5,478,745.

"AAV terminal repeat" or "AAV TR" means, without limitation, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any now known or later discovered It can be derived from any AAV, including any other AAV (see, eg, Table 1). AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences may be altered by insertions, deletions, truncations and/or missense mutations).

AAV proteins VP1, VP2, and VP3 are capsid proteins that interact together to form an AAV capsid of icosahedral symmetry. VP1.5 is an AAV capsid protein described in US Patent Application Publication No. 2014/0037585.

The viral vectors of the present invention may also be "targeted" viral vectors, as described in WO 00/28004 and Chao et al., (2000) Molecular Therapy 2:619. For example, it can be a parvovirus (with directional tropism) and/or a "hybrid" parvovirus (ie, the viral TR and the viral capsid are from different parvoviruses).

The viral vector of the invention is further a double-stranded parvovirus particle as described in WO 01/92551, the disclosure of which is incorporated herein by reference in its entirety. I can do it. Thus, in some embodiments, double-stranded (duplex) genomes can be packaged into viral capsids of the invention.

Additionally, viral capsids or genomic elements may contain other modifications including insertions, deletions and/or substitutions.

A "chimeric" viral structural protein, as used herein, refers to one or more of the amino acid sequences of the capsid protein (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) are modified by substitution, as well as one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9) amino acid residues in the amino acid sequence compared to the wild type. , 10, etc.) amino acid residues have been modified by insertions and/or deletions. In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype are combined in any combination with corresponding wild-type domains, functional regions, epitopes, etc. from different AAV serotypes. Alternatively, chimeric capsid proteins of the invention can be produced. In other embodiments, all substitutions are from the same serotype. In other embodiments, all substitutions are from AAV or synthetics. Production of chimeric capsid proteins can be carried out according to protocols well known in the art, and a large number of chimeric capsid proteins have been described both in the literature and herein, and these can be included in the capsids of the present invention. I can do it.

In an alternative embodiment, at least one viral protein from the group consisting of AAV capsid proteins VP1, VP2, and VP3 necessary to form virion particles capable of encapsidating the AAV genome is combined with other viral proteins. A virion particle can be constructed that differs from at least one of the following. For each viral protein present (VP1, VP2, and/or VP3), the proteins are of the same type (eg, all AAV2 VP1). As an example, at least one of the viral proteins is a chimeric viral protein and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric and only VP3 is non-chimeric. For example, optimally, viral particles composed of chimeric AAV2/8-derived VP1/VP2 (N-terminus of AAV2 and C-terminus of AAV8) paired only with VP3 from AAV2; Chimeric VP1/VP2 28m-2P3 paired with VP3 only (N-terminus from AAV8 and C-terminus from AAV2, no VP3 start codon mutation). In another embodiment, only VP3 is chimeric and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, optimal is chimeric VP1/VP2 28m-2P3 paired with VP3 derived only from AAV3. As another example, chimeras do not exist.

As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids.

Naturally occurring levorotatory (L-) amino acids are shown in Table 2.

Alternatively, the amino acids can be modified amino acid residues (non-limiting examples are shown in Table 4) and/or post-translationally modified (e.g., acetylation, amidation, formylation, hydroxylation, methylation). , phosphorylation or sulfation).

Additionally, the non-naturally occurring amino acid can be a "non-natural" amino acid as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link molecules of interest with AAV capsid proteins.

As used herein, the term "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or equivalent DNA molecules. Homologous recombination also generates new combinations of DNA sequences. These new combinations of DNA represent genetic mutations. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.

As used herein, the terms "gene editing," "genome editing," or "genome manipulation" refer to the use of engineered nucleases or "molecular scissors" to insert or delete DNA in the genome of a living organism. Refers to a type of genetic manipulation in which a gene is deleted or replaced. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.

The term "gene delivery" as used herein refers to the process by which foreign DNA is transferred into host cells for gene therapy applications.

The term "CRISPR" as used herein refers to the clustered, regularly arranged short palindromic repeats that are characteristic of the bacterial defense system that forms the basis of the CRISPR-Cas9 genome editing technique.

The term "zinc finger" as used herein refers to a small protein structural motif characterized by the coordination of one or more zinc ions to stabilize the fold.

In some embodiments, the AAV particles of the invention can be synthetic viral vectors designed to exhibit a range of desirable phenotypes suitable for different in vitro and in vivo applications. Accordingly, in one aspect, the invention provides AAV particles comprising adeno-associated virus (AAV).

The present invention provides an array of synthetic viral vectors exhibiting a range of desirable phenotypes suitable for different in vitro and in vivo applications. In particular, the present invention provides an unexpected finding that combining capsid proteins from different AAV serotypes into individual capsids allows for the development of improved AAV capsids with multiple desirable phenotypes in each individual capsid. Based on the findings of Such chimeric or shuffled virions are sometimes referred to as polyploids, haploids, or triploids to indicate that their capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes. A new method for producing such virions is described herein. By preventing translation of undesired open reading frames, these methods result in the production of homogeneous populations of generated virions.

The ability to generate homogeneous (e.g., substantially or completely) populations of recombinant virions dramatically reduces or eliminates the inheritance of undesirable/contaminating virion properties (e.g., transduction specificity or antigenicity). do.

AAV virions have T=1 icosahedral symmetry and consist of three structural viral proteins VP1, VP2, and VP3. Sixty copies of three viral proteins in a ratio of 1:1:8-10 (VP1:VP2:VP3, respectively) form the virion (Rayaprolu, V., et al., J. Virol. 87(24): 13150-13160 (2013).

In one embodiment, the AAV virion is an isolated virion that has at least one of the viral structural proteins VP1, VP2, and VP3 from a different serotype than other VPs, and each VP is from only one serotype. . For example, VP1 is derived only from AAV2, VP2 is derived only from AAV4, and VP3 is derived only from AAV8.

In one embodiment, AAV virions encapsidating an AAV genome containing heterologous genes between two AAV ITRs can be formed with only two viral structural proteins, VP1 and VP3. In one embodiment, the virion is conformationally correct, ie, has T=1 icosahedral symmetry. In one embodiment, the virion is infectious.

Infectious virions contain VP1/VP3 VP1/VP2/VP3. Typically, VP2/VP3-only and VP3-only virions are not infectious.

The viral structural proteins used to generate these virion populations include the 12 AAV serotypes isolated for gene therapy, other species of such genes, mutant serotypes, and shuffled serotypes. , for example, AAV2 VP1.5 and AAV4 VP2, AAV4 VP3, or any other AAV serotype desired.

For example, the triploid AAV2/8/9 vectors described herein, generated by co-transfection of AAV helper plasmids from serotypes 2, 8, and 9, are much more potent than AAV2 and similar to AAV8 in the mouse liver. with transduction. Importantly, triploid AAV2/8/9 vectors have an improved ability to evade neutralizing antibodies from sera immunized with the parent serotype. Although AAV3 is less efficiently transduced throughout the mouse body after systemic administration, the haploid vectors H-AAV83 or H -AAV93 or H-rh10-3 not only induces systemic transduction, but also much higher transduction in liver and other tissues compared to AAV3.

Accordingly, in one aspect, the present invention provides an adeno-associated virus (AAV) having a viral capsid, wherein the capsid contains a protein VP1 derived from one or more first AAV serotypes and one or more proteins VP1 from a first AAV serotype. an adeno-associated virus ( AAV). When at least one viral structural protein is derived from two or more serotypes, we refer to a phenomenon sometimes called cross-dressing, resulting in mosaic capsids. On the other hand, when each of the viral capsid proteins is derived from the same serotype, a mosaic capsid will not result, even if at least one of the viral proteins is derived from a different serotype. For example, VP1 from AAV2, VP2 from AAV6, and VP3 from AAV8.

In some embodiments, a capsid of the invention comprises capsid protein VP2 from one or more third AAV serotypes in any combination, wherein one or more third at least one of the AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsids described herein can include capsid protein VP1.5. VP1.5 is described in US Patent Publication No. 2014/0037585, and the amino acid sequence of VP1.5 is provided herein.

In some embodiments, only virions containing at least one viral protein that is different from other viral proteins are produced. For example, optimally VP1 and VP2 are derived from the same serotype and VP3 is derived from an alternate serotype. In other embodiments, optimally VP1 is derived from one serotype and VP2 and VP3 are derived from another serotype. In another embodiment, only particles are produced in which VP1 is derived from one serotype, VP2 is derived from a second serotype, and VP3 is derived from yet another serotype.

This can be done, for example, by changing splice donor sites, splice acceptor sites, initiation codons, and combinations thereof, by site-specific deletions and/or additions.

This allows a method for producing a population of substantially homogeneous polyploid virion populations, such as haploid or triploid virus particles.

In some embodiments, AAV virions can be formed from the three typical viral structural proteins VP1, VP2, and VP3 (e.g., Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids also fall within the scope of this invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion encapsidating an AAV genome, wherein at least one of the viral capsid structural proteins is different from other viral capsid structural proteins, and , isolated AAV virions in which each viral capsid structural protein is exclusively of the same type. In a further embodiment, the isolated AAV virion has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3, wherein the two viral proteins are derived from the AAV genome. is sufficient to form AAV virions that encapsidate the virus, at least one of the viral structural proteins present is derived from a different serotype than the other viral structural proteins, and VP1 is derived from only one serotype; VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype. For example, VP1.5 can be derived from AAV serotype 2 and VP3 can be derived from AAV serotype 8.

In some embodiments, a capsid of the invention comprises capsid protein VP1.5 in any combination, wherein the capsid protein VP1.5 is from one or more than one fourth AAV serotype. and at least one of the one or more fourth AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV viral structural proteins described herein can include viral structural protein VP2.

The invention also provides an AAV capsid comprising capsid protein VP1 and capsid protein VP2 in any combination, wherein capsid protein VP1 is from one or more first AAV serotypes, and capsid protein VP2 is derived from one or more second AAV serotypes, and at least one of the first AAV serotypes is different from at least one of the second AAV serotypes. provide. In some embodiments, no chimeric viral structural proteins are present in the virion.

In some embodiments, the AAV particles of the invention can include capsids that include capsid protein VP3 in any combination, where capsid protein VP3 is associated with one or more third AAV serum at least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsids described herein can include capsid protein VP1.5.

The present invention provides an AAV particle comprising an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1 and capsid protein VP1.5 in any combination, wherein capsid protein VP1 is one or more than one. the capsid protein VP1.5 is from one or more second AAV serotypes, and at least one of the first AAV serotypes is from a first AAV serotype; Further provided are AAV particles that differ from at least one of the two AAV serotypes.

In some embodiments, the capsid comprises capsid protein VP3 in any combination, wherein the capsid protein VP3 is from one or more third AAV serotypes, and one or more at least one of the third AAV serotypes that is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsids described herein can include capsid protein VP1.5.

The present invention provides adeno-associated virus (AAV) capsids comprising capsid protein VP1 and capsid protein VP1.5 in any combination, wherein the capsid protein VP1 is derived from one or more than one first AAV serotype. and the capsid protein VP1.5 is from one or more second AAV serotypes, and at least one of the first AAV serotypes is derived from one of the second AAV serotypes. Further providing an AAV capsid different from at least one.

In some embodiments, an AAV capsid of the invention comprises capsid protein VP3 in any combination, wherein the capsid protein VP3 is from one or more than one third AAV serotype; At least one of the one or more third AAV serotypes is different from the first AAV serotype and/or the second AAV serotype. In some embodiments, the AAV capsid proteins described herein can include capsid protein VP2.

Some embodiments of the capsids of the invention include one or more first AAV serotypes, one or more second AAV serotypes, one or more second AAV serotypes, and one or more second AAV serotypes. The three AAV serotypes and one or more fourth AAV serotypes are selected in any combination from the group consisting of the AAV serotypes listed in Table 1.

In some embodiments of the invention, the AAV capsids described herein lack capsid protein VP2.

Some embodiments of the capsids of the invention include a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein, and/or a chimeric capsid VP1.5 protein.

In some embodiments, the AAV capsids of the invention are AAV AAV2/8/9, H-AAV82, H-AAV92, H-AAV82G9, AAV2/8 3:1, AAV2/8 1:1, AAV2/8 1 :3, or AAV8/9, all of which are described in the Examples section provided herein.

A non-limiting list of AAV capsid proteins that can be included in the capsids of the invention in any combination with other capsid proteins described herein and/or with other capsid proteins now known or later developed. Examples include LK3, LK01-19, AAV-DJ, Olig001, rAAV2-Retro, AAV-LiC, AAV0Kera1, AAV-Kera2, AAV-Kera3, AAV 7m8, AAV1,9, AAVr3.45, AAV Clone 32, AAV Clone 83, AAV-U87R7-C5, AAV ShH13, AAV ShH19, AAV L1-12, AAV HAE-1, AAV HAE-2, AAV variant ShH10, AAV2.5T, AAV LS1-4, AAV Lsm, AAV1289, AAVHSC 1 -17, AAV2 Rec 1-4, AAV8BP2, AAV-B1, AAV-PHP.B, AAV9.45, AAV9.61, AAV9.47, AAVM41, AAV2 display type peptide, AAV2-GMN, AAV9-peptide display type, AAV8 and AAV9 peptide display type, AAVpo2.1, AAVpo4, AAVpo5, AAVpo6, AAV rh, AAV Hu, AAV-Go.1, AAV-mo.1, BAAV, AAAV, AAV8 K137R, AAV Anc80L65, AAV2G9, AAV2 265 insertion Types include AAV2/265D, AAV2.5, AAV3 SASTG, AAV2i8, AAV8G9, AAV2 tyrosine variants AAV2 Y-F, AAV8 Y-F, AAV9 Y-F, AAV6 Y-F, AAV6.2 and any combination thereof.

By way of non-limiting example, the AAV capsid proteins and viral capsids of the invention may be derived from another virus, optionally another parvovirus or AAV, as described, for example, in WO 00/28004. It can be chimeric in that it can contain all or a portion of the capsid subunits.

The following publications describe chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the invention in any combination with wild-type capsid proteins and/or other chimeric or variant capsid proteins now known or later identified. is listed.

The following biological sequence files, which are listed in the U.S. Patent and Trademark Office's package of issued patents and published applications, include the wild-type capsid protein and/or other chimeras now known or later identified. describes chimeric or variant capsid proteins that can be incorporated into the AAV capsids of the invention in any combination with variant capsid proteins (for illustrative purposes, U.S. Patent Application No. 11/486,254 (corresponding to the issue): 11486254.raw, 11932017.raw, 12172121.raw, 12302206.raw, 12308959.raw, 12679144.raw, 13036343.raw, 13121532.raw, 13172915.raw, 13583920.raw, 13668 120.raw, 13673351.raw, 13679684.raw, 14006954.raw, 14149953.raw, 14192101.raw, 14194538.raw, 14225821.raw, 14468108.raw, 14516544.raw, 14603469.raw, 14680836.raw, 14695644.raw, 14878703. raw, 14956934.raw, 15191357.raw, 15284164.raw, 15368570.raw, 15371188.raw, 15493744.raw, 15503120.raw, 15660906.raw, and 15675677.raw.

It is understood that from any combination of AAV serotypes, any combination of VP1 and VP3, and if present, VP1.5 and VP2, can be employed to generate the AAV capsids of the invention. For example, VP1 proteins from any combination of AAV serotypes can be combined with VP3 proteins from any combination of AAV serotypes, and each VP1 protein can be present in any ratio of different serotypes. Each VP3 protein can be present in any ratio of different serotypes, and the VP1 and VP3 proteins can be present in any ratio of different serotypes. If present, VP1.5 and/or VP2 proteins from any combination of AAV serotypes can be combined with VP1 and VP3 proteins from any combination of AAV serotypes, with each VP1.5 protein comprising: Each VP2 protein can be present in any ratio of different serotypes, and each VP1 protein can be present in any ratio of different serotypes. Each VP3 protein can be present in any ratio of different serotypes, and VP1.5 and/or VP2 proteins can be present in combination with VP1 and VP3 proteins in any ratio of different serotypes. It is further understood that it is possible to

For example, each viral protein and/or each AAV serotype is A:B, A:B:C, A:B:C:D, A:B:C:D:E, A:B:C: D:E:F, A:B:C:D:E:F:G, A:B:C:D:E:F:G:H, A:B:C:D:E:F:G: Can be combined in any ratio, which can be in the ratio H:I or A:B:C:D:E:F:G:H:I:J, where A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. B can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, C can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; D can be 1, 2, 3, 4, 5, 6, 7, 8, 9; 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; E can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. F is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, G can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, H can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc.; I can be 1, 2, 3, 4, Can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, etc. ;J is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 50, 60, 70, It can be 80, 90, 100, etc.

Any combination of VP1, VP1.5, VP2 and/or VP3 capsid proteins may be present in the capsids of the invention as chimeric capsid proteins compared to the same protein type and/or compared to different capsid proteins. It is understood that there can be ratios.

In a further aspect, the invention provides a viral vector comprising, consisting essentially of, and/or consisting of (a) an AAV capsid of the invention; and (b) a nucleic acid molecule comprising at least one terminal repeat sequence. Further provided are viral vectors, wherein the nucleic acid molecule is enveloped by an AAV capsid. In some embodiments, the viral vector can be an AAV particle.

In some embodiments, the viral vectors of the invention can have systemic or selective tropism for skeletal muscle, cardiac muscle, and/or diaphragm muscle. In some embodiments, the viral vectors of the invention can have reduced tropism for the liver.

The invention further provides a composition that can be a pharmaceutical formulation comprising a capsid protein, capsid, viral vector, AAV particle composition and/or pharmaceutical formulation of the invention and a pharmaceutically acceptable carrier. .

In some non-limiting examples, the present invention provides an AAV capsid protein comprising a modification in the amino acid sequence in threefold axis loop 4 (Opie et al., J. Viral. 77: 6995-7006 (2003)). (VP1, VP1.5, VP2 and/or VP3) and modified AAV capsid proteins. We have demonstrated that modifications in this loop result in viral vectors containing modified AAV capsid proteins that exhibit (i) reduced transduction of the liver, (ii) enhanced migration across endothelial cells, and (iii) systemic transduction. introduction; including but not limited to (iv) enhanced transduction of muscle tissue (e.g., skeletal muscle, cardiac muscle and/or diaphragm muscle), and/or (v) reduced transduction of brain tissue (e.g., neurons). We have discovered that one or more desirable properties can be imparted. Accordingly, the present invention addresses some of the limitations associated with traditional AAV vectors. For example, vectors based on AAV8 and rAAV9 vectors are attractive for systemic nucleic acid delivery because they readily cross the endothelial cell barrier, but systemic administration of rAAV8 or rAAV9 is difficult because most vectors are delivered to the liver. , thereby leading to reduced transduction of other important target tissues such as skeletal muscle.

In certain embodiments, the modified AAV capsid consists of VP1, VP2, and/or VP3 created through DNA shuffling, and cell type-specific vectors can be developed through directed evolution methods. DNA shuffling by AAV is generally described in Li, W. et al., Mol. Ther. 16(7): 1252-12260 (2008), which is incorporated herein by reference. In certain embodiments, DNA shuffling is used to create VP1, VP2, and/or VP3 using DNA sequences for capsid genes from two or more different AAV serotypes, AAV chimeras, or other AAVs. be able to. In certain embodiments, the haploid AAV can consist of VP1 created by DNA shuffling, VP2 created by DNA shuffling, and/or VP3 created by DNA shuffling.

In certain embodiments, haploid AAV-derived VP1 can also be generated by randomly fragmenting the capsid genomes of AAV2, AAV8, and AAV9 using restriction enzymes and/or DNase, and A VP1 capsid protein library consisting of parts is generated. In this embodiment, AAV2/8/9 VP1 capsid proteins produced by DNA shuffling can also be combined with VP2 and/or VP3 proteins from different serotypes, and in some embodiments from AAV3. This allows the capsid to contain VP1, which contains amino acids derived from AAV2, AAV8, and AAV9, randomly connected together through DNA shuffling, and VP2, which contains only amino acids derived from native AAV3 VP2 and/or VP3. This will result in a haploid AAV consisting of VP3 and VP3. In certain embodiments, the donor for producing VP1, VP2, and/or VP3 is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV chimera or other AAV, or It can be any AAV, including those selected from Table 1 or Table 3. In certain embodiments, shuffled VP1 expresses only VP1, or only VP1/VP2, or only VP3, for example.

In another embodiment, nucleic acids encoding VP1, VP2, and/or VP3 can be generated through DNA shuffling. In one embodiment, the first nucleic acid produced by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid created by DNA shuffling will encode VP2 and VP3. In another embodiment, the first nucleic acid produced by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid created by DNA shuffling will encode VP2 and the third nucleic acid will encode VP3. In a further embodiment, the first nucleic acid produced by DNA shuffling will encode VP1 and VP2, and the second nucleic acid produced by DNA shuffling will encode VP3. In additional embodiments, the first nucleic acid produced by DNA shuffling will encode VP1 and VP3, and the second nucleic acid produced by DNA shuffling will encode VP2.

In aspects of the invention, the transduction of myocardial and/or skeletal muscle (determined on an individual skeletal muscle, multiple skeletal muscles, or any type of skeletal muscle) is compared to the level of transduction in the liver. at least about 5x, 10x, 50x, 100x, 1000x or higher.

In certain embodiments, the modified AAV capsid protein of the invention comprises three-fold axial loop 4 (e.g., amino acid positions 575-600 [inclusive] of native AAV2 VP1 capsid protein or the corresponding containing one or more modifications in the amino acid sequence of the region). As used herein, "modifications" in an amino acid sequence can each include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Substitutions, insertions and/or deletions are included. In certain embodiments, the modification is a substitution. For example, in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 from one AAV-derived three-fold axis loop 4 , 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at amino acid positions 575-600 of the native AAV2 capsid protein or the corresponding positions of another AAV-derived capsid protein. can be replaced in However, the modified viral capsids of the invention are not limited to AAV capsids in which amino acids from one AAV capsid are substituted into another AAV capsid, and the substituted and/or inserted amino acids may be from any source. Furthermore, it can be naturally occurring or partially or completely synthetic.

The nucleic acid and amino acid sequences of several AAV-derived capsid proteins described herein are known in the art. Therefore, amino acids that "correspond" to amino acid positions 575-600 (inclusive) or amino acid positions 585-590 (inclusive) of the native AAV2 capsid protein are ) can be easily determined.

In some embodiments, the invention envisions that modified capsid proteins of the invention can be produced by modifying any AAV capsid protein, now known or later discovered. Additionally, the AAV capsid protein to be modified may include naturally occurring AAV capsid proteins (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins or any of the AAV capsid proteins shown in Table 3). ), but is not limited thereto. Those skilled in the art will appreciate that a variety of manipulations to AAV capsid proteins are known in the art and the invention is not limited to modifications of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may already have changes compared to naturally occurring AAV (e.g. naturally occurring AAV capsid proteins, e.g. AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7). , AAV8, AAV9, AAV10, AAV11 and/or AAV12 or any other AAV now known or later discovered). Such AAV capsid proteins are also within the scope of the invention.

For example, in some embodiments, the AAV capsid protein to be modified can include an amino acid insertion immediately following amino acid 264 of the native AAV2 capsid protein sequence (e.g., PCT Publication WO2006/066066) and/or can be an AAV with an altered HI loop as described in WO2009/108274 and/or be an AAV modified to contain a poly-His sequence to facilitate purification Can be done. As another illustrative example, an AAV capsid protein can have a peptide targeting sequence incorporated therein as an insertion or replacement. Additionally, the AAV capsid protein can include a large domain from another AAV substituted and/or inserted into the capsid protein.

Thus, in certain embodiments, the AAV capsid protein to be modified can be obtained from naturally occurring AAV, but with one or more foreign sequences inserted and/or substituted into the capsid protein (e.g., native (exogenous to the virus) and/or modified by deletion of one or more amino acids.

Therefore, herein certain AAV capsid proteins (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 capsid proteins or any of the AAVs shown in Table 1, etc. When referring to a capsid protein derived from a capsid protein, it is intended to include the native capsid protein as well as capsid proteins with alterations other than the modifications of the present invention. Such changes include substitutions, insertions and/or deletions. In certain embodiments, the capsid protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 inserted therein as compared to the native AAV capsid protein sequence. , 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids (other than insertions of the invention) )include. In embodiments of the invention, the capsid protein is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to the native AAV capsid protein sequence. Contains less than 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acid substitutions (other than amino acid substitutions according to the invention). In embodiments of the invention, the capsid protein is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, compared to the native AAV capsid protein sequence. Deficiency of 16, 17, 18, 19 or 20, more than 20, more than 30, more than 40, more than 50, more than 60, or more than 70 amino acids deletions (other than the amino acid deletions of the invention).

Using AAV serotype 2 as an exemplary virus, M11 is the VP1 start codon, M138 is the VP2 start codon, and M203 is the VP3 start codon. Deletion of the start codon, typically by substitution of M11 and M138, renders expression of VP1 and VP2 inoperable, while a similar deletion of the VP3 start codon is not sufficient. This is because the viral capsid ORF contains multiple ATG codons with varying strengths as initiation codons. Therefore, care must be taken when designing constructs that do not express VP3 to ensure that alternative VP3 species are not produced. For VP3, deletion of M138 is required, or if VP2 but not VP3 is desired, deletion of M203 plus M211 and 235 is typically the best approach (Warrington, K. H. Jr., et al., J. of Virol. 78(12): 6595-6609 (June 2004)). This can be done by mutation such as substitution or other means known in the art. Corresponding start codons in other serotypes can also be readily determined, and it can also be determined whether additional ATG sequences in eg VP3 can function as alternative start codons.

Thus, for example, the term "AAV2 capsid protein" includes AAV capsid proteins having the native AAV2 capsid protein sequence (see GenBank Accession No. AAC03780) as well as substitutions, insertions and/or deletions into the native AAV2 capsid protein sequence. (described in the preceding paragraph).

In certain embodiments, the AAV capsid protein has a native AAV capsid protein sequence, or is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, have a 98% or 99% similar or identical amino acid sequence. For example, in certain embodiments, the "AAV2" capsid protein is at least about 75%, 80% < 85%, 90%, 95%, 97%, 98% of the native AAV2 capsid protein sequence and the native AAV2 capsid protein sequence. or 99% similar or identical sequences.

Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity may be determined using the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2,482 (1981), the sequence similarity or identity of Needleman & Wunsch, J. Mol. Similarity search methods for identity alignment algorithms, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), computer implementations of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, GAP, BESTFIT, FASTA, and TFASTA) in Madison, WI, the Best Fit sequence program or test described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984); It can be determined using standard techniques known in the art.

Another suitable algorithm is Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993) This is the BLAST algorithm described in . A particularly useful BLAST program is the WU-BLAST-2 program, available from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html. be. WU-BLAST-2 uses several search parameters that are optionally set to default values. The parameters are dynamic values, established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is being searched. However, these values can be adjusted to increase sensitivity.

Furthermore, an additional useful algorithm is gapped BLAST as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402.

In some embodiments of the invention, modifications are made in the region of amino acid positions 585-590 (inclusive) of the native AAV2 capsid protein (using VP1 numbering) or in the region of other AAV (native AAV2 VP1 capsid proteins). protein: GenBank accession numbers AAC03780 or YP680426), ie, the amino acids corresponding to amino acid positions 585-590 (VP1 numbering) of the native AAV2 capsid protein. Amino acid positions in other AAV serotypes or modified AAV capsids that “correspond” to positions 585-590 of the native AAV2 capsid protein will be apparent to those skilled in the art and can be determined using sequence alignment techniques (e.g., WO 2006/2006). 066066) and/or crystal structure analysis (Padron et al., (2005) J. Virol. 79: 5047-58).

To illustrate, modifications can be introduced into an AAV capsid protein that already contains insertions and/or deletions such that the position of all downstream sequences is shifted. In this situation, the amino acid positions corresponding to amino acid positions 585-590 in the AAV2 capsid protein are still readily identifiable to those skilled in the art. To illustrate, the capsid protein can be an AAV2 capsid protein containing an insertion after amino acid position 264 (see, eg, WO 2006/066066). The amino acids found from positions 585 to 590 (eg, RGNRQA (SEQ ID NO: 1) in the native AAV2 capsid protein), now from positions 586 to 591, are still identifiable to one skilled in the art.

The invention also provides viral capsids comprising, consisting essentially of, or consisting of a modified AAV capsid protein of the invention. In certain embodiments, the viral capsid is a parvovirus capsid, which can further be an autonomous parvovirus capsid or a dependent virus capsid. Optionally, the viral capsid is an AAV capsid. In certain embodiments, the AAV capsid is AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or as shown in Table 1 or otherwise known or later. and/or derived from any of the foregoing by one or more insertions, substitutions and/or deletions.

In embodiments of the invention, the isolated AAV virions or substantially homogeneous AAV virion population comprises one nucleic acid helper plasmid expressing VP1, VP2, and VP3 of one serotype and VP1 of another serotype; Such expression, rather than the expression product of a mixture of VP2 and another nucleic acid helper plasmid expressing VP3, is referred to as "cross-dressing."

In embodiments of the invention, the isolated AAV virions are free of mosaic capsids, and the substantially homogeneous population of AAV virions is free of substantially homogeneous mosaic capsid populations.

Any disclosure in PCT/US18/22725, filed March 15, 2018, falls within the scope of the invention as defined in any one or more of the claims of this application, or as long as it falls within the scope of any invention to be defined in any amended claim that may be filed in the future in this application or any patent derived therefrom, as well as any one or more claims to which that or those claims apply. To the extent that the relevant country's law provides that the disclosure of PCT/US18/22725 is part of the state of the art for the claims in or for that country, the inventors , hereby has the right to disclaim said disclosure from the claims of this application or any patent derived therefrom only to the extent necessary to prevent invalidation of this application or any patent derived therefrom.

By way of example and without limitation, the inventors disclaim from any claim of this application or any patent derived therefrom, now or hereafter amended, any one or more of the following subject matter: have the right to:
A. Any subject matter disclosed in Example 9 of PCT/US18/22725; or
B. Polyploid, produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes. Vector virions called vector virions; or
C. Produced or producible by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes , vector virions called polyploid vector virions; or
D. Produced or producible by transfection of three AAV helper plasmids, AAV2, AAV8, and AAV9, to produce individual polyploid vector virions composed of different capsid subunits from different serotypes. vector virions, called polyploid vector virions; or
E. VP1/VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, e.g. VP1/VP2 is derived from only one AAV serotype (from its capsid) and VP3 vector virions, called haploid vectors, derived from only one alternative AAV serotype; or
F. Any one or more AAV vector virions selected from:
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP2/VP3 capsid subunit from AAV2; or
A vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82), comprising a VP1/VP2 capsid subunit derived from AAV8 and a VP1/VP2 capsid subunit derived from AAV2. a vector having a VP3 capsid subunit of; or
Vectors in which VP1/VP2 are derived from different serotypes; or
A vector (termed haploid AAV92 or H-AAV92) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV2; or
A vector having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2G9 (referred to as haploid AAV2G9 or H-AAV2G9), wherein the AAV9 glycan receptor binding site is grafted into AAV2. ;or
A vector (termed haploid AAV83 or H-AAV83) having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAV93 or H-AAV93) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAVrh10-3 or H-AAVrh10-3) having a VP1/VP2 capsid subunit from AAVrh10 and a VP3 capsid subunit from AAV3; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP2/VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2 capsid subunits from AAV2 and VP3 capsid subunits from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV8; or
A vector called 28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N terminus derived from AAV2 and a C terminus derived from AAV8, and the VP3 capsid subunit is derived from AAV2; or a vector referred to as chimeric AAV8/2 or chimeric AAV82, in which the chimeric VP1/VP2 capsid subunit has an AAV8-derived N terminus and an AAV2-derived C terminus, and the VP3 initiation codon a vector without mutations and in which the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus derived from AAV2 and a C-terminus derived from AAV8; or
G. a population, e.g. a substantially homogeneous population, e.g. a population of 1010 particles, e.g. a population of 1010 substantially homogeneous particles; or
H. A method of producing any one of the vectors or vector populations of A and/or B and/or C and/or D and/or E and/or F and/or G; or
I. Any combination thereof.

Without limitation, the inventors believe that the foregoing reservation of disclaimer applies at least to claims 1-30 as appended to this application and paragraphs 1-83 as set forth at [00437]. express that Modified viral capsids can be used as "capsid vehicles", for example, as described in US Pat. No. 5,863,541. Molecules that can be packaged by modified viral capsids and imported into cells include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

A heterologous molecule is defined as a molecule that is not naturally found in AAV infection, eg, a molecule that is not encoded by the wild-type AAV genome. Additionally, therapeutically useful molecules can be associated with the outside of the viral capsid for transfer of the molecule to host target cells. Such related molecules can include DNA, RNA, small organic molecules, metals, carbohydrates, lipids and/or polypeptides. In one aspect of the invention, the therapeutically useful molecule is covalently linked (ie, conjugated or chemically linked) to the capsid protein. Methods for covalently linking molecules are known to those skilled in the art.

The modified viral capsids of the invention are also useful for generating antibodies against the novel capsid structure. As a further alternative, the exogenous amino acid sequence can be inserted into a modified viral capsid for antigen presentation to cells, eg, for administration to a subject to generate an immune response against the exogenous amino acid sequence.

In other embodiments, the viral capsid is administered prior to and/or concurrently with (e.g., within minutes or hours of each other) administration of a viral vector that delivers a nucleic acid or functional RNA encoding a polypeptide of interest. Can be administered to block certain cellular sites. For example, the capsids of the invention can be delivered to block cellular receptors on liver cells, and the delivery vectors can be administered subsequently or simultaneously, which results in transduction of liver cells. and may enhance transduction of other targets (eg, skeletal, cardiac and/or diaphragm muscle).

According to an exemplary embodiment, a modified viral capsid can be administered to a subject before and/or simultaneously with a modified viral vector according to the invention. Additionally, the present invention provides compositions and pharmaceutical formulations comprising modified viral capsids of the present invention; optionally, the compositions also include modified viral vectors of the present invention.

The invention also provides nucleic acid molecules (optionally isolated nucleic acid molecules) encoding the modified viral capsids and capsid proteins of the invention. Furthermore, vectors containing the nucleic acid molecules and cells (in vivo or in culture) containing the nucleic acid molecules and/or vectors of the invention are provided. Suitable vectors include, but are not limited to, viral vectors (e.g., adenoviruses, AAVs, herpesviruses, alphaviruses, vaccinia, poxviruses, baculoviruses, and others), plasmids, phages, YACs, BACs, and others. included. Such nucleic acid molecules, vectors and cells can be used, for example, as reagents (eg, helper packaging constructs or packaging cells) for the production of modified viral capsids or viral vectors as described herein.

Viral capsids according to the invention can be produced using any method known in the art, for example by expression from baculovirus (Brown et al., (1994) Virology 198:477-488).

In some embodiments, modifications to AAV capsid proteins of the invention are "selective" modifications. This approach is similar to previous studies using whole subunits or large domain exchanges between AAV serotypes (e.g., WO 00/28004 and Hauck et al., (2003) J. Virology 77:2768-2774 (see ). In certain embodiments, "selective" modifications include insertions and/or substitutions of less than about 20, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 contiguous amino acids and / or lead to deletions.

The modified capsid proteins and capsids of the invention can further include any other modifications now known or later identified.

Viral capsids are targeted viral capsids that contain targeting sequences (e.g., substituted or inserted into the viral capsid) that direct the viral capsid to interact with cell surface molecules present in the desired target tissue. (e.g., WO 00/28004 and Hauck et al., (2003) J. Virology 77:2768-2774); Shi et al., Human Gene Therapy 17:353-361 (2006) [ describes insertion of the integrin receptor binding motif RGD at positions 520 and/or 584 of the AAV capsid subunit]; and US Pat. No. 7,314,912 [amino acid positions 447, 534, and 573 of the AAV2 capsid subunit and describes the insertion of a P1 peptide containing an RGD motif following position 587). Other positions within the AAV capsid subunit that permit insertion are known in the art (e.g., positions 449 and 588 as described by Grifman et al., Molecular Therapy 3:964-975 (2001)). ).

For example, some of the viral capsids of the present invention are targeted to most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestines, skin, endothelial cells, and/or lungs). ) has a relatively inefficient tropism. Targeting sequences can be advantageously incorporated into these low transduction vectors, thereby conferring a desired tropism on the viral capsid and optionally a selective tropism towards particular tissues. AAV capsid proteins, capsids and vectors containing targeting sequences are described, for example, in WO 00/28004. Another possibility is to use 1 as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) as a means of redirecting the low-transduction vector to the desired target tissue. One or more non-naturally occurring amino acids can be incorporated into an orthogonal site in an AAV capsid subunit. These unnatural amino acids can be advantageously used to produce, without limitation, glycans (mannose-dendritic cell targeting); RGD, bombesin or neuropeptides for targeted delivery to specific cancer cell types; growth factors; Molecules of interest can be chemically linked to AAV capsid proteins, including RNA aptamers or peptides selected by phage display targeting specific cell surface receptors such as receptors, integrins, and others. Methods for chemically modifying amino acids are known in the art (see, eg, Greg T. Hermanson, Bioconjugate Techniques, 1st edition, Academic Press, 1996).

In typical embodiments, the targeting sequence can be a viral capsid sequence (eg, an autonomous parvovirus capsid sequence, an AAV capsid sequence, or any other viral capsid sequence) that directs infection to a particular cell type.

As another non-limiting example, a heparin-binding domain (e.g., the heparin-binding domain of respiratory syncytial virus) typically binds to the HS receptor to confer heparin-binding properties to the resulting mutant. (e.g., AAV4, AAV5) may be inserted or substituted into capsid subunits.

B19 infects primary erythroid progenitor cells using globoside as its receptor (Brown et al., (1993) Science 262:114). The structure of B19 has been determined at a resolution of 8 Å (Agbandje-McKenna et al., (1994) Virology 203:106). The region of the B19 capsid that binds globoside has been mapped between amino acids 399 and 406, the loop-out region between β-barrel structures E and F (Chapman et al., (1993) Virology 194:419). (Chipman et al., (1996) Proc. Nat. Acad. Sci. USA 93:7502). Therefore, the globoside receptor binding domain of the B19 capsid may be substituted into the AAV capsid protein to target the viral capsid or viral vector containing it to red blood cells.

In typical embodiments, the exogenous targeting sequence can be any amino acid sequence that encodes a peptide that alters the tropism of a viral capsid or a viral vector, including a modified AAV capsid protein. In certain embodiments, the targeting peptide or protein can be naturally occurring, or alternatively completely or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind cell surface receptors and glycoproteins, such as RGD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblasts). growth factors, platelet-derived growth factors, insulin-like growth factors I and II, etc.), cytokines, melanocyte-stimulating hormones (e.g., alpha, beta, or gamma), neuropeptides and endorphins, and others, as well as their cognates of target cells. Includes fragments thereof that retain the capacity for receptors. Other exemplary peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin-releasing peptide, interleukin 2, chicken egg white lysozyme, erythropoietin, gonadoliverin, corticostatin, beta-endorphin, leu-enkephalin, Included are limorphin, alpha-neo-enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and the fragments thereof mentioned above. As yet a further alternative, binding domains from toxins (eg, tetanus toxin or snake toxins, such as alpha-bungarotoxin, and others) can be substituted into the capsid protein as targeting sequences. In yet a further exemplary embodiment, the AAV capsid protein contains a "non-classical" import/export signal peptide (e.g., fibroblast growth factor-1) as described by Cleves (Current Biology 7:R318 (1997)). and -2, interleukin 1, HIV-1 Tat protein, herpesvirus VP22 protein, and others) into the AAV capsid protein. Peptide motifs that direct uptake by specific cells are also included, eg, the FVFLP peptide motif triggers uptake by liver cells.

Phage display techniques and other techniques known in the art can be used to identify peptides that recognize any cell type of interest.

The targeting sequence may encode any peptide that targets a cell surface binding site, including a receptor (eg, a protein, carbohydrate, glycoprotein or proteoglycan). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate, and other glycosaminoglycans, sialic acid moieties found on mucins, glycoproteins, and gangliosides, MHC I glycoproteins, membrane sugars. Includes carbohydrate components found on proteins, including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and others.

In certain embodiments, heparan sulfate (HS) or heparin binding domains are substituted into the viral capsid (eg, in AAV that would not otherwise bind HS or heparin). It is known in the art that HS/heparin binding is mediated by "basic patches" rich in arginine and/or lysine. In an exemplary embodiment, a sequence following the motif BXXB, in which "B" is a basic residue and X is neutral and/or hydrophobic. As one non-limiting example, BXXB is RGNR. In certain embodiments, BXXB is substituted across amino acid positions 262-265 in the native AAV2 capsid protein or at the corresponding position in the capsid protein of another AAV.

）；Grifman et al., Molecular Therapy 3:964-975 (2001)により記載されているような腫瘍ターゲティングペプチド（例えば、NGR、NGRAHA（SEQ ID NO：5)）；Work et al., Molecular Therapy 13:683-693 (2006)により記載されているような肺または脳ターゲティング配列

；Hajitou et al., TCM 16:80-88 (2006)により記載された血管ターゲティング配列

；Koivunen et al., J. Nucl. Med. 40:883-888 (1999)により記載されているようなターゲティングペプチド

、XXXY^＊XXX［式中、Y^＊はホスホ-Tyrである］（SEQ ID NO：41)、

、およびGSL）；ならびにNewton & Deutscher, Phage Peptide Display in Handbook of Experimental Pharmacology, pages 145-163, Springer-Verlag, Berlin (2008)により記載されているような腫瘍ターゲティングペプチド

が含まれる。 Other non-limiting examples of suitable targeting sequences include the coronary endothelial cell targeting peptides (consensus sequence

); tumor targeting peptides (e.g., NGR, NGRAHA (SEQ ID NO: 5)); Work et al., Molecular Therapy 13 as described by Grifman et al., Molecular Therapy 3:964-975 (2001); Lung or brain targeting sequences such as those described by :683-693 (2006)

; vascular targeting sequence described by Hajitou et al., TCM 16:80-88 (2006)

; targeting peptides as described by Koivunen et al., J. Nucl. Med. 40:883-888 (1999)

, XXXY ^* XXX [where Y ^* is phospho-Tyr] (SEQ ID NO: 41),

, and GSL); and tumor targeting peptides as described by Newton & Deutscher, Phage Peptide Display in Handbook of Experimental Pharmacology, pages 145-163, Springer-Verlag, Berlin (2008).

is included.

As yet a further alternative, the targeting sequence can be used for chemical conjugation with another molecule targeting entry into the cell (e.g. arginine and/or lysine, which can be chemically conjugated via its R group). may be a peptide (which may contain residues).

As another option, the AAV capsid protein or viral capsid of the invention can include mutations as described in WO 2006/066066. For example, the capsid protein can include selective amino acid substitutions at amino acid positions 263, 705, 708, and/or 716 of the native AAV2 capsid protein or corresponding changes in another AAV-derived capsid protein. Additionally or alternatively, in representative embodiments, the capsid protein, viral capsid or vector contains a selective amino acid insertion immediately following amino acid position 264 of the AAV2 capsid protein or a corresponding change in other AAV-derived capsid proteins. including. By "immediately after amino acid position For example, positions 265-268). The aforementioned aspects of the invention can be used to deliver heterologous nucleic acids to cells or subjects as described herein. For example, modified vectors can be used to generate mucopolysaccharidoses described herein (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [α-L-iduronidase], Scheie syndrome [α-L-iduronidase], Hurler syndrome). - Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA: α-glucosaminide acetyltransferase] ], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfatase], B [β-galactosidase], Maroteau-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], others), Lysosomal storage diseases such as Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage diseases (eg, Pompe disease; lysosomal acid α-glucosidase) can be treated.

Those skilled in the art will appreciate that for some AAV capsid proteins, corresponding modifications can be inserted and/or or will recognize that it is a substitution. Similarly, when modifying an AAV other than AAV2, particular amino acid positions may differ from those in AAV2 (see, eg, Table 3). Corresponding amino acid positions will be readily apparent to those skilled in the art using well-known techniques, as described elsewhere herein.

In typical embodiments, insertions and/or substitutions and/or deletions in the capsid protein result in amino acids that (i) maintain the hydrophilic loop structure in that region; (ii) alter the configuration of the loop structure. (iii) charged amino acids; and/or (iv) amino acids that can be phosphorylated or sulfated after 264 in the AAV2 capsid protein, or otherwise charged due to post-translational modification (e.g., glycosylation). or amino acid insertions, substitutions and/or rearrangements that can result in corresponding changes in the capsid protein of another AAV. Amino acids suitable for insertion/substitution include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine or glutamine. In certain embodiments, threonine is inserted or substituted within the capsid subunit. Non-limiting examples of corresponding positions in some other AAVs are shown in Table 3 (position 2). In certain embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid, or phenylalanine (excluding AAVs that have threonine, glutamic acid, or phenylalanine, respectively, at this position).

According to this aspect of the invention, in some embodiments, the AAV capsid protein is modified to include a non-AAV2, AAV3a or AAV3b sequence after amino acid position 264 in the AAV2, AAV3a or AAV3b capsid protein, respectively; and /or contains an amino acid insertion at the corresponding position in an AAV2, AAV3a or AAV3b capsid protein modified (ie obtained from AAV2, AAV3a or AAV3b) by deletion of one or more amino acids. The amino acid corresponding to position 264 in the capsid subunit of AAV2 (or AAV3a or AAV3b) can be easily identified from the starting virus obtained from AAV2 (or AAV3a or AAV3b) and this subunit then Further modifications can be made. Amino acids suitable for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine or glutamine.

In other embodiments, the AAV capsid protein comprises a non-AAV1, non-AAV8, or non-AAV9 sequence at amino acid position 265 of the AAV1 capsid protein, amino acid position 266 of the AAV8 capsid protein, or amino acid position 265 of the AAV9 capsid protein, or respectively. and/or amino acid substitutions at the corresponding positions of the AAV1, AAV8 or AAV9 capsid protein modified by deletion of one or more amino acids (i.e. deletions obtained from AAV1, AAV8 or AAV9). including. The amino acid corresponding to position 265 of the AAV1 and AAV9 capsid subunits and position 266 of the AAV8 capsid subunit can be easily identified from the starting virus obtained from AAV1, AAV8 or AAV9, and this amino acid can then be added according to the invention. Further modifications can be made. Amino acids suitable for insertion include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine or glutamine.

In an exemplary embodiment of the invention, the capsid protein comprises a threonine, aspartate, glutamic acid, or phenylalanine after amino acid position 264 of the AAV2 capsid protein or the corresponding position of another capsid protein (i.e., an insertion).

In other representative embodiments, the modified capsid proteins or viral capsids of the invention further comprise one or more mutations as described in WO 2007/089632 (e.g., of the AAV2 capsid protein). E7K mutation at amino acid position 531 or the corresponding position in another AAV-derived capsid protein).

In a further aspect, the modified capsid protein or capsid can include mutations as described in WO 2009/108274.

Another possibility is that the AAV capsid protein is mutated as described by Zhong et al. (Virology 381: 194-202 (2008); Proc. Nat. Acad. can be included. For example, an AAV capsid protein can contain a YF mutation at amino acid position 730.

The above modifications can be incorporated into the capsid proteins or capsids of the invention in combination with each other and/or with any other modifications now known or later discovered.

The invention also encompasses viral vectors containing the modified capsid proteins and capsids of the invention. In certain embodiments, the viral vector is a parvovirus vector (eg, comprising a parvovirus capsid and/or vector genome), such as an AAV vector (eg, comprising an AAV capsid and/or vector genome). In typical embodiments, the viral vector comprises a modified AAV capsid comprising a modified capsid protein subunit of the invention and a vector genome.

For example, in typical embodiments, a viral vector comprises (a) a modified viral capsid comprising a modified capsid protein of the invention (e.g., a modified AAV capsid); and (b) a nucleic acid comprising a terminal repeat sequence (e.g., AAV TR). The nucleic acid comprising a terminal repeat sequence comprises a nucleic acid that is enveloped by a modified viral capsid. The nucleic acid can optionally include two terminal repeats (eg, two AAV TRs).

In typical embodiments, the viral vector is a recombinant viral vector that includes a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant viral vectors are described in more detail below.

In some embodiments, a viral vector of the invention (i) has reduced hepatic transduction compared to the level of transduction by a viral vector without a modified capsid protein of the invention; (ii) (iii) exhibits enhanced systemic transduction by viral vectors in animal subjects compared to levels observed with viral vectors without modified capsid proteins of the invention; (iii) by viral vectors without modified capsid proteins of the invention; demonstrate enhanced migration across endothelial cells compared to the level of migration and/or (iv) muscle tissue (compared to the level of transduction by a viral vector without the modified capsid protein of the invention) (eg, skeletal, cardiac and/or diaphragm muscles) and/or (v) reduced transduction of brain tissue (eg, neurons). In some embodiments, the viral vector has muscle-directed systemic transduction, e.g., the viral vector transduces multiple skeletal muscle groups throughout the body, optionally transducing cardiac and/or diaphragm muscles. Introduce.

Furthermore, in some embodiments of the invention, the modified viral vectors exhibit efficient transduction of target tissues.

It will be understood by those skilled in the art that the modified capsid proteins, viral capsids, viral vectors and AAV particles of the present invention exclude capsid proteins, capsids, viral vectors and AAV particles as present or found in their native state. Dew.

Methods of Producing Viral Vectors The invention further provides methods of producing the inventive viral vectors of the invention as AAV particles. Accordingly, the invention provides a method of making AAV particles comprising an AAV capsid of the invention, comprising: (a) one or more species that together provide all the functions and genes necessary for assembling the AAV particle; transfecting the plasmid into a host cell; (b) introducing one or more nucleic acid constructs into a packaging or producer cell line to bring together all the functions and genes necessary to assemble the AAV particle; (c) introducing into a host cell one or more recombinant baculovirus vectors that together provide all the functions and genes necessary to assemble AAV particles; and/or (d) A method is provided comprising introducing into a host cell one or more recombinant herpesvirus vectors that together provide all the functions and genes necessary to assemble an AAV particle. Conditions for forming AAV virions are standard conditions for producing AAV vectors in cells (e.g. mammalian or insect cells), including, by way of non-limiting example, Ad helper plasmids or HSV. Includes transfection of cells in the presence of other helper viruses.

Non-limiting examples of various methods of making viral vectors of the invention include Clement and Grieger ("Manufacturing of recombinant adeno-associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev. 3:16002 (2016) ) and Grieger et al. ("Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector" Mol Ther 24(2):287-297 (2016)) , the entire contents of which are incorporated herein by reference.

In one exemplary aspect, the invention provides a method of producing a viral vector, comprising: providing a cell with (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence); and (b) a nucleic acid template. provides sufficient AAV sequences (eg, AAV rep sequences and AAV cap sequences encoding AAV capsids of the invention) for replication and packaging into AAV capsids. Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In certain embodiments, the nucleic acid template comprises two AAV ITR sequences located 5' and 3' to the heterologous nucleic acid sequence (if present), although they need not be directly contiguous with the heterologous nucleic acid sequence. .

The nucleic acid template and AAV rep and cap sequences are provided in conditions such that a viral vector containing the nucleic acid template packaged within an AAV capsid is produced in the cell. The method can further include collecting the viral vector from the cell. Viral vectors can be harvested from the culture medium and/or by lysing the cells.

In one embodiment, the nucleic acid template is modified such that the cap sequence is unable to express all three viral structural proteins VP1, VP2, and VP3 from a nucleic acid sequence derived from only one serotype (the first nucleic acid sequence). . This modification can be, for example, by removing the initiation codon for at least one of the viral structural proteins. The template also includes at least one additional nucleic acid sequence (second nucleic acid sequence) derived from a different serotype that encodes and is capable of expressing viral structural proteins that the first nucleic acid sequence is unable to express. , wherein the second nucleic acid sequence is incapable of expressing a viral structural protein that the first nucleic acid sequence is capable of expressing. In one embodiment, a first nucleic acid sequence is capable of expressing two of the viral structural proteins, while a second nucleic acid sequence is capable of expressing only the remaining viral sequences. For example, a first nucleic acid sequence can express VP1 and VP2 from one serotype, but not VP3, and a second nucleic acid sequence can express VP3 from an alternate serotype. but cannot express VP1 or VP2. The template is incapable of expressing any other of the three viral structural proteins. In one embodiment, the first nucleic acid sequence is capable of expressing only one of the three viral structural proteins and the second nucleic acid sequence is capable of expressing only two other viral structural proteins other than the first. Can be done.

In another embodiment, there is a third nucleic acid sequence from a third serotype. In this embodiment, each of the three nucleic acid sequences is capable of expressing only one of the three capsid virus structural proteins VP1, VP2, and VP3, and each of the three nucleic acid sequences is capable of expressing a virus expressed by another of the sequences. without expressing structural proteins, resulting in the production of capsids containing VP1, VP2, and VP3 in which each of the viral structural proteins in the capsid are all derived exclusively from the same serotype; in this embodiment, VP1, VP2, and VP3 are produced together; VP2, and VP3 are all derived from different serotypes.

Modifications to prevent expression can be by any means known in the art. For example, removing initiation codons, splice acceptors, splice donors, and combinations thereof. Deletions and additions as well as site-directed changes that alter the reading frame can be used. Nucleic acid sequences can also be produced synthetically. These helper templates typically do not contain ITRs.

The cell can be a cell permissive for AAV virus replication. Any suitable cell known in the art may be employed. In certain embodiments, the cell is a mammalian cell. Alternatively, the cell can be a trans-complementing packaging cell line that provides the function deleted from the replication-defective helper virus, such as 293 cells or other Ela trans-complementing cells.

AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep/cap gene on a single plasmid. The AAV replication and packaging sequences do not need to be provided together, although it may be advantageous to do so. AAV rep and/or cap sequences may be provided by any viral or non-viral vector. For example, the rep/cap sequence may be provided by a hybrid adenovirus or herpesvirus vector (eg, inserted into the Ela or E3 region of a deleted adenovirus vector). EBV vectors may also be employed to express AAV cap and rep genes. One advantage of this method is that, although EBV vectors are episomal, they maintain high copy numbers throughout continuous cell division (i.e., as extrachromosomal elements termed "EBV-based nuclear episomes"). (see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

As a further alternative, the rep/cap sequence may be stably integrated into the cell. Typically, AAV rep/cap sequences are not in close proximity to TRs to prevent rescue and/or packaging of these sequences.

Nucleic acid templates can be provided to cells using any method known in the art. For example, the template can be provided by a non-viral (eg, plasmid) or viral vector. In certain embodiments, the nucleic acid template is provided by a herpesvirus or adenovirus vector (eg, inserted into the Ela or E3 region of a deleted adenovirus). As another illustration, Palombo et al., (1998) J. Virology 72:5025 describes a baculovirus vector with a reporter gene in close proximity to the AAV TR. EBV vectors may also be employed to deliver templates, as described above for the rep/cap gene.

In another exemplary embodiment, the nucleic acid template is provided by a replicating rAAV virus. In still other embodiments, the AAV provirus containing the nucleic acid template is stably integrated into the chromosome of the cell.

To increase viral titer, cells can be provided with helper virus function (eg, adenovirus or herpesvirus) that promotes productive AAV infection. Helper virus sequences required for AAV replication are known in the art. Typically these sequences are provided by a helper adenovirus or herpesvirus vector. Alternatively, the adenovirus or herpesvirus sequences can be expressed by another non-viral or viral vector, such as those described by Ferrari et al., (1997) Nature Med. 3:1295, and US Pat. Nos. 6,040,183 and 6,093,570. As such, it can be provided as a non-infectious adenoviral miniplasmid with all helper genes to promote efficient AAV production.

Additionally, helper virus functions can be provided by the packaging cell, in which the helper sequences are embedded in the chromosome or maintained as stable extrachromosomal elements. Generally, helper virus sequences cannot be packaged into AAV virions, eg, are not in close proximity to the TR.

Those skilled in the art will recognize that it may be advantageous to provide AAV replication and capsid sequences as well as helper virus sequences (eg, adenovirus sequences) on a single helper construct. This helper construct can be a non-viral or viral construct. As a non-limiting example, a helper construct can be a hybrid adenovirus or a hybrid herpesvirus that includes an AAV rep/cap gene.

In one particular embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The vector can further include a nucleic acid template. AAV rep/cap sequences and/or rAAV templates can be incorporated into deletion regions (eg, E1a or E3 regions) of the adenovirus.

In further embodiments, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. According to this embodiment, the rAAV template can be provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element (eg, as an EBV-based nuclear episome).

In a further exemplary embodiment, the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper. rAAV templates can be provided as separate replicating viral vectors. For example, the rAAV template can be provided by an rAAV particle or a second recombinant adenoviral particle.

According to the methods described above, hybrid adenoviral vectors typically contain adenoviral 5' and 3' cis sequences (ie, adenoviral terminal repeats and PAC sequences) sufficient for adenoviral replication and packaging. The AAV rep/cap sequences and, if present, the rAAV template are embedded in the adenoviral backbone and in close proximity to the 5' and 3' cis sequences, so that these sequences are packaged into the adenoviral capsid. may be done. As mentioned above, adenovirus helper sequences and AAV rep/cap sequences are generally not in close proximity to the TR so that these sequences are not packaged into AAV virions.

Zhang et al. ((2001) Gene Ther. 18:704-12) describe a chimeric helper containing both an adenovirus and the AAV rep and cap genes.

Herpesviruses may also be used as helper viruses in AAV packaging methods.

Hybrid herpesviruses encoding AAV Rep proteins can advantageously facilitate scalable AAV vector generation schemes. Hybrid herpes simplex virus type I (HSV-1) vectors expressing AAV-2 rep and cap genes have been described (Conway et al., (1999) Gene Therapy 6:986 and WO 00/17377). ).

As a further alternative, baculovirus vectors can be used to produce viral vectors of the invention in insect cells, e.g. /cap gene and rAAV template can be delivered.

AAV vector stocks free of contaminating helper virus can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be separated from helper viruses based on its affinity for heparin substrates (Zolotukhin et al. (1999) Gene Therapy 6:973). Deleted replication-defective helper viruses can be used so that any contaminating helper virus is replication-incompetent. As a further alternative, adenoviral helpers lacking late gene expression may be employed. This is because only adenoviral early gene expression is required to mediate packaging of the AAV virus. Adenovirus mutants deficient in expression of late genes are known in the art (eg, ts100K and ts149 adenovirus mutants).

Recombinant Viral Vectors The invention provides a method of administering a nucleic acid molecule to a cell, the method comprising contacting the cell with a viral vector, AAV particle and/or composition or pharmaceutical formulation of the invention.

The invention further provides a method of delivering a nucleic acid to a subject, the method comprising administering to the subject a viral vector, AAV particle and/or a composition or pharmaceutical formulation of the invention.

In certain embodiments, the subject is a human, and in some embodiments, the subject has or is at risk for a disorder that can be treated by a gene therapy protocol. Non-limiting examples of such disorders include muscular dystrophies, including Duchenne or Becker muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Gaucher disease, Fabry disease, Pompe disease, cancer. , arthritis, muscle wasting, heart disease including congestive heart failure or peripheral artery disease, intimal thickening, epilepsy, neurological disorders including Huntington's disease, Parkinson's disease or Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Hurler syndrome , Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteau-Lamy syndrome, Krabbe disease, phenylketonuria, Batten disease, spinal cerebral ataxia, These include LDL receptor deficiencies, hyperammonemia, anemia, arthritis, retinal degenerative disorders including macular degeneration, adenosine deaminase deficiencies, metabolic disorders, and cancers including tumorigenic cancers.

In some embodiments of the methods of the invention, the viral vectors, AAV particles and/or compositions or pharmaceutical formulations of the invention can be administered to skeletal, cardiac and/or diaphragm muscles.

In the methods described herein, the viral vectors, AAV particles and/or compositions or pharmaceutical formulations of the invention are administered to the subject of the invention via systemic routes (e.g., intravenous, intraarterial, intraperitoneal, etc.). can be administered/delivered to In some embodiments, viral vectors and/or compositions can be administered to a subject via intraventricular, intracisternal, intraparenchymal, intracranial, and/or intrathecal routes. In certain embodiments, the viral vectors and/or pharmaceutical formulations of the invention are administered intravenously.

Viral vectors of the invention are useful for in vitro, ex vivo, and in vivo delivery of nucleic acid molecules to cells. In particular, viral vectors can be advantageously employed to deliver or transfer nucleic acid molecules into animal cells, including mammalian cells.

Any heterologous nucleic acid sequence of interest can be delivered with the viral vectors of the invention. Nucleic acid molecules of interest include nucleic acid molecules encoding polypeptides, including therapeutic polypeptides (e.g., for medical or veterinary use) and/or immunogenic polypeptides (e.g., for vaccines). It will be done.

Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulatory protein (CFTR), dystrophin (mini- and micro-dystrophin), e.g., Vincent et al., (1993) Nature Genetics 5: 130; U.S. Patent Application Publication No. 2003/017131; International Publication No. 2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevich et al., Mol. Ther. 16:657-64 (2008)), anti-inflammatory polypeptides such as myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, IkappaB dominant variant, sarcospan (sarcospan), utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, clotting factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endo Statins, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α ₁ -antitrypsin, adenosine deaminase, hypoxanthine guanine Phosphoribosyltransferase, glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched chain keto acid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, lymphotoxin, and others), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor) , fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, bone morphogenetic protein [including RANKL and VEGF], glial-derived growth factor, transforming growth factor-α and - modulates lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis growth factor-α soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, calcium handling (e.g., SERCA _2A , PP1 inhibitor 1 and fragments thereof [e.g., WO 2006/029319 and WO 2007/100465]), molecules that affect G protein-coupled receptor kinase type 2 knockdown , anti-inflammatory factors such as truncated constitutively active bARKct, IRAP, anti-myostatin protein, aspartoacylase, monoclonal antibodies (including single chain monoclonal antibodies; an exemplary Mab is the Herceptin® Mab), Neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see US Pat. No. 7,071,172), angiogenesis inhibitors such as vasohibin and other VEGF inhibitors (e.g., vasohibin 2 [see WOJP 2006/073052]). . Other exemplary heterologous nucleic acid sequences include suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer treatment, tumor suppressor genes. encodes a product (eg, p53, Rb, Wt-1), TRAIL, FAS-ligand, as well as any other polypeptide that has a therapeutic effect in a subject in need thereof. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, such as antibodies or antibody fragments against myostatin (see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005) (want to be).

Heterologous nucleic acid sequences that encode polypeptides include those that encode reporter polypeptides (eg, enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein (GFP), luciferase, β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase genes. It will be done.

Optionally, the heterologous nucleic acid molecule is a secreted polypeptide (e.g., is a secreted polypeptide in its native state or is secreted, e.g., by functional association with a secretion signal sequence, as known in the art). encodes a polypeptide engineered to

Alternatively, in certain embodiments of the invention, heterologous nucleic acid molecules include antisense nucleic acid molecules, ribozymes (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (Puttaraju et al., ( 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNA (RNAi) including siRNA, shRNA or miRNA that mediate gene silencing (Sharp et al., (2000) ) Science 287:2431), and other non-coding RNAs such as "guide" RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; Yuan et al. US Pat. No. 5,869,248 ), and others. Exemplary untranslated RNAs include RNAi against multidrug resistance (MDR) gene products (e.g., for administration to the heart to treat and/or prevent tumors and/or to prevent chemotherapy damage); , RNAi against myostatin (e.g. for Duchenne muscular dystrophy), RNAi against VEGF (e.g. to treat and/or prevent tumors), RNAi against phospholamban (e.g. to treat cardiovascular diseases, e.g. Andino et al. al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)); Ranban S16E (e.g., to treat cardiovascular diseases, see e.g. Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi against adenosine kinases (e.g., for epilepsy), and pathogenic organisms and Includes RNAi against viruses (eg, hepatitis B and/or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

Additionally, nucleic acid sequences that direct alternative splicing can be delivered. To illustrate, an antisense sequence (or other inhibitory sequence) complementary to the 5' and/or 3' splice site of exon 51 of dystrophin is delivered in conjunction with the U1 or U7 small nuclear (sn) RNA promoter. , can induce skipping of this exon. For example, a DNA sequence containing the U1 or U7 snRNA promoter located 5' to the antisense/inhibition sequence can be packaged and delivered into the modified capsid of the invention.

Viral vectors may also contain heterologous nucleic acid molecules that share homology with and recombine with host cell chromosomal loci. This approach can be used, for example, to correct genetic defects in host cells.

The invention also provides viral vectors expressing immunogenic polypeptides, peptides and/or epitopes, eg, for vaccination. Nucleic acid molecules may be derived from, without limitation, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and others. Any immunogen of interest known in the art may be encoded, including immunogens of.

The use of parvoviruses as vaccine vectors is known in the art (e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; Young et al. US Pat. No. 5,916,563; Mazzara et al. No. 5,905,040, Samulski et al., US Pat. No. 5,882,652, and US Pat. No. 5,863,541). Antigens may be provided in parvovirus capsids. Alternatively, the immunogen or antigen may be expressed from a heterologous nucleic acid molecule introduced into a recombinant vector genome. Any immunogen or antigen of interest described herein and/or known in the art can be provided by the viral vectors of the invention.

Immunogenic polypeptides are intended to induce an immune response and/or to protect a subject from infection and/or disease, including but not limited to microbial, bacterial, protozoal, parasitic, fungal and/or viral infections and diseases. It can be any polypeptide, peptide, and/or epitope suitable for protection. For example, the immunogenic polypeptide may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen such as influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentiviral immunogen ( For example, equine infectious anemia virus immunogen, simian immunodeficiency virus (SIV) immunogen, or human immunodeficiency virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix/capsid protein, and HIV or SIV gag, pol and env gene products). Immunogenic polypeptides also include arenavirus immunogens (e.g., Lassa fever virus immunogens such as Lassa fever virus nucleocapsid protein and Lassa fever envelope glycoprotein), poxvirus immunogens (e.g., vaccinia L1 or L8 gene products). vaccinia virus immunogens such as), flavivirus immunogens (e.g. yellow fever virus immunogen or Japanese encephalitis virus immunogen), filovirus immunogens (e.g. Ebola virus immunogen, or Marburg virus immunogen, e.g. NP and GP gene products), bunyavirus immunogens (e.g., RVFV, CCHF, and/or SFS virus immunogens), or coronavirus immunogens (e.g., infectious human coronavirus immunogens, such as human coronavirus envelope glycoprotein, or pig It can also be an infectious gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). Immunogenic polypeptides may further include polio immunogens, herpes immunogens (e.g., CMV, EBV, HSV immunogens), mumps immunogens, measles immunogens, rubella immunogens, diphtheria toxin or other diphtheria immunogens, pertussis immunogens, etc. antigen, hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any other vaccine immunogen now known or later identified in the art as an immunogen. be able to.

Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of cancer cells. Exemplary cancer and tumor cell antigens are described by S.A. Rosenberg (Immunity 10:281 (1991)). Other exemplary cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO-1. , CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE- 2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product ( U.S. Patent No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (SialylTn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigen (WO 90/05142); telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigen; and/or antigens now known or later discovered to be associated with the following cancers: melanoma, glandular Cancer, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer , bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified (e.g., Rosenberg, (1996) Ann. Rev. Med 47:481-91).

As a further alternative, the heterologous nucleic acid molecule can encode any polypeptide, peptide and/or epitope that is desirably produced in vitro, ex vivo, or in vivo in a cell. For example, a viral vector can be introduced into cultured cells and the expressed gene product isolated therefrom.

It will be understood by those skilled in the art that a heterologous nucleic acid molecule of interest can be functionally associated with appropriate control sequences. For example, the heterologous nucleic acid molecule may be functionally associated with expression control elements such as transcriptional/translational control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and/or enhancers, and the like. Can be done.

Furthermore, the regulated expression of a heterologous nucleic acid molecule of interest can be controlled at the post-transcriptional level, e.g. by the presence or absence of oligonucleotides, small molecules and/or other compounds that selectively block splicing activity at specific sites. (eg as described in WO2006/119137).

Those skilled in the art will recognize that a variety of promoter/enhancer elements can be used depending on the desired level and tissue-specific expression. Promoters/enhancers can be constitutive or inducible depending on the desired expression pattern. Promoters/enhancers can be native or foreign, and can be natural or synthetic sequences. By foreign it is intended that the transcription initiation region is not found in the wild type host into which it is introduced.

In certain embodiments, promoter/enhancer elements can be native to the target cell or subject to be treated. In typical embodiments, promoter/enhancer elements can be native to the heterologous nucleic acid sequence.

The promoter/enhancer element is generally selected such that it functions in the target cell of interest. Furthermore, in certain embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. Promoter/enhancer elements may be constitutive or inducible.

Inducible expression control elements are typically advantageous in ideal applications to provide regulation of the expression of heterologous nucleic acid sequences. Inducible promoter/enhancer elements for gene delivery can be tissue-specific or preferential promoters/enhancer elements, including muscle-specific or preferential (myocardial, skeletal and/or smooth muscle-specific) neural tissue-specific or preferential (including brain-specific or preferential); ocular-specific or preferential (including retina-specific and cornea-specific); liver-specific or preferential; Included are bone marrow specific or preferred, pancreas specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter/enhancer elements include, but are not limited to, Tet on/off elements, RU486 inducible promoters, ecdysone inducible promoters, rapamycin inducible promoters, and metallothionein promoters.

In embodiments where a heterologous nucleic acid sequence is transcribed and then translated in a target cell, specific initiation signals are generally included for efficient translation of the inserted protein coding sequence. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

Viral vectors according to the invention provide a means for delivering heterologous nucleic acid molecules into a wide range of cells, including dividing and non-dividing cells. For example, viral vectors can be employed to produce polypeptides in vitro or for ex vivo or in vivo gene therapy to deliver nucleic acid molecules of interest to cells in vitro. Viral vectors are additionally useful in methods of delivering nucleic acids to a subject in need thereof to express, for example, immunogenic or therapeutic polypeptides or functional RNA. In this way, polypeptides or functional RNA can be produced in vivo in a subject. The subject may be one in need of the polypeptide due to having a deficiency in the polypeptide.

Additionally, this method can be practiced because the production of polypeptides or functional RNA in a subject may have some beneficial effect.

Viral vectors can also be used to transport polypeptides or functional RNA of interest in cultured cells or subjects (e.g., to produce polypeptides or to detect the effects of functional RNA on a subject, e.g. in connection with screening methods). (using the object as a bioreactor for observation).

In general, a viral vector of the invention may be employed to deliver a heterologous nucleic acid molecule encoding a polypeptide or functional RNA for any disorder or disease condition in which it would be beneficial to deliver a therapeutic polypeptide or functional RNA. can be treated and/or prevented. Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulatory protein) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor VIII), factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [ GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, Cytokines, including FAS-ligand, interferon; mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g. α, β, γ], RNAi for myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides, e.g. IkappaB Dominant mutants, sarcospans, utrophin, mini-utrophin, antisense or RNAi against splice junctions in the dystrophin gene to induce exon skipping [see e.g. WO2003/095647], anti against U7 snRNA to induce exon skipping myostatin or myostatin propeptide) and Becker's disease, Gaucher's disease (glucocerebrosidase), Hurler's disease (α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase ), glycogen storage diseases (e.g. Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain keto acid dehydrogenase), retinal degenerative diseases (and other diseases; e.g. PDGF for macular degeneration and/or vasohibin or other inhibitors of VEGF or other angiogenesis inhibitors for example to treat/prevent retinal damage in type I diabetes), parenchyma such as the brain. Organ diseases (including Parkinson's disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin and/or VEGF], glioblastoma [RNAi against endostatin, angiostatin and/or VEGF]), liver modulate the serca2a, phospholamban gene by delivering protein phosphatase inhibitor I (I-1) and its fragments (e.g. I1C), kidney, heart, including congestive heart failure or peripheral artery disease (PAD). Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1S100Al, parvalbumin, adenylyl cyclase type 6, truncated constitutively active bARKct Molecules that cause G protein-coupled receptor kinase type 2 knockdown such as calsarcin, RNAi against phospholamban; phospholamban inhibitory molecules or dominant negative molecules such as phospholamban S16E, and others), arthritis (insulin-like growth factors) , joint disorders (insulin-like growth factors 1 and/or 2), intimal thickening (e.g. by delivering enos, inos), improving survival of transplanted hearts (superoxide dismutase), AIDS (soluble CD4), Muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFα soluble receptors), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor ), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, spinal cerebral ataxia including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmunity Includes diseases, as well as others. The present invention provides a method for increasing transplant success after organ transplantation and/or for reducing the negative effects of organ transplantation or adjunctive therapy (e.g., by administering immunosuppressive drugs or inhibitory nucleic acids to block cytokine production). It can further be used to reduce side effects. As another example, bone morphogenetic proteins (including BNP2, 7, etc., RANKL and/or VEGF) can be administered with bone allografts, eg, after bone fractures or surgical removal in cancer patients.

The invention can also be used to generate induced pluripotent stem cells (iPS). For example, the viral vectors of the invention can be used to generate non-pluripotent cells such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, heart cells, nervous system cells, epithelial cells, endothelial cells, and others. Stem cell-associated nucleic acids can be delivered into cells. Nucleic acids encoding factors associated with stem cells are known in the art. Non-limiting examples of such factors associated with stem cells and pluripotency include Oct-3/4, the SOX family (e.g., SOX1, SOX2, SOX3 and/or SOX15), the Klf family (e.g., Klf1, Klf2, Klf4 and/or Klf5), the Myc family (eg C-myc, L-myc and/or N-myc), NANOG and/or LIN28.

The invention may also be practiced to improve metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or VIII), mucopolysaccharidoses (e.g., Sly syndrome [β-glucuronidase], Hurler's syndrome [α-L-iduronidase], Scheie syndrome [α-L-iduronidase], Hurler-Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA: α-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfatase], B [β-galactosidase] ], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], others), Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage diseases (e.g., Pompe disease; lysosomal acidic α- lysosomal storage diseases such as glucosidase) can be treated and/or prevented.

Gene transfer has substantial potential utility for understanding and providing treatments for disease states. There are several genetic diseases for which defective genes are known and have been cloned. In general, the disease states are divided into two classes: deficiencies of enzymes, which are generally recessively inherited, and unbalanced conditions, which may involve regulatory or structural proteins, which are typically dominantly inherited. For diseases that are defective, gene transfer can be used to introduce normal genes into affected tissues for replacement therapy and antisense mutations to generate animal models for the disease. For unbalanced disease states, gene transfer can be used to generate a disease state in a model system, and this model can then be used in an effort to combat this disease state. The viral vector according to the invention therefore allows the treatment and/or prevention of genetic diseases.

Viral vectors according to the invention may also be employed to provide functional RNA to cells in vitro or in vivo. Expression of functional RNA in a cell can, for example, reduce expression of a particular target protein by the cell. Thus, functional RNA can be administered to reduce the expression of a particular protein in a subject in need thereof. Functional RNA can also be administered to cells in vitro to modulate gene expression and/or cell physiology, eg, to optimize cell or tissue culture systems or screening methods.

In addition, the viral vectors according to the invention find use in diagnostic and screening methods in which the nucleic acids of interest are expressed transiently or stably in cell culture systems or alternatively in transgenic animal models.

The viral vectors of the invention may also be used for a variety of non-therapeutic purposes, including, but not limited to, use in protocols for determining gene targeting, clearance, transcription, translation, etc., as will be apparent to those skilled in the art. You can also do that. Viral vectors can also be used to assess safety (diffusion, toxicity, immunogenicity, etc.). Such data is considered, for example, by the US Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.

As a further aspect, the viral vectors of the invention may be used to generate an immune response in a subject. With this embodiment, a viral vector containing a heterologous nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is mounted by the subject against the immunogenic polypeptide. Immunogenic polypeptides are as described herein above. In some embodiments, a protective immune response is elicited.

Alternatively, the viral vector may be administered to cells ex vivo and the modified cells administered to the subject. A viral vector containing a heterologous nucleic acid can be introduced into a cell, the cell can be administered to a subject, where the heterologous nucleic acid encoding the immunogen is expressed, and an immune response against the immunogen can be induced in the subject. In certain embodiments, the cell is an antigen presenting cell (eg, a dendritic cell).

An “active immune response” or “active immunity” is defined as “an encounter with an immunogen that involves the differentiation and proliferation of immunocompetent cells within the lymphoreticular tissue, leading to the synthesis of antibodies and/or the generation of cell-mediated reactivity.” characterized by 'subsequent involvement of host tissues and cells'. Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Stated differently, an active immune response is initiated by the host after exposure to an immunogen by infection or vaccination. Active immunity is acquired via “transfer of preformed substances (antibodies, transfer factors, thymus grafts, and interleukin 2) from an actively immunized host to a nonimmunized host” (ibid.) This can be contrasted with passive immunization.

A "protective" immune response or "protective" immunity, as used herein, indicates that the immune response confers some benefit to the subject in that it prevents or reduces the occurrence of disease. Alternatively, a protective immune response or immunity can be used (e.g., by preventing the formation of a cancer or tumor, by causing regression of a cancer or tumor, and/or by preventing metastasis, and/or by preventing metastasis). may be useful in the treatment and/or prevention of diseases, particularly cancers or tumors (by preventing nodule growth). The protective effect may be complete or partial, so long as the benefits of the treatment outweigh any drawbacks thereof.

In certain embodiments, viral vectors or cells containing heterologous nucleic acid molecules can be administered in an immunogenically effective amount as described below.

Viral vectors of the invention also include viral vectors expressing one or more cancer cell antigens (or immunologically similar molecules) or any other immunogen that generates an immune response against cancer cells. The administration can be administered for cancer immunotherapy. Illustratively, an immune response may generate a viral vector containing a heterologous nucleic acid encoding a cancer cell antigen, e.g., to treat a patient with cancer and/or to prevent cancer from developing in a subject. The cancer cell antigen can be generated in the subject by administering the cancer cell antigen. Viral vectors may be administered to a subject in vivo or by using ex vivo methods, as described herein. Alternatively, the cancer antigen can be expressed as part of or otherwise associated with the viral capsid (eg, as described above).

As another alternative, any other therapeutic nucleic acid (eg, RNAi) or polypeptide (eg, cytokine) known in the art can be administered to treat and/or prevent cancer.

As used herein, the term "cancer" encompasses tumorigenic cancers.

Similarly, the term "cancerous tissue" includes tumors. "Cancer cell antigen" includes tumor antigens.

The term "cancer" has the meaning it is understood in the art, eg, uncontrolled tissue growth that has the potential to spread (ie, metastasize) to distant sites in the body. Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer. breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition now known or later identified. is included. In representative aspects, the invention provides methods of treating and/or preventing tumorigenic cancer.

The term "tumor" is also understood in the art as, for example, an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

The terms "treating cancer", "treatment of cancer" and equivalent terms mean that the severity of cancer is reduced or at least partially eliminated and/or the progression of the disease is slowed and/or or to control and/or to stabilize the disease. In certain aspects, these terms include cancer metastasis is prevented or reduced or at least partially eliminated and/or metastatic nodule growth is prevented or reduced or at least partially eliminated. Indicates that it will be removed.

The terms "cancer prevention" or "preventing cancer" and equivalent terms mean that the method at least partially eliminates or reduces the incidence and/or severity of cancer occurrence; and/or or intended to be delayed. Stated otherwise, the likelihood or probability of developing cancer in a subject may be reduced and/or delayed.

In certain embodiments, cells may be removed from a subject with cancer and contacted with a viral vector expressing a cancer cell antigen according to the invention. The modified cells are then administered to the subject, thereby eliciting an immune response against the cancer cell antigen. This method can be advantageously employed in immunocompromised subjects who are unable to mount a sufficient immune response in vivo (ie, unable to produce stimulatory antibodies in sufficient quantities).

The immune response is triggered by immunomodulatory cytokines (e.g., α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin-2, interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin-12, Interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-α, tumor necrosis factor-β, monocyte chemotactic protein-1, granulocyte-macrophage colony-stimulating factor, and lymphotoxin) It is known in the art that it can be enhanced. Therefore, immunomodulatory cytokines (preferably CTL-inducing cytokines) may be administered to the subject in conjunction with the viral vector.

Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, nucleic acids encoding the cytokines may be delivered to the subject using a suitable vector and the cytokines produced in vivo.

Subjects, Pharmaceutical Formulation, and Mode of Administration The viral vectors, AAV particles and capsids according to the invention find use in both veterinary and medical applications. Suitable subjects include both birds and mammals. The term "bird" as used herein includes, without limitation, chickens, ducks, geese, quail, turkeys, pheasants, parrots, parakeets, and others. As used herein, the term "mammal" includes, without limitation, humans, non-human primates, cows, sheep, goats, horses, cats, dogs, rabbits, and the like.

Human subjects include neonatal, infant, juvenile, adult and geriatric subjects.

In typical embodiments, the subject is "in need" of the methods of the invention.

In certain embodiments, the invention provides viral vectors and/or capsids and/or AAV particles of the invention in a pharmaceutically acceptable carrier and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, Pharmaceutical compositions including adjuvants, diluents, etc. are provided. For injections, the carrier will typically be a liquid. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier is respirable and can optionally be in solid or liquid particulate form. For administration to a subject or other pharmaceutical use, the carrier is sterile and/or physiologically compatible.

By "pharmaceutically acceptable" is meant a substance that is not toxic or otherwise unnecessary, ie, a substance that can be administered to a subject without causing any undesirable biological effects.

One aspect of the invention is a method of introducing a nucleic acid molecule into a cell in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infection by standard transduction methods appropriate for the particular target cell. The titer of viral vector to be administered will vary depending on the type and number of target cells and the particular viral vector, and can be determined by one of ordinary skill in the art without undue experimentation. In typical embodiments, at least about 10 ³ infectious units, optionally at least about 10 ⁵ infectious units, are introduced into the cell.

Cells into which the viral vector is introduced include, but are not limited to, nervous system cells (including cells of the peripheral and central nervous system, in particular brain cells such as neurons and oligodendrocytes), lung cells, ocular cells (retinal cells). epithelial cells (e.g., intestinal and respiratory epithelial cells); muscle cells (e.g., including skeletal muscle cells, cardiomyocytes, smooth muscle cells, and/or diaphragm muscle cells); cells, pancreatic cells (including islet cells), hepatocytes, cardiomyocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, and others. Can be of any type, including. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cells can be stem cells (eg neural stem cells, liver stem cells). As yet a further alternative, the cell can be a cancer or tumor cell. Moreover, the cells can be from any species of origin, as described above.

To administer the modified cells to a subject, the viral vector can be introduced into the cells in vitro. In certain embodiments, the cells are removed from the subject, the viral vector is introduced into them, and the cells are then returned to the subject. Methods for removing cells from a subject for ex vivo manipulation and then introducing them back into the subject are known in the art (see, eg, US Pat. No. 5,399,346). Alternatively, a recombinant viral vector can be introduced into cells from a donor subject, into cultured cells, or into cells of any other suitable origin, and the cells can be transferred to a subject in need of it (i.e., a "recipient" subject). ).

Cells suitable for ex vivo nucleic acid delivery are described above. The amount of cells to be administered to a subject will vary depending on the age, condition and species of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, and so on. Typically, at least about 10 ² to about 10 ⁸ cells or at least about 10 ³ to about 10 ⁶ cells are administered per dose in a pharmaceutically acceptable carrier. In certain embodiments, cells transduced with a viral vector are administered to a subject in a therapeutically or prophylactically effective amount together with a pharmaceutical carrier.

In some embodiments, the viral vector is introduced into a cell and the cell is administered to a subject to generate an immunogenic response to the delivered polypeptide (e.g., expressed as a transgene or in a capsid). Can be triggered. Typically, an amount of cells expressing an immunogenically effective amount of the polypeptide is administered together with a pharmaceutically acceptable carrier. An "immunogenically effective amount" is an amount of expressed polypeptide sufficient to elicit an active immune response against the polypeptide in a subject to whom the pharmaceutical formulation is administered. In certain embodiments, the dosage is sufficient to generate a protective immune response (as defined above).

The degree of protection conferred need not be complete or permanent, so long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.

A further aspect of the invention is a method of administering a viral vector and/or a viral capsid to a subject. Administration of the viral vectors and/or capsids according to the invention to a human subject or animal in need thereof can be by any means known in the art. Optionally, the viral vector and/or capsid is delivered in a therapeutically or prophylactically effective dose in a pharmaceutically acceptable carrier.

Viral vectors and/or capsids of the invention can be further administered to elicit an immunogenic response (eg, as a vaccine). Typically, an immunogenic composition of the invention comprises an immunogenically effective amount of a viral vector and/or capsid together with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to generate a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, so long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof. Subjects and immunogens are as described above.

The dosage of viral vector and/or capsid to be administered to a subject will depend on the mode of administration, the disease or condition to be treated and/or prevented, the individual subject's condition, the particular viral vector or capsid, and the nucleic acid to be delivered. and others, and can be determined in a routine manner. Exemplary doses to achieve a therapeutic effect include at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ transducing units , optionally at a titer of about 10 ⁸ to about 10 ¹³ transducing units.

In certain embodiments, more than one administration (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., or more doses) may be employed. Dosing can be a single dose or cumulative (a series of doses) and can be readily determined by one of ordinary skill in the art. For example, treatment of a disease or disorder may include a single administration of an effective dose of a viral vector of the pharmaceutical compositions disclosed herein. Alternatively, the treatment of a disease or disorder is carried out over a series of time periods, such as once a day, twice a day, three times a day, once every few days, or once a week, using a viral vector. may include multiple administrations of effective doses of. The timing of administration can vary from individual to individual depending on factors such as the severity of the individual's symptoms. For example, an effective dose of a viral vector disclosed herein can be administered to an individual once every six months for an indefinite period of time or until the individual no longer requires treatment. One of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of the viral vectors disclosed herein administered can be adjusted accordingly.

In one embodiment, the administration period of the viral vector is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months , 10 months, 11 months, 12 months, or longer. In further embodiments, the period during which administration is stopped is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days. , 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine. (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration into skeletal, diaphragm, and/or cardiac muscle]), intradermal, intrapleural, intracerebral, and intraarticular. ), local (e.g., to both skin and mucosal surfaces, including respiratory tract surfaces, and transdermal administration), intralymphatic, and other, as well as direct tissue or organ injection (e.g., liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain). ) is included. Administration can also be to the tumor (eg, within or near a tumor or lymph node). The most appropriate route in any given case will depend on the nature and severity of the condition being treated and/or prevented and the nature of the particular vector used.

Administration to skeletal muscle according to the invention includes, but is not limited to, the extremities (e.g., upper arms, forearms, thighs, and/or lower legs), back, neck, head (e.g., tongue), chest, abdomen, pelvis. / perineum, and/or skeletal muscle administration in the fingers. Appropriate skeletal muscles include, but are not limited to, abductor digiti minimi (of the hand), abductor digiti minimi (of the foot), abductor pollicis of the foot, and abductor ossis metatarsi (of the foot). quinti), abductor pollicis brevis of the hand, abductor pollicis longus of the hand, adductor brevis, adductor pollicis of the foot, adductor pollicis longus, adductor magnus, adductor pollicis of the hand Muscles, elbow muscles, anterior scalenes, knee joint muscles, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator, deltoid, depressor angular oris, inferior depressor labii, digastrics, dorsal interosseous muscles (hands), dorsal interosseous muscles (legs), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, Extensor digiti minimi, extensor digitorum, extensor digitorum brevis, extensor digitorum longus, extensor pollicis brevis, extensor pollicis longus, extensor pollicis, extensor pollicis brevis of the hand, Extensor pollicis longus of the hand, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum brevis (hand), flexor digitorum brevis (foot), flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus. , flexor digitorum superficialis, flexor pollicis brevis of the foot, flexor pollicis longus of the foot, flexor pollicis brevis of the hand, flexor pollicis longus of the hand, frontalis muscle, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, cervicoiliolicostalis, lumbar ilicostalis, thoracoiliocas, iliacus, inferior twins, inferior oblique, inferior rectus, infraspinatus, interspinatus, transverse intertransversi, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superior, levator labii nasolini superior, levator palpebrae superioris, levator scapulae, long rotators, head longissimus muscle, longissimus cervix, longissimus thoracis, longus capitis, longus cervix, vermiformis (hand), vermiformis (foot), masseter, medial pterygoid, medial rectus, middle scalene, Multifidus, mylohyoid, inferior oblique, superior oblique, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, mouth Orbicularis muscle, palmaris brevis, palmaris longus, pubis muscle, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertiary, piriformis, plantar interosseus , plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantaris, rectus capitis frontalis, rectus capitis lateralis, major Rectus capitis, Rectus capitis minor, Rectus femoris, Rhomboid major, Rhomboid minor, Laughter, Sartorius, Scalene minimalis, Semimembranosus, Semispinalis capitis, Semispinalis cervicis, Half thoracic muscle spinas, semitendinosus, serratus anterior, short rotators, soleus, spinae capitis, spinae cervicis, spinae thoracic, splenius capitis, splenius cervix, sternocleidomastoid, Sternohyoid muscle, sternothyroid muscle, stylohyoid muscle, subclavian muscle, subscapularis muscle, gemini superior muscle, superior oblique muscle, superior rectus muscle, supinator muscle, supraspinatus muscle, temporalis muscle, femoris muscle Tensor membranus, teres major, teres minor, chest muscles, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps, vastus intermedius, vastus lateralis, vastus medialis, Included are the zygomaticus major and minor, as well as any other suitable skeletal muscles known in the art.

Viral vectors and/or capsids can be administered intravenously, intraarterially, intraperitoneally, by extremity perfusion (optionally, by isolated extremity perfusion of the legs and/or arms; e.g. Arruda et al., (2005) Blood 105: 3458 -3464) and/or to skeletal muscle by direct intramuscular injection. In certain embodiments, the viral vector and/or capsid is administered to a limb (arm and/or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally by limb isolated perfusion (e.g., intravenously or jointly). (by intravenous administration). In aspects of the invention, the viral vectors and/or capsids of the invention can advantageously be administered without employing "hydrodynamic" techniques. Tissue delivery (e.g., to muscle) of prior art vectors is often enhanced by hydrodynamic techniques (e.g., high volume intravenous/intravenous administration), which increases pressure in the vasculature and facilitates the ability of cells to cross the endothelial cell barrier. In certain embodiments, the viral vectors and/or capsids of the invention are administered by bolus infusion and/or elevated intravascular pressure (e.g., greater than normal systolic pressure, e.g., 5%, 10% from normal systolic pressure). %, 15%, 20%, 25% increase in intravascular pressure), etc., can be administered in the absence of hydrodynamic techniques. Such methods may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage and/or compartment syndrome.

Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum. Viral vectors and/or capsids can be delivered to the myocardium by intravenous administration, intraarterial administration such as intraaortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and/or coronary perfusion. can be delivered to.

Administration to the diaphragm muscle can be by any suitable method, including intravenous, intraarterial, and/or intraperitoneal administration.

Delivery to target tissues can also be achieved by delivering depots containing viral vectors and/or capsids. In typical embodiments, the depot containing the viral vector and/or capsid is implanted into skeletal, cardiac and/or diaphragm muscle tissue, or the tissue is surrounded by a film or other matrix containing the viral vector and/or capsid. can be brought into contact with. Such implantable matrices or substrates are described in US Pat. No. 7,201,898.

In certain embodiments, the viral vectors and/or viral capsids according to the invention are used to treat and/or prevent skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g. muscular dystrophy, heart disease [e.g. PAD or congestive heart failure)]. (for) to be administered.

In representative embodiments, the invention is used to treat and/or prevent disorders of skeletal, cardiac and/or diaphragm muscles.

In an exemplary embodiment, the invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof, comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector of the invention. wherein the viral vector contains dystrophin, mini-dystrophin, micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, an anti-inflammatory polypeptide, e.g. Antibodies or antibodies against B-dominant mutants, sarcospans, utrophin, micro-dystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, myostatin or myostatin propeptide fragment, and/or a heterologous nucleic acid encoding RNAi against myostatin. In certain embodiments, viral vectors can be administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle as described elsewhere herein.

Alternatively, the invention can be practiced to deliver nucleic acids to skeletal, cardiac, or diaphragm muscle, which may be used in disorders such as metabolic disorders such as diabetes [e.g., insulin], hemophilia [e.g. factor IX or factor VIII], mucopolysaccharidoses [e.g. Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteau-Lamy syndrome, etc. ] or glycogen storage diseases such as Gaucher disease [glucocerebrosidase] or Fabry disease [alpha-galactosidase A] or Pompe disease [lysosomal acid alpha-glucosidase]). Used as a platform for the production of polypeptides (eg, enzymes) or functional RNA (eg, RNAi, microRNA, antisense RNA) that circulate in the blood or for systemic delivery to other tissues. Other suitable proteins for treating and/or preventing metabolic disorders are described herein. The use of muscle as a platform to express nucleic acids of interest is described in US Patent Application Publication No. 2002/0192189.

Therefore, in one aspect, the present invention further provides a method for treating and/or preventing metabolic disorders in a subject in need thereof, the method comprising administering a therapeutically or prophylactically effective amount of the viral vector of the present invention to the skeletal muscle of the subject. wherein the viral vector comprises a heterologous nucleic acid encoding a polypeptide, and the metabolic disorder is a result of a defect and/or defect in the polypeptide. Metabolic disorders and heterologous nucleic acids encoding exemplary polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., is a secreted polypeptide in its native state, or engineered to be secreted, e.g., by functional association with a secreted signal sequence known in the art). polypeptide). Without being limited by any particular theory of the invention, according to this embodiment, administration to skeletal muscle can result in secretion of the polypeptide into the systemic circulation and delivery to the target tissue. Methods of delivering viral vectors to skeletal muscle are described in more detail herein.

The invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (eg, ribozymes) for systemic delivery.

The present invention also provides a method of treating and/or preventing congenital heart defect or PAD in a subject in need thereof, the method comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of a viral vector of the present invention. ^Also provided are methods, wherein the viral vector contains, e.g. I1C), RNAi against phospholamban; phospholamban inhibitory or dominant negative molecules such as phospholamban S16E, zinc finger proteins that regulate the phospholamban gene, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, β-adrenergic receptor kinase inhibitor (βARKct), protein phosphatase 1 inhibitor 1 and its fragments (e.g., I1C), S100A1, parvalbumin, adenylyl cyclase type 6, truncated constitutively active bARKct Molecules that cause G protein-coupled receptor kinase type 2 knockdown, such as Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir- 133, mir-206, mir-208 and/or mir-26a.

Injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the viral vectors and/or viral capsids of the invention may be administered locally rather than systemically, eg, in a depot or sustained release formulation. Additionally, viral vectors and/or viral capsids can be delivered attached to a surgically implantable matrix (e.g., as described in U.S. Patent Application Publication No. 2004/0013645; herein The disclosed viral vectors and/or viral capsids can be administered to a subject's lungs by any suitable means, optionally by administering an aerosol suspension of respirable particles comprised of the viral vectors and/or viral capsids. The respirable particles can be liquid or solid. Aerosols of liquid particles containing viral vectors and/or viral capsids are known to those skilled in the art. may be generated by any suitable means, such as a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as described in US Pat. No. 4,501,729. may similarly be produced using any solid particulate pharmaceutical aerosol generator by techniques known in the pharmaceutical art.

Viral vectors and capsids can be administered to tissues of the CNS (eg, brain, eye), which may advantageously give a wider distribution of viral vectors or capsids than would be observed in the absence of the present invention.

In certain embodiments, the delivery vectors of the invention can be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, Progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, spinal cord or head trauma, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, stroke, and mood disorders. Mental disorders including (e.g. depression, bipolar affective disorder, persistent affective disorder, secondary mood disorders), schizophrenia, drug dependence (e.g. alcoholism and other substance dependence), neurosis (e.g. anxiety, obsessive-compulsive disorder, somatoform disorders, dissociation, grief, postpartum depression), psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention-deficit disorder, psychosexual disorders, sleep disorders, pain disorders, Includes eating or weight disorders (eg, obesity, cachexia, anorexia nervosa, and bulimia) and cancers and tumors of the CNS (eg, pituitary tumors).

Disorders of the CNS include ocular disorders that involve the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

Most, if not all, ocular diseases and disorders are associated with one or more of three types of manifestations: (1) angiogenesis, (2) inflammation, and (3) degeneration. Delivery vectors of the invention can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that slow cell degeneration, promote cell sparing, or promote cell growth, and combinations thereof. .

For example, diabetic retinopathy is characterized by neovascularization. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intraocularly (e.g., in the vitreous) or periocularly (e.g., in the sub-Tenon's region). . One or more neurotrophic factors may also be co-delivered either intraocularly (eg, intravitreally) or periocularly.

Uveitis involves inflammation. One or more anti-inflammatory factors can be administered by intraocular (eg, intravitreal or anterior chamber) administration of a delivery vector of the invention.

Retinitis pigmentosa, by comparison, is characterized by retinal degeneration. In typical embodiments, retinitis pigmentosa can be treated by intraocular (eg, intravitreal) administration of a delivery vector encoding one or more neurotrophic factors.

Age-related macular degeneration involves both neovascularization and retinal degeneration. This disorder is characterized by the delivery vectors of the invention encoding one or more neurotrophic factors into the eye (e.g., the vitreous) and/or the delivery vectors of the invention encoding one or more anti-angiogenic factors. The delivery vector can be administered intraocularly or periocularly (eg, in the sub-Tenon's region).

Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatments for glaucoma include administering one or more neuroprotective agents that protect cells from excitotoxic damage using the delivery vectors of the invention. Such agents include intraocularly and optionally intravitreally delivered N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors.

In other aspects, the invention may be used to treat stroke, eg, to reduce the occurrence, incidence, or severity of stroke. Efficacy of therapeutic treatment for seizures can be judged by behavior (e.g., tremors, ocular or oral tics) and/or electrographic measures (most seizures have a signature of electrographic abnormalities). can. Accordingly, the present invention may also be used to treat epilepsy, which is characterized by multiple seizures over time.

In one exemplary embodiment, somatostatin (or an active fragment thereof) is administered to the brain using a delivery vector of the invention to treat pituitary tumors. According to this embodiment, a delivery vector encoding somatostatin (or an active fragment thereof) is administered by microinjection into the pituitary gland. Similarly, such treatments can be used to treat acromegaly (abnormal secretion of growth hormone from the pituitary gland). The nucleic acid (eg, GenBank Accession No. J00306) and amino acid (eg, GenBank Accession No. P01166; containing processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.

In certain embodiments, the vector can include a secretion signal as described in US Pat. No. 7,071,172.

In typical embodiments of the invention, viral vectors and/or viral capsids are administered to the CNS (eg, brain or eye). The viral vector and/or capsid can be transmitted to the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, hypothalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, The cerebrum, including the occipital, temporal, parietal, and frontal lobes; may be introduced into the cortex, basal ganglia, hippocampus, and amygdala (portaamygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. be. Viral vectors and/or capsids may also be administered to different areas of the eye, such as the retina, cornea and/or optic nerve.

Viral vectors and/or capsids may be delivered into the cerebrospinal fluid (eg, by lumbar puncture) for more dispersed administration of the delivery vector.

Viral vectors and/or capsids may also be administered intravascularly into the CNS in situations where the blood-brain barrier is perturbed (eg, brain tumors or cerebral infarction).

The viral vector and/or capsid can be administered, without limitation, intrathecally, intraocularly, intracerebrally, intracerebroventricularly, intravenously (e.g., in the presence of a sugar such as mannitol), intranasally, intraaurally, intraocularly. by any route known in the art, including intravitreal (e.g., subretinal, anterior chamber) and periocular (e.g., subtenon region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons. It can be administered to the desired area of the CNS.

In certain embodiments, the viral vector and/or capsid are administered as a liquid formulation by direct injection (eg, stereotaxic injection) into the desired region or compartment in the CNS. In other embodiments, the viral vector and/or capsid may be provided by topical application to the desired area or by intranasal administration of an aerosol formulation. Administration to the eye can be by topical application of droplets. As a further alternative, the viral vector and/or capsid can be administered as a solid, sustained release formulation (see, eg, US Pat. No. 7,201,898).

In still additional embodiments, the viral vectors are used to treat and/or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). Can be used for retrograde transport. For example, a viral vector can be delivered to muscle tissue, from where it can translocate into neurons.

In other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least Reduce by 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In still other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder by, for example, about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30%. ～about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90% , about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, reduced by about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70%.

The viral vectors disclosed herein may include a solvent, emulsion or other diluent in an amount sufficient to solubilize the viral vectors disclosed herein. In other aspects of this embodiment, the viral vectors disclosed herein contain a solvent, emulsion or diluent, e.g., less than about 90% (v/v), less than about 80% (v/v), about 70% (v/v), less than about 65% (v/v), less than about 60% (v/v), less than about 55% (v/v), less than about 50% (v/v), about 45% (v/v), less than about 40% (v/v), less than about 35% (v/v), less than about 30% (v/v), less than about 25% (v/v), about 20% (v/v), less than about 15% (v/v), less than about 10% (v/v), less than about 5% (v/v), or less than about 1% (v/v) may be included. In other aspects of this embodiment, the viral vectors disclosed herein contain a solvent, emulsion or other diluent, e.g., about 1% (v/v) to 90% (v/v), about 1% (v/v) v/v) ~ 70% (v/v), approximately 1% (v/v) ~ 60% (v/v), approximately 1% (v/v) ~ 50% (v/v), approximately 1% (v/v) ~ 40% (v/v), approximately 1% (v/v) ~ 30% (v/v), approximately 1% (v/v) ~ 20% (v/v), approximately 1 % (v/v) ~ 10% (v/v), approx. 2% (v/v) ~ 50% (v/v), approx. 2% (v/v) ~ 40% (v/v), approx. 2% (v/v) ~ 30% (v/v), approximately 2% (v/v) ~ 20% (v/v), approximately 2% (v/v) ~ 10% (v/v), Approx. 4% (v/v) ~ 50% (v/v), Approx. 4% (v/v) ~ 40% (v/v), Approx. 4% (v/v) ~ 30% (v/v) , about 4% (v/v) to 20% (v/v), about 4% (v/v) to 10% (v/v), about 6% (v/v) to 50% (v/v) ), approximately 6% (v/v) to 40% (v/v), approximately 6% (v/v) to 30% (v/v), approximately 6% (v/v) to 20% (v/v) v), approximately 6% (v/v) to 10% (v/v), approximately 8% (v/v) to 50% (v/v), approximately 8% (v/v) to 40% (v /v), approximately 8% (v/v) to 30% (v/v), approximately 8% (v/v) to 20% (v/v), approximately 8% (v/v) to 15% ( v/v), or in an amount ranging from about 8% (v/v) to 12% (v/v).

Aspects herein disclose partially treating an individual suffering from a disease or disorder. As used herein, the term "treating" refers to reducing or eliminating clinical symptoms of a disease or disorder in an individual; or delaying or preventing the onset of clinical symptoms of a disease or disorder in an individual. . For example, the term "treating" refers to reducing symptoms of a condition characterized by a disease or disorder by, for example, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%. % can mean reduce by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% can. The actual symptoms associated with a particular disease or disorder are well known and depend on, but are not limited to, the location of the disease or disorder, the cause of the disease or disorder, the severity of the disease or disorder, and/or the person affected by the disease or disorder. This can be determined by one skilled in the art by considering factors including the tissue or organ involved. One of ordinary skill in the art would know the appropriate symptoms or indicators associated with a particular type of disease or disorder and how to determine whether an individual is a candidate for the treatment disclosed herein. There is.

In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces symptoms associated with a disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 % or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces symptoms associated with a disease or disorder by, for example, up to 10%, up to 15%, up to 20%, up to 25% %, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75 %, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%. In still other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces symptoms associated with a disease or disorder, such as by about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% ~90%, approximately 20% to approximately 80%, approximately 20% to approximately 20%, approximately 20% to approximately 60%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 30% to approximately 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

In one aspect, the viral vectors disclosed herein increase the level and/or amount of protein encoded in the viral vector administered to a patient, e.g., by at least 10 %, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, It is possible to increase by at least 75%, at least 80%, at least 85%, at least 90% or at least 95%. In other aspects of this embodiment, the viral vector reduces the severity of the disease or disorder in an individual suffering from the disease or disorder compared to a patient not receiving the same treatment, e.g., by about 10% to about 100%, by about 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% ~100%, approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80% , about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% It is possible to do so.

In aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein increases the amount of protein encoded within the viral vector in an individual by, e.g., at least 10% compared to an individual not receiving the same treatment. , at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least Increase by 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces the severity of a disease or disorder in an individual by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to reduce by 75%, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%, or maintain the severity of a disease or disorder in an individual. In still other aspects of this embodiment, a therapeutically effective amount of a viral vector disclosed herein reduces the severity of a disease or disorder in an individual, e.g., from about 10% to about 100%, from about 10% to about 90%, Approximately 10% to approximately 80%, approximately 10% to approximately 70%, approximately 10% to approximately 60%, approximately 10% to approximately 50%, approximately 10% to approximately 40%, approximately 20% to approximately 100%, approximately 20 % to approximately 90%, approximately 20% to approximately 80%, approximately 20% to approximately 20%, approximately 20% to approximately 60%, approximately 20% to approximately 50%, approximately 20% to approximately 40%, approximately 30% to Reduce or maintain about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

Viral vectors are administered to individuals or patients. The individual or patient is typically a human, but includes, but is not limited to, dogs, cats, birds, cows, horses, sheep, goats, reptiles, and other animals, whether domesticated or not. It can be an animal.

In one embodiment, the viral vectors of the invention can be used to generate AAVs that target specific tissues including, but not limited to, the central nervous system, retina, heart, lungs, skeletal muscle, and liver. These targeted viral vectors can be used to target tissue-specific diseases or endogenous proteins in specific normal tissues such as factor IX (FIX), factor VIII, FVIII and other proteins known in the art. can be treated for the production of sexually produced proteins.

Diseases of the Central Nervous System In one aspect, diseases of the central nervous system can be treated using AAV, where the AAV can be of any AAV serotype, as well as AAV1, AAV2. , AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10. In one embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.

Retinal Diseases In one embodiment, retinal diseases can be treated using AAV, where the AAV can be any AAV serotype, and AAV1, AAV2, AAV3, AAV4, AAV5. , AAV7, AAV8, AAV9 or AAV10. In one embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9 or AAV10.

Heart Disease In a further aspect, heart disease can be treated using AAV, wherein the AAV can be any AAV serotype, and the recipient AAV and AAV1, AAV3, AAV4, AAV6 or A donor capsid selected from one or more of AAV9. In additional embodiments, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9. In another embodiment, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV3, AAV4, AAV6 or AAV9.

Lung Disease In one aspect, the lung disease can be treated using AAV, where the AAV serotype is the recipient AAV, which can be any AAV serotype, and AAV1, AAV5, AAV6, AAV9. or AAV10. In another embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10. In further embodiments, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV5, AAV6, AAV9 or AAV10.

Skeletal Muscle Diseases In a further aspect, diseases of skeletal muscle can be treated using AAV, where the AAV serotype is the recipient AAV, which can be any AAV serotype, and AAV1, AAV2, A donor capsid selected from one or more of AAV6, AAV7, AAV8, or AAV9. In another embodiment, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9. In embodiments, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9.

Liver Disease In one aspect, liver disease can be treated using AAV, where the AAV serotype is the recipient AAV, which can be any AAV, and AAV2, AAV3, AAV6, AAV7, A donor capsid selected from one or more of AAV8, or AAV9. In additional embodiments, the recipient AAV is AAV2 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9. In further embodiments, the recipient AAV is AAV3 and the donor capsid is selected from one or more of AAV2, AAV3, AAV6, AAV7, AAV8, or AAV9.

In some embodiments, this application may be defined in any of the following paragraphs:
1. An isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, comprising:
Two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome;
at least one of the viral structural proteins present is from a different serotype than other viral structural proteins;
VP1 is derived from only one serotype, VP2 is derived from only one serotype, and VP3 is derived from only one serotype,
The isolated AAV virions.
2. The isolated AAV virion of paragraph 1 in which all three viral structural proteins are present.
3. The isolated AAV virion of paragraph 2, in which all three viral structural proteins are derived from different serotypes.
4. The isolated AAV virion of paragraph 2 in which only one of the three structural proteins is derived from a different serotype.
5. The isolated AAV virion of paragraph 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP1.
6. The isolated AAV virion of paragraph 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP2.
7. The isolated AAV virion of paragraph 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP3.
8. A substantially homogeneous population of virions according to any of paragraphs 1 to 7, which is at least 10 ¹ virions.
9. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 ⁷ virions.
10. A substantially homogeneous population of virions of paragraph 8 that is at least 10 ⁷ to 10 ¹⁵ virions.
11. A substantially homogeneous population of the virions of paragraph 8 that is at least ¹⁰⁹ virions.
12. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 ¹⁰ virions.
13. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 to ¹¹ virions.
14. A substantially homogeneous population of virions of paragraph 10 that is at least 95% homogeneous.
15. A substantially homogeneous population of virions of paragraph 10 that is at least 99% homogeneous.
16. A method of producing adeno-associated virus (AAV) virions, comprising:
contacting the cell with the first nucleic acid sequence and the second nucleic acid sequence under conditions for the formation of AAV virions;
AAV virions are formed from at least VP1 and VP3 viral structural proteins;
a first nucleic acid encodes VP1 derived only from a first AAV serotype, but is incapable of expressing VP3;
the second nucleic acid sequence encodes VP3 derived exclusively from a second AAV serotype different from the first AAV serotype, and is further incapable of expressing VP1;
the AAV virion comprises VP1 derived only from the first serotype and VP3 derived only from the second serotype, and
When VP2 is expressed, it is derived from only one serotype,
Said method.
17. The first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, and 17. The method of paragraph 16, wherein the VP1 has a mutation in its start codon that prevents translation of VP1 from RNA transcribed from the nucleic acid.
18. The method of paragraph 16, wherein VP2 from only one serotype is expressed.
19. The method of paragraph 18, wherein VP2 is derived from a different serotype than VP1 and a different serotype than VP3.
20. The method of paragraph 18, wherein VP2 is derived from the same serotype as VP1.
21. The method of paragraph 18, wherein VP2 is derived from the same serotype as VP3.
22. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or Table 1 or Table 3, or any of each AAV. The method of paragraph 16 which is a chimera.
23. an AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 16, which is a chimera of.
24. AAV virions are formed from VP1, VP2, and VP3 capsid proteins;
a viral structural protein is encoded by a first nucleic acid derived exclusively from a first AAV serotype and a second nucleic acid derived exclusively from a second AAV serotype different from the first AAV serotype;
the first nucleic acid has a mutation in the A2 splice acceptor site, and the second nucleic acid has a mutation in the A1 splice acceptor site, and
the polyploid AAV virion comprises VP1 derived only from a first serotype and VP2 and VP3 derived only from a second serotype;
Paragraph 18 method.
25. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 24, which is a chimera of.
26. An AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 24, which is a chimera of.
27. A first nucleic acid sequence whose viral structural proteins are derived exclusively from a first AAV serotype that is different from a second and third serotype; a second AAV that is different from the first and third AAV serotypes; encoded by a second nucleic acid sequence derived only from a serotype and a third nucleic acid sequence derived only from a third AAV serotype that is different from the first and second AAV serotypes, and further
the first nucleic acid sequence has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid; VP1 and VP3 have mutations in the start codons of VP1 and VP3 that prevent translation of VP1 and VP3 from RNA transcribed from the nucleic acid sequence; and have mutations in the start codons of VP1 and VP2 that prevent translation of VP2,
the AAV virion comprises VP1 derived only from a first serotype, VP2 derived only from a second serotype, and VP3 derived only from a third serotype;
Paragraph 18 method.
28. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 27, which is a chimera of.
29. an AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3; or any of each AAV. The method of paragraph 27, which is a chimera of.
30. an AAV in which the third AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 27, which is a chimera of.
31. the first nucleic acid sequence has mutations in the start codons of VP2 and VP3 and mutations in the A2 splice acceptor site that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence; further, the second nucleic acid sequence has a mutation in the start codon of VP1 and a mutation in the A1 splice acceptor site that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence, and 19. The method of paragraph 18, wherein the sexual capsid comprises VP1 derived only from a first serotype and VP2 and VP3 derived only from a second serotype.
32. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or Table 1 or Table 3, or any of each AAV. The method of paragraph 31, which is a chimera of.
33. An AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 31, which is a chimera of.
34. A viral structural protein is encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and
the start codons for VP2 and VP3 have been mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence;
The capsid protein is encoded by a second nucleic acid derived from only a single AAV serotype, and the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid. having in it, and
the polyploid AAV capsid comprises VP1 from a first nucleic acid sequence created through DNA shuffling and VP2 and VP3 from only a second serotype;
Paragraph 18 method.
35. A viral structural protein is encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and
the start codons for VP2 and VP3 have been mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and
the A2 splice acceptor site of the first nucleic acid is mutated;
the capsid protein is encoded by a second nucleic acid sequence derived from only a single AAV serotype, and the second nucleic acid is in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid. and a mutation in the A1 splice acceptor site, and
the polyploid AAV capsid comprises VP1 from a first nucleic acid created through DNA shuffling and VP2 and VP3 from only a second serotype;
Paragraph 18 method.
36. A group consisting of an AAV whose AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Table 1 or Table 3, and any chimera of each AAV. Selected from Paragraph 15 Billion.
37. A substantially homogeneous population of virions produced by the method of paragraph 16.
38. A substantially homogeneous population of virions produced by the method of paragraph 18.
39. The AAV virion of paragraph 38, wherein the heterologous gene encodes a protein for treating a disease.
40. The disease is caused by a lysosomal storage disease, such as a mucopolysaccharidosis (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [aL-iduronidase], Scheie syndrome [aL-iduronidase], Hurler-Scheie syndrome [aL-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase] ], Morquio syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebro 39.
41. The isolated AAV virion of any of paragraphs 1-7, wherein at least one of the viral structural proteins is a chimeric viral structural protein.
42. The isolated AAV virion of paragraph 41, wherein the chimeric viral structural protein is derived from an AAV serotype but is different from other viral structural proteins.
43. The isolated AAV virion of any of paragraphs 1-7, wherein none of the viral structural proteins are chimeric viral structural proteins.
44. The isolated AAV virion of paragraph 41, wherein there is no serotype overlap between the chimeric viral structural protein and at least one other viral structural protein.
45. A method of modulating transduction using any of the methods of paragraphs 16-35.
46. The method of paragraph 45 for enhancing transduction.
47. A method of altering the tropism of an AAV virion comprising using any of the methods of paragraphs 16-35.
48. A method of altering the immunogenicity of an AAV virion comprising using the method of any of paragraphs 16-35.
49. A method of increasing vector genome copy number in a tissue comprising using any of the methods of paragraphs 16-35.
50. A method for increasing transgene expression comprising using the method of any of paragraphs 16-35.
51. An effective amount of the virions of any of paragraphs 1-7, 36, 43, and 44, or a substantially homogeneous population of the virions of any of paragraphs 8-15, 37-42, and 44, or of paragraphs 16-- 35. A method of treating a disease comprising administering a virion produced by any of the methods of
the heterologous gene encodes a protein for treating a disease that is suitable for treatment by gene therapy in a subject having the disease;
Said method.
52. The method of paragraph 51, wherein the disease is selected from genetic diseases, cancer, immunological diseases, inflammation, autoimmune diseases, and degenerative diseases.
53. The method of any of paragraphs 51 and 52, wherein multiple administrations are performed.
54. The method of paragraph 53, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration.
55. Transducing, copying into an AAV vector having particles having viral structural proteins all derived from only one serotype, comprising administering AAV virions of any of paragraphs 1-15 and 36-44. and transgene expression.
56. An isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion encapsidating an AAV genome, wherein at least one of the viral capsid structural proteins is different from other viral capsid structural proteins, and , an isolated AAV virion, said virion containing only each structural protein of the same type.
57. has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3;
The two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome, and at least one of the other viral structural proteins present is different from the other viral structural proteins, and the virions are the same. containing only each structural protein of the type,
Isolated AAV virions of paragraph 56.
58. The isolated AAV virion of paragraph 57 in which all three viral structural proteins are present.
59. The isolated AAV virion of paragraph 58 further comprising a fourth AAV structural protein.
60. has at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3;
the two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome, and at least one of the viral structural proteins present is from a different serotype than the other viral structural proteins;
VP1 is derived from only one serotype, VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype,
Isolated AAV virions of paragraph 56.
61. The isolated AAV virion of any of paragraphs 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one other viral structural protein.
62. The virion of paragraph 61, in which only VP3 is chimeric and VP1 and VP2 are non-chimeric.
63. The virion of paragraph 61, in which only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
64. The virion of paragraph 63, wherein the chimera is composed of subunits from AAV serotypes 2 and 8, and VP3 is derived from AAV serotype 2.
65. The isolated AAV virion of any of paragraphs 56-64, wherein all viral structural proteins are derived from different serotypes.
66. The isolated AAV virion of any of paragraphs 56-64, wherein only one of the structural proteins is from a different serotype.
67. A substantially homogeneous population of virions according to any of paragraphs 56 to 66, which is at least 10 ⁷ virions.
68. A substantially homogeneous population of virions of paragraph 67 that is at least 10 ⁷ to 10 ¹⁵ virions.
69. A substantially homogeneous population of virions of paragraph 67 that is at least 10 ⁹ virions.
70. A substantially homogeneous population of virions of paragraph 67 that is at least 10 ¹⁰ virions.
71. A substantially homogeneous population of the virions of paragraph 67 that is at least 10 ^{to 11} virions.
72. A substantially homogeneous population of virions according to any of paragraphs 67 to 71, which is at least 95% homogeneous.
73. A substantially homogeneous population of virions of paragraph 72 that is at least 99% homogeneous.
74. A group consisting of an AAV whose AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Table 1 or Table 3, and any chimera of each AAV. A virion according to any of paragraphs 56 to 73, selected from:
75. Substantially homogeneous population of virions of paragraph 73.
76. The AAV virion of any of paragraphs 56-74, wherein the heterologous gene encodes a protein for treating a disease.
77. The disease is associated with lysosomal storage diseases, such as mucopolysaccharidoses (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [aL-iduronidase], Scheie syndrome [aL-iduronidase], Hurler-Scheie syndrome [aL-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase] ], Morquio syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebro 77.
78. The isolated AAV virion of any of paragraphs 56-60 and 66-77, wherein none of the viral structural proteins are chimeric viral structural proteins.
79. The isolated AAV virion of any of paragraphs 57-78, wherein there is no serotype overlap between the chimeric viral structural protein and at least one other viral structural protein.
80. Treating a disease comprising administering an effective amount of the virions of any of paragraphs 56-66, 74, 76-79, or a substantially homogeneous population of the virions of any of paragraphs 67-73 and 75. A method of
the heterologous gene encodes a protein for treating a disease that is suitable for treatment by gene therapy in a subject having the disease;
Said method.
81. The method of paragraph 80, wherein the disease is selected from genetic diseases, cancer, immunological diseases, inflammation, autoimmune diseases, and degenerative diseases.
82. The method of any of paragraphs 80 and 81, wherein multiple administrations are performed.
83. The method of paragraph 82, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration.
84. Isolated AAV virions of paragraphs 1-7, 36, 39-44, 56-66, 74, 76-79; Substantially Homogenous Populations and Methods 16-35, 45-55, and 80-83: Any disclosure in PCT/US18/22725, filed March 15, 2018, may be included in the claims of this application. Any invention that falls within the scope of the invention as defined in any one or more thereof or is to be defined in any amended claim that may be filed in the future in this application or in any patent derived therefrom. and to the extent that the disclosure of PCT/US18/22725 falls within the scope of the law of any relevant country or countries to which that or those claims apply. To the extent necessary to prevent invalidation of this application or any patent derived therefrom, the inventors hereby provide that: We reserve the right to disclaim such disclosure from the claims of this application or any patents derived therefrom.

By way of example and without limitation, the inventors disclaim from any claim of this application or any patent derived therefrom, now or hereafter amended, any one or more of the following subject matter: have the right to:
A. Any subject matter disclosed in Example 9 of PCT/US18/22725; or
B. Polyploid, produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes. Vector virions called vector virions; or
C. Produced or producible by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes , vector virions called polyploid vector virions; or
D. Produced or producible by transfection of three AAV helper plasmids, AAV2, AAV8, and AAV9, to produce individual polyploid vector virions composed of different capsid subunits from different serotypes. vector virions, called polyploid vector virions; or
E. VP1/VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, e.g. VP1/VP2 is derived from only one AAV serotype (from its capsid) and VP3 vector virions, called haploid vectors, derived from only one alternative AAV serotype; or
F. Any one or more AAV vector virions selected from:
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP2/VP3 capsid subunit from AAV2; or
A vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82), comprising a VP1/VP2 capsid subunit derived from AAV8 and a VP1/VP2 capsid subunit derived from AAV2. a vector having a VP3 capsid subunit of; or
Vectors in which VP1/VP2 are derived from different serotypes; or
A vector (termed haploid AAV92 or H-AAV92) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV2; or
A vector having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2G9 (referred to as haploid AAV2G9 or H-AAV2G9), wherein the AAV9 glycan receptor binding site is grafted into AAV2. ;or
A vector (termed haploid AAV83 or H-AAV83) having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAV93 or H-AAV93) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAVrh10-3 or H-AAVrh10-3) having a VP1/VP2 capsid subunit from AAVrh10 and a VP3 capsid subunit from AAV3; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP2/VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2 capsid subunits from AAV2 and VP3 capsid subunits from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV8; or
28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N terminus derived from AAV2 and a C terminus derived from AAV8, and the VP3 capsid subunit is derived from AAV2; or a vector called chimeric AAV8/2 or chimeric AAV82, in which the chimeric VP1/VP2 capsid subunit has an AAV8-derived N terminus and an AAV2-derived C terminus, and a vector without mutations and in which the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus derived from AAV2 and a C-terminus derived from AAV8; or
G. a population, such as a substantially homogeneous population, such as a population of 1010 particles, such as a population of 1010 particles, of substantially homogeneous; or
H. A method of producing any one of the vectors or vector populations of A and/or B and/or C and/or D and/or E and/or F and/or G; or
I. Any combination thereof.

Without limitation, the inventors believe that the foregoing reservation of disclaimer applies at least to claims 1-30 as appended to this application and paragraphs 1-83 as set forth at [00437]. express that Modified viral capsids can be used as "capsid vehicles" as described, for example, in US Pat. No. 5,863,541. Molecules that can be packaged and transported into cells by modified viral capsids include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

In some embodiments, this application may be defined in any of the following paragraphs:
1. An isolated AAV virion having three viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, wherein the viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome. and the VP1 and VP2 viral structural proteins present are derived from the same serotype, the VP3 serotype is derived from an alternate serotype, and VP1 and VP2 are derived from only a single serotype, and VP3 is derived from a single serotype. Isolated AAV virions derived from only a single serotype.
2. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are derived from AAV serotype 8 or 9 and VP3 is derived from AAV serotype 3 or 2.
3. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are derived from AAV serotype 8 and VP3 is derived from AAV serotype 2G9.
4. An isolated AAV virion having three viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, wherein the viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome. and the VP1 and VP2 viral structural proteins present are derived from the same chimeric serotype, the VP3 serotype is not a chimeric serotype, and VP1 and VP2 are derived from only a single chimeric serotype, and the VP3 An isolated AAV virion in which VP1 and VP2 are derived from chimeric AAV serotype 28m and VP3 is derived from AAV serotype 2.
5. The isolated AAV virion of paragraph 1, wherein VP1 and VP2 are derived from AAV serotype AAV rh10 and VP3 is derived from AAV serotype 2G9.
6. A method of making adeno-associated virus (AAV) virions, the method comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, the method comprising: , VP1, VP2, and VP3 viral structural proteins, a first nucleic acid encoding VP1 and VP2 derived only from a first AAV serotype but unable to express VP3; The nucleic acid sequence of 2 encodes VP3 from an alternative AAV serotype different from the first AAV serotype, and neither VP1 nor VP2 can be expressed, and the AAV virions are exclusively derived from the first serotype. VP1 and VP2 derived from VP1 and VP3 derived only from a second serotype.
7. AAV virions produced by the method of paragraph 6.
8. The method of paragraph 2, wherein VP1 and VP2 are derived from AAV serotype 8 or 9 and VP3 is derived from AAV serotype 3 or 2.
9. The method of paragraph 2, wherein VP1 and VP2 are derived from AAV serotype 8 and VP3 is derived from AAV serotype 2G9.
10. A method of making adeno-associated virus (AAV) virions, the method comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, the method comprising: , formed from VP1, VP2, and VP3 viral structural proteins, the first nucleic acid encoding VP1 and VP2 derived only from a first chimeric AAV serotype but incapable of expressing VP3, and a second nucleic acid sequence encodes VP3 from an alternative AAV serotype; further, neither VP1 nor VP2 can be expressed; VP1 and VP2 are derived from AAV serotype 28m; and VP3 is derived from AAV serotype 2. how to.
11. The method of paragraph 2, wherein VP1 and VP2 are derived from AAV serotype AAV rh10 and VP3 is derived from AAV serotype 2G9.
12. Haploid vectors, where VP1/VP2 are derived from one AAV vector capsid and VP3 is derived from an alternate.
13. Haploid vector AAV82 (H-AAV82), in which VP1/VP2 is derived from AAV8 and VP3 is derived from AAV2.
14. Haploid vector AAV92 (H-AAV92), in which VP1/VP2 is derived from AAV9 and VP3 is derived from AAV2.
15. Haploid vector AAV82 G9 (H-AAV82G9), in which VP1/VP2 are derived from AAV8, VP3 is derived from AAV2G9, and AAV2G9 has an AAV9 glycan receptor binding site grafted into AAV2.
16. Haploid vector AAV83 (H-AAV83), where VP1/VP2 is derived from AAV8 and VP3 is derived from AAV3.
17. Haploid vector AAV93 (H-AAV93), where VP1/VP2 are derived from AAV9 and VP3 is derived from AAV3.
18. Haploid vector AAVrh10-3 (H-AAVrh10-3), where VP1/VP2 are derived from AAVrh10 and VP3 is derived from AAV3.
19. Vector 28m-2VP3 (H-28m-2VP3), in which the chimeric VP1/VP2 capsid subunit has an N terminus derived from AAV2 and a C terminus derived from AAV8, and the VP3 capsid subunit is derived from AAV2.
20. A vector referred to as chimeric AAV8/2 or chimeric AAV82, wherein the chimeric VP1/VP2 capsid subunit has an N terminus derived from AAV8 and a C terminus derived from AAV2, and has no mutation of the VP3 start codon; and , a vector in which the VP3 capsid subunit is derived from AAV2.

In some embodiments, this application may be defined in any of the following paragraphs:
1. A method of making a polyploid adeno-associated virus (AAV) capsid, the method comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, the method comprising: a capsid is formed from VP1, VP2, and VP3 capsid proteins, and the capsid protein is derived from a first nucleic acid derived only from the first AAV serotype and a second AAV serotype that is different from the first AAV serotype. wherein the first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid; Furthermore, the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and polyploid AAV capsids are present only in the first serotype. and VP2 and VP3 derived only from a second serotype.
2. an AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or Table 1 or Table 3, or any of each AAV; The method of paragraph 1, which is a chimera.
3. an AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV; The method of paragraph 1, which is a chimera of.
4. A method of making a polyploid adeno-associated virus (AAV) capsid comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, the method comprising: a capsid is formed from VP1, VP2, and VP3 capsid proteins, and the capsid protein is derived from a first nucleic acid derived only from the first AAV serotype and a second AAV serotype that is different from the first AAV serotype. further derived from a second nucleic acid, further the first nucleic acid has a mutation in the A2 splice acceptor site, further the second nucleic acid has a mutation in the A1 splice acceptor site, and , wherein the polyploid AAV capsid comprises VP1 derived only from a first serotype and VP2 and VP3 derived only from a second serotype.
5. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 4, which is a chimera of.
6. an AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV; The method of paragraph 4, which is a chimera of.
7. A polyploid adeno-associated virus (AAV) capsid comprising contacting a cell with a first nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence under conditions for the formation of AAV virions. A method of making a first AAV capsid, wherein the AAV capsid is formed from VP1, VP2, and VP3 capsid proteins, and the capsid protein is derived only from a first AAV serotype that is different from a second and a third serotype. a second nucleic acid derived solely from a second AAV serotype that is different from the first and third AAV serotypes; and a third AAV serotype that is different from the first and second AAV serotypes. wherein the first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid. , further, the second nucleic acid has a mutation in the start codon of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid, and further, the third nucleic acid has a mutation in the start codon of VP1 and VP3 that prevents translation of VP1 and VP3 from RNA transcribed from the second nucleic acid; VP1 and VP2 have mutations in the initiation codons of VP1 and VP2 that prevent translation of VP1 and VP2 from RNA transcribed from the nucleic acids of VP2 derived only from a serotype and VP3 derived only from a third serotype.
8. An AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 7, which is a chimera of.
9. An AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV. The method of paragraph 7, which is a chimera of.
10. an AAV in which the third AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV; The method of paragraph 7, which is a chimera of.
11. A method of making a polyploid adeno-associated virus (AAV) capsid, the method comprising contacting a cell with a first nucleic acid sequence and a second nucleic acid sequence under conditions for the formation of AAV virions, the method comprising: a capsid is assembled from VP1, VP2, and VP3 capsid proteins, and the capsid protein is derived from a first nucleic acid derived only from the first AAV serotype and a second AAV serotype different from the first AAV serotype. The first nucleic acid contains mutations in the start codons of VP2 and VP3 that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid, as well as mutations encoded only in the second nucleic acid derived from the first nucleic acid. in the A2 splice acceptor site, and the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid as well as in the A1 splice acceptor site. A method having a mutation and wherein the AAV polyploid capsid comprises VP1 derived only from a first serotype and VP2 and VP3 derived only from a second serotype.
12. an AAV in which the first AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV; The method of paragraph 11, which is a chimera of.
13. an AAV in which the second AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or from Table 1 or Table 3, or any of each AAV; The method of paragraph 11, which is a chimera of.
14. A method of making a polyploid adeno-associated virus (AAV) capsid, the method comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions for the formation of AAV virions. is formed from VP1, VP2, and VP3 capsid proteins, the capsid protein being encoded by and further transcribed from a first nucleic acid created through DNA shuffling of two or more different AAV serotypes. The start codons for VP2 and VP3 have been mutated to prevent translation of VP2 and VP3 from RNA, and the capsid protein is encoded by a second nucleic acid derived from only a single AAV serotype and a second the nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid, and the polyploid AAV capsid is derived from the first nucleic acid produced through DNA shuffling. A method comprising VP1 and VP2 and VP3 derived only from a second serotype.
15. A method of making a polyploid adeno-associated virus (AAV) capsid, the method comprising contacting a cell with a first nucleic acid and a second nucleic acid under conditions for the formation of AAV virions. is formed from VP1, VP2, and VP3 capsid proteins, the capsid protein being encoded by and further transcribed from a first nucleic acid created through DNA shuffling of two or more different AAV serotypes. The initiation codons for VP2 and VP3 have been mutated so that they cannot be translated from RNA, the A2 splice acceptor site of the first nucleic acid has been mutated, and the capsid protein has been mutated into a single Mutations in the start codon of VP1 encoded by a second nucleic acid derived exclusively from the AAV serotype that prevent translation of VP1 from RNA transcribed from the second nucleic acid as well as the A1 splice acceptor site and the polyploid AAV capsid comprises VP1 from a first nucleic acid created through DNA shuffling and VP2 and VP3 from only a second serotype.
16. An AAV whose AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV. The method of paragraphs 14 and 15.
17. The method of any of paragraphs 1-16, wherein the AAV capsid has a substantially homologous capsid protein.
18. The method of paragraph 17, wherein the substantially homologous capsid protein of polyploid adeno-associated virus (AAV) is VP1.
19. The method of paragraph 17, wherein the substantially homologous capsid protein is VP2.
20. The method of paragraph 17, wherein the substantially homologous capsid protein is VP3.
21. The method of paragraph 17, wherein the substantially homologous capsid proteins are VP1 and VP2, VP1 and VP3, VP2 and VP3, or VP1 and VP2 and VP3.
22. The method of any of paragraphs 1-21, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV capsids.
23. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions containing the capsid protein VP1 of only one serotype.
24. The method of paragraph 22, the method of paragraph 17, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions comprising the capsid protein VP2 of only one serotype.
25. The method of paragraph 22, wherein the polyploid adeno-associated virus (AAV) is in a substantially homogeneous population of AAV virions containing the capsid protein VP3 of only one serotype.
26. Polyploid adeno-associated virus (AAV) has the capsid proteins VP1 and VP2 of only one serotype, or VP1 and VP3 of only one serotype, or VP2 and VP3 of only one serotype, or one serotype The method of paragraph 22, wherein the AAV virion population is in a substantially homogeneous population containing only type VP1.
27. A polyploid AAV prepared using the method of any of paragraphs 1-26.
28. A polyploid AAV according to any of paragraphs 1-27, constructed from only VP1 and VP3.
29. A polyploid AAV prepared using the method of any of paragraphs 1-28, further comprising a heterologous gene.
30. The polyploid AAV of paragraph 29, wherein the heterologous gene encodes a protein for treating a disease.
31. The disease is caused by a lysosomal storage disease, such as a mucopolysaccharidosis (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [aL-iduronidase], Scheie syndrome [aL-iduronidase], Hurler-Scheie syndrome [aL-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D[N-acetylglucosamine 6-sulfatase] ], Morquio syndrome A [galactose-6-sulfatase], B[-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebro 31.

In some embodiments, this application may be defined in any of the following paragraphs:
1. An isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, wherein the two viral proteins form an AAV virion that encapsidates an AAV genome. an isolated AAV virion, wherein at least one of the other viral structural proteins present is different from other viral structural proteins, and the virion contains only each structural protein of the same type.
2. The isolated AAV virion of paragraph 1 in which all three viral structural proteins are present.
3. The isolated AAV virion of paragraphs 1 and 2, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one other viral structural protein.
4. The virion of paragraph 3, in which only VP3 is chimeric and VP1 and VP2 are non-chimeric.
5. The virion of paragraph 3, in which only VP1 and VP2 are chimeric and only VP3 is non-chimeric.
6. The virion of paragraph 5, wherein the chimera consists of subunits from AAV serotypes 2 and 8, and VP3 is derived from AAV serotype 2.
7. The isolated AAV virion of paragraphs 1-6, wherein all three viral structural proteins are from different serotypes.
8. The isolated AAV virion of paragraphs 1-6, in which only one of the three structural proteins is derived from a different serotype.
9. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 ⁷ virions.
10. A substantially homogeneous population of virions of paragraph 8 that is at least 10 ⁷ to 10 ¹⁵ virions.
11. A substantially homogeneous population of the virions of paragraph 8 that is at least ¹⁰⁹ virions.
12. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 ¹⁰ virions.
13. A substantially homogeneous population of the virions of paragraph 8 that is at least 10 to ¹¹ virions.
14. A substantially homogeneous population of virions according to paragraphs 9 to 13 that is at least 95% homogeneous.
15. A substantially homogeneous population of virions of paragraph 14 that is at least 99% homogeneous.
16. An AAV whose AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV. Billion in paragraphs 1-15.
17. A substantially homogeneous population of the virions of paragraph 16.
18. The AAV virion of paragraphs 1-17, wherein the heterologous gene encodes a protein for treating a disease.
19. The disease is caused by a lysosomal storage disease, such as a mucopolysaccharidosis (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [aL-iduronidase], Scheie syndrome [aL-iduronidase], Hurler-Scheie syndrome [aL-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase] ], Morquio syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebro 19.
20. The isolated AAV virion of any of paragraphs 1-2 and 8-19, wherein none of the viral structural proteins are chimeric viral structural proteins.
21. The isolated AAV virion of paragraphs 1-19, wherein there is no serotype overlap between the chimeric viral structural protein and at least one other viral structural protein.
22. Treating a disease comprising administering an effective amount of the virions of any of paragraphs 1-9, 16, 18-21 or a substantially homogeneous population of the virions of any of paragraphs 10-15 and 17. A method,
the heterologous gene encodes a protein for treating a disease that is suitable for treatment by gene therapy in a subject having the disease;
Said method.
23. The method of paragraph 22, wherein the disease is selected from genetic diseases, cancer, immunological diseases, inflammation, autoimmune diseases, and degenerative diseases.
24. The method of any of paragraphs 22 and 23, wherein multiple administrations are performed.
25. The method of paragraph 24, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration.
26. Isolated AAV virions in paragraphs 1-25, Applicant disclaims as follows: Any disclosure in PCT/US18/22725, filed on March 15, 2018, does not constitute any one of the claims of this application. or within the scope of the present invention as defined herein or within the scope of any invention as defined in any amended claim that may be filed in the future in this application or in any patent derived therefrom. To the extent that the disclosure of PCT/US18/22725 is within the art of the art for such claims in or for that country, the law of any relevant country or countries to which the or those claims apply applies. The inventors hereby intend to use this application or any patent derived therefrom only to the extent necessary to prevent invalidation of this application or any patent derived therefrom. reserves the right to disclaim such disclosure from any patent claims derived therefrom.

By way of example and without limitation, the inventors disclaim from any claim of this application or any patent derived therefrom, now or hereafter amended, any one or more of the following subject matter: have the right to:
A. Any subject matter disclosed in Example 9 of PCT/US18/22725; or
B. Polyploid, produced or producible by transfection of two AAV helper plasmids or three plasmids to produce individual polyploid vector virions composed of different capsid subunits from different serotypes. Vector virions called vector virions; or
C. Produced or producible by transfection of two AAV helper plasmids, AAV2 and AAV8 or AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes , vector virions called polyploid vector virions; or
D. Produced or producible by transfection of three AAV helper plasmids, AAV2, AAV8 and AAV9, to produce individual polyploid vector virions consisting of different capsid subunits from different serotypes. , vector virions called polyploid vector virions; or
E. VP1/VP2 is derived from one AAV vector capsid or AAV serotype and VP3 is derived from an alternative, e.g. VP1/VP2 is derived from only one AAV serotype (from its capsid) and VP3 vector virions, called haploid vectors, derived from only one alternative AAV serotype; or
F. Any one or more AAV vector virions selected from:
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP2/VP3 capsid subunit from AAV2; or
A vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8 or haploid AAV8/2 or haploid AAV82 or H-AAV82), comprising a VP1/VP2 capsid subunit derived from AAV8 and a VP1/VP2 capsid subunit derived from AAV2. a vector having a VP3 capsid subunit of; or
Vectors in which VP1/VP2 are derived from different serotypes; or
A vector (termed haploid AAV92 or H-AAV92) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV2; or
A vector having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2G9 (referred to as haploid AAV2G9 or H-AAV2G9), wherein the AAV9 glycan receptor binding site is grafted into AAV2. ;or
A vector (termed haploid AAV83 or H-AAV83) having a VP1/VP2 capsid subunit from AAV8 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAV93 or H-AAV93) having a VP1/VP2 capsid subunit from AAV9 and a VP3 capsid subunit from AAV3; or
A vector (termed haploid AAVrh10-3 or H-AAVrh10-3) having a VP1/VP2 capsid subunit from AAVrh10 and a VP3 capsid subunit from AAV3; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP2/VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2 capsid subunits from AAV2 and VP3 capsid subunits from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV8 and a VP3 capsid subunit from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having a VP1 capsid subunit from AAV2 and a VP3 capsid subunit from AAV8; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (referred to as haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV2; or
a vector produced by transfection of AAV2 helper and AAV8 helper plasmids (termed haploid AAV2/8), having VP1/VP2/VP3 capsid subunits from AAV8; or
28m-2VP3 or haploid 2m-2VP3 or haploid vector 28m-2VP3, wherein the chimeric VP1/VP2 capsid subunit has an N terminus derived from AAV2 and a C terminus derived from AAV8, and the VP3 capsid subunit is derived from AAV2; or a vector called chimeric AAV8/2 or chimeric AAV82, in which the chimeric VP1/VP2 capsid subunit has an AAV8-derived N terminus and an AAV2-derived C terminus, and a vector without mutations and in which the VP3 capsid subunit is derived from AAV2; or a vector in which the chimeric VP1/VP2 capsid subunit has an N-terminus derived from AAV2 and a C-terminus derived from AAV8; or
G. a population, such as a substantially homogeneous population, such as a population of 1010 particles, such as a population of 1010 particles, of substantially homogeneous; or
H. A method of producing any one of the vectors or vector populations of A and/or B and/or C and/or D and/or E and/or F and/or G; or
I. Any combination thereof.

Without limitation, the inventors believe that the foregoing reservation of disclaimer applies at least to claims 1-30 as appended to this application and paragraphs 1-83 as set forth at [00437]. express that Modified viral capsids can be used as "capsid vehicles" as described, for example, in US Pat. No. 5,863,541. Molecules that can be packaged and transported into cells by modified viral capsids include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof.

Example 1: Application of polyploid adeno-associated virus vectors for enhanced transduction and evasion of neutralizing antibodies Adeno-associated virus (AAV) vectors have been used successfully in clinical trials in hemophiliacs and blind patients. . The exploration of effective strategies to enhance AAV transduction and avoid neutralizing antibody activity remains essential. Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites for AAV Nabs located on different capsid subunits of one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and demonstrate their transduction and Nab Avoidance activity was studied. Haploid virus yield was similar to that of the parent, and heparin sulfate binding capacity was positively correlated with AAV2 capsid input. To determine whether mixing capsid proteins altered the tropism of these haploid vectors, we analyzed the transduction efficacy of haploid viruses by transducing human Huh7 and mouse C2Cl2 cell lines. (Figure 1). Haploid vector transduction was lower than AAV2 in Huh7 cells, but haploid vector AAV2/8 3:1 induced 3-fold higher transduction than AAV2 in C2C12 cells.

After intramuscular injection, all haploid viruses induced higher transduction than the parental AAV vector (2-9 times more than AAV2), highest with the haploid vector AAV2/8 1:3. After systemic administration, 4-fold higher transduction was observed in the liver with haploid AAV2/8 1:3 than with AAV8 alone. Haploid AAV2/89 and their parental vectors were injected directly into the hind limb muscles of C57Bl6 mice. As a control, mixtures of AAV2 and AAV8 viruses in ratios of 3:1, 1:1 and 1:3 were also investigated. For simple comparison, one leg was injected with AAV2 and the opposite leg was injected with haploid vector. Similar muscle transduction was achieved with the parental AAV8 capsid compared to AAV2 (Figure 2). Contrary to the results in C2C12 cells, enhanced muscle transduction was observed from all of the haploid viruses (Fig. 2). Haploid vectors AAV2/9 1:1 and AAV2/8 1:3 achieved 4-fold and 2-fold higher transduction than AAV2, respectively. Notably, muscle transduction of haploid vector AAV2/8 3:1 was more than 6-fold higher than that of AAV2. However, all controls (injections with physical mixing of the parental vectors) had similar transduction efficiencies as the AAV2 vector.

Additionally, we packaged therapeutic factor IX cassettes into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Compared to AAV8 viral vectors, higher FIX expression and improved phenotypic correction were achieved using haploid AAV2/8 1:3 viral vectors. In addition, haploid virus AAV2/8 1:3 was able to evade AAV2 neutralization and had very low Nab cross-reactivity with AAV2.

To improve the Nab evasion ability of polyploid viruses, we constructed the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids in a 1:1:1 ratio. produced a vector. After systemic administration, two-fold higher transduction was observed in the liver with triploid vector AAV2/8/9 than with AAV8. Neutralizing antibody analysis demonstrated that the AAV2/8/9 vector was able to evade neutralizing antibody activity from mouse serum immunized with the parental serotype. These results indicate that polyploid viruses can potentially acquire significance from their parental serotypes for enhanced transduction and evasion of Nab recognition. This strategy should be considered in future clinical trials in patients positive for neutralizing antibodies.

The number of helper plasmids with different cap genes can be mixed and matched based on the specific requirements of a particular treatment regimen, without limitation.

cell line
HEK293, Huh7 and C2C12 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 10% penicillin-streptomycin at 37°C, 5% CO2.

Recombinant AAV virus production Recombinant AAV was produced by a triple plasmid transfection system. HEK293 cells in a 15 cm dish were transfected with 9 μg of AAV transgene plasmid pTR/CBA-Luc, 12 μg of AAV helper plasmid and 15 μg of Ad helper plasmid XX680. To generate triploid AAV2/8 virions, the amount of each helper plasmid to transfected AAV2 or AAV8 was co-transfected at three different ratios: 1:1, 1:3 and 3:1. To generate haploid AAV2/8/9 vectors, the ratio of helper plasmids for each serotype was 1:1:1. Sixty hours after transfection, HEK293 cells were harvested and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Virus titer was determined by quantitative PCR.

Western and Immunoblots According to viral titer, the same amount of virions was loaded into each lane and subsequently electrophoresed on NuPage 4-10% polyacrylamide Bis-Tris gels (Invitrogen, Carlsbad, CA) and then iBlot ( 2 dry blotting system (Invitrogen, Carlsbad, Calif.) onto a PVDF membrane. The membrane was incubated with B1 antibody specific for AAV capsid protein.

Native immunoblot assays were performed as previously described. Briefly, purified capsids were transferred to Hybond-ECL membranes (Amersham, Piscataway, NJ) by using a vacuum dot blotter. Membranes were blocked in 10% milk PBS for 1 hour and then incubated with monoclonal antibodies A20 or ADK8. The membrane was incubated with peroxidase-conjugated goat anti-mouse antibody for l hours. Proteins were visualized with an Amersham Imager 600 (GE Healthcare Biosciences, Pittsburg, PA).

In vitro transduction assay Huh7 and C2C12 cells were transduced with recombinant viruses at 1×10 ⁴ vg/cell in flat-bottomed 24-well plates. After 48 hours, cells were harvested and evaluated by luciferase assay system (Promega, Madison, WI).

Heparin inhibition assay
The ability of soluble heparin to inhibit binding of recombinant virus to Huh7 or C2C12 cells was assayed. Briefly, AAV2, AAV8, haploid viruses AAV2/8 1:1, AAV2/8 1:3 and AAV2/8 3:1 were mixed at 37°C in the presence or absence of soluble HS in DMEM. Incubated for hours. After preincubation, the mixture of recombinant viruses and soluble HS were added to Huh7 or C2C12 cells. Forty-eight hours after transduction, cells were harvested and evaluated by luciferase assay.

Antigen presentation from haploid AAV capsids is similar to that of AAV8 in vivo. To study the efficacy of capsid antigen presentation, we tested pXR2-OVA and pXR8-OVA in a 1:3 ratio. A haploid AAV2/8 OVA 1:3 vector was produced by transfection. 1×10 ¹¹ vg of AAV2/8-OVA and AAV8-OVA vector were administered to C57BL/6 mice via retroorbital injection. Three days later, CFSE-labeled OT-1 mouse spleen cells were transferred into C57BL/6 mice. Ten days after transfer of OT-1 splenocytes, T cell proliferation was measured by flow cytometry. OT-1 T cell proliferation was significantly increased in mice receiving AAV2/8-OVA 1:3 or AAV8-OVA compared to control mice without AAV vector administration (Figure 5). However, there was no difference between the AAV2/8-OVA 1:3 and AAV8-OVA groups regarding OT-1 cell proliferation.

Animal studies The animal experiments performed in this study were performed on C57BL/6 mice and FIX-/- mice. Mice were maintained according to NIH guidelines as approved by the UNC Animal Care and Use Committee (IACUC). Six-week-old female C57BL/6 mice were injected with 3×10 ¹⁰ vg of recombinant virus via retroorbital injection. After intraperitoneal (ip) injection of D-luciferin substrate (Nanolight Pinetop, AZ), luciferase expression was imaged 1 week after injection using a Xenogen IVIS Lumina (Caliper Lifesciences, Waltham, MA). Bioluminescence images were analyzed using Living Image (PerkinElmer, Waltham, MA). For muscle transduction, 1×10 ¹⁰ AAV/Luc particles were injected into the gastrocnemius muscle of 6-week-old female C57BL/6. Mice were imaged at the indicated time points.

Next, we evaluated the transduction efficiency of haploid virus in mouse liver. A mixture of AAV2 and AAV8 viruses was also injected as a control. A dose of 3×10 ¹⁰ vg of recombinant virus was injected into C57BL/6 mice via the retroorbital vein, and imaging was performed 3 days after AAV injection. Haploid virus AAV2/8 1:3 induced the highest transduction efficiency in mouse liver against other haploid combinations, a mixture of parental viruses and parental AAV8 (Figures 3A and 3B). The transduction efficiency of haploid vector AAV2/8 1:3 was approximately 4 times higher than that of AAV8 (Fig. 3B). Liver transduction from other haploid viruses was lower than that from the parental AAV8, but higher than that from AAV2 (Figures 3A and 3B). Seven days after injection, mice were sacrificed, livers were removed, and genomic DNA was isolated. The luciferase gene copy number in the liver was determined by qPCR. In contrast to the liver transduction efficiency results, similar AAV vector genome copy numbers were found in the liver regardless of viral composition (Fig. 3C). When transgene expression was normalized to gene copy number, haploid vector AAV2/8 1:3 induced the highest relative transgene expression compared to any other haploid vector combination or parental serotype (Figure 3D).

FIX knockout male mice (FIX KO mice) were given 1 × ¹⁰ vg via tail vein injection. Blood was drawn from the retroorbital plexus at various times post-injection. At 6 weeks, mouse bleeding analysis was performed.

Quantification of Luciferase Expression in the Liver Animals used in imaging studies were sacrificed 4 weeks after recombinant virus injection and livers were collected. The liver was minced and homogenized in passive lysis buffer. After centrifuging the liver lysate, luciferase activity was detected in the supernatant. Total protein concentration in tissue lysates was measured using the Bradford assay (BioRad, Hercules, CA).

Detection of AAV genome copy number in liver Finely minced liver was treated with protease K. Total genomic DNA was isolated by PureLink Genomic DNA mini Kit (Invitrogen, Carlsbad, CA). Luciferase gene was detected by qPCR assay. The mouse lamin gene served as an internal control.

Human FIX Expression, Function and Tail Bleeding Time Assay Human FIX expression, one-step hFIX activity assay and tail bleeding time assay were performed as previously described. For neutralization assays, Huh7 cells were seeded in 48-well plates at a density of 10 ⁵ cells per well. Two-fold dilutions of mouse antibodies were incubated with AAV-Luc (1×10 ⁸ vg) for 1 hour at 37°C. The mixture was added to the cells and incubated at 37°C for 48 hours. Cells were lysed with passive lysis buffer (Promega, Madison, WI) and luciferase activity was measured. Nab titer was defined as the highest dilution at which luciferase activity was 50% lower than the serum-free control.

Statistical analysis Data were presented as mean ± SD. All statistical analyzes were performed using Student's t-test. A P value <0.05 was considered a statistically significant difference.

We tested whether AAV2/8 1:3 could increase therapeutic transgene expression in animal disease models. Using human FIX (hFIX or human factor IX) as a therapeutic gene, the haploid vector AAV2/8 1:3/hFIX was administered via the tail vein to FIX knockout (KO) mice at a dose of 1 × ¹⁰ vg/mouse. Injected with. The haploid vector encodes the human-optimized FIX transgene and is driven by the liver-specific promoter TTR. At 1, 2 and 4 weeks post-injection, hFIX expression and activity in the blood circulation was analyzed by ELISA and one-step factor activity, respectively. At week 6, blood loss with respect to in vivo hFIX function was assessed using a tail clipping assay. Consistent with the observation of high liver transduction with haploid AAV vectors in wild-type C57BL/6 mice, haploid vector AAV2/8 1:3 liver targeting resulted in significantly more hFIX than AAV8 vectors two weeks after injection. was produced (Figure 4A). Higher hFIX protein expression in AAV2/8 1:3 was associated with higher FIX activity, as expected (Fig. 4B). Blood loss in mice receiving AAV2/8 1:3/hFIX injections was similar to that in wild-type C57BL/6 mice and much lower than in KO mice (Figure 4C). There was no statistically significant difference in blood loss between mice receiving AAV8 and AAV2/8 1:3/hFIX injections, although AAV8 mice lost slightly more blood than AAV2/8 1:3 mice ( Figure 4C).

Ability of haploid virus AAV2/8 to evade Nabs To study whether haploid viruses are able to evade Nabs generated in response to the parental vector, we performed Nab binding assays using monoclonal antibodies by immunoblot assays. carried out. Three dilutions of viral genome-containing particles were adsorbed onto nitrocellulose membranes and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. The haploid virus neutralization profile for A20 and ADK9 was similar to native immunoblot data (Table 5). Haploid AAV2/8 1:3 almost completely evaded AAV2 serum and A20 neutralization, suggesting that this haploid virus has the potential to be used in individuals with anti-AAV2 Nabs. (Table 5).

In vitro characterization of haploid viruses. Our previous studies have demonstrated capsid compatibility between AAV1, 2, 3 and 5 capsids. Haploid viruses were produced by transfection of different ratios of AAV helper plasmids from the two serotypes using the AAV transgene and adenovirus helper pXX6-80. Enhanced transduction from haploid viruses was observed in several cell lines compared to the parental vector. AAV2 is well characterized regarding its biology and as a gene delivery vehicle, and AAV8 has received much attention because of its high transduction in mouse liver. Both serotypes have been used in several clinical trials in hemophilia patients. To investigate the potential of AAV serotypes 2 and 8 capsids to form haploid viruses and their transduction profiles, we constructed helper plasmids for AAV2 and AAV8 at 3:1, 1:1 and 1:1. Haploid vectors were generated by transfecting at a ratio of 3:3. All haploid viruses were purified using cesium gradients and titered by Q-PCR. There were no significant differences in virus yield between haploid viruses and parental AAV2 or AAV8. To determine whether haploid virus capsid proteins were expressed, Western blot analysis was performed on equivalent viral genomes from purified haploid viruses using monoclonal antibody B1, which recognizes AAV2 and AAV8 capsid proteins. carried out. A mixture of AAV2- and AAV8-derived VP2 capsids was observed in all haploid viruses, and the strength of AAV2- or AAV8-derived VP2 capsids in haploid viruses was related to the ratio of the two helper plasmids. These results suggested that AAV2- and AAV8-derived capsids were compatible and able to assemble into AAV virions.

To determine whether the tropism of haploid viruses was altered by mixing capsid proteins, we analyzed the transduction efficacy of haploid viruses by transducing human Huh7 and mouse C2Cl2 cell lines. The transduction efficiency of AAV8 was much lower than AAV2 in both cell lines. Transduction from all haploid vectors was higher than for AAV8, and the efficiency correlated positively with addition of AAV2 capsids in both cell lines. Haploid vector transduction was lower than AAV2 in Huh7 cells, whereas haploid vector AAV2/8 3:1 induced 3-fold higher transduction than AAV2 in C2C12 cells.

This in vitro transduction data confirms that the virus preparation consists of haploid vectors rather than a mixture of individual serotype vectors, indicating that haploid vectors can enhance AAV transduction. Heparin sulfate proteoglycans have been identified as the main receptor for AAV2. Next, we investigated whether inhibition of heparin binding ability altered haploid virus transduction. Preincubation of AAV vector with soluble heparin blocked AAV2 transduction by almost 100% in both Huh7 and C2C12 cells, and AAV8 transduction by 37% and 56% in Huh7 and C2C12 cells, respectively. Inhibition of haploid vector transduction by soluble heparin was dependent on AAV2 capsid input in both cell lines. Higher inhibition of transduction was observed with higher AAV2 capsid input. This result suggests that haploid viruses can use both primary receptors from the parental vector for effective transduction [Figure 1].

Increased Haploid Virus Muscle Transduction As mentioned above, the transduction efficiency of haploid virus AAV2/8 3:1 is higher than that of AAV2 and AAV8 in the muscle cell line C2C12. Next, we investigated whether the high transduction in vitro was transferred to mouse muscle tissue. AAV2/8 haploid and parental vector were injected directly into the hind limb muscles of C57BL/6 mice. As a control, mixtures of AAV2 and AAV8 viruses in ratios of 3:1, 1:1 and 1:3 were also investigated. For simple comparison, one leg was injected with AAV2 and the other leg was injected with the test vector. A total of 1×10 ¹⁰ vg of vector was administered for each virus. Similar muscle transduction was achieved with AAV8 compared to AAV2. Contrary to the results with C2C12 cells, enhanced muscle transduction was observed from all of the haploid viruses [Figure 2].

Haploid vectors AAV2/8 1:1 and AAV2/8 1:3 achieved 4-fold and 2-fold higher transduction than AAV2, respectively. Of note, muscle transduction of haploid vector AAV2/8 3:1 was more than 6-fold higher than that of AAV2. However, all mixed viruses had similar transduction efficiency to AAV2. These results suggest that haploid viruses can increase muscle transduction and further support that the virus produced from cotransfection of two capsid plasmids is haploid.

Enhanced liver transduction of haploid viruses
AAV2 and AAV8 have been used for liver targeting in several clinical trials in hemophilia B patients. We also evaluated the transduction efficiency of haploid viruses in mouse liver. Viruses mixed with AAV2 and AAV8 were also injected as controls. A dose of 3×10 ¹⁰ vg of AAV/luc vector was administered to C57BL mice via the retroorbital vein; imaging was performed on day 3 after AAV injection. Haploid virus AAV2/8 1:3 induced the highest transduction efficiency in mouse liver compared to other haploids, mixed viruses and even parental AAV8 [Figures 3A and 3B]. The transduction efficiency of haploid vector AAV2/8 1:3 was approximately 4 times higher than that of AAV8 [Figure 3B]. Liver transduction from other haploid viruses was lower than from the parental vector AAV8, but higher than AAV2 [Figures 3A and 3B]. Seven days after injection, mice were sacrificed, livers were harvested, and genomic DNA was isolated. The luciferase gene copy number in the liver was determined by qPCR. In contrast to the liver transduction efficiency results, similar AAV vector genome copy numbers were found in the liver regardless of haploid virus or AAV serotypes 2 and 8 [Figure 3C]. Consistent with transgene expression in the liver, haploid vector AAV2/8 1:3 has the highest relative transduction compared to all other haploid vectors and serotypes when transgene expression is normalized to gene copy number. induced gene expression [Figure 3D]. The transduction profile of haploid viruses in the liver was different from that in muscle transduction, with all haploid viruses inducing higher transgene expression than for the parental serotype, and The one was the best.

Increased therapeutic FIX expression and improved bleeding phenotype correction by haploid vectors in a hemophilia B mouse model. Based on the above results, haploid vector AAV2/8 1:3 has much higher hepatic transduction than AAV8. was induced. Next, we further tested whether the haploid vector AAV2/8 1:3 could increase therapeutic transgene expression in animal disease models. We used human FIX (hFIX) as a therapeutic agent and used the haploid vector AAV2/8 1:3 encoding a human optimized FIX transgene and driven by the liver-specific promoter TTR. /hFIX was injected into FIX knockout (KO) mice via the tail vein at a dose of 1×10 ¹⁰ vg/mouse. At 1, 2 and 4 weeks post-injection, hFIX expression and activity in the blood circulation was analyzed by ELISA and one-step factor activity, respectively. At week 6, blood loss with respect to in vivo hFIX function was assessed using a tail clipping assay. Consistent with the observation of high liver transduction with haploid AAV vectors in wild-type C57BL/6 mice, haploid vector AAV2/8 1:3 liver targeting resulted in significantly more hFIX than AAV8 vectors two weeks after injection. [Figure 4A]. Higher hFIX protein expression in AAV2/8 1:3 was closely associated with higher FIX activity [Figure 4B]. Blood loss in mice receiving AAV2/8 1:3/hFIX injections was similar to that in wild-type C57BL/6 mice and lower than in KO mice [Figure 4C]. However, AAV8-treated mice lost more blood than wild-type mice [Figure 4C]. These data show that haploid vector AAV2/8 1:3 increases therapeutic transgene expression from the liver and improves disease phenotype correction.

Ability of Haploid Virus AAV2/8 to Evade Neutralizing Antibodies Each individual haploid virus virion consists of 60 subunits from different AAV serotype capsids. Insertion of several capsid subunits from one serotype into other capsid subunits from a different serotype can change the virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of one single virion. To study whether haploid viruses are able to evade Nabs generated from the parental vector, we first performed Nab binding assays using monoclonal antibodies by immunoblot assays. Three dilutions of viral genome-containing particles were adsorbed onto nitrocellulose membranes and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. All haploid viruses and viruses with a mixture of AAV2 and AAV8 were recognized by monoclonal antibodies ADK8 or A20. The reactivity of haploid viruses with A20 was increased by incorporating more AAV2 capsids into haploid virus virions. However, there were no obvious changes in anti-AAV8 Nab ADK8 recognition among haploid viruses, regardless of capsid ratio. Notably, the binding of haploid AAV2/8 1:3 to A20 is much weaker than that of the parental AAV2 and the virus of a mixture of AAV2 and AAV8 (1:3 ratio), indicating that haploid AAV2/8 1:3 indicates a depletion of A20 binding sites on the virion surface.

Next, we analyzed the immunological profile of haploid viruses on serum from AAV-immunized mice. Nab titers were used to assess the ability of serum to inhibit vector transduction. Four weeks after injection, serum was collected from mice treated with parental virus. As shown in Table 5, the haploid virus neutralization profile against A20 or ADK8 was similar to data from native immunoblots. There was no Nab cross-reactivity between AAV8 and AAV2. Interestingly, serum from AAV8-immunized mice had similar neutralizing activity against all AAV8 and haploid viruses, regardless of the amount of AAV8 capsid incorporation, but against viruses mixed with AAV2 and AAV8. However, it did not have one. The lack of inhibition of AAV8 serum against mixed viruses may be explained by the superior transduction of AAV2 to AAV8 in the tested cell lines. However, haploid viruses partially evaded neutralization from AAV2 serum. Haploid AAV2/8 1:1 transduction was reduced 16-fold compared to parental AAV2 after incubation of virus and anti-AAV2 serum. The ability to evade AAV2 serum Nab for haploid viruses was much higher than for viruses mixed with AAV2 and AAV8. Remarkably, haploid AAV2/8 1:3 almost completely evaded AAV2 serum and A20 neutralization, indicating that haploid viruses have the potential to be used in individuals with anti-AAV2 Nabs. This suggests that (Table 5).

Improved ability to evade neutralizing antibodies using triploid vectors generated from three serotypes Our data above indicate that haploid AAV2/8 viruses were unable to evade AAV8 neutralizing antibody activity; demonstrated that AAV8 had the ability to evade AAV2 neutralizing antibodies, which was dependent on the amount of capsid integration. To study whether polyploid viruses made from more serotype capsids had improved Nab evasion ability, we tested polyploid viruses AAV2/8/9 (1:1:1 ratio). was created. After injecting the triploid vector AAV2/8/9 into mice, compared to AAV2, the triploid virus AAV2/8/9 induced 2-fold higher transduction in the liver than AAV8. No differences in liver transduction were observed among AAV8 and the haploid vectors AAV2/9 and AAV8/9 (triploid vectors were generated from two AAV helper plasmids in a 1:1 ratio). Systemic administration of AAV9 was shown to induce higher liver transduction than AAV8. When performing neutralizing antibody assays, the haploid AAV2/8/9 vector improved its Nab evasion ability by approximately 20-fold, 32-fold and 8-fold compared to AAV2, 8 and 9, respectively (Table 6).

In this study, polyploid AAV virions were assembled from capsids of two or three serotypes. The ability of haploid viruses to bind to the AAV2 major receptor heparin was dependent on the amount of AAV2 capsid input. While all haploid viruses achieved higher transduction efficacy in mouse muscle and liver than the parental AAV2 vector, the haploid virus AAV2/8 1:3 showed more pronounced liver transduction than the parental AAV8 vector. It had an enhancement. Compared to AAV8, systemic administration of haploid virus AAV2/8 1:3 to deliver human FIX induced much higher FIX expression and improved hemophilic phenotype correction in FIX-/- mice . Importantly, haploid virus AAV2/8 1:3 was able to evade neutralization of anti-AAV2 serum. Integration of AAV9 capsids into haploid AAV2/8 virions further improved their ability to evade neutralizing antibodies.

The main receptor for AAV2 is HSPG, but the main receptor for AAV8 remains unknown. To study whether haploid viruses can use receptors from both AAV2 and AAV8, we performed a heparin inhibition assay to test the ability of haploid viruses to bind to heparin receptor motifs. did. Heparin inhibition results in Huh7 and C2C12 cell lines confirm that haploid viruses use the heparin receptor motif of the AAV2 capsid for efficient transduction. To some extent, AAV8 also showed reduced transduction efficiency in the presence of heparin, but the transduction efficiency was still higher than that of AAV2.

One of the most challenging aspects of efficient transduction in clinical trials is the widespread incidence of neutralizing antibodies against AAV vectors. Nab-mediated clearance of AAV vectors has been a limiting factor for repeated administration of AAV gene transfer. Several studies have considered genetically modifying AAV capsids for Nab evasion by rational mutation of neutralizing antibody recognition sites or directed evolution approaches. Capsid mutations can alter AAV tropism and transduction efficiency. In addition, the identification of Nab binding sites on AAV virions has lagged behind vector application in clinical trials, and it is not possible to find all Nab binding sites from poly sera. Previous studies have demonstrated that the recognition sites of some AAV monoclonal antibodies are spread over different subunits of one virion. When AAV8 capsids were introduced into AAV2 virions, A20 binding capacity and neutralizing activity from AAV2-immunized sera was dramatically reduced against haploid viruses. Integration of AAV2 capsids into AAV8 virions did not reduce their ability to bind the intact AAV8 monoclonal antibody ADK8 or circumvent the neutralizing activity of anti-AAV8 serum (Table 5). This suggests that all Nab recognition sites from polyserum may be located on the same subunit of the AAV8 virion. The results also suggest that AAV8 capsids integrated into AAV2 virions may play a major role in the intracellular trafficking of the virus.

When triploid viruses were generated from capsids of three serotypes AAV2, 8 and 9, unlike the triploid vector AAV2/8, haploid AAV2/8/9 viruses were generated from serum from AAV2, 8 or 9 immunized mice. AAV8 and AAV9 share a similar transduction pathway.

Several lines of evidence from this study support polyploid virion assembly from transfection of two or three AAV helper plasmids. (1) Using Western blot analysis, two VP2 bands of different sizes were displayed from the haploid virus. These VP2s are consistent in size from different serotypes. (2) The transduction profile was different in C2C12 cells versus Huh7 cells. The haploid AAV2/8 3:1 vector specifically demonstrated lower transduction in Huh7 cells than with AAV2, but higher in C2C12 cells. (3) Higher muscle transduction was demonstrated with all haploid AAV2/8 viruses compared to viruses with parental vectors AAV2 and AAV8 and a mixture of AAV2 and AAV8. (4) Triploid virus AAV2/8 1:3 had enhanced hepatotropism compared to AAV8. (5) The binding pattern of haploid viruses to A20 and ADK8 is different from that of viruses with a mixture of AAV2 and AAV8. (6) The profile of AAV2 serum neutralizing activity differs between haploid viruses and mixed viruses. (7) Triploid AAV2/8/9 viruses evade neutralizing antibody activity in sera from mice immunized with any parental serotype.

These polyploid viruses enhance transduction efficiency in vitro and in vivo and even evade neutralization from parental vector immunizing sera. Application of a polyploid virus to deliver the therapeutic transgene FIX was able to increase FIX expression and improve hemophilic phenotype correction in FIX-deficient mice. These results demonstrate that haploid AAV vectors have the ability to enhance transduction and evade Nabs.

Example 2: Enhanced AAV transduction from haploid AAV vectors by assembly of AAV virions with VP1/VP2 from one AAV vector and VP3 from an alternative by application of theoretical polyploid methodology In the above studies, the present invention They demonstrated that increased AAV transduction was achieved using polyploid vectors produced by transfection of two AAV helper plasmids (AAV2 and AAV8 or AAV9) or three plasmids (AAV2, AAV8 and AAV9). It has been demonstrated that These individual polyploid vector virions may be composed of different capsid subunits from different serotypes. For example, haploid AAV2/8 produced by transfection of AAV2 helper and AAV8 helper plasmids may have different combinations of capsid subunits in one virion for effective transduction: VP1 from AAV8 and VP1 from AAV2. VP2/VP3 from AAV8, or VP1/VP2 from AAV8 and VP3 from AAV2, or VP1 from AAV2 and VP2/VP3 from AAV8, or VP1/VP2 from AAV2 and VP3 from AAV8, or VP1 from AAV8 and VP3 from AAV2, or VP1 from AAV2 and VP3 from AAV8, or VP1/VP2/VP3 from AAV2, or VP1/VP2/VP3 from AAV8. In the following studies, we found that enhanced transduction can be achieved from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from an alternative.

The production of VP1, VP2, and VP3 by different AAV serotypes provides two different strategies to produce these different proteins. Interestingly, the VP protein is translated from a single CAP nucleotide sequence with overlapping sequences for VP1, VP2, and VP3.

The Cap gene encodes three proteins VP1, VP2, and VP3. As shown in the above figure, VP1 contains VP2 and VP3 proteins, and VP2 contains VP3 protein. Thus, the Cap gene has three segments: the start of VP1 - the start of VP2 - the start of VP3 - the ends of all three VP proteins.

When sourced from Cap genes from two different AAV serotypes (designated A and B), there are six possible combinations of three Cap proteins. In one case, VP1 identified as serotype A, which can be any serotype (or a chimeric or other non-naturally occurring AAV), is derived only from the first serotype A and serotype VP2/VP3, identified as B, is derived solely from serotype B and is a different serotype from the VP1 serotype (or chimeric or other non-naturally occurring AAV). In one case, both VP1 and VP2 are derived only from the first serotype A and VP3 is derived only from serotype B. Methods of producing VP1 of a first serotype and VP2/VP3 of a second serotype; or VP1/VP2 from a first serotype and VP3 from a second serotype are provided herein. Disclosed in the Examples. In one case, VP1 and VP3 are derived only from the first serotype and VP2 is derived only from the second serotype.

When sourced from Cap genes from three different AAV serotypes (designated A, B and C), there are six possible combinations of the three Cap proteins. In this case, VP1, identified as serotype A, which can be any serotype (or chimeric or other non-naturally occurring AAV), is the first serotype distinct from serotypes VP2 and VP3. VP2, identified as serotype B, which is a different serotype from VP1 and VP3 serotypes (or chimeric or other non-naturally occurring AAVs), is derived from a second serotype; and VP1 (or chimeric or other non-naturally occurring AAV) and the serotype of VP3, identified as serotype C, which is a different serotype than the serotype of VP2, is derived from a third serotype. Methods of making a first serotype VP1, a second serotype VP2 and a third serotype VP3 are disclosed in the Examples presented herein.

In some embodiments, when VP1 is identified as a first serotype A and VP2 and VP3 are identified as a second serotype B, in one embodiment this means that VP1 is derived only from serotype A. This is understood to mean that VP2 and VP3 are derived only from serotype B. In another embodiment, when VP1 is identified as a first serotype A, VP2 as a second serotype B, and VP3 as a third serotype C, in one embodiment this means that VP1 is identified as a serotype It is understood to mean that VP2 is derived only from serotype B; and VP3 is derived only from serotype C. As described in more detail in the Examples below, in one embodiment, to generate haploid vectors using two different serotypes, serotype A (or chimeric or other a nucleotide sequence for VP1 from a non-naturally occurring AAV) and VP2 and/or VP3 from only a second serotype, or alternatively VP2 from only a second serotype, and a third serotype. It is also possible to include a second nucleotide sequence for VP3 that is derived solely from the type (see, eg, Figures 13-15). In one embodiment, VP1/VP2 is derived only from the first serotype and VP3 is derived only from the second serotype.

In the case of three different Cap genes, helper plasmids can be generated with complete copies of the nucleotide sequences for specific VP proteins from the three AAV serotypes. Each Cap gene produces the VP protein associated with that particular AAV serotype (designated A, B, and C).

In some embodiments, when VP1 is identified as a first serotype A, VP2 is identified as a second serotype B, and VP3 is identified as a third serotype C, in one embodiment this is understood to mean that it is derived only from serotype A; VP2 is derived only from serotype B, and VP3 is derived only from serotype C. As described in more detail in the Examples below, to generate such haploid vectors, VP1 from serotype A is used, which expresses only VP1 from serotype A and not VP2 or VP3 from serotype A. a second nucleotide sequence that expresses serotype B VP2 and not serotype B VP3; and a third nucleotide sequence that expresses serotype C VP3.

In certain embodiments, the haploid virion contains only VP1 and VP3 capsid proteins. In certain embodiments, the haploid virion comprises VP1, VP2, and VP3 capsid proteins.

Nucleotide sequences expressing capsid proteins in each of these embodiments of various combinations of VP1 and VP3 to form haploid virions; or various serotype combinations of VP1/VP2/VP3 to form haploid virions. It should be noted that the vector can be expressed from one or more vectors, such as plasmids. In one embodiment, the nucleic acid sequence expressing VP1 or VP2 or VP3 is codon-optimized so that recombination between nucleotide sequences is significantly reduced, especially when expressed from one vector, such as a plasmid. Ru.

A theoretical haploid vector with the C terminus of VP1/VP2 from AAV8 and VP3 from AAV2 enhances AAV transduction
It has been demonstrated that the haploid vector AAV2/8 with any ratio of AAV2 to AAV8 capsids induced higher liver transduction than viruses with AAV2 or a mixture of AAV2 and AAV8 vectors at the same ratio. To elucidate which AAV subunits in individual haploid AAV2/8 vectors contribute to higher transduction than AAV2, we analyzed AAV8 VP1/VP2 only, AAV2 VP3 only, AAV2-derived Expressing a chimeric VP1/VP2 (28m-2VP3) with an N-terminus derived from AAV8 and a C-terminus derived from AAV8 (28m-2VP3), or a chimeric AAV8/2 with an N-terminus derived from AAV8 and a C-terminus derived from AAV2 without the VP3 start codon mutation. Different constructs were created. These plasmids were used to produce different combinations of haploid AAV vectors. Liver transduction efficiency was evaluated after injecting 1 × 10 ¹⁰ of these haploid vector particles into mice via the retroorbital vein. The chimeric AAV82 vector (AAV82) induced slightly higher liver transduction than AAV2. However, haploid AAV82 (H-AAV82) had much higher liver transduction than AAV2. A further increase in liver transduction was observed using haploid vector 28m-2vp3. We also administered these haploid vectors into the muscles of mice. To facilitate comparison, the AAV2 vector was injected into the right leg and the haploid vector was injected into the left leg while the mouse was supine. Images were taken 3 weeks after AAV injection. Consistent with the observations in the liver, all haploid and chimeric vectors had higher muscle transduction, with the one from haploid vector 28m-2vp3 being the best. This result indicates that the chimeric VP1/VP2 with an N terminus derived from AAV2 and a C terminus derived from AAV8 is responsible for the high liver transduction of the haploid AAV82 vector.

Enhanced AAV liver transduction from haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 The inventors demonstrated that the haploid vector AAV82, which has VP1/VP2 from AAV8 and VP3 from AAV2, As described above, it has been shown to increase liver transduction. Next, we will investigate whether VP1/VP2 also increases transduction in other haploid virions derived from different serotypes. In preclinical studies, AAV9 has been shown to efficiently transduce different tissues. The present inventors created a haploid AAV92 vector (H-AAV92) in which VP1/VP2 is derived from AAV9 and VP3 is derived from AAV2. Imaging was performed 1 week after systemic administration. Approximately 4-fold higher liver transduction was achieved with H-AAV92 than with AAV2. This data indicates that VP1/VP2 from other serotypes can also increase AAV2 transduction.

Enhanced AAV liver transduction from haploid vectors carrying VP3 from AAV2 mutants or other serotypes
AAV9 uses glycans as its primary receptor for effective transduction. In our previous studies, we grafted the AAV9 glycan receptor binding site into AAV2 to create AAV2G9 and found that AAV2G9 has higher liver tropism than AAV2. Herein, the present inventors created a haploid vector (H-AAV82G9) in which VP1/VP2 is derived from AAV8 and VP3 is derived from AAV2G9. After systemic injection into mice, >10-fold higher liver transduction was observed at both 1 and 2 weeks after H-AAV82G9 application compared to AAV2G9. To study haploid vectors in which VP3 is derived from other serotypes and VP1/VP2 are derived from different serotypes or variants, we constructed other constructs: AAV3 VP3 only, AAV rh10 VP1/VP2 only. and generated different haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-3) in various combinations. Imaging was performed 1 week after systemic injection into mice. Consistent with results obtained with other haploid vectors, higher liver transduction was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-3) than with AAV3. Interestingly, these haploid vectors also induced systemic transduction based on the imaging profile, which is different from the results for haploid vector 5 with AAV2-derived VP3, which only efficiently transduced the liver. In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from an alternative can enhance transduction and possibly change tropism.

Haploid vectors with VP1/VP3 from one AAV serotype and VP2 from another AAV serotype enhance AAV transduction and avoid antibody neutralization
Generate several constructs to study haploid vectors in which VP2 is derived from one serotype and VP1/VP3 are derived from different serotypes. Generate a construct expressing only AAV2 VP2. This is achieved by incorporating a mutation in the AAV2 VP1 start codon and/or a mutation in the AAV2 VP1 splice acceptor site, combined with a mutation in the VP3 start codon, as shown for example in FIG. 10. A construct expressing only AAV8 VP1/3 is also generated. This is achieved by incorporating mutations in the AAV8 VP2 start codon. Similarly, constructs expressing only AAV2 VP1/3 and constructs expressing only AAV8 VP2 are generated.

Substantially homogeneous haploid vector populations encoding the luciferase transgene and having AAV2VP1 and AAV8VP1/3 or having AAV8VP1 and AAV2 VP1/3 are generated from these constructs using appropriate plasmids and helper viruses. . Inject 1 x ¹⁰ of these haploid vector particles into mice via the retroorbital vein and assess liver transduction efficiency by imaging 1 week later. Higher liver transduction was achieved using a homogeneous haploid vector population than with AAV2, and much lower Nab cross-reactivity was observed with haploid vectors compared to activity with AAV2 or AAV8. It is expected that it will be seen. Furthermore, homogeneous haploid vector populations can also induce systemic transduction (eg, as identified based on imaging profiles), but this differs from the results using either AAV2 or AAV8.

In these examples, we demonstrate that haploid viruses made from VP1/VP2 and VP3 from compatible serotypes also increase transduction. After systemic injection of 2 × 10 ¹⁰ vg of AAV vector into mice, haploid AAV vectors consisting of VP1/VP2 from serotypes 7, 8, 9 and rh10 and VP3 from AAV2 or AAV3 were isolated from AAV2-only and AAV3-only. It was found to exhibit a 2-7 fold increase in transduction compared to capsids across multiple tissue types including liver, heart and brain. These tissues additionally had higher vector genome copy numbers in these tissues, indicating that non-cognate VP1/VP2 integration may influence AAV receptor binding and intracellular trafficking. It shows that you can get it. In addition, chimeric and haploid capsids were created by combining either AAV2 or AAV8 VP1/VP2 with AAV2 or AAV8 VP3. When these haploid AAV vectors were injected into mice, the haploid AAV vector consisting of AAV8 VP1/2 and AAV2 VP3 had 5-fold higher transduction than the virus consisting of AAV2 VP alone. Remarkably, a haploid vector consisting of chimeric AAV2/8 (N-terminus of AAV2 and C-terminus of AAV8)-derived VP1/VP2 paired with AAV2-derived VP3 was found to contain AAV8 VP1 paired with AAV2 VP3. had a 50-fold increase in transgene expression compared to capsids consisting of /VP2. Considering the same proportion of AAV8 VP3-derived capsids, there is a difference in the VP1/2 N-terminal region between AAV2 and AAV8, which is similar to the VP1/2 N-terminus of AAV2 and its cognate VP3. It can indicate "interaction" between Taken together, the work presented herein provides insight into current AAV production strategies that can increase transduction across multiple tissue types.

The haploid vector is also injected into the muscle of the mouse. For a simple comparison, inject the AAV2 vector into the right leg and the haploid vector into the left leg while the mouse is supine. Images will be taken 3 weeks after AAV injection. Enhanced transduction in muscle by haploid vectors is also expected.

Ability to evade neutralizing antibodies from homogeneous haploid virus populations To study whether haploid viruses are able to evade Nabs generated from parental vectors, we performed Nab binding assays using monoclonal antibodies by immunoblot assays. implement. Three dilutions of viral genome-containing particles are adsorbed onto nitrocellulose membranes and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. A homogeneous haploid virus population would be expected to greatly reduce recognition by monoclonal antibodies ADK8 or A20 to undetectable.

Next, generate an immunological profile of a homogeneous haploid virus population using serum from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Four weeks after injection, collect serum from mice treated with parental virus. We compared the neutralization profile of haploid viruses against A20 or ADK8, which is expected to be similar to the data obtained from native immunoblots. It is expected that no Nab cross-reactivity will be observed between AAV8 and AAV2. Homogeneous haploid virus populations are expected to at least partially, and perhaps completely evade, neutralization from either AAV2 or AAV8 sera.

Haploid vectors with VP2/VP3 from one AAV serotype and VP1 from another AAV serotype enhance AAV transduction and avoid antibody neutralization
Generate several constructs to study haploid vectors in which VP1 is derived from one serotype and VP2/VP3 are derived from different serotypes. Generate a construct expressing only AAV2 VP1. This may include mutations in the AAV2 VP2 start codon, e.g. mutations in the AAV2 VP3 start codon as shown in Figures 7 and 21, or mutations in the VP2 and VP3 splice acceptor sites as shown in Figure 9, or mutations in the AAV2 VP3 start codon, e.g. This is achieved by the incorporation of both mutations as shown in Figure 11. Generate a construct expressing only AAV8 VP2/3. This is achieved by incorporating mutations in the AAV8 VP1 start codon, eg, see FIG. 21, and/or the splice acceptor site, eg, see FIG. 12. Similarly, constructs are generated that express only AAV2 VP2/3, and constructs that express only AAV8 VP1.

A substantially homogeneous haploid vector population encoding a luciferase transgene and having AAV2 VP1 and AAV8 VP2/3 or having AAV8 VP1 and AAV2 VP2/3 is generated from these constructs using appropriate plasmids and helper viruses. Make it. Inject 1 x ¹⁰ of these haploid vector particles into mice via the retroorbital vein and assess liver transduction efficiency by imaging 1 week later. Higher liver transduction was achieved using a homogeneous haploid vector population than with AAV2, and much lower Nab cross-reactivity was observed with haploid vectors compared to activity with AAV2 or AAV8. It is expected that it will be seen. Furthermore, homogeneous haploid vector populations may also induce systemic transduction (eg, as identified based on imaging profiles), but this differs from the results using either AAV2 or AAV8.

The haploid vector is also injected into the muscle of the mouse. For a simple comparison, inject the AAV2 vector into the right leg and the haploid vector into the left leg while the mouse is supine. Images will be taken 3 weeks after AAV injection. Enhanced transduction in muscle by haploid vectors is also expected.

Ability to evade neutralizing antibodies from homogeneous haploid virus populations To study whether haploid viruses are able to evade Nabs generated from parental vectors, we performed Nab binding assays using monoclonal antibodies by immunoblot assays. implement. Three dilutions of viral genome-containing particles are adsorbed onto nitrocellulose membranes and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. A homogeneous haploid virus population would be expected to greatly reduce recognition by monoclonal antibodies ADK8 or A20 to undetectable.

Next, generate an immunological profile of a homogeneous haploid virus population using serum from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Four weeks after injection, collect serum from mice treated with parental virus. We compared the neutralization profile of haploid viruses against A20 or ADK8, which is expected to be similar to the data obtained from native immunoblots. It is expected that no Nab cross-reactivity will be observed between AAV8 and AAV2. A homogeneous haploid virus population is expected to at least partially, and perhaps completely evade neutralization from either AV2 or AAV8 sera.

Triploid vectors with VP1 from one AAV serotype, VP2 from another AAV serotype and VP3 from a third AAV serotype enhance AAV transduction and avoid antibody neutralization
Several constructs are generated to study triploid vectors in which VP1, VP2, and VP3 are each derived from a different AAV serotype. Generate a construct expressing only AAV2 VP1. This is achieved, for example, by incorporating mutations in the AAV2 VP2 start codon and VP3 start codon, as shown in Figure 7, or by incorporating mutations in the splice acceptor site for VP2/3, as shown in Figure 9, for example. Ru. Generate a construct expressing only AAV9 VP2. This is accomplished by incorporating mutations in the AAV9 VP1 start codon and/or in the AAV9 VP1 splice acceptor site, as well as mutations in the VP3 start codon. Alternatively, this is achieved by synthesizing a fragment of the AAV9 Cap coding sequence excluding the upstream coding sequence for VP1 and mutating the VP3 start codon. Generate a construct expressing only AAV8 VP3. This is achieved by incorporating mutations in the AAV8 VP1 start codon and/or splice acceptor site, as well as mutations in the AAV8 VP2 start codon. Alternatively, this is accomplished by synthesizing a fragment of the AAV8 Cap coding sequence excluding the upstream coding sequences for VP1 and VP2.

A substantially homogeneous triploid vector population encoding the luciferase transgene and having AAV2 VP1, AAV9 VP2 and AAV8 VP3 is generated from these constructs using appropriate plasmids and helper viruses (e.g., Figure 13, 14 and 15). Inject 1 x ¹⁰ of these triploid vector particles into mice via the retroorbital vein and assess liver transduction efficiency by imaging 1 week later. Higher liver transduction is achieved using a homogeneous triploid vector population than with AAV2, AAV9 or AAV8, and much greater activity compared to that with either AAV2, AAV8 or AAV8. It is expected that lower Nab cross-reactivity will be seen using triploid vectors. Furthermore, homogeneous triploid vector populations can also induce systemic transduction (eg, as identified based on imaging profiles).

The triploid vector is also injected into the muscle of the mouse. For a simple comparison, inject the AAV2 vector, AAV9 vector, or AAV8 vector into the right leg and the triploid vector into the left leg while the mouse is supine. Images will be taken 3 weeks after AAV injection. Enhanced transduction in muscle by triploid vectors is expected.

Ability to Evade Neutralizing Antibodies of Homogenous Triploid Virus Populations Each individual haploid virus virion consists of 60 subunits, each derived from a different AAV serotype capsid. Combining serotype capsid proteins from three different serotypes is expected to alter virion surface structure. It is well known that most AAV monoclonal antibodies recognize residues on different subunits of one single virion. To study whether triploid viruses are able to evade Nabs generated from the parental vector, perform Nab binding assays using monoclonal antibodies by immunoblot assays. Three dilutions of viral genome-containing particles are adsorbed onto nitrocellulose membranes and probed with Nab A20 or ADK8, which recognize intact AAV2 or AAV8, respectively. Homogeneous triploid virus populations are expected to greatly reduce recognition by monoclonal antibodies ADK8 or A20 to undetectable.

Next, generate an immunological profile of a homogeneous triploid virus population using serum from AAV-immunized mice. Nab titers are used to assess the ability of serum to inhibit vector transduction. Four weeks after injection, collect serum from mice treated with parental virus. Compare the neutralization profile of triploid viruses against A20 or ADK8, which is expected to be similar to the data obtained from native immunoblots. No Nab cross-reactivity is expected between AAV8 and AAV2. Homogeneous triploid virus populations are expected to at least partially, and perhaps completely, evade neutralization from either AAV2, AAV9 or AAV8 sera.

Example 3: Polyploid adeno-associated virus vectors enhance transduction and evade neutralizing antibodies Adeno-associated virus (AAV) vectors have been used successfully in clinical trials in hemophiliacs and blind patients. There is. Although the application of AAV vectors has proven to be safe and has shown therapeutic efficacy in these clinical trials, one of the major challenges is its low infectivity, which requires a relatively large amount of viral genome. It is. In addition, a large proportion of the population has neutralizing antibodies (Nab) against AAV in their blood and other body fluids. The presence of Nab poses another major challenge to broader AAV application in future clinical trials. Effective strategies to enhance AAV transduction and circumvent neutralizing antibody activity are highly needed. Previous studies have shown the compatibility of capsids from AAV serotypes and recognition sites for AAV Nabs located on different capsid subunits of one virion. In this study, we investigated whether polyploid AAV viruses produced from co-transfection of different AAV helper plasmids have the capacity for enhanced AAV transduction and Nab evasion. propose. We co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids. Haploid virus yield was similar to that of the parent, suggesting that these two AAV capsids were compatible. In Huh7 and C2Cl2 cell lines, the transduction efficiency of AAV8 was much lower than that from AAV2; however, transduction from all haploid vectors was higher than that from AAV8. The transduction efficiency and heparin sulfate binding capacity of haploid vectors were positively correlated with the amount of AAV2 capsids integrated. These results indicate that haploid virus vectors retain the viral properties of their parents and take advantage of the parental vectors for enhanced transduction. After intramuscular injection, all haploid viruses induced higher transduction than the parental AAV vector (2-9 times more than AAV2), highest with the haploid vector AAV2/8 3:1.

After systemic administration, 4-fold higher transduction was observed in the liver with haploid vector AAV2/8 1:3 than with AAVS alone. Importantly, we packaged therapeutic factor IX cassettes into haploid vector AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved using the haploid vector AAV2/8 1:3 viral vector compared to the case of AAVS. Remarkably, haploid virus AAV2/8 1:3 was able to evade AAV2 neutralization and had very low Nab cross-reactivity with AAV2. However, AAVS neutralizing antibodies can inhibit haploid vector AAV2/8 transduction with the same efficiency as AAV8. Next, we produced the triploid vector AAV2/8/9 vector by co-transfecting AAV2, AAV8 and AAV9 helper plasmids in a 1:1:1 ratio. After systemic administration, two-fold higher transduction was observed in the liver with the triploid vector AAV2/8/9 than with AAV8 (Figure 6). Neutralizing antibody analysis showed that the AAV2/8/9 vector, unlike the AAV2/8 triploid vector, was able to evade neutralizing antibody activity from mouse sera immunized with the parental serotype. Proven. The results indicate that polyploid viruses can potentially acquire significance from their parental serotypes for enhanced transduction and have the capacity for evasion of Nab recognition. This strategy should be considered in future clinical trials in patients positive for neutralizing antibodies.

Example 4: Substitution of AAV Capsid Subunits Enhances Transduction and Evades Neutralizing Antibodies Demonstrates Therapeutic Efficacy Using Adeno-Associated Virus (AAV) Vectors in Clinical Trials of Patients with Blood Diseases and Blinding Disorders was achieved. However, two concerns limit the expansion of AAV vector applications: AAV capsid-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies (Nabs). Enhancement of AAV transduction using low-dose AAV vectors potentially reduces capsid antigen loading and, hopefully, increases capsid CTL-mediated clearance of AAV-transduced target cells without compromising transgene expression. Remove. Currently, 12 serotypes and over 100 variants or mutants are being considered for gene delivery due to their different tissue tropisms and transduction efficiencies. It has been demonstrated that there is capsid compatibility between AAV serotypes and that integration of specific amino acids from one serotype into the AAV capsid of another enhances AAV transduction. By exploiting different mechanisms for effective AAV transduction from different serotypes, enhanced AAV transduction is achieved using mosaic viruses in vitro and in vivo in which AAV capsid subunits are derived from different serotypes. It was done. Recent structural studies of the interaction of AAV vectors with monoclonal neutralizing antibodies have demonstrated that Nab binds to residues on several different subunits on a single virion surface, suggesting that It is suggested that changes in subunit assembly of AAV virions may remove the AAV Nab binding site and then evade Nab activity. We demonstrate that mosaic AAV vectors can circumvent Nab activity. These results indicate that substitution of AAV capsid subunits has the potential to enhance the ability of AAV transduction and neutralizing antibody evasion.

Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for patients with hematological and blinding disorders. Two concerns limit widespread AAV vector application: AAV capsid-specific cytotoxic T lymphocyte (CTL) response-mediated elimination of AAV-transduced target cells and neutralizing antibody (Nab)-mediated Blocking AAV transduction. It has been demonstrated that capsid antigen presentation is dose-dependent, indicating that enhancing AAV transduction with low doses of AAV vectors potentially reduces capsid antigen load and, hopefully, increases transduction. We show that capsid CTL-mediated clearance of AAV-transduced target cells eliminates capsid CTL-mediated clearance of AAV-transduced target cells without compromising gene expression. Several approaches have been considered for this purpose, including optimization of transgene cassettes, modification of AAV capsids and interference with AAV trafficking with pharmacological agents. Modifications of AAV capsids can alter AAV tropism; in particular, AAV transduction efficiency is unknown in human tissues. Although several clinical trials are underway, AAV vectors were chosen empirically based on observations from animal models. Pharmacological reagents to enhance AAV transduction usually have unwanted side effects. It is essential to develop ideal strategies to enhance AAV transduction without altering its tropism from capsid modifications and without side effects from pharmacological treatments. There are currently 12 serotypes and over 100 variants or variants being considered for gene delivery. Effective AAV transduction involves receptor- and co-receptor-mediated binding on the target cell surface, endocytosis to endosomes, escape from endosomes, nuclear entry, and uncoating of AAV virions with subsequent transgene expression. It involves a process including. To rationally design novel AAV vectors for enhanced transduction, we developed chimeric viruses AAV2.5 (AAV2 mutant with 5 aa substitution from AAV1) and AAV2G9 (galactose-accepting AAV9-derived galactose receptor). The body was transplanted into an AAV2 capsid). Both chimeric mutants induce much higher transduction in mouse muscle and liver than AAV2, respectively. These observations indicate that these chimeric viruses can use properties from both AAV serotypes for enhanced transduction (e.g., AAV2G9 has two major receptors, heparin and galactose). for effective cell surface binding). Compatibility between capsid subunits from different AAV serotypes for virus assembly and integration of specific amino acids from other serotypes (1 or 9) into AAV serotype 2 may inhibit AAV2 transduction in muscle and liver. Based on our preliminary results demonstrating that substitution of several capsid subunits from other serotypes enhanced effective AAV from different serotypes, we We reason that AAV transduction can be enhanced by exploiting different mechanisms for transduction. In addition, pre-existing antibodies against naturally occurring AAV have influenced the success of hemophilia B and other AAV gene transfer studies. In the general human population, around 50% possess neutralizing antibodies. To design Nab-evading AAV vectors, chemical modification, in situ rational design and combinatorial mutagenesis of AAV vectors of different serotypes, capsids and biological depletion of Nab titers (empty capsid utilization, B cell depletion and plasma Several approaches have been considered, including apheresis. These approaches have low efficiency or side effects or changes in AAV tropism. Recent structural studies of the interaction of AAV vectors with monoclonal neutralizing antibodies have demonstrated that Nab binds to residues on several different subunits on a single virion surface, suggesting that It is suggested that changes in the subunit assembly of AAV virions may remove the AAV Nab binding site and then evade Nab activity. We have results that strongly support the concept that novel mosaic AAV vectors have the potential to enhance transduction in various tissues and can circumvent neutralizing antibody activity.

Treatment of diseases such as diseases of the central nervous system, heart, lungs, skeletal muscle, and liver; such as Parkinson's disease, Alzheimer's disease, cystic fibrosis, ALS, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, myasthenia gravis, In each of Examples 5-6 below for the treatment of diseases, including hemophilia A or B, the capsid virions described herein are produced using a particular AAV serotype and mosaic. , a haploid capsid alternatively produced using the rational polyploid method of Example 2, in which VP1 is derived only from a first serotype and VP2 and/or VP3 are derived only from a second serotype; Or, for example, generate haploid capsids in which VP1, VP2, and VP3 are each derived from a different serotype. Alternative methods for making such virions are also described, eg, in Examples 7-15.

Example 5: Treatment of diseases of the central nervous system (CNS) using VP1/VP2/VP3 from two or more different AAV serotypes In the initial experiments, two helper plasmids are used. The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV4. The third plasmid encodes the nucleotide sequence for glutamate decarboxylase 65 (GAD65) and/or glutamate decarboxylase 67 (GAD67), which nucleotide sequence is inserted between the two ITRs. Polyploid virions can be used to encapsidate nucleic acid sequences containing GAD65 and/or GAD67 for therapeutic use. In the following examples, capsids are prepared using, for example, the rational polyploid method of Example 2, such that VP1 is derived from only one serotype, VP3 is derived only from an alternate serotype, and VP2 is derived from only one serotype. Haploid capsids can be produced with or without the presence of . When VP2 is present, it can be derived from only one serotype, which can be the same as either VP1 or VP3, or can be derived from a third serotype, or a cross of the above resulting in a mosaic haploid capsid. Capsids can be prepared by dressing methodologies. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes delivering proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for central nervous system tissues associated with Parkinson's disease. Contains nucleotide sequences for GAD65 and/or GAD67 proteins for treating Parkinson's disease by partially increasing . Indeed, haploid viruses produced by this method to treat Parkinson's disease can have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV4.

Further experiments will again use two helper plasmids, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV5. The third plasmid encodes the nucleotide sequence for CLN2 for treating Batten disease, contained in the third plasmid and inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes delivering proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for central nervous system tissues associated with Parkinson's disease. Contains a nucleotide sequence for treating Batten disease by partially increasing . Indeed, haploid viruses produced by this method to treat Batten disease have higher specificity for relevant central nervous system tissues than viral vectors consisting only of AAV3 or AAV5.

Another experiment uses three helper plasmids, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV4. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV5. The fourth plasmid encodes the nucleotide sequence for nerve growth factor (NGF) for treating Alzheimer's disease, contained in the third plasmid and inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV4, and AAV5) to deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat Alzheimer's disease. Contains nucleotide sequences for treating Alzheimer's disease, in part by increasing specificity for relevant central nervous system tissues. Indeed, triploid viruses produced by this method to treat Alzheimer's disease have higher specificity for relevant central nervous system tissues than viral vectors consisting only of AAV3, AAV4 or AAV5.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV4 and VP3 from AAV5. The second plasmid encodes the nucleotide sequence for AAC inserted between the two ITRs to treat Canavan disease. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV2, AAV4, and AAV5) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat Canavan disease. Contains nucleotide sequences for treating Canavan disease, in part by increasing specificity for relevant central nervous system tissues. Indeed, triploid viruses produced by this method to treat Canavan disease have higher specificity for relevant central nervous system tissues than viral vectors consisting only of AAV2, AAV4 or AAV5.

Treatment of heart diseases using VP1/VP2/VP3 from two or more different AAV serotypes In one experiment, two helper plasmids are used but with different AAV serotypes as sources of Rep and Cap genes. . The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV6. The third plasmid encodes the nucleotide sequence for a protein for treating heart disease, contained in the third plasmid and inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to achieve specificity for cardiac tissues associated with cardiac diseases. Contains nucleotide sequences for treating heart disease by partially increasing the number of nucleotide sequences. Indeed, haploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissues than viral vectors consisting only of AAV2 or AAV6.

Further experiments use two helper plasmids, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The third plasmid encodes the nucleotide sequence for a protein for treating heart disease, contained in the third plasmid and inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to achieve specificity for cardiac tissues associated with cardiac diseases. Contains a nucleotide sequence that encodes a protein for treating heart disease by increasing the portion. Indeed, haploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissues than viral vectors consisting only of AAV3 or AAV9.

In one experiment, three helper plasmids are used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The fourth plasmid contains a nucleotide sequence, contained in the third plasmid and inserted between the two ITRs, encoding a protein for treating heart disease. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV9) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to improve cardiac disease. Contains nucleotide sequences for treating heart disease, in part by increasing specificity for heart tissue associated with heart disease. In fact, triploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissues than viral vectors consisting only of AAV3, AAV6 or AAV9.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV2, AAV3, and AAV9) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat cardiac diseases. It encodes a nucleotide sequence for treating heart disease, in part by increasing specificity for the relevant heart tissue. In fact, triploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissue than viral vectors consisting only of AAV2, AAV3 or AAV9.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV6. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Haploid AAV generated from two plasmids is associated with heart disease by utilizing multiple AAV serotypes (e.g., AAV3 and AAV6) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention. Encodes a nucleotide sequence for treating heart disease, in part by increasing specificity for heart tissue. Indeed, haploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissues than viral vectors consisting only of AAV2 or AAV6.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV9. The second plasmid contains a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV9) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to improve cardiovascular disease. encodes a nucleotide sequence for treating heart disease, in part by increasing specificity for heart tissue associated with heart disease. In fact, triploid viruses produced by this method to treat heart diseases have higher specificity for relevant heart tissues than viral vectors consisting only of AAV3, AAV6 or AAV9.

Treatment of lung diseases using VP1/VP2/VP3 from two or more different AAV serotypes In one experiment, again using two helper plasmids, but with VP1/VP2/VP3 as a source of Rep and Cap genes. Different AAV serotypes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Cap gene from AAV9. The third plasmid encodes the nucleotide sequence for CFTR to treat cystic fibrosis inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes delivering proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for cystic fibrosis-associated lung tissues. Contains a nucleotide sequence for CFTR to treat cystic fibrosis by partially increasing CFTR. Indeed, haploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV9.

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV10. The third plasmid encodes the nucleotide sequence for CFTR to treat cystic fibrosis inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes delivering proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for cystic fibrosis-associated lung tissues. Contains a nucleotide sequence for CFTR to treat cystic fibrosis by partially increasing CFTR. Indeed, haploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV10.

In one experiment, three helper plasmids are used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV10. A fourth plasmid encodes a nucleotide sequence for CFTR for treating cystic fibrosis, contained in the third plasmid and inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV9, and AAV10) to provide proteins encoding VP1, VP2, and VP3 according to the method of the present invention to induce cystic fibrosis. Contains a nucleotide sequence for CFTR for treating cystic fibrosis, in part by increasing specificity for lung tissue associated with the disease. Indeed, triploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV9 or AAV10.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV9. The second plasmid encodes the nucleotide sequence for CFTR inserted between the two ITRs to treat cystic fibrosis. Haploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV2 and AAV9) that deliver proteins encoding VP1, VP2, and VP3 to treat cystic fibrosis according to the methods of the present invention. Contains nucleotide sequences for treating cystic fibrosis, in part by increasing specificity for relevant central nervous system tissues. Indeed, haploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV9.

Further experiments use one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV10. The second plasmid encodes the nucleotide sequence for CFTR inserted between the two ITRs to treat cystic fibrosis. Haploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3 and AAV10) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat cystic fibrosis. Contains nucleotide sequences for treating cystic fibrosis, in part by increasing specificity for relevant central nervous system tissues. Indeed, haploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV10.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV10. The second plasmid encodes the nucleotide sequence for CFTR inserted between the two ITRs to treat cystic fibrosis. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV2, AAV9, and AAV10) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat Canavan disease. Contains nucleotide sequences for treating cystic fibrosis, in part by increasing specificity for relevant central nervous system tissues. Indeed, triploid viruses produced by this method to treat cystic fibrosis have higher specificity for relevant tissues than viral vectors consisting only of AAV2, AAV9 or AAV10.

Treatment of skeletal muscle diseases using VP1/VP2/VP3 from two or more different AAV serotypes For the following experiments, skeletal muscle diseases including, but not limited to, Duchenne muscular dystrophy, limb-girdle muscular dystrophy, cerebral palsy, severe It can be myasthenia and amyotrophic lateral sclerosis (ALS).

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV8. The third plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes that deliver proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for skeletal muscle associated with skeletal muscle diseases. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by partially increasing . Indeed, haploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant skeletal muscle tissue than viral vectors consisting only of AAV2 or AAV8.

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene from AAV3 and the Cap gene, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The third plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes that deliver proteins encoding VP1, VP2, and VP3 according to the method of the present invention to achieve specificity for skeletal muscle associated with skeletal muscle diseases. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by partially increasing . Indeed, haploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant skeletal muscle tissue than viral vectors consisting only of AAV3 or AAV9.

In one experiment, three helper plasmids are used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV8. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV9. The fourth plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV8, and AAV9) to deliver proteins encoding VP1, VP2, and VP3 according to the method of the present invention to stimulate skeletal muscle production. Contains nucleotide sequences for proteins for treating skeletal muscle diseases, in part by increasing specificity for skeletal muscle associated with the disease. Indeed, triploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV8 or AAV9.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9. The second plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Haploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3 and AAV9) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to be associated with skeletal muscle diseases. contains nucleotide sequences for treating diseases of skeletal muscle, in part by increasing specificity for skeletal muscle tissue. Indeed, haploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant skeletal muscle tissue than viral vectors consisting only of AAV3 or AAV9.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8. The second plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Haploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3 and AAV8) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to be associated with skeletal muscle diseases. contains nucleotide sequences for treating diseases of skeletal muscle, in part by increasing specificity for skeletal muscle tissue. Indeed, haploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant skeletal muscle tissue than viral vectors consisting only of AAV3 or AAV8.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV9. The second plasmid encodes the nucleotide sequence for a protein for treating diseases of skeletal muscle inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV8, and AAV9) that deliver proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat skeletal muscle diseases. contains nucleotide sequences for treating diseases of skeletal muscle, in part by increasing the specificity for skeletal muscle tissue associated with skeletal muscle tissue. Indeed, triploid viruses produced by this method to treat skeletal muscle diseases have higher specificity for relevant skeletal muscle tissue than viral vectors consisting only of AAV3, AAV8 or AAV9.

Treatment of liver diseases using VP1/VP2/VP3 from two or more different AAV serotypes In one experiment, again using two helper plasmids, but as a source of the Rep and Cap genes. Different AAV serotypes. The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV6. The third plasmid encodes the nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between the two ITRs. Haploid AAV generated from three plasmids has specificity for FIX associated with hemophilia B by utilizing multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the method of the present invention. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by increasing the portion. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV6. .

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep and Cap genes from AAV2, and the second helper plasmid has the Rep and Cap genes from AAV3 and AAV7. The third plasmid encodes the nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between the two ITRs. Haploid AAV generated from three plasmids has specificity for FIX associated with hemophilia B by utilizing multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the method of the present invention. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by increasing the portion. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV7. .

In one experiment, three helper plasmids are used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7. The fourth plasmid encodes the nucleotide sequence for factor IX (FIX) for treating hemophilia B inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat hemophilia. Contains a nucleotide sequence for a protein for treating hemophilia B, in part by increasing its specificity for liver tissue associated with hemophilia B. Indeed, triploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV6 or AAV7. have sex.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6. The second plasmid encodes the nucleotide sequence for FIX to treat hemophilia B inserted between the two ITRs. Haploid AAV generated from two plasmids can be used to treat hemophilia B by utilizing multiple AAV serotypes (e.g., AAV2 and AAV6) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention. Contains nucleotide sequences for treating hemophilia B, in part by increasing specificity for relevant liver tissue. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV6. .

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7. The second plasmid encodes the nucleotide sequence for FIX to treat hemophilia B inserted between the two ITRs. Haploid AAV generated from two plasmids can be used to treat hemophilia B by utilizing multiple AAV serotypes (e.g., AAV3 and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention. Contains nucleotide sequences for treating hemophilia B, in part by increasing specificity for relevant liver tissue. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV7. .

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7. The second plasmid encodes the nucleotide sequence for FIX to treat hemophilia B inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat hemophilia. Contains a nucleotide sequence for treating hemophilia B, in part by increasing specificity for liver tissues associated with B. Indeed, triploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia B have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV6 or AAV7. have sex.

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV2 and the Cap gene from AAV6. The third plasmid encodes the nucleotide sequence for factor VIII (FVIII) to treat hemophilia A inserted between the two ITRs. Haploid AAV generated from three plasmids utilizes multiple AAV serotypes delivering proteins encoding VP1, VP2, and VP3 to achieve specificity for FVIII associated with hemophilia A according to the method of the present invention. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by increasing the portion. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV6. .

In one experiment, two helper plasmids are again used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV2, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7. The third plasmid encodes the nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Haploid AAV generated from three plasmids has specificity for FVIII associated with hemophilia A by utilizing multiple AAV serotypes that supply proteins encoding VP1, VP2, and VP3 according to the method of the present invention. Contains nucleotide sequences for proteins for treating diseases of skeletal muscle by increasing the portion. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV7. .

In one experiment, three helper plasmids are used, but with different AAV serotypes as sources of Rep and Cap genes. The first helper plasmid has the Rep gene and Cap gene from AAV3, and the second helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV6. The third helper plasmid has the Rep gene from AAV3 and the Cap gene from AAV7. The fourth plasmid encodes the nucleotide sequence for FVIII to treat hemophilia A inserted between the two ITRs. Triploid AAV generated from four plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat hemophilia. Contains a nucleotide sequence for the FVIII protein to treat hemophilia A, in part by increasing its specificity for liver tissue associated with B. Indeed, triploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV6 or AAV7. have sex.

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. The helper plasmid has Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6. The second plasmid encodes the nucleotide sequence for FVIII for treating hemophilia B inserted between the two ITRs. Haploid AAV generated from two plasmids can be used to treat hemophilia A by utilizing multiple AAV serotypes (e.g., AAV2 and AAV6) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention. Contains nucleotide sequences for FVIII for treating hemophilia A, in part by increasing specificity for the relevant liver tissue. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV2 or AAV6. .

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7. The second plasmid encodes the nucleotide sequence for FVIII for treating hemophilia A inserted between the two ITRs. Haploid AAV generated from two plasmids can be used to treat hemophilia B by utilizing multiple AAV serotypes (e.g., AAV3 and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention. Contains nucleotide sequences for FVIII for treating hemophilia A, in part by increasing specificity for the relevant liver tissue. Indeed, haploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV3 or AAV7. .

Another experiment uses one helper plasmid but different AAV serotypes as sources of Rep and Cap genes. Helper plasmids have Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7. The second plasmid encodes the nucleotide sequence for FVIII for treating hemophilia A inserted between the two ITRs. Triploid AAV generated from two plasmids utilizes multiple AAV serotypes (e.g., AAV3, AAV6, and AAV7) that supply proteins encoding VP1, VP2, and VP3 according to the methods of the present invention to treat hemophilia. Contains a nucleotide sequence for FVIII to treat hemophilia B, in part by increasing specificity for liver tissue associated with A. Indeed, triploid viruses produced by this method to treat liver tissue of patients suffering from hemophilia A have higher specificity for relevant tissues than viral vectors consisting only of AAV3, AAV6 or AAV7. have sex.

Example 6: Use of an AAV of the invention to treat the disease treatment of Parkinson's disease A 45-year-old male patient suffering from Parkinson's disease was treated with a first helper plasmid carrying the Rep and Cap genes from AAV2, and , a second helper plasmid carrying a Rep gene from AAV2 and a Cap gene from AAV4, and a nucleotide sequence encoding glutamate decarboxylase 65 (GAD65) and/or glutamate decarboxylase 67 (GAD67), the nucleotide sequence of which is AAV produced from a cell line, e.g., the HEK293 isolated cell line of ATCC No. PTA 13274 (see e.g., U.S. Patent No. 9,441,206), containing a third plasmid inserted between the two ITRs. Treat with. Haploid AAV generated from three plasmids contains nucleotide sequences for GAD65 and/or GAD67 proteins for treating Parkinson's disease. AAV is administered to a patient and immediately after administration the patient exhibits a reduction in the frequency of tremors and an improvement in the patient's balance. Over time, patients also experience a reduction in the number and severity of the hallucinations and delusions they were suffering from prior to administration of AAV.

Treatment of Batten Disease An 8-year-old male patient suffering from Batten disease was treated with a first helper plasmid carrying the Rep gene and Cap gene from AAV3 and a second helper plasmid carrying the Rep gene from AAV3 and the Cap gene from AAV5. AAV produced from a cell line, eg, the HEK293 isolated cell line of ATCC No. PTA 13274 (see, eg, US Pat. No. 9,441,206), containing a helper plasmid of . The third plasmid encodes the nucleotide sequence for CLN2 to treat Batten disease, where the CLN2 gene is inserted between the two ITRs. Haploid AAV produced from three plasmids contains nucleotide sequences for treating Batten disease. AAV is administered to a patient and immediately after administration the patient exhibits increased mental acuity. In addition, the patient experiences a reduction in seizures and improvement in symptoms and motor skills that the patient suffered from prior to administration of AAV.

Treatment of Alzheimer's Disease A 73-year-old female patient suffering from Alzheimer's disease was treated with a first helper plasmid carrying the Rep gene and Cap gene from AAV3; a second helper plasmid carrying the Rep gene from AAV3 and the Cap gene from AAV4. a cell line, e.g., the HEK293 isolated cell line of ATCC No. PTA 13274 (e.g., U.S. Patent No. 9,441,206 (see ). The fourth plasmid encodes the nucleotide sequence for nerve growth factor (NGF) to treat Alzheimer's disease, where NGF is inserted between two ITRs. Triploid AAV is administered to patients, and immediately after administration, patients exhibit increased mental acuity and short-term memory. Patients are also able to communicate better with others and begin to function more independently than before administration of AAV.

Treatment of Heart Disease A 63-year-old male patient suffering from heart disease was treated with a cell line containing any of the following, such as the HEK293 isolated cell line of ATCC No. PTA 13274 (e.g., U.S. Patent No. 9,441,206). Treat with AAV generated from (see):
(1) A first helper plasmid containing an AAV2-derived Rep gene and a Cap gene; a second helper plasmid containing an AAV2-derived Rep gene and an AAV6-derived Cap gene; and a third plasmid containing two a third plasmid inserted between the ITRs encoding a nucleotide sequence for a protein for treating heart disease;
(2) A first helper plasmid containing an AAV3-derived Rep gene and a Cap gene; a second helper plasmid containing an AAV3-derived Rep gene and an AAV9-derived Cap gene; and a third plasmid containing two a third plasmid inserted between the ITRs encoding a nucleotide sequence for a protein for treating heart disease;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV6-derived Cap gene; and a fourth plasmid containing a nucleotide sequence encoding a protein for treating heart disease, contained in the third plasmid and inserted between the two ITRs;
(4) a helper plasmid with Rep from AAV2 and VP1 from AAV2, VP2 from AAV3 and VP3 from AAV9; and a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs a second plasmid containing;
(5) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV6; and a nucleotide sequence encoding a protein for treating heart disease inserted between the two ITRs or (6) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV9; and a second plasmid inserted between the two ITRs to treat heart disease. A second plasmid containing a nucleotide sequence encoding a protein for.
Here, polyploid AAV is administered to a patient, and immediately after administration, the patient exhibits a reduction in symptoms associated with heart disease and a comparable improvement in the patient's heart health.

Treatment of Cystic Fibrosis A 19-year-old female suffering from cystic fibrosis was treated with a cell line, such as the HEK293 isolated cell line of ATCC No. PTA 13274 (e.g., U.S. Pat. 9,441,206)):
(1) A first helper plasmid with AAV3-derived Rep gene and Cap gene; a second helper plasmid with AAV3-derived Rep and AAV10-derived Cap genes; and a nucleotide for CFTR inserted between two ITRs a third plasmid encoding the sequence;
(2) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV9-derived Cap gene; and a fourth plasmid encoding a nucleotide sequence for CFTR inserted between the two ITRs;
(3) a helper plasmid with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV9; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs;
(4) a helper plasmid having Rep from AAV3 and VP1 from AAV2, VP2 from AAV10 and VP3 from AAV10; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs; or (7) A helper plasmid with Rep from AAV2 and VP1 from AAV2, VP2 from AAV9 and VP3 from AAV10; and a second plasmid encoding the nucleotide sequence for CFTR inserted between the two ITRs.
Here, AAV is administered to a patient, and immediately after administration, the patient receives: a delay in the increase in damage to the patient's lungs; a decrease in the increase in loss of lung function; and a decrease in the rate at which the liver becomes damaged and the severity of cirrhosis. Indicating a slowdown in growth. The same patient also experiences a reduction in the severity of cystic fibrosis-related diabetes that the patient had begun to suffer from.

Skeletal Muscle Disease - Treatment of Amyotrophic Lateral Sclerosis (ALS) A 33-year-old man suffering from amyotrophic lateral sclerosis (ALS) was treated with a cell line, e.g., ATCC, containing one of the following: Treat with AAV produced from the HEK293 isolated cell line of No. PTA 13274 (see, e.g., U.S. Patent No. 9,441,206):
(1) A first helper plasmid having Rep gene and Cap gene derived from AAV2; Second helper plasmid having Rep gene derived from AAV2 and Cap gene derived from AAV8; and superoxide dismutase inserted between two ITRs a third plasmid encoding the nucleotide sequence for 1 (SOD1);
(2) A first helper plasmid with AAV3-derived Rep gene and Cap gene; a second helper plasmid with AAV3-derived Rep and AAV9-derived Cap gene; and a nucleotide for SOD1 inserted between two ITRs a third plasmid encoding the sequence;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV8-derived Cap gene; and a fourth plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs;
(4) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs;
(5) a helper plasmid having Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs; or (6) A helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding the nucleotide sequence for SOD1 inserted between the two ITRs.
Here, AAV is administered to a patient, and immediately after administration, the patient is diagnosed with ALS, which includes slowing down or stopping the progression of damage to motor neurons in the brain and spinal cord and maintaining communication between the patient's brain and muscles. Shows reduction in associated symptoms.

Treatment of Duchenne Muscular Dystrophy A 5-year-old male suffering from Duchenne muscular dystrophy (DMD) was treated with a cell line containing either: AAV generated from the HEK293 isolated cell line of ATCC No. PTA 13274; Treat with:
(1) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV2-derived Rep and AAV8-derived Cap genes; and a nucleotide for dystrophin inserted between two ITRs. a third plasmid encoding the sequence;
(2) A first helper plasmid with AAV3-derived Rep gene and Cap gene; a second helper plasmid with AAV3-derived Rep gene and AAV9-derived Cap gene; and a nucleotide for dystrophin inserted between two ITRs. a third plasmid encoding the sequence;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV8-derived Cap gene; and a fourth plasmid encoding the nucleotide sequence for dystrophin inserted between the two ITRs;
(4) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and a second plasmid encoding the nucleotide sequence for dystrophin inserted between the two ITRs;
(5) a helper plasmid having Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and a second plasmid encoding a nucleotide sequence for dystrophin inserted between the two ITRs; or (6) A helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and a second plasmid encoding the nucleotide sequence for dystrophin inserted between the two ITRs.
Here, AAV is administered to a patient, and immediately after administration, the patient experiences a delay in damage to the patient's skeletal muscles and increased wasting, as well as a delay or cessation of damage to the heart and lungs as a result of Duchenne muscular dystrophy. show.

Treatment of Myasthenia Gravis A 33-year-old woman suffering from myasthenia gravis (MG) was treated with a cell line, e.g., generated from the HEK293 isolated cell line of ATCC No. PTA 13274, containing either: Treat with AAV:
(1) A first helper plasmid having Rep gene and Cap gene derived from AAV2; a second helper plasmid having Rep gene derived from AAV2 and Cap gene derived from AAV8; and a patient inserted between two ITRs. a third plasmid encoding a nucleotide sequence for a gene that no longer causes MG;
(2) a first helper plasmid having a Rep gene and a Cap gene derived from AAV3; a second helper plasmid having a Rep gene derived from AAV3 and a Cap gene derived from AAV9; and a patient inserted between two ITRs. A third plasmid encoding a gene that no longer causes MG;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV8-derived Cap gene; and a fourth plasmid inserted between the two ITRs, encoding a gene such that the patient no longer suffers from MG;
(4) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and a gene inserted between the two ITRs such that the patient no longer suffers from MG. a second plasmid encoding;
(5) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and a gene inserted between the two ITRs such that the patient no longer suffers from MG. or (6) a helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV; and inserted between the two ITRs so that the patient no longer has MG A second plasmid encodes a gene that does not cause disease.
Here, AAV is administered to a patient and immediately after administration the patient exhibits a slowing in the increase in the disconnection of communication between the muscles and nerves of the patient's body, resulting in a slowing in the severity of the loss of muscle control. or bring about a suspension. After administration of AAV, the patient's motor function stabilizes and no longer worsens, and the patient's breathing also does not worsen after administration of AAV.

Treatment of Limb-Girdle Muscular Dystrophy A 13-year-old male suffering from Limb-Girdle Muscular Dystrophy (LGMD) was treated with a cell line generated from the HEK293 isolated cell line of ATCC No. PTA 13274 containing either: Treat with AAV:
(1) A first helper plasmid having an AAV2-derived Rep gene and a Cap gene; a second helper plasmid having an AAV2-derived Rep gene and an AAV8-derived Cap gene; and myotilin, which was inserted between the two ITRs. a third plasmid encoding a nucleotide sequence for one of 15 genes with mutations associated with LGMD, including but not limited to teretonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan;
(2) a first helper plasmid having an AAV3-derived Rep gene and a Cap gene; a second helper plasmid having an AAV3-derived Rep gene and an AAV9-derived Cap gene; and myotilin, which was inserted between the two ITRs. a third plasmid encoding a nucleotide sequence for one of 15 genes with mutations associated with LGMD, including but not limited to teretonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV8-derived Cap gene; and a third helper plasmid with LGMD-associated mutations inserted between the two ITRs, including but not limited to myotilin, teretonin, calpain-3, alpha-sarcoglycan, and beta-sarcoglycan. a fourth plasmid encoding the nucleotide sequence for one of the genes;
(4) Helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV9 and VP3 from AAV9; and myotillin, teretonin, calpain-3, alpha-sarcoglycan inserted between the two ITRs and a second plasmid encoding a nucleotide sequence for one of the 15 genes with mutations associated with LGMD, including but not limited to beta-sarcoglycan;
(5) Helper plasmid with Rep from AAV3 and VP1 from AAV3, VP2 from AAV8 and VP3 from AAV8; and myotillin, teretonin, calpain-3, alpha-sarcoglycan inserted between the two ITRs and (6) Rep from AAV3 and VP1 from AAV3, AAV8 helper plasmid with VP2 from AAV and VP3 from AAV; and LGMD containing, but not limited to, myotilin, teretonin, calpain-3, alpha-sarcoglycan and beta-sarcoglycan inserted between the two ITRs. A second plasmid encodes the nucleotide sequence for one of the 15 genes carrying mutations.
Here, one or more AAVs, each encoding one of 15 different genes associated with LGMD, are administered to a patient, and immediately after administration, the patient exhibits slowed or stopped additional muscle wasting and atrophy.

Liver diseases – treatment of hemophilia B
A 9-year-old male suffering from hemophilia B, which results from a deficiency in factor IX (FIX), was treated with a cell line generated from a cell line, e.g., the HEK293 isolated cell line of ATCC No. PTA 13274, containing any of the following: Treat with AAV:
(1) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV2-derived Rep and AAV6-derived Cap gene; and hemophilia inserted between two ITRs a third plasmid encoding a nucleotide sequence for FIX to treat B;
(2) a first helper plasmid with Rep gene and Cap gene derived from AAV2; a second helper plasmid with Rep gene derived from AAV3 and Cap gene derived from AAV7; and hemophilia inserted between two ITRs a third plasmid encoding a nucleotide sequence for FIX to treat B;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV6-derived Cap gene; and a fourth plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs;
(4) a helper plasmid with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6; and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs;
(5) a helper plasmid having Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7; and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs; or (6) A helper plasmid with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7', and a second plasmid encoding the nucleotide sequence for FIX inserted between the two ITRs.
Here, AAV is administered to a patient and immediately after administration the patient exhibits a reduction in the severity of hemophilia B, including a reduction in bleeding episodes.

Treatment of hemophilia A
An 8-year-old male suffering from hemophilia A resulting from a deficiency of factor VIII (FVIII) was treated with a cell line generated from a cell line, e.g., the HEK293 isolated cell line of ATCC No. PTA 13274, containing any of the following: Treat with AAV:
(1) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV2-derived Rep and AAV6-derived Cap genes; and a nucleotide for FVIII inserted between two ITRs a third plasmid encoding the sequence;
(2) A first helper plasmid with AAV2-derived Rep gene and Cap gene; a second helper plasmid with AAV3-derived Rep and AAV7-derived Cap genes; and a nucleotide for FVIII inserted between two ITRs a third plasmid encoding the sequence;
(3) A first helper plasmid that has an AAV3-derived Rep gene and a Cap gene; a second helper plasmid that has an AAV3-derived Rep gene and an AAV6-derived Cap gene; and a fourth plasmid encoding a nucleotide sequence for FVIII inserted between the two ITRs;
(4) a helper plasmid with Rep from AAV2 and VP1 from AAV2, VP2 from AAV6 and VP3 from AAV6; and a second plasmid encoding the nucleotide sequence for FVIII inserted between the two ITRs;
(5) a helper plasmid having Rep from AAV2 and VP1 from AAV3, VP2 from AAV7 and VP3 from AAV7; and a second plasmid encoding a nucleotide sequence for FVIII inserted between the two ITRs; or (6) A helper plasmid with Rep from AAV2 and VP1 from AAV3, VP2 from AAV6 and VP3 from AAV7', and a second plasmid encoding the nucleotide sequence for FVIII inserted between the two ITRs.
Here, AAV is administered to a patient, and immediately after administration, the patient exhibits a reduction in the severity of hemophilia A, including a reduction in bleeding episodes.

Example 7. Generation of haploid capsids from two different serotypes and initiation codon mutations In this example, polyploid AAV virions are assembled from capsids of two different serotypes. Ligating the nucleotide sequences for VP1, VP2, and VP3 from only the first AAV serotype into the helper plasmid so that the helper plasmid contains nucleic acid sequences for VP1, VP2, and VP3 capsid proteins from two different serotypes. and ligate the nucleotide sequences for VP1, VP2, and VP3 from only the second AAV serotype into the same or a different helper plasmid. Either before or after ligation of the first and second serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins into the helper plasmid, VP1 derived only from the first serotype and the second The capsid nucleotide sequence is altered to provide VP2 and VP3 derived only from serotypes of In this example, as shown in Figure 7, the VP1 nucleotide sequence of the first serotype is altered by mutating the initiation codons for the VP2 and VP3 capsid proteins. In this example, we used the ACG start site of VP2 and the three ATG start sites of VP3 to ensure that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of the VP2 and VP3 capsid proteins from the first serotype. mutate it like this. Similarly, as shown in Figure 8, the ATG start site of VP1 has been modified such that this codon cannot initiate translation of RNA encoding VP1, but can initiate translation for both VP2 and VP3. mutated in the nucleotide sequence encoding the capsid protein of serotype 2. Therefore, in this example, polypoid AAV virions containing VP1 but not VP2 or VP3 from only a first serotype, and VP2 and VP3 but not VP1 from only a second serotype, Create.

In applying this technique to generate polyploid AAV virions through mutation of the start codon, the start codons of VP2 and VP3 of AAV2 are modified such that only VP1 is translated from the RNA transcribed from the plasmid shown in Figure 19. were mutated as highlighted in . In a further application of this technique, the start codon of VP1 of AAV2 was mutated as highlighted in Figure 18 so that VP2 and VP3 rather than VP1 were translated from the RNA transcribed from the plasmid shown in Figure 19. . Mutation of the start codon therefore provides a way to knock out expression of one or more of VP1, VP2, and VP3.

Example 8. Generation of haploid capsids from two different serotypes and start codon mutations In this example, polyploid AAV virions are assembled from capsids of two different serotypes. ligate the nucleotide sequences for VP1, VP2, and VP3 from only the first AAV serotype into the helper plasmid so that the helper plasmid contains VP1, VP2, and VP3 capsid proteins from two different serotypes, and , ligate VP1, VP2, and VP3 from only the second AAV serotype into the same or different helper plasmids. VP1 and VP3 derived from only the first serotype, either before or after ligation of the first and second serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins into the helper plasmid; The capsid nucleotide sequence is altered to provide VP2 derived only from the second serotype. In this example, the ACG initiation site of VP2 is mutated such that this codon is unable to initiate translation of RNA transcribed from the nucleotide sequence of the VP2 capsid protein from the first serotype. Similarly, the ATG start sites for VP1 and VP3 were modified in a second serotype such that these codons cannot initiate translation of RNA encoding VP1 and VP3, but can initiate translation for both VP2. mutated in the nucleotide sequence encoding the capsid protein. Therefore, in this example, polypoid AAV virions containing VP1 and VP3 but not VP2 from only a first serotype and VP2 but not VP1 and VP3 from only a second serotype Create.

In applying this technique to generate polyploid AAV virions through mutation of the start codon, the start codon of VP2 of AAV2 was changed to Figure 20 such that VP1 and VP3 are translated from RNA transcribed from the plasmid shown in Figure 20. Mutations were made as highlighted. Mutation of the start codon therefore provides a way to knock out expression of one or more of VP1, VP2, and VP3.

Example 9. Generation of haploid capsids from two different serotypes and splice acceptor site mutations In this example, polyploid AAV virions are assembled from capsids of two different serotypes. ligate the nucleotide sequences for VP1, VP2, and VP3 from only the first AAV serotype into the helper plasmid so that the helper plasmid contains VP1, VP2, and VP3 capsid proteins from two different serotypes, and , ligate VP1, VP2, and VP3 from only the second AAV serotype into the same or different helper plasmid. Either before or after ligation of the first and second serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins into the helper plasmid, VP1 derived only from the first serotype and the second The capsid nucleotide sequence is altered to provide VP2 and VP3 derived only from serotypes of In this example, as shown in Figure 9, the nucleotide sequence of the first serotype has been altered by mutating the A2 splice acceptor site. In this example, by mutating the A2 splice acceptor site, VP2 and VP3 capsid proteins from the first serotype are not produced. Similarly, as shown in Figure 10, by mutating the A1 splice acceptor site, VP1 capsid protein from the second serotype is not produced, while VP2 and VP3 capsid proteins are produced. Therefore, in this example, polypoid AAV virions containing VP1 but not VP2 or VP3 from only a first serotype, and VP2 and VP3 but not VP1 from only a second serotype, Create.

Example 10. Generation of haploid capsids from two different serotypes and mutations in initiation codons and splice acceptor sites In this example, polyploid AAV virions are assembled from capsids of two different serotypes. ligate the nucleotide sequences for VP1, VP2, and VP3 from only the first AAV serotype into the helper plasmid so that the helper plasmid contains VP1, VP2, and VP3 capsid proteins from two different serotypes, and , ligate VP1, VP2, and VP3 derived only from the second AAV serotype into the same or different plasmid. Either before or after ligation of the first and second serotype nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins into the helper plasmid, VP1 derived only from the first serotype and the second The capsid nucleotide sequence is altered to provide VP2 and VP3 derived only from serotypes of In this example, as shown in Figure 11, the nucleotide sequence of the first serotype has been altered by mutating the initiation codons for the VP2 and VP3 capsid proteins and mutating the A2 splice acceptor site. In this example, the ACG start site of VP2 and the three ATG start sites of VP3 are mutated together with the A2 splice acceptor site. As a result, only VP1 capsid protein of the first serotype is produced. Neither VP2 nor VP3 capsid proteins from the first serotype are produced. Similarly, the ATG start site of VP1 is mutated together with the A1 splice acceptor site, as shown in Figure 12. As a result, only the second serotype VP2 and VP3 capsid proteins are produced. VP1 capsid protein from the second serotype is not produced. Therefore, in this example, polypoidal AAV virions containing VP1 but no VP2 or VP3 from only a first serotype and containing VP2 and VP3 but no VP1 from only a second serotype were used. Produce.

Example 11. Generation of haploid capsids from two different serotypes using two plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2/VP3 from AAV9 were generated using two plasmids. Make it. A helper plasmid containing a plasmid backbone together with the Ad early gene and Rep (eg, from AAV2) is created as shown in Figure 13. Ligated to this helper plasmid are a nucleotide sequence encoding a capsid protein derived only from AAV5 and a separate nucleotide sequence encoding a capsid protein derived only from AAV9. Regarding the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has the initiation codon for VP2/VP3 mutated to prevent translation and/or the A2 splice acceptor site prevents splicing. It has been mutated as follows. Regarding the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has the initiation codon for VP1 mutated to prevent translation and/or the A1 splice acceptor site is mutated to prevent splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line, ATCC No. PTA 13274, along with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). Virus is purified from the supernatant and characterized. As shown in Figure 13, the viral capsid contains VP2/VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray) as seen in the virion shown at the bottom of Figure 13. .

Example 12. Generation of haploid capsids from two different serotypes using three plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2/VP3 from AAV9 were generated using three plasmids. Make it. A first helper plasmid containing the Ad early gene is created as shown in Figure 14. Create a second helper plasmid containing the plasmid backbone together with Rep (e.g. AAV2). To this second helper plasmid are ligated a nucleotide sequence encoding a capsid protein derived only from AAV5 and a separate nucleotide sequence encoding a capsid protein derived only from AAV9. Regarding the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has the initiation codon for VP2/VP3 mutated to prevent translation and/or the A2 splice acceptor site prevents splicing. It has been mutated as follows. Regarding the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has the initiation codon for VP1 mutated to prevent translation and/or the A1 splice acceptor site is mutated to prevent splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line, ATCC No. PTA 13274, along with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). Virus is purified from the supernatant and characterized. As shown in Figure 14, the viral capsid contains VP2/VP3 of AAV9 (shown in light gray) and VP1 of AAV5 (shown in dark gray) as seen in the virion shown at the bottom of Figure 13. .

Example 13. Generation of haploid capsids from two different serotypes using four plasmids In this example, haploid AAV virions containing VP1 from AAV5 and VP2/VP3 from AAV9 were generated using four plasmids. Make it. A first helper plasmid containing the Ad early gene is created as shown in Figure 15. Create a second helper plasmid containing the plasmid backbone together with Rep (e.g. AAV2). A nucleotide sequence encoding a capsid protein derived only from AAV5 is ligated to this second helper plasmid. Create a third helper plasmid containing the plasmid backbone together with Rep. A nucleotide sequence encoding a capsid protein derived only from AAV9 is ligated to this third helper plasmid. The fourth plasmid contains the transgene and two ITRs. Regarding the nucleotide sequence encoding the capsid protein of AAV5, this nucleotide sequence has the initiation codon for VP2/VP3 mutated to prevent translation and/or the A2 splice acceptor site prevents splicing. It has been mutated as follows. Regarding the nucleotide sequence encoding the capsid protein of AAV9, this nucleotide sequence has the initiation codon for VP1 mutated to prevent translation and/or the A1 splice acceptor site is mutated to prevent splicing. It has been mutated. The helper plasmid is transfected into the HEK293 cell line, ATCC No. PTA 13274, along with a plasmid encoding a transgene with two ITRs (see, eg, US Pat. No. 9,441,206). Virus is purified from the supernatant and characterized. As shown in Figure 14, the viral capsid contains VP2/VP3 of AA9 (shown in light gray) and VP1 of AAV5 (shown in dark gray) as seen in the virion shown at the bottom of Figure 13. .

Example 14. Generation of haploid capsids from three different serotypes and initiation codon mutations In this example, polyploid AAV virions are assembled from capsids of three different serotypes. VP1, VP2, and VP3 from only the first AAV serotype, such that the helper plasmid contains nucleic acid sequences for VP1, VP2, and VP3 capsid proteins from three different serotypes, and VP1, VP2, and VP3 from the second AAV serotype. Helper plasmids are constructed by ligating the nucleotide sequences for VP1, VP2, and VP3 from only the AAV serotype and VP1, VP2, and VP3 from only the third AAV serotype into the helper plasmid. Either before or after ligating the nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins from each of the three different serotypes into the helper plasmid, the VP1, VP3, and VP3 capsid proteins from only the first serotype are added to the helper plasmid. The capsid nucleotide sequence is altered to provide VP2 from only one serotype and VP3 from only a third serotype. In this example, the VP1 nucleotide sequence of the first serotype is altered by mutating the start codons for the VP2 and VP3 capsid proteins. In this example, we used the ACG start codon of VP2 and the three ATG start codons of VP3, which are incapable of initiating the translation of RNA transcribed from the nucleotide sequences of the VP2 and VP3 capsid proteins from the first serotype. mutate like this. Similarly, the VP1 and VP3 nucleotide sequences of the second serotype have been altered by mutating the start codons for the VP1 and VP3 capsid proteins. In this example, the ATG start site of VP1 and the three ATG start codons of VP3 are mutated such that these codons cannot initiate translation of RNA transcribed from the nucleotide sequences of the VP1 and VP3 capsid proteins. Additionally, the VP1 and VP2 nucleotide sequences of the third serotype have been altered by mutating the initiation codons for the VP1 and VP2 capsid proteins. In this example, the ATG initiation codon of VP1 and the ACG initiation codon of VP2 are mutated such that these codons are unable to initiate translation of RNA transcribed from the nucleotide sequences of the VP1 and VP2 capsid proteins. Thus, this example includes VP1 derived only from the first serotype but not VP2 or VP3; VP2 derived only from the second serotype but not VP1 or VP2; Polypoid AAV virions are generated that contain only serotype-derived VP3 but no VP1 or VP2.

Example 15. Generation of haploid capsids from two different serotypes using DNA shuffling. Generate polyploid AAV virions. In this example, the nucleotide capsid protein sequences for AAV1, AAV2 and AAV8 are subjected to treatment with one or more restriction enzymes and/or DNase to cut the DNA into 50-100 bp long DNA fragments. The mixture of DNA fragments is then subjected to primerless polymerase chain reaction (PCR). PCR is repeated multiple times or until the DNA molecule produced by PCR reaches the size of the nucleic acid encoding the capsid gene. At this point, another round of PCR is performed in which primers containing sequences for the restriction enzyme recognition sites are added to allow ligation of the newly created DNA to the helper plasmid. Prior to ligation into the helper plasmid, sequence the AAV1/2/8 nucleotide sequence and translate any start codon within the nucleotide sequence that can initiate translation of the VP2 and VP3 capsid proteins from RNA transcribed from this sequence. mutate to prevent In this way, AAV1/2/8 can produce only VP1, and the AAV1/2/8 nucleotide sequence is ligated into the helper plasmid. In this experiment, the nucleotide sequences encoding the AAV9 capsid proteins (VP1, VP2, and VP3) are also ligated into the same or different helper plasmids. To generate polyploid AAV virions with VP1 derived from the AAV1/2/8 nucleotide sequence generated by DNA shuffling and VP2 and VP3 derived only from AAV9, the ATG start codon of VP1 of AAV9, encoding VP1 Mutations are made so that RNA cannot be translated. Therefore, this example contains VP1, but no VP2 or VP3, derived from a nucleotide sequence created by DNA shuffling the capsid protein nucleotide sequence of AAV1/2/8, and VP2 and VP3 derived only from AAV9. Generate polypoid AAV virions containing but not VP1.

An example of DNA shuffling is shown in Figure 16, starting with nucleic acids encoding VP1, VP2, and VP3 from eight AAV serotypes, processing the nucleic acids first through DNase I fragmentation, and then Assemble and amplify various fragments of AAV-derived nucleic acids. A DNA shuffled nucleic acid encoding the AAV capsid protein is generated, which is then expressed to create a library of capsids. These capsids are then tested on animals to screen for capsids that exhibit specific tissue tropism and/or reduced immunogenicity, and those that show promise are selected for further development (Figure 16) .

Example 16. Liver transduction of haploid vector H-AAV829
Experiments were performed using three AAVs. In Figure 22A, the composition of AAV capsid subunits is shown. A hybrid AAV (AAV82) is shown that combines the amino acids of only VP1 derived from AAV8 and the amino acids encoding VP2 and VP3 derived from AAV2. Two haploid AAV viruses were produced from cotransfection of two plasmids, one encoding VP1 and VP2 and another encoding VP3, into HEK293 cells. Three AAVs, AAV82, 28m-2vp3 and H-AAV82, along with parental AAV2 controls, were injected into C57BL6 mice via the retroorbital vein at a dose of 3 x 10 ¹⁰ particles (Figure 22B). Imaging was performed one week later (Figure 22B). Liver transduction was quantified based on data representing the mean and standard deviation of 5 mice (Figure 22C).

Example 17. Muscle transduction of haploid vector H-AAV82 The three AAVs from Example 23 (AAV82, H-AAV82 and 28m-vp3) were then injected into the hind limb muscles of mice at a dose of 1 x 10 ⁹ AAV/luc particles. Injected. Three weeks after injection, images were taken for 3 minutes as seen in Figure 23A. Imaging was performed supine: left leg - AAV82, H-AAV82 or 28m-vp3 and right leg - AAV2 parent AAV. Figure 23B provides data for four mice after intramuscular injection and calculated fold increase in transduction by transduction from AAV82, H-AAV82 or 28m-vp3 versus parental AAV2.

Example 18. Liver transduction of haploid vector H-AAV92 In this experiment, haploid AAV92 is generated in which VP1 and VP2 are derived only from AAV9 and VP3 is derived only from AAV3 (Figure 24A). H-AAV92 was produced from co-transfection of two plasmids, one encoding AAV9 VP1 and VP2 and another encoding AAV2 VP3, into HEK293 cells. H-AAV92 and parental AAV2 were injected into C57BL6 mice via the retroorbital vein at a dose of 3 x 10 ¹⁰ particles (Figure 24B). Imaging was performed one week later (Figure 24B). Liver transduction was quantified based on data representing the mean and standard deviation of 5 mice (Figure 24C).

Example 19. Liver transduction of haploid vector H-AAV82G9 In this experiment, haploid AAV82G9 is generated in which VP1 and VP2 are derived only from AAV8 and VP3 is derived only from AAV2G9 (Figure 25A). H-AAV82G9 was produced from co-transfection of two plasmids, one encoding AAV8 VP1 and VP2 and another encoding AAV2G9 VP3, into HEK293 cells. H-AAV82G9 and AAV2G9 were injected into C57BL6 mice via the retroorbital vein at a dose of 3 x 10 ¹⁰ particles (Figure 25B). Imaging was performed one week later (Figure 25B). Liver transduction was quantified based on data representing the mean and standard deviation of 5 mice (Figure 25C).

Finally, although aspects of the specification are illustrated by reference to specific embodiments, those skilled in the art will appreciate that these disclosed embodiments simply embody the principles of the subject matter disclosed herein. It should be understood that these are merely illustrative examples. Therefore, it should be understood that the disclosed subject matter is in no way limited to the particular methodologies, protocols and/or reagents, etc. described herein. As such, various modifications or changes to the disclosed subject matter or alternative arrangements thereof may be made in accordance with the teachings herein without departing from the spirit thereof. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. isn't it. Accordingly, the invention is not limited to what has been precisely shown and described.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. It will be appreciated that variations to these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will employ such variations as appropriate, and the inventors intend that the invention may be modified otherwise than as specifically described herein. intended to be implemented. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of all possible variations of the embodiments described above is encompassed by the present invention, unless otherwise indicated herein or the context clearly indicates otherwise, any combination of all possible variations of the above embodiments are encompassed by the invention.

Groupings of alternative aspects, elements or steps of the invention should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and/or patentability. In the event of such inclusion or deletion, the specification is deemed to include that group as modified and therefore as fulfilling the description of any Markush group as used in the appended claims. .

Unless indicated otherwise, all numerical values expressing features, items, quantities, parameters, properties, time periods, etc., as used in this specification and the claims, are modified in all cases by the term "about." It should be understood that As used herein, the term "about" means that the feature, item, amount, parameter, property or period so conditioned is less than or equal to the stated feature, item, amount, parameter, characteristic or period of time. It means to cover the range of ±10% above and below the value. Accordingly, unless indicated to the contrary, the numerical parameters set forth herein and in the appended claims are approximations that may vary. At a minimum, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical representation shall, at a minimum, take into account the number of significant digits to be reported and apply normal rounding techniques. should be interpreted by Although the numerical ranges and values describing the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as accurately as possible. However, any numerical range or value inherently contains some error that necessarily results from the standard deviation found in each test measurement. The recitation of numerical ranges of values herein only serves as a way to omit individual reference to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated herein as if individually recited herein.

All methods described herein can be performed in any suitable order, unless otherwise indicated herein or the context is clear to the contrary. Any and all examples provided herein or the use of exemplary language (e.g., "such as") are only intended to better illustrate the invention. No limitation on the scope of the invention as otherwise claimed is intended. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited using the language "consisting of" or "consisting essentially of" in the claims. When used in a claim, the transitional term ``consisting of'' refers to any element not specified in the claim, whether filed or added by amendment. , eliminate a step or component. The transitional term "consisting essentially of" limits the scope of the claim to those materials or steps specified and to those that do not substantially affect the essential and novel features. Aspects of the invention as claimed are described essentially or expressly herein and are enabled herein.

All patents, patent publications, and other publications referenced and identified herein describe and disclose, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. , which are individually and expressly incorporated herein by reference in their entirety. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or otherwise. All statements regarding the dates or contents of these documents are based on information available to the applicants and do not constitute an admission as to the accuracy of the dates or contents of these documents.

Example 20. Chimeric Capsid Protein and AAV Haploid Viral Vector Transduction As described above, we generated a series of constructs of AAV helper plasmids with mutations in the start code of the capsid ORF in which only one or two viral VP proteins were expressed. did. We also generated chimeric AAV helper constructs in which VP1/2 proteins are driven from two different serotypes (AAV2 and AAV8). These constructs were used to produce a panel of haploid virus vectors and evaluate transduction efficacy in mice. It was found that enhanced transduction was achieved from haploid vectors with VP1/VP2 from serotypes 7, 8, 9 and rh10 and VP3 from AAV2 or AAV3 compared to AAV2-only and AAV3-only vectors. It was. It was further shown that chimeric VP1/VP2 capsids with an N-terminus from AAV2 and a C-terminus from AAV8 as well as AAV vectors made from AAV2-derived VP3 induced much higher transduction. The data provided herein demonstrate a simple and effective method to enhance AAV transduction for further applications of AAV vectors.

Haploid vectors with VP1/VP2 from other serotypes and VP3 from AAV2 enhance AAV liver transduction
Haploid viruses were produced by co-transfecting plasmids expressing AAV8 VP1/2 and AAV2 VP3 at a 1:1 ratio. The results showed that haploid vector AAV82 with VP1/VP2 from AAV8 and VP3 from AAV2 increased liver transduction (Figures 22B and 22C).

A haploid AAV92 vector (H-AAV92) was produced using VP1/VP2 of AAV9 and VP3 of AAV2 (Figure 24A). Imaging was performed 1 week after systemic administration. Approximately 4-fold higher liver transduction than with AAV2 was achieved with H-AAV92 (Figures 24B and 24C). This data indicates that VP1/VP2 from other serotypes can increase AAV2 transduction.

Enhanced AAV liver transduction from haploid vectors with VP3 derived from AAV2 mutants
AAV9 vectors use glycans as their primary receptors for their efficient transduction. In a previous study, we grafted the AAV9 glycan receptor binding site onto the AAV2 capsid to create AAV2G9 and found that AAV2G9 has higher liver tropism than AAV2. A haploid vector (H-AAV82G9) in which VP1/VP2 is derived from AAV8 and VP3 is derived from AAV2G9 is described herein (Figure 25A). After systemic injection into mice, >10-fold higher liver transduction was observed at both 1 and 2 weeks after H-AAV82G9 application compared to AAV2G9 (Figures 25B and 25C). This data shows that integrating VP1/VP2 from other serotypes into AAV2 mutant VP3 could increase liver transduction.

Enhanced AAV liver transduction from haploid vectors with AAV3-derived VP3
A haploid vector in which VP3 is derived from another serotype and VP1/VP2 is derived from a different serotype or variant, the start code of which is mutated to express only AAV3 VP3 or AAV rh10 VP1/VP2. Haploid vector from which protein constructs were created. Different haploids H-AAV83 (VP1/VP2 from AAV8 and VP3 from AAV3), H-AAV93 (VP1/VP2 from AAV9 and VP3 from AAV3) and H-AAVrh10-3 (VP1/VP2 from AAV rh10 and AAV3-derived VP3) vector was produced (Figure 26A) and injected into mice via systemic administration. Imaging was performed in the first week. As shown in Figures 26B and 26C, higher liver transduction was achieved with haploid vectors (H-AAV83, H-AAV93 and H-AAVrh10-3) than with AAV3. This is consistent with results obtained with other haploid vectors. Furthermore, these haploid vectors also enhanced transduction from other tissues, as shown in Figures 26B and 26D. Interestingly, these haploid vectors also induced systemic transduction based on the imaging profile, which is different from the results for haploid vectors with AAV2-derived VP3, which only efficiently transduced the liver (Figure 22 and 24). In summary, haploid vectors with VP1/VP2 from one serotype and VP3 from an alternative were able to enhance transduction and possibly change their tropism.

Haploid vectors with C-terminus of VP1/VP2 from AAV8 and VP3 from AAV2 enhance AAV transduction
AAV8 VP1/VP2 only, AAV2 VP3 only, chimeric VP1/VP2 (28m-2VP3) with N terminus derived from AAV2 and C terminus derived from AAV8, or N terminus derived from AAV8 and AAV2 without mutation of the VP3 start codon A series of constructs were generated that expressed chimeric AAV8/2 with a derived C-terminus (Figure 27A). These plasmids were used to produce different combinations of haploid AAV vectors at a 1:1 plasmid ratio (Figure 27B). Liver transduction efficiency was assessed after injecting 1×10 ¹⁰ of these haploid vector particles into mice via the retroorbital vein (FIG. 27C). The chimeric AAV82 vector (AAV82) induced slightly higher liver transduction than AAV2. However, haploid AAV82 (H-AAV82) had much higher liver transduction than AAV2. A further increase in liver transduction was observed using haploid vector 28m-2vp3. These haploid vectors were administered into the muscles of mice. For a simple comparison, the AAV2 vector was injected into the right leg and the haploid vector was injected into the left leg while the mouse was supine. Images were taken 3 weeks after AAV injection. Consistent with the observations in liver, all haploid and chimeric vectors had higher muscle transduction, with the one from haploid vector 28m-2vp3 being the best (Figure 27D). This result indicates that the chimeric VP1/VP2 with an N terminus derived from AAV2 and a C terminus derived from AAV8 is responsible for the high liver transduction of the haploid AAV82 vector.

Increased virion trafficking to the nucleus from chimeric haploid vectors
AAV transduction involves many steps. After binding, AAV virions are taken up into endosomes via endocytosis. After escaping from endosomes, AAV virions translocate to the nucleus for transgene expression. We determined which steps resulted in high transduction from haploid vectors. First, we performed an AAV vector binding assay and found that fewer 28m-2VP3 virions bound to Huh7 cells than other vectors (Figure 28). Next, we detected the AAV genome copy number in the nucleus and found no differences between different AAV vectors. Interestingly, more AAV virions were observed in the nucleus compared to virions with combined AAV genome copy number (Figure 28). These results indicate that the AAV vector 28m-2VP3 is more efficient in trafficking.

High transduction of haploid AAV vectors does not result from virion stability The following experiments were performed by heating viral virions. Viruses were heated for half an hour at different temperatures and then applied to Western blots using primary antibodies A20 ADK8 or B1 that recognize intact or non-intact virions. As shown in Figure 29, all virus virions collapsed when the virus was heated at 70°C. With the exception of the AAV82 vector, there was no difference in heating stability among AAV haploid vectors regardless of different temperatures. This data indicates that enhanced transduction may not be associated with haploid virion stability.

Effect of acidic conditions on VP1 N-terminal exposure of haploid vectors
It has been demonstrated that the VP1/VP2 N terminus is exposed on the virion surface in acidic endosomes after endocytosis of AAV vectors. The VP1/VP2 terminus contains the phospholipase A2 and NLS domains for AAV vectors, which help the AAV virus escape from endosomes and translocate to the nucleus. AAV haploid vectors were incubated with PBS at different pH values for 30 min and then applied to Western blot analysis to detect the N-terminus of VP1 using antibody A1. The results showed that no VP1 N-terminus was exposed when the virus was treated at different pHs (Figure 30).

The data presented herein demonstrate that enhanced transduction can be achieved from haploid vectors with VP1/VP2 from one AAV vector capsid and VP3 from an alternative.

Plasmid and site-directed mutagenesis
All plasmids used to express VP12 and VP3 were generated by site-directed mutagenesis. Mutagenesis was performed using the QuikChange II XL Site-Directed mutagenesis Kit (Agilent) according to the manufacturer's manual. A fragment containing the N-terminus of the AAV2 capsid (1201 aa) and the C-terminus of the AAV8 capsid was generated by overlapping PCR. The fragment was then cloned into the SwaI and NotI sites of pXR. All mutations and constructs were verified by DNA sequencing.

Virus Production Recombinant AAV was produced by a triple plasmid transfection system. HEK293 cells in a 15 cm dish were transfected with 9 ug of AAV transgene plasmid pTR/CBA-Luc, 12 ug of AAV helper plasmid containing AAV Rep and Cap genes and 15 ug of Ad helper plasmid pXX6-80. Sixty hours after transfection, HEK293 cells were harvested and lysed. The supernatant was subjected to CsCl gradient ultracentrifugation. Virus titer was determined by quantitative PCR.

In vitro transduction assay Huh7 and C2C12 cells were transduced with recombinant viruses at 1×10 ⁴ vg/cell in flat-bottomed 24-well plates. After 48 hours, cells were harvested and evaluated by luciferase assay system (Promega, Madison, WI).

Animal studies The animal experiments performed in this study were performed on C57BL/6 mice and FIX-/- mice. Mice were maintained according to NIH guidelines as approved by the UNC Animal Care and Use Committee (IACUC). Six-week-old female C57BL/6 mice were injected with 1×10 ¹⁰ vg of recombinant virus via injection. After intraperitoneal (ip) injection of D-luciferin substrate (Nanolight Pinetop, AZ), luciferase expression was imaged 1 week after injection using a Xenogen IVIS Lumina (Caliper Lifesciences, Waltham, MA). Bioluminescence images were analyzed using Living Image (PerkinElmer, Waltham, MA). For muscle transduction, 5×10 ⁹ AAV/Luc particles were injected into the gastrocnemius muscle of 6-week-old female C57BL/6. Mice were imaged at the indicated time points.

Detection of AAV genome copy number in liver Minced liver was treated with protease K, and total genomic DNA was isolated by Pure Link Genomic DNA mini Kit (Invitrogen, Carlsbad, CA). Luciferase gene was detected by qPCR assay. The mouse lamin gene served as an internal control.

Statistical analysis Data were presented as mean ± SD. All statistical analyzes were performed using Student's t-test. A P value <0.05 was considered a statistically significant difference.

References

(Table 1)

(Table 2) Amino acid residues and abbreviations

表中、（X）→任意のアミノ酸への変異
（-）→任意のアミノ酸の挿入
注記：位置2の挿入は、挿入の部位によって示される (Table 3)

In the table, (X) → Mutation to any amino acid (-) → Insertion of any amino acid Note: Insertion at position 2 is indicated by the site of insertion

(Table 4)

(Table 5) Neutralizing antibody titer and cross-reactivity against triploid virus AAV2/8

(Table 6) Neutralizing antibody titer and cross-reactivity against haploid virus AAV2/8/9

array

Sequence information
SEQUENCE LISTING
<110> The University of North Carolina at Chapel Hill
<120> RATIONAL POLYPLOID ADENO-ASSOCIATED VIRUS VECTORS AND METHODS OF
MAKING AND USING THE SAME
<150> US 62/678,675
<151> 2018-05-31
<150> US 62/668,056
<151> 2018-05-07
<160> 142
<170> PatentIn version 3.5

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<211> 8
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 56
Cys Leu Arg Ser Gly Arg Gly Cys
1 5

<210> 57
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 57
Cys His Trp Met Phe Ser Pro Trp Cys
1 5

<210> 58
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (2)..(3)
<223> Xaa can be any naturally occurring amino acid
<400> 58
Trp Xaa Xaa Phe
1

<210> 59
<211> 8
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 59
Cys Ser Ser Arg Leu Asp Ala Cys
1 5

<210> 60
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 60
Cys Leu Pro Val Ala Ser Cys
1 5

<210> 61
<211> 13
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 61
Cys Gly Phe Glu Cys Val Arg Gln Cys Pro Glu Arg Cys
1 5 10

<210> 62
<211> 13
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 62
Cys Val Ala Leu Cys Arg Glu Ala Cys Gly Glu Gly Cys
1 5 10

<210> 63
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 63
Ser Trp Cys Glu Pro Gly Trp Cys Arg
1 5

<210> 64
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 64
Tyr Ser Gly Lys Trp Gly Trp
1 5

<210> 65
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 65
Gly Leu Ser Gly Gly Arg Ser
1 5

<210> 66
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 66
Leu Met Leu Pro Arg Ala Asp
1 5

<210> 67
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 67
Cys Ser Cys Phe Arg Asp Val Cys Cys
1 5

<210> 68
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 68
Cys Arg Asp Val Val Ser Val Ile Cys
1 5

<210> 69
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 69
Met Ala Arg Ser Gly Leu
1 5

<210> 70
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 70
Met Ala Arg Ala Lys Glu
1 5

<210> 71
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 71
Met Ser Arg Thr Met Ser
1 5

<210> 72
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 72
Lys Cys Cys Tyr Ser Leu
1 5

<210> 73
<211> 14
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 73
Met Tyr Trp Gly Asp Ser His Trp Leu Gln Tyr Trp Tyr Glu
1 5 10

<210> 74
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 74
Met Gln Leu Pro Leu Ala Thr
1 5

<210> 75
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 75
Glu Trp Leu Ser
1

<210> 76
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 76
Ser Asn Glu Trp
1

<210> 77
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 77
Thr Asn Tyr Leu
1

<210> 78
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 78
Trp Asp Leu Ala Trp Met Phe Arg Leu Pro Val Gly
1 5 10

<210> 79
<211> 13
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 79
Cys Thr Val Ala Leu Pro Gly Gly Tyr Val Arg Val Cys
1 5 10

<210> 80
<211> 13
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 80
Cys Val Ala Tyr Cys Ile Glu His His Cys Trp Thr Cys
1 5 10

<210> 81
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 81
Cys Val Phe Ala His Asn Tyr Asp Tyr Leu Val Cys
1 5 10

<210> 82
<211> 10
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 82
Cys Val Phe Thr Ser Asn Tyr Ala Phe Cys
1 5 10

<210> 83
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 83
Val His Ser Pro Asn Lys Lys
1 5

<210> 84
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 84
Cys Arg Gly Asp Gly Trp Cys
1 5

<210> 85
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> Xaa can be any naturally occurring amino acid
<400> 85
Xaa Arg Gly Cys Asp Xaa
1 5

<210> 86
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (1)..(2)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is S or T
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa can be any naturally occurring amino acid
<400> 86
Pro Xaa Xaa Xaa
1

<210> 87
<211> 11
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 87
Ser Gly Lys Gly Pro Arg Gln Ile Thr Ala Leu
1 5 10

<210> 88
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa is A or Q
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa is N or A
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa is L or Y
<220>
<221> MISC_FEATURE
<222> (14)..(14)
<223> Xaa is N, M or R
<220>
<221> MISC_FEATURE
<222> (15)..(15)
<223> Xaa is R or K
<400> 88
Ala Ala Ala Ala Ala Ala Ala Ala Ala Xaa Xaa Xaa Thr Xaa Xaa
1 5 10 15

<210> 89
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 89
Val Tyr Met Ser Pro Phe
1 5

<210> 90
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 90
Ala Thr Trp Leu Pro Pro Arg
1 5

<210> 91
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 91
His Thr Met Tyr Tyr His His Tyr Gln His His Leu
1 5 10

<210> 92
<211> 19
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 92
Ser Glu Val Gly Cys Arg Ala Gly Pro Leu Gln Trp Leu Cys Glu Lys
1 5 10 15
Tyr Phe Gly

<210> 93
<211> 18
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 93
Cys Gly Leu Leu Pro Val Gly Arg Pro Asp Arg Asn Val Trp Arg Trp
1 5 10 15
Leu Cys

<210> 94
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 94
Cys Lys Gly Gln Cys Asp Arg Phe Lys Gly Leu Pro Trp Glu Cys
1 5 10 15

<210> 95
<211> 5
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 95
Ser Gly Arg Ser Ala
1 5

<210> 96
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 96
Trp Gly Phe Pro
1

<210> 97
<211> 5
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (3)..(4)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is Y, W, F or H
<400> 97
Leu Trp Xaa Xaa Xaa
1 5

<210> 98
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (3)..(4)
<223> Xaa can be any naturally occurring amino acid
<400> 98
Xaa Phe Xaa Xaa Tyr Leu Trp
1 5

<210> 99
<211> 17
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 99
Ala Glu Pro Met Pro His Ser Leu Asn Phe Ser Gln Tyr Leu Trp Tyr
1 5 10 15
Thr

<210> 100
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> Xaa is W or F
<400> 100
Trp Ala Tyr Xaa Ser Pro
1 5

<210> 101
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 101
Ile Glu Leu Leu Gln Ala Arg
1 5

<210> 102
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 102
Asp Ile Thr Trp Asp Gln Leu Trp Asp Leu Met Lys
1 5 10

<210> 103
<211> 16
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 103
Ala Tyr Thr Lys Cys Ser Arg Gln Trp Arg Thr Cys Met Thr Thr His
1 5 10 15

<210> 104
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 104
Pro Gln Asn Ser Lys Ile Pro Gly Pro Thr Phe Leu Asp Pro His
1 5 10 15

<210> 105
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 105
Ser Met Glu Pro Ala Leu Pro Asp Trp Trp Trp Lys Met Phe Lys
1 5 10 15

<210> 106
<211> 16
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 106
Ala Asn Thr Pro Cys Gly Pro Tyr Thr His Asp Cys Pro Val Lys Arg
1 5 10 15

<210> 107
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 107
Thr Ala Cys His Gln His Val Arg Met Val Arg Pro
1 5 10

<210> 108
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 108
Val Pro Trp Met Glu Pro Ala Tyr Gln Arg Phe Leu
1 5 10

<210> 109
<211> 8
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 109
Asp Pro Arg Ala Thr Pro Gly Ser
1 5

<210> 110
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 110
Phe Arg Pro Asn Arg Ala Gln Asp Tyr Asn Thr Asn
1 5 10

<210> 111
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 111
Cys Thr Lys Asn Ser Tyr Leu Met Cys
1 5

<210> 112
<211> 11
<212>PRT
<213> Artificial
<220>
<223> peptide
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa is R or Q
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> Xaa is L or R
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa is G or N
<220>
<221> MISC_FEATURE
<222> (6)..(7)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa is A or N
<400> 112
Cys Xaa Xaa Thr Xaa Xaa Xaa Gly Xaa Gly Cys
1 5 10

<210> 113
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 113
Cys Pro Ile Glu Asp Arg Pro Met Cys
1 5

<210> 114
<211> 12
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 114
His Glu Trp Ser Tyr Leu Ala Pro Tyr Pro Trp Phe
1 5 10

<210> 115
<211> 9
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 115
Met Cys Pro Lys His Pro Leu Gly Cys
1 5

<210> 116
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 116
Arg Met Trp Pro Ser Ser Thr Val Asn Leu Ser Ala Gly Arg Arg
1 5 10 15

<210> 117
<211> 20
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 117
Ser Ala Lys Thr Ala Val Ser Gln Arg Val Trp Leu Pro Ser His Arg
1 5 10 15
Gly Gly Glu Pro
20

<210> 118
<211> 20
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 118
Lys Ser Arg Glu His Val Asn Asn Ser Ala Cys Pro Ser Lys Arg Ile
1 5 10 15
Thr Ala Ala Leu
20

<210> 119
<211> 4
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 119
Glu Gly Phe Arg
1

<210> 120
<211> 6
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 120
Ala Gly Leu Gly Val Arg
1 5

<210> 121
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 121
Gly Thr Arg Gln Gly His Thr Met Arg Leu Gly Val Ser Asp Gly
1 5 10 15

<210> 122
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 122
Ile Ala Gly Leu Ala Thr Pro Gly Trp Ser His Trp Leu Ala Leu
1 5 10 15

<210> 123
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 123
Ser Met Ser Ile Ala Arg Leu
1 5

<210> 124
<211> 7
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 124
His Thr Phe Glu Pro Gly Val
1 5

<210> 125
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 125
Asn Thr Ser Leu Lys Arg Ile Ser Asn Lys Arg Ile Arg Arg Lys
1 5 10 15

<210> 126
<211> 15
<212>PRT
<213> Artificial
<220>
<223> peptide
<400> 126
Leu Arg Ile Lys Arg Lys Arg Arg Lys Arg Lys Lys Thr Arg Lys
1 5 10 15

<210> 127
<211> 4636
<212> DNA
<213> Artificial
<220>
<223> AAV1
<400> 127
ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag 60
agctgccaga cgacggccct ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa 120
cgcgacaggg gggagagtgc cacactctca agcaaggagg ttttgtaagt ggtgatgtca 180
tatagttgtc acgcgatagt taatgattaa cagtcaggtg atgtgtgtta tccaatagga 240
tgaaagcgcg cgcatgagtt ctcgcgagac ttccggggta taaaggggtg agtgaacgag 300
cccgccgcca ttctctgctc tgaactgcta gaggaccctc gctgccatgg ctaccttcta 360
cgaagtcatt gttcgcgtcc catttgacgt ggaggaacat ctgcctggaa tttctgacag 420
ctttgtggac tgggtaactg gtcaaatttg ggagctgcct ccccgagtcag atttgaattt 480
gactctgatt gagcagcctc agctgacggt tgctgacaga attcgccgcg tgttcctgta 540
cgagtggaac aaattttcca agcaggaatc caaattcttt gtgcagtttg aaaagggatc 600
tgaatatttt catctgcaca cgcttgtgga gacctccggc atctcttcca tggtcctagg 660
ccgctacgtg agtcagattc gcgcccagct ggtgaaagtg gtcttccagg gaatcgagcc 720
acagatcaac gactgggtcg ccatcaccaa ggtaaagaag ggcggagcca ataaggtggt 780
ggattctggg tatattcccg cctacctgct gccgaaggtc caaccggagc ttcagtgggc 840
gtggacaaac ctggacgagt ataaattggc cgccctgaac ctggaggac gcaaacggct 900
cgtcgcgcag tttctggcag aatcctcgca gcgctcgcag gaggcggctt cgcagcgtga 960
gttctcggct gacccggtca tcaaaagcaa gacttcccag aaatacatgg cgctcgtcaa 1020
ctggctcgtg gagcacggca tcacttccga gaagcagtgg atccaggaga atcaggagag 1080
ctacctctcc ttcaactcca cgggcaactc tcggagccaa atcaaggccg cgctcgacaa 1140
cgcgaccaaa atcatgagtc tgacaaaaag cgcggtggac tacctcgtgg ggagctccgt 1200
tcccgaggac atttcaaaaa acagaatctg gcaaattttt gagatgaacg gctacgaccc 1260
ggcctacgcg ggatccatcc tctacggctg gtgtcagcgc tccttcaaca agaggaacac 1320
cgtctggctc tacggacccg ccacgaccgg caagaccaac atcgcggagg ccatcgccca 1380
cactgtgccc ttttacggct gcgtgaactg gaccaatgaa aactttccct ttaatgactg 1440
tgtggacaaa atgctcattt ggtgggagga gggaaagatg accaacaagg tggttgaatc 1500
cgccaaggcc atcctggggg gctccaaggt gcgggtcgat cagaaatgta aatcctctgt 1560
tcaaattgat tctacccccg tcattgtaac ttccaataca aacatgtgtg tggtggtgga 1620
tgggaattcc acgacctttg aacaccagca gccgctggag gaccgcatgt tcaaatttga 1680
actgactaag cggctcccgc cagattttgg caagattact aagcaggaag tcaaagactt 1740
ttttgcttgg gcaaaggtca atcaggtgcc ggtgactcac gagtttaaag ttcccaggga 1800
attggcggga actaaagggg cggagaaatc tctaaaacgc ccactgggtg acgtcaccaa 1860
tactagctat aaaagtccag agaagcggggc ccggctctca tttgttcccg agacgcctcg 1920
cagttcagac gtgactgtcg atcccgctcc tctgcgaccg ctcaattgga attcaaggta 1980
tgattgcaaa tgtgaccatc atgctcaatt tgacaacatt tctgacaaat gtgatgaatg 2040
tgaatatttg aatcggggca aaaatggatg tatctgtcac aatgtaactc actgtcaaat 2100
ttgtcacggg attcccccct gggagaagga aaacttgtca gattttgggg attttgacga 2160
tgccaataaa gaacagtaaa taaagcgagt agtcatgtct tttgttgatc accctccaga 2220
ttggttggaa gaagttggtg aaggtcttcg cgagtttttg ggccttgaag cgggcccacc 2280
gaaaccgaaa cccaatcagc agcatcaaga tcaagcccgt ggtcttgtgc tgcctggtta 2340
taactatctc ggacccggaa acggtctcga tcgaggagag cctgtcaaca gggcagacga 2400
ggtcgcgcga gagcacgaca tctcgtacaa cgagcagctt gaggcgggag acaaccccta 2460
cctcaagtac aaccacgcgg acgccgagtt tcaggagaag ctcgccgacg acacatcctt 2520
cgggggaaac ctcggaaagg cagtctttca ggccaagaaa agggttctcg aaccttttgg 2580
cctggttgaa gagggtgcta agacggcccc taccggaaag cggatagacg accactttcc 2640
aaaaagaaag aaggctcgga ccgaagagga ctccaagcct tccacctcgt cagacgccga 2700
agctggaccc agcggatccc agcagctgca aatcccagca caaccagcct caagtttggg 2760
agctgataca atgtctgcgg gaggtggcgg cccattgggc gacaataacc aaggtgccga 2820
tggagtgggc aatgcctcgg gagattggca ttgcgattcc acgtggatgg gggacagagt 2880
cgtcaccaag tccacccgca cctgggtgct gcccagctac aacaaccacc agtaccgaga 2940
gatcaaaagc ggctccgtcg acggaagcaa cgccaacgcc tactttggat acagcacccc 3000
ctgggggtac tttgacttta accgcttcca cagccactgg agccccccgag actggcaaag 3060
actcatcaac aactattggg gcttcagacc ccggtctctc agagtcaaaa tcttcaacat 3120
ccaagtcaaa gaggtcacgg tgcaggactc caccaccacc atcgccaaca acctcacctc 3180
caccgtccaa gtgtttacgg acgacgacta ccaactcccg tacgtcgtcg gcaacgggac 3240
cgagggatgc ctgccggcct tccccccgca ggtctttacg ctgccgcagt acggctacgc 3300
gacgctgaac cgagacaacg gagacaaccc gacagagcgg agcagcttct tttgcctaga 3360
gtactttccc agcaagatgc tgaggacggg caacaacttt gagtttacct acagctttga 3420
agaggtgccc ttccactgca gcttcgcccc gagccagaac ctctttaagc tggccaaccc 3480
gctggtggac cagtacctgt accgcttcgt gagcacctcg gccacgggcg ccatccagtt 3540
ccaaaagaac ctggcgggca gatacgccaa cacctacaaa aactggttcc cggggcccat 3600
gggccgaacc cagggctgga aacacgagctc tggcagcagc accaacagag tcagcgtcaa 3660
caacttttcc gtctcaaacc ggatgaacct ggagggggcc agctaccaag tgaacccca 3720
gcccaacggg atgacaaaca cgctccaagg cagcaaccgc tacgcgctgg aaaacaccat 3780
gatcttcaac gctcaaaacg ccacgccggg aactacctcg gtgtacccag aggacaatct 3840
actgctgacc agcgagagcg agactcagcc cgtcaaccgg gtggcttaca aacacgggcgg 3900
tcagatggcc accaacgccc agaacgccac cacggctccc acggtcggga cctacaacct 3960
ccaggaagtg cttcctggca gcgtatggat ggagagggac gtgtacctcc aaggacccat 4020
ctgggccaag atcccagaga cgggggcgca ctttcacccc tctccggcca tgggcggatt 4080
cggactcaaa cacccgccgc ccatgatgct catcaaaaac acgccggtgc ccggcaacat 4140
caccagcttc tcggacgtgc ccgtcagcag cttcatcacc cagtacagca ccgggcaggt 4200
caccgtggag atggaatggg agctcaaaaa ggaaaactcc aagaggtgga acccagagat 4260
ccagtacacc aacaactaca acgaccccca gtttgtggac tttgctccag acggctccgg 4320
cgaatacaga accacgag ccatcggaac ccgatacctc acccgacccc tttaacccat 4380
tcatgtcgca taccctcaat aaaccgtgta ttcgtgtcag tgaaatactg cctcttgtgg 4440
tcattcaatg aacatcagct tacaacatct acaaaacccc cttgcttgag agtgtggcac 4500
tctcccccct gtcgcgttcg ctcgctcgct ggctcgtttg ggggggtggc agctcaaaga 4560
gctgccagac gacggccctc tggccgtcgc ccccccaaac gagccagcga gcgagcgaac 4620
gcgacaggggggagag 4636

<210> 128
<211> 4679
<212> DNA
<213> Artificial
<220>
<223>AAV2
<400> 128
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180
ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240
gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300
ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg 360
accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg 420
aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga 480
ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc 540
cggaggccct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc 600
tcgtggaaac caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg 660
aaaaactgat tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg 720
tcacaaagac cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc 780
ccaattactt gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac 840
agtatttaag cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga 900
cgcacgtgtc gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc 960
cggtgatcag atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca 1020
aggggattac ctcggagaag cagtggatcc aggaggacca ggcctcatac atctctccttca 1080
atgcggcctc caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta 1140
tgagcctgac taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacattt 1200
ccagcaatcg gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt 1260
ccgtctttct gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg 1320
ggcctgcaac taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct 1380
acgggtgcgt aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg 1440
tgatctggtg ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc 1500
tcggaggaag caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga 1560
ctcccgtgat cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga 1620
ccttcgaaca ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc 1680
tggatcatga ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa 1740
aggatcacgt ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa 1800
gacccgcccc cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc 1860
agccatcgac gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat 1920
gttctcgtca cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga 1980
atcagaattc aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg 2040
tgtcagaatc tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc 2100
atcatatcat gggaaaggtg ccagacgctt gcactgcctg cgatctggtc aatgtggatt 2160
tggatgactg catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat 2220
cttccagatt ggctcgagga cactctctct gaaggaataa gacagtggtg gaagctcaaa 2280
cctggcccac caccaccaaa gcccgcagag cggcataagg acgacagcag gggtcttgtg 2340
cttcctgggt acaagtacct cggacccttc aacggactcg acaagggaga gccggtcaac 2400
gaggcagacg ccgcggccct cgagcacgac aaagcctacg accggcagct cgacagcgga 2460
gacaacccgt acctcaagta caaccacgcc gacgcggagt ttcaggagcg ccttaaagaa 2520
gatacgtctt ttgggggcaa cctcggacga gcagtcttcc aggcgaaaaa gagggttctt 2580
gaacctctgg gcctggttga ggaacctgtt aagacggctc cgggaaaaaa gaggccggta 2640
gagcactctc ctgtggagcc agactcctcc tcgggaaccg gaaaggcggg ccagcagcct 2700
gcaagaaaaa gattgaattt tggtcagact ggagacgcag actcagtacc tgacccccag 2760
cctctcggac agccaccagc agccccctct ggtctgggaa ctaatacgat ggctacaggc 2820
agtggcgcac caatggcaga caataacgag ggcgccgacg gagtgggtaa ttcctcggga 2880
aattggcatt gcgattccac atggatgggc gacagagtca tcaccaccag cacccgaacc 2940
tgggccctgc ccacctacaa caaccacctc tacaaacaaa tttccagcca atcaggagcc 3000
tcgaacgaca atcactactt tggctacagc accccttggg ggtattttga cttcaacaga 3060
ttccactgcc acttttcacc acgtgactgg caaagactca tcaacaacaa ctggggattc 3120
cgacccaaga gactcaactt caagctcttt aacattcaag tcaaagaggt cacgcagaat 3180
gacggtacga cgacgattgc caataacctt accagcacgg ttcaggtgtt tactgactcg 3240
gagtaccagc tcccgtacgt cctcggctcg gcgcatcaag gatgcctccc gccgttccca 3300
gcagacgtct tcatggtgcc acagtatgga tacctcaccc tgaacaacgg gagtcaggca 3360
gtaggatgct cttcatttta ctgcctggag tactttcctt ctcagatgct gcgtaccgga 3420
aacaacttta ccttcagcta cacttttgag gacgttcctt tccacagcag ctacgctcac 3480
agccagagtc tggaccgtct catgaatcct ctcatcgacc agtacctgta ttacttgagc 3540
agaacaaaca ctccaagtgg aaccaccacg cagtcaaggc ttcagttttc tcaggccgga 3600
gcgagtgaca ttcgggacca gtctaggaac tggcttcctg gaccctgtta ccgccagcag 3660
cgagtatcaa agacatctgc ggataacaac aacagtgaat actcgtggac tggagctacc 3720
aagtaccacc tcaatggcag agactctctg gtgaatccgg gcccggccat ggcaagccac 3780
aaggacgatg aagaaaagtt ttttcctcag agcggggttc tcatctttgg gaagcaaggc 3840
tcagagaaaa caaatgtgga cattgaaaag gtcatgatta cagacgaaga ggaaatcagg 3900
acaaccaatc ccgtggctac ggagcagtat ggttctgtat ctaccaacct ccagagaggc 3960
aacagacaag cagctacgc agatgtcaac acacaaggcg ttcttccagg catggtctgg 4020
caggacagag atgtgtacct tcaggggccc atctgggcaa agattccaca cacggacgga 4080
cattttcacc cctctcccct catgggtgga ttcggactta aacaccctcc tccacagatt 4140
ctcatcaaga acaccccggt acctgcgaat ccttcgacca ccttcagtgc ggcaaagttt 4200
gcttccttca tcacacagta ctccacggga caggtcagcg tggagatcga gtgggagctg 4260
cagaaggaaa acagcaaacg ctggaatccc gaaattcagt acacttccaa ctacaacaag 4320
tctgttaatg tggactttac tgtggacact aatggcgtgt attcagagcc tcgccccatt 4380
ggcaccagat acctgactcg taatctgtaa ttgcttgtta atcaataaac cgtttaattc 4440
gtttcagttg aactttggtc tctgcgtatt tctttcttt ctagtttcca tggctacgta 4500
gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 4560
actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 4620
cggggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaa 4679

<210> 129
<211> 4726
<212> DNA
<213> Artificial
<220>
<223>AAV3
<400> 129
ttggccactc cctctatgcg cactcgctcg ctcggtgggg cctggcgacc aaaggtcgcc 60
agacggacgt gctttgcacg tccggcccca ccgagcgagc gagtgcgcat agagggagtg 120
gccaactcca tcactagagg tatggcagtg acgtaacgcg aagcgcgcga agcgagacca 180
cgcctaccag ctgcgtcagc agtcaggtga cccttttgcg acagtttgcg acaccacgtg 240
gccgctgagg gtatatattc tcgagtgagc gaaccaggag ctccattttg accgcgaaat 300
ttgaacgagc agcagccatg ccggggttct acgagattgt cctgaaggtc ccgagtgacc 360
tggacgagcg cctgccgggc atttctaact cgtttgttaa ctgggtggcc gagaaggaat 420
gggacgtgcc gccggattct gacatggatc cgaatctgat tgagcaggca cccctgaccg 480
tggccgaaaa gcttcagcgc gagttcctgg tggagtggcg ccgcgtgagt aaggccccgg 540
aggccctctt ttttgtccag ttcgaaaagg gggagaccta cttccacctg cacgtgctga 600
ttgagaccat cggggtcaaa tccatggtgg tcggccgcta cgtgagccag attaaagaga 660
agctggtgac ccgcatctac cgcggggtcg agccgcagct tccgaactgg ttcgcggtga 720
ccaaaacgcg aaatggcgcc gggggcggga acaaggtggt ggacgactgc tacatcccca 780
actacctgct ccccaagacc cagcccgagc tccagtggggc gtggactaac atggaccagt 840
atttaagcgc ctgtttgaat ctcgcggagc gtaaacggct ggtggcgcag catctgacgc 900
acgtgtcgca gacgcaggag cagaacaaag agaatcagaa ccccaattct gacgcgccgg 960
tcatcaggtc aaaaacctca gccaggtaca tggagctggt cgggtggctg gtggaccgcg 1020
ggatcacgtc agaaaagcaa tggattcagg aggaccaggc ctcgtacatc tccttcaacg 1080
ccgcctccaa ctcgcggtcc cagatcaagg ccgcgctgga caatgcctcc aagatcatga 1140
gcctgacaaa gacggctccg gactacctgg tgggcagcaa ccgccggag gacattacca 1200
aaaatcggat ctaccaaatc ctggagctga acgggtacga tccgcagtac gcggcctccg 1260
tcttcctggg ctgggcgcaa aagaagttcg ggaagaggaa caccatctgg ctctttgggc 1320
cggccacgac gggtaaaacc aacatcgcgg aagccatcgc ccacgccgtg cccttctacg 1380
gctgcgtaaa ctggaccaat gagaactttc ccttcaacga ttgcgtcgac aagatggtga 1440
tctggtggga ggagggcaag atgacggcca aggtcgtgga gagcgccaag gccattctgg 1500
gcggaagcaa ggtgcgcgtg gaccaaaagt gcaagtcatc ggcccagatc gaacccactc 1560
ccgtgatcgt cacctccaac accaacatgt gcgccgtgat tgacgggaac agcaccacct 1620
tcgagcatca gcagccgctg caggacgga tgtttgaatt tgaacttacc cgccgtttgg 1680
accatgactt tgggaaggtc accaaacagg aagtaaagga ctttttccgg tgggcttccg 1740
atcacgtgac tgacgtggct catgagttct acgtcagaaa gggtggagct aagaaacgcc 1800
ccgcctccaa tgacgcggat gtaagcgagc caaaacggga gtgcacgtca cttgcgcagc 1860
cgacaacgtc agacgcggaa gcaccggcgg actacgcgga caggtaccaa aacaaatgtt 1920
ctcgtcacgt gggcatgaat ctgatgcttt ttccctgtaa aacatgcgag agaatgaatc 1980
aaatttccaa tgtctgtttt acgcatggtc aaagagactg tggggaatgc ttccctggaa 2040
tgtcagaatc tcaacccgtt tctgtcgtca aaaagaagac ttatcagaaa ctgtgtccaa 2100
ttcatcatat cctgggaagg gcacccgaga ttgcctgttc ggcctgcgat ttggccaatg 2160
tggacttgga tgactgtgtt tctgagcaat aaatgactta aaccaggtat ggctgctgac 2220
ggttatcttc cagattggct cgaggacaac ctttctgaag gcattcgtga gtggtgggct 2280
ctgaaacctg gagtccctca acccaaagcg aaccaacaac accaggacaa ccgtcggggt 2340
cttgtgcttc cgggttacaa atacctcgga cccggtaacg gactcgacaa aggagagccg 2400
gtcaacgagg cggacgcggc agccctcgaa cacgacaaag cttacgacca gcagctcaag 2460
gccggtgaca acccgtacct caagtacaac cacgccgacg ccgagtttca ggagcgtctt 2520
caagaagata cgtcttttgg gggcaacctt ggcagagcag tcttccaggc caaaaagagg 2580
atccttgagc ctcttggtct ggttgaggaa gcagctaaaa cggctcctgg aaagaagggg 2640
gctgtagatc agtctcctca ggaaccggac tcatcatctg gtgtggcaa atcgggcaaa 2700
cagcctgcca gaaaaagact aaatttcggt cagactggag actcagagtc agtcccagac 2760
cctcaacctc tcggagaacc accagcagcc cccacaagtt tgggatctaa tacaatggct 2820
tcaggcggtg gcgcaccaat ggcagacaat aacgagggtg ccgatggagt gggtaattcc 2880
tcaggaaatt ggcattgcga ttcccaatgg ctgggcgaca gagtcatcac caccagcacc 2940
agaacctggg ccctgcccac ttacaacaac catctctaca agcaaatctc cagccaatca 3000
ggagcttcaa acgacaacca ctactttggc tacagcaccc cttgggggta ttttgacttt 3060
aacagattcc actgccactt ctcaccacgt gactggcagc gactcattaa caacaactgg 3120
ggattccggc ccaagaaact cagcttcaag ctcttcaaca tccaagttag aggggtcacg 3180
cagaacgatg gcacgacgac tattgccaat aaccttacca gcacggttca agtgtttacg 3240
gactcggagt atcagctccc gtacgtgctc gggtcggcgc accaaggctg tctcccgccg 3300
tttccagcgg acgtcttcat ggtccctcag tatggatacc tcaccctgaa caacggaagt 3360
caagcggtgg gacgctcatc cttttactgc ctggagtact tcccttcgca gatgctaagg 3420
actggaaata acttccaatt cagctatacc ttcgaggatg taccttttca cagcagctac 3480
gctcacagcc agagtttgga tcgcttgatg aatcctctta ttgatcagta tctgtactac 3540
ctgaacagaa cgcaaggaac aacctctgga acaaccaacc aatcacggct gctttttagc 3600
caggctgggc ctcagtctat gtctttgcag gccagaaatt ggctacctgg gccctgctac 3660
cggcaacaga gactttcaaa gactgctaac gacaacaaca acagtaactt tccttggaca 3720
gcggccagca aatatcatct caatggccgc gactcgctgg tgaatccagg accagctatg 3780
gccagtcaca aggacgatga agaaaaattt ttccctatgc acggcaatct aatatttggc 3840
aaagaaggga caacggcaag taacgcagaa ttagataatg taatgattac ggatgaagaa 3900
gagattcgta ccaccaatcc tgtggcaaca gagcagtatg gaactgtggc aaataacttg 3960
cagagctcaa atacagctcc cacgactgga actgtcaatc atcagggggc cttacctggc 4020
atggtgtggc aagatcgtga cgtgtacctt caaggaccta tctgggcaaa gattcctcac 4080
acggatggac actttcatcc ttctcctctg atgggaggct ttggactgaa acatccgcct 4140
cctcaaatca tgatcaaaaa tactccggta ccggcaaatc ctccgacgac tttcagcccg 4200
gccaagtttg cttcatttat cactcagtac tccactggac aggtcagcgt ggaaattgag 4260
tgggagctac agaaagaaaa cagcaaacgt tggaatccag agattcagta cacttccaac 4320
tacaacaagt ctgttaatgt ggactttact gtagacacta atggtgttta tagtgaacct 4380
cgccctattg gaacccggta tctcacacga aacttgtgaa tcctggttaa tcaataaacc 4440
gtttaattcg tttcagttga actttggctc ttgtgcactt ctttatcttt atcttgtttc 4500
catggctact gcgtagataa gcagcggcct gcggcgcttg cgcttcgcgg tttacaactg 4560
ctggttaata tttaactctc gccatacctc tagtgatgga gttggccact ccctctatgc 4620
gcactcgctc gctcggtggg gcctggcgac caaaggtcgc cagacggacg tgctttgcac 4680
gtccggcccc accgagcgag cgagtgcgca tagagggagt ggccaa 4726

<210> 130
<211> 4767
<212> DNA
<213> Artificial
<220>
<223> AAV4
<400> 130
ttggccactc ccctctatgcg cgctcgctca ctcactcggc cctggagacc aaaggtctcc 60
agactgccgg cctctggccg gcagggccga gtgagtgagc gagcgcgcat agagggagtg 120
gccaactcca tcatctaggt ttgcccactg acgtcaatgt gacgtcctag ggttagggag 180
gtccctgtat tagcagtcac gtgagtgtcg tatttcgcgg agcgtagcgg agcgcatacc 240
aagctgccac gtcacagcca cgtggtccgt ttgcgacagt ttgcgacacc atgtggtcag 300
gagggtatat aaccgcgagt gagccagcga ggagctccat tttgcccgcg aattttgaac 360
gagcagcagc catgccgggg ttctacgaga tcgtgctgaa ggtgcccagc gacctggacg 420
agcacctgcc cggcatttct gactcttttg tgagctgggt ggccgagaag gaatgggagc 480
tgccgccgga ttctgacatg gacttgaatc tgattgagca ggcacccctg accgtggccg 540
aaaagctgca acgcgagttc ctggtcgagt ggcgccgcgt gagtaaggcc ccggaggccc 600
tcttctttgt ccagttcgag aagggggaca gctacttcca cctgcacatc ctggtggaga 660
ccgtgggcgt caaatccatg gtggtgggcc gctacgtgag ccagattaaa gagaagctgg 720
tgacccgcat ctaccgcggg gtcgagccgc agcttccgaa ctggttcgcg gtgaccaaga 780
cgcgtaatgg cgccggaggc gggaacaagg tggtggacga ctgctacatc cccaactacc 840
tgctccccaa gacccagccc gagctccagt gggcgtggac taacatggac cagtatataa 900
gcgcctgttt gaatctcgcg gagcgtaaac ggctggtggc gcagcatctg acgcacgtgt 960
cgcagacgca ggagcagaac aaggaaaacc agaaccccaa ttctgacgcg ccggtcatca 1020
ggtcaaaaac ctccgccagg tacatggagc tggtcgggtg gctggtggac cgcgggatca 1080
cgtcagaaaa gcaatggatc caggaggacc aggcgtccta catctccttc aacgccgcct 1140
ccaactcgcg gtcacaaatc aaggccgcgc tggacaatgc ctccaaaatc atgagcctga 1200
caaagacggc tccggactac ctggtgggcc agaacccgcc ggaggacatt tccagcaacc 1260
gcatctaccg aatcctcgag atgaacgggt acgatccgca gtacgcggcc tccgtcttcc 1320
tgggctgggc gcaaaagaag ttcgggaaga ggaacaccat ctggctcttt gggccggcca 1380
cgacgggtaa aaccaacatc gcggaagcca tcgcccacgc cgtgcccttc tacggctgcg 1440
tgaactggac caatgagaac tttccgttca acgattgcgt cgacaagatg gtgatctggt 1500
gggaggaggg caagatgacg gccaaggtcg tagagagcgc caaggccatc ctgggcggaa 1560
gcaaggtgcg cgtggaccaa aagtgcaagt catcggccca gatcgaccca actcccgtga 1620
tcgtcacctc caacaccaac atgtgcgcgg tcatcgacgg aaactcgacc accttcgagc 1680
accaacaacc actccaggac cggatgttca agttcgagct caccaagcgc ctggagcacg 1740
actttggcaa ggtcaccaag caggaagtca aagacttttt ccggtgggcg tcagatcacg 1800
tgaccgaggt gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc 1860
ccaatgacgc agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga 1920
cgtcagacgc ggaagctccg gtggactacg cggacaggta ccaaaacaaa tgttctcgtc 1980
acgtgggtat gaatctgatg ctttttccct gccggcaatg cgagagaatg aatcagaatg 2040
tggacatttg cttcacgcac ggggtcatgg actgtgccga gtgcttcccc gtgtcagaat 2100
ctcaacccgt gtctgtcgtc agaaagcgga cgtatcagaa actgtgtccg attcatcaca 2160
tcatggggag ggcgcccgag gtggcctgct cggcctgcga actggccaat gtggacttgg 2220
atgactgtga catggaacaa taaatgactc aaaccagata tgactgacgg ttaccttcca 2280
gattggctag aggacacacct ctctgaaggc gttcgagagt ggtgggcgct gcaacctgga 2340
gcccctaaac ccaaggcaaa tcaacaacat caggacaacg ctcggggtct tgtgcttccg 2400
ggttacaaat acctcggacc cggcaacgga ctcgacaagg gggaacccgt caacgcagcg 2460
gacgcggcag ccctcgagca cgacaaggcc tacgaccagc agctcaaggc cggtgacaac 2520
ccctacctca agtacaacca cgccgacgcg gagttccagc agcggcttca gggcgacaca 2580
tcgtttgggg gcaacctcgg cagagcagtc ttccaggcca aaaagagggt tcttgaacct 2640
cttggtctgg ttgagcaagc gggtgagacg gctcctggaa agaagagacc gttgattgaa 2700
tccccccagc agcccgactc ctccacggggt atcggcaaaa aaggcaagca gccggctaaa 2760
aagaagctcg ttttcgaaga cgaaactgga gcaggcgacg gaccccctga gggatcaact 2820
tccggagcca tgtctgatga cagtgagatg cgtgcagcag ctggcggagc tgcagtcgag 2880
ggcggacaag gtgccgatgg agtgggtaat gcctcgggtg attggcattg cgattccacc 2940
tggtctgagg gccacgtcac gaccaccagc accagaacct gggtcttgcc cacctacaac 3000
aaccacctct acaagcgact cggagagagc ctgcagtcca acacctacaa cggattctcc 3060
accccctggg gatactttga cttcaaccgc ttccactgcc acttctcacc acgtgactgg 3120
cagcgactca tcaacaacaa ctggggcatg cgacccaaag ccatgcgggt caaaatcttc 3180
aacatccagg tcaaggaggt cacgacgtcg aacggcgaga caacggtggc taataacctt 3240
accagcacgg ttcagatctt tgcggactcg tcgtacgaac tgccgtacgt gatggatgcg 3300
ggtcaagagg gcagcctgcc tccttttccc aacgacgtct ttatggtgcc ccagtacggc 3360
tactgtggac tggtgaccgg caacacttcg cagcaacaga ctgacagaaa tgccttctac 3420
tgcctggagt actttccttc gcagatgctg cggactggca acaactttga aattacgtac 3480
agttttgaga aggtgccttt ccactcgatg tacgcgcaca gccagagcct ggaccggctg 3540
atgaaccctc tcatcgacca gtacctgtgg ggactgcaat cgaccaccac cggaaccacc 3600
ctgaatgccg ggactgccac caccaacttt accaagctgc ggcctaccaa cttttccaac 3660
tttaaaaaga actggctgcc cgggccttca atcaagcagc agggcttctc aaagactgcc 3720
aatcaaaact acaagatccc tgccaccggg tcagacagtc tcatcaaata cgagacgcac 3780
agcactctgg acggaagatg gagtgccctg acccccggac ctccaatggc cacggctgga 3840
cctgcggaca gcaagttcag caacagccag ctcatctttg cggggcctaa acagaacggc 3900
aacacggcca ccgtacccgg gactctgatc ttcacctctg aggaggagct ggcagccacc 3960
aacgccaccg atacggacat gtggggcaac ctacctggcg gtgaccagag caacagcaac 4020
ctgccgaccg tggacagact gacagccttg ggagccgtgc ctggaatggt ctggcaaaac 4080
agagacattt actaccaggg tcccatttgg gccaagattc ctcataccga tggacacttt 4140
cacccctcac cgctgattgg tgggtttggg ctgaaacacc cgcctcctca aatttttatc 4200
aagaacaccc cggtacctgc gaatcctgca acgaccttca gctctactcc ggtaaactcc 4260
ttcattactc agtacagcac tggccaggtg tcggtgcaga ttgactggga gatccagaag 4320
gagcggtcca aacgctggaa ccccgaggtc cagtttacct ccaactacgg acagcaaaac 4380
tctctgttgt gggctcccga tgcggctggg aaatacactg agcctagggc tatcggtacc 4440
cgctacctca cccaccact gtaataacct gttaatcaat aaaccggttt attcgtttca 4500
gttgaacttt ggtctccgtg tccttcttat cttatctcgt ttccatggct actgcgtaca 4560
taagcagcgg cctgcggcgc ttgcgcttcg cggtttacaa ctgccggtta atcagtaact 4620
tctggcaaac cagatgatgg agttggccac attagctatg cgcgctcgct cactcactcg 4680
gccctggaga ccaaaggtct ccagactgcc ggcctctggc cggcagggcc gagtgagtga 4740
gcgagcgcgc atagagggag tggccaa 4767

<210> 131
<211> 4642
<212> DNA
<213> Artificial
<220>
<223>AAV5
<400> 131
ctctcccccc tgtcgcgttc gctcgctcgc tggctcgttt gggggggtgg cagctcaaag 60
agctgccaga cgacggccct ctggccgtcg cccccccaaa cgagccagcg agcgagcgaa 120
cgcgacaggg gggagagtgc cacactctca agcaaggggg ttttgtaagc agtgatgtca 180
taatgatgta atgcttattg tcacgcgata gttaatgatt aacagtcatg tgatgtgttt 240
tatccaatag gaagaaagcg cgcgtatgag ttctcgcgag acttccgggg tataaaagac 300
cgagtgaacg agccccgccgc cattctttgc tctggactgc tagaggaccc tcgctgccat 360
ggctaccttc tatgaagtca ttgttcgcgt cccatttgac gtggaggaac atctgcctgg 420
aatttctgac agctttgtgg actgggtaac tggtcaaatt tgggagctgc ctccagagtc 480
agatttaaat ttgactctgg ttgaacagcc tcagttgacg gtggctgata gaattcgccg 540
cgtgttcctg tacgagtgga acaaattttc caagcaggag tccaaattct ttgtgcagtt 600
tgaaaaggga tctgaatatt ttcatctgca cacgcttgtg gagacctccg gcatctcttc 660
catggtcctc ggccgctacg tgagtcagat tcgcgcccag ctggtgaaag tggtcttcca 720
gggaattgaa ccccagatca acgactgggt cgccatcacc aaggtaaaga agggcggagc 780
caataaggtg gtggattctg ggtatattcc cgcctacctg ctgccgaagg tccaccgga 840
gcttcagtgg gcgtggacaa acctggacga gtataaattg gccgccctga atctggagga 900
gcgcaaacgg ctcgtcgcgc agtttctggc agaatcctcg cagcgctcgc aggaggcggc 960
ttcgcagcgt gagttctcgg ctgacccggt catcaaaagc aagacttccc agaaatacat 1020
ggcgctcgtc aactggctcg tggagcacgg catcacttcc gagaagcagt ggatccagga 1080
aaatcaggag agctacctct cctcaactc caccggcaac tctcggagcc agatcaaggc 1140
cgcgctcgac aacgcgacca aaattatgag tctgacaaaa agcgcggtgg actacctcgt 1200
ggggagctcc gttcccgagg acatttcaaa aaacagaatc tggcaaattt ttgagatgaa 1260
tggctacgac ccggcctacg cgggatccat cctctacggc tggtgtcagc gctccttcaa 1320
caagaggaac accgtctggc tctacggacc cgccacgacc ggcaagacca acatcgcgga 1380
ggccatcgcc cacactgtgc ccttttacgg ctgcgtgaac tggaccaatg aaaactttcc 1440
ctttaatgac tgtgtggaca aaatgctcat ttggtgggag gagggaaaga tgaccaacaa 1500
ggtggttgaa tccgccaagg ccatcctggg gggctcaaag gtgcgggtcg atcagaaatg 1560
taaatcctct gttcaaattg attctacccc tgtcattgta acttccaata caaacatgtg 1620
tgtggtggtg gatgggaatt ccacgacctt tgaacaccag cagccgctgg aggaccgcat 1680
gttcaaattt gaactgacta agcggctccc gccagatttt ggcaagatta ctaagcagga 1740
agtcaaggac ttttttgctt gggcaaaggt caatcaggtg ccggtgactc acgagtttaa 1800
agttcccagg gaattggcgg gaactaaagg ggcggagaaa tctctaaaac gcccactggg 1860
tgacgtcacc aatactagct ataaaagtct ggagaagcgg gccaggctct catttgttcc 1920
cgagacgcct cgcagttcag acgtgactgt tgatcccgct cctctgcgac cgctcaattg 1980
gaattcaagg tatgattgca aatgtgacta tcatgctcaa tttgacaaca tttctaacaa 2040
atgtgatgaa tgtgaatatt tgaatcgggg caaaaatgga tgtatctgtc acaatgtaac 2100
tcactgtcaa atttgtcatg ggattccccc ctgggaaaag gaaaacttgt cagattttgg 2160
ggattttgac gatgccaata aagaacagta aataaagcga gtagtcatgt cttttgttga 2220
tcaccctcca gattggttgg aagaagttgg tgaaggtctt cgcgagtttt tgggccttga 2280
agcgggccca ccgaaaccaa aacccaatca gcagcatcaa gatcaagccc gtggtcttgt 2340
gctgcctggt tataactatc tcggacccgg aaacggtctc gatcgaggag agcctgtcaa 2400
cagggcagac gaggtcgcgc gagagcacga catctcgtac aacgagcagc ttgaggcggg 2460
agacaacccc tacctcaagt acaaccacgc ggacgccgag tttcaggaga agctcgccga 2520
cgacacatcc ttcgggggaa acctcggaaa ggcagtcttt caggccaaga aaagggttct 2580
cgaacctttt ggcctggttg aagagggtgc taagacggcc cctaccggaa agcggataga 2640
cgaccacttt ccaaaaagaa agaaggctcg gaccgaagag gactccaagc cttccacctc 2700
gtcagacgcc gaagctggac ccagcggatc ccagcagctg caaatcccag cccaaccagc 2760
ctcaagtttg ggagctgata caatgtctgc gggaggtggc ggcccattgg gcgacaataa 2820
ccaaggtgcc gatggagtgg gcaatgcctc gggagattgg cattgcgatt ccacgtggat 2880
gggggacaga gtcgtcacca agtccacccg aacctgggtg ctgcccagct acaacaacca 2940
ccagtaccga gagatcaaaa gcggctccgt cgacggaagc aacgccaacg cctactttgg 3000
atacagcacc ccctgggggt actttgactt taaccgcttc cacagccact ggagcccccg 3060
agactggcaa agactcatca acaactactg gggcttcaga ccccggtccc tcagagtcaa 3120
aatcttcaac attcaagtca aagaggtcac ggtgcaggac tccaccacca ccatcgccaa 3180
caacctcacc tccaccgtcc aagtgtttac ggacgacgac taccagctgc cctacgtcgt 3240
cggcaacggg accgagggat gcctgccggc cttccctccg caggtcttta cgctgccgca 3300
gtacggttac gcgacgctga accgcgacaa cacagaaaat cccaccgaga ggagcagctt 3360
cttctgccta gagtactttc ccagcaagat gctgagaacg ggcaacaact ttgagtttac 3420
ctacaacttt gaggaggtgc ccttccactc cagcttcgct cccagtcaga acctgttcaa 3480
gctggccaac ccgctggtgg accagtactt gtaccgcttc gtgagcacaa ataacactgg 3540
cggagtccag ttcaacaaga acctggccgg gagatacgcc aacacctaca aaaactggtt 3600
cccggggccc atgggccgaa cccagggctg gaacctggggc tccggggtca accgcgccag 3660
tgtcagcgcc ttcgccacga ccaataggat ggagctcgag ggcgcgagtt accaggtgcc 3720
cccgcagccg aacggcatga ccaacaacct ccagggcagc aacacctatg ccctggagaa 3780
cactatgatc ttcaacagcc agccggcgaa ccggggcacc accgccacgt acctcgaggg 3840
caacatgctc atcaccagcg agagcgagac gcagccggtg aaccgcgtgg cgtacaacgt 3900
cggcgggcag atggccacca acaaccagag ctccaccact gcccccgcga ccggcacgta 3960
caacctccag gaaatcgtgc ccggcagcgt gtggatggag agggacgtgt acctccaagg 4020
acccatctgg gccaagatcc cagagacggg ggcgcacttt cacccctctc cggccatggg 4080
cggattcgga ctcaaacacc caccgcccat gatgctcatc aagaacacgc ctgtgcccgg 4140
aaatatcacc agcttctcgg acgtgcccgt cagcagcttc atcacccagt acagcaccgg 4200
gcaggtcacc gtggagatgg agtgggagct caagaaggaa aactccaaga ggtggaaccc 4260
agagatccag tacacaaaca actacaacga cccccagttt gtggactttg ccccggacag 4320
caccggggaa tacagaacca ccagacctat cggaacccga taccttaccc gaccccttta 4380
acccattcat gtcgcatacc ctcaataaac cgtgtattcg tgtcagtaaa atactgcctc 4440
ttgtggtcat tcaatgaata acagcttaca acatctacaa aacctccttg cttgagagtg 4500
tggcactctc ccccctgtcg cgttcgctcg ctcgctggct cgtttggggg ggtggcagct 4560
caaagagctg ccagacgacg gccctctggc cgtcgccccc ccaaacgagc cagcgagcga 4620
gcgaacgcga caggggggag ag 4642

<210> 132
<211> 4683
<212> DNA
<213> Artificial
<220>
<223>AAV6
<400> 132
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 60
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg 120
gccaactcca tcactagggg ttcctggagg ggtggagtcg tgacgtgaat tacgtcatag 180
ggttagggag gtcctgtatt agaggtcacg tgagtgtttt gcgacatttt gcgacaccat 240
gtggtcacgc tgggtattta agcccgagtg agcacgcagg gtctccattt tgaagcggga 300
ggtttgaacg cgcagcgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga 360
ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg ccgagaagga 420
atgggagttg ccgccagatt ctgacatgga tctgaatctg attgagcagg cacccctgac 480
cgtggccgag aagctgcagc gcgacttcct ggtccagtgg cgccgcgtga gtaaggcccc 540
ggaggccctc ttctttgttc agttcgagaa gggcgagtcc tacttccacc tccatattct 600
ggtggagacc acggggggtca aatccatggt gctgggccgc ttcctgagtc agattaggga 660
caagctggtg cagaccatct accgcgggat cgagccgacc ctgcccaact ggttcgcggt 720
gaccaagacg cgtaatggcg ccggaggggg gaacaaggtg gtggacgagt gctacatccc 780
caactacctc ctgcccaaga ctcagcccga gctgcagtgg gcgtggacta acatggagga 840
gtatataagc gcgtgtttaa acctggccga gcgcaaacgg ctcgtggcgc acgacctgac 900
ccacgtcagc cagacccagg agcagaacaa ggagaatctg aaccccaatt ctgacgcgcc 960
tgtcatccgg tcaaaaacct ccgcacgcta catggagctg gtcgggtggc tggtggaccg 1020
gggcatcacc tccgagaagc agtggatcca ggaggaccag gcctcgtaca tctccttcaa 1080
cgccgcctcc aactcgcggt ccgatcaa ggccgctctg gacaatgccg gcaagatcat 1140
ggcgctgacc aaatccgcgc ccgactacct ggtaggcccc gctccgcccg ccgacattaa 1200
aaccaaccgc atttaccgca tcctggagct gaacggctac gaccctgcct acgccggctc 1260
cgtctttctc ggctgggccc agaaaaggtt cggaaaacgc aacaccatct ggctgtttgg 1320
gccggccacc acgggcaaga ccaacatcgc ggaagccatc gcccacgccg tgcccttcta 1380
cggctgcgtc aactggacca atgagaactt tcccttcaac gattgcgtcg acaagatggt 1440
gatctggtgg gaggagggca agatgacggc caaggtcgtg gagtccgcca aggccattct 1500
cggcggcagc aaggtgcgcg tggaccaaaa gtgcaagtcg tccgcccaga tcgatcccac 1560
ccccgtgatc gtcacctcca acaccaacat gtgcgccgtg attgacggga acagcaccac 1620
cttcgagcac cagcagccgt tgcaggaccg gatgttcaaa tttgaactca cccgccgtct 1680
ggagcatgac tttggcaagg tgacaaagca ggaagtcaaa gagttcttcc gctgggcgca 1740
ggatcacgtg accgaggtgg cgcatgagtt ctacgtcaga aagggtggag ccaacaagag 1800
acccgccccc gatgacgcgg ataaaagcga gcccaagcgg gcctgcccct cagtcgcgga 1860
tccatcgacg tcagacgcgg aaggagctcc ggtggacttt gccgacaggt accaaaacaa 1920
atgttctcgt cacgcgggca tgcttcagat gctgtttccc tgcaaaacat gcgagagaat 1980
gaatcagaat ttcaacattt gcttcacgca cgggaccaga gactgttcag aatgtttccc 2040
cggcgtgtca gaatctcaac cggtcgtcag aaagaggacg tatcggaaac tctgtgccat 2100
tcatcatctg ctggggcggg ctcccgagat tgcttgctcg gcctgcgatc tggtcaacgt 2160
ggatctggat gactgtgttt ctgagcaata aatgacttaa accaggtatg gctgccgatg 2220
gttatcttcc agattggctc gaggacacc tctctgaggg cattcgcgag tggtgggact 2280
tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac ggccggggtc 2340
tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg 2400
tcaacgcggc ggatgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag 2460
cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc 2520
aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg 2580
ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc 2640
cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag acaggccagc 2700
agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgacc 2760
cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt 2820
caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct 2880
caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc 2940
gaacatgggc cttgcccacc tataacacc acctctaca gcaaatctcc agtgcttcaa 3000
cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt 3060
tcaacagatt ccactgccat ttctcaccac gtgactggca gcgactcatc aacaacaatt 3120
ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc aaggaggtca 3180
cgacgaatga tggcgtcacg accatcgcta ataaccttac cagcacggtt caagtcttct 3240
cggactcgga gtaccagttg ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc 3300
cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca 3360
gccaggcagt gggacggtca tccttttact gcctggaata tttcccatcg cagatgctga 3420
gaacgggcaa taactttacc ttcagctaca ccttcgagga cgtgcctttc cacagcagct 3480
acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag tacctgtatt 3540
acctgaacag aactcagaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc 3600
gggggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc 3660
ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg 3720
gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc actgctatgg 3780
cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg atttttggaa 3840
aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca gacgaagagg 3900
aaatcaaagc cactaaccc gtggccaccg aaagatttgg gactgtggca gtcaatctcc 3960
agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa 4020
tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca 4080
cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag cacccgcctc 4140
ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag ttttcggcta 4200
caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg gagattgaat 4260
gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat acatctaact 4320
atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc 4380
gccccattgg cacccgttac ctcacccgtc ccctgtaatt gtgtgttaat caataaaccg 4440
gttaattcgt gtcagttgaa ctttggtctc atgtcgttat tatcttatct ggtcaccata 4500
gcaaccggtt acacattaac tgcttagttg cgcttcgcga ataccccctag tgatggagtt 4560
gcccactccc tctatgcgcg ctcgctcgct cggtggggcc ggcagagcag agctctgccg 4620
tctgcggacc tttggtccgc aggccccacc gagcgagcga gcgcgcatag agggagtggg 4680
caa4683

<210> 133
<211> 4721
<212> DNA
<213> Artificial
<220>
<223> AAV7
<400> 133
ttggccactc cctctatgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc 60
agacggcaga gctctgctct gccggcccca ccgagcgagc gagcgcgcat agagggagtg 120
gccaactcca tcactagggg taccgcgaag cgcctcccac gctgccgcgt cagcgctgac 180
gtaaatcacg tcatagggga gtggtcctgt attagctgtc acgtgagtgc ttttgcgaca 240
ttttgcgaca ccacgtggcc atttgaggta tatatggccg agtgagcgag caggatctcc 300
attttgaccg cgaaatttga acgagcagca gccatgccgg gtttctacga gatcgtgatc 360
aaggtgccga gcgacctgga cgagcacctg ccgggcattt ctgactcgtt tgtgaactgg 420
gtggccgaga aggaatggga gctgcccccg gattctgaca tggatctgaa tctgatcgag 480
caggcacccc tgaccgtggc cgagaagctg cagcgcgact tcctggtcca atggcgccgc 540
gtgagtaagg ccccggaggc cctgttcttt gttcagttcg agaagggcga gagctacttc 600
caccttcacg ttctggtgga gaccacgggg gtcaagtcca tggtgctagg ccgcttcctg 660
agtcagattc gggagaagct ggtccagacc atctaccgcg gggtcgagcc cacgctgccc 720
aactggttcg cggtgaccaa gacgcgtaat ggcgccggcg gggggaacaa ggtggtggac 780
gagtgctaca tccccaacta cctcctgccc aagacccagc ccgagctgca gtgggcgtgg 840
actaacatgg aggagtatat aagcgcgtgt ttgaacctgg ccgaacgcaa acggctcgtg 900
gcgcagcacc tgacccacgt cagccagacg caggagcaga acaaggagaa tctgaacccc 960
aattctgacg cgccccgtgat caggtcaaaa acctccgcgc gctacatgga gctggtcggg 1020
tggctggtgg accggggcat cacctccgag aagcagtgga tccaggagga ccaggcctcg 1080
tacatctcct tcaacgccgc ctccaactcg cggtcccaga tcaaggccgc gctggacaat 1140
gccggcaaga tcatggcgct gaccaaatcc gcgcccgact acctggtggg gccctcgctg 1200
cccgcggaca ttaaaaccaa ccgcatctac cgcatcctgg agctgaacgg gtacgatcct 1260
gcctacgccg gctccgtctt tctcggctgg gcccagaaaa agttcgggaa gcgcaacacc 1320
atctggctgt ttgggcccgc caccaccggc aagaccaaca ttgcggaagc catcgcccac 1380
gccgtgccct tctacggctg cgtcaactgg accaatgaga actttccctt caacgattgc 1440
gtcgacaaga tggtgatctg gtgggaggag ggcaagatga cggccaaggt cgtggagtcc 1500
gccaaggcca ttctcggcgg cagcaaggtg cgcgtggacc aaaagtgcaa gtcgtccgcc 1560
cagatcgacc ccacccccgt gatcgtcacc tccaacacca acatgtgcgc cgtgattgac 1620
gggaacagca ccaccttcga gcaccagcag ccgttgcagg accggatgtt caaatttgaa 1680
ctcacccgcc gtctggagca cgactttggc aaggtgacga agcaggaagt caaagagttc 1740
ttccgctggg ccagtgatca cgtgaccgag gtggcgcatg agttctacgt cagaaagggc 1800
ggagccagca aaagacccgc ccccgatgac gcggatataa gcgagcccaa gcgggcctgc 1860
ccctcagtcg cggatccatc gacgtcagac gcggaaggag ctccggtgga ctttgccgac 1920
aggtaccaaa acaaatgttc tcgtcacgcg ggcatgattc agatgctgtt tccctgcaaa 1980
acgtgcgaga gaatgaatca gaatttcaac atttgcttca cacacggggt cagagactgt 2040
ttagagtgtt tccccggcgt gtcagaatct caaccggtcg tcagaaaaaa gacgtatcgg 2100
aaactctgcg cgattcatca tctgctgggg cgggcgcccg agattgcttg ctcggcctgc 2160
gacctggtca acgtggacct ggacgactgc gtttctgagc aataaatgac ttaaaccagg 2220
tatggctgcc gatggttatc ttccagattg gctcgaggac aacctctctg agggcattcg 2280
cgagtggtgg gacctgaaac ctggagcccc gaaacccaaa gccaaccagc aaaagcagga 2340
caacggccgg ggtctggtgc ttcctggcta caagtacctc ggacccttca acggactcga 2400
caagggggag cccgtcaacg cggcggacgc agcggccctc gagcacgaca aggcctacga 2460
ccagcagctc aaagcgggtg acaatccgta cctgcggtat aaccacgccg acgccgagtt 2520
tcaggagcgt ctgcaagaag atacgtcatt tgggggcaac ctcgggcgag cagtcttcca 2580
ggccaagaag cgggttctcg aacctctcgg tctggttgag gaaggcgcta agacggctcc 2640
tgcaaagaag agaccggtag agccgtcacc tcagcgttcc cccgactcct ccacgggcat 2700
cggcaagaaa ggccagcagc ccgccagaaa gagactcaat ttcggtcaga ctggcgactc 2760
agagtcagtc cccgaccctc aacctctcgg agaacctcca gcagcgccct ctagtgtggg 2820
atctggtaca gtggctgcag gcggtggcgc accaatggca gacaataacg aaggtgccga 2880
cggagtgggt aatgcctcag gaaattggca ttgcgattcc acatggctgg gcgacagagt 2940
cattaccacc agcacccgaa cctgggccct gcccacctac aacaaccacc tctacaagca 3000
aatctccagt gaaactgcag gtagtaccaa cgacaacacc tacttcggct acagcacccc 3060
ctgggggtat tttgacttta acagattcca ctgccacttc tcaccacgtg actggcagcg 3120
actcatcaac aacaactggg gattccggcc caagaagctg cggttcaagc tcttcaacat 3180
ccaggtcaag gaggtcacga cgaatgacgg cgttacgacc atcgctaata accttaccag 3240
cacgattcag gtattctcgg actcggaata ccagctgccg tacgtcctcg gctctgcgca 3300
ccagggctgc ctgcctccgt tcccggcgga cgtcttcatg attcctcagt acggctacct 3360
gactctcaac aatggcagtc agtctgtggg acgttcctcc ttctactgcc tggagtactt 3420
cccctctcag atgctgagaa cgggcaacaa ctttgagttc agctacagct tcgaggacgt 3480
gcctttccac agcagctacg cacacagcca gagcctggac cggctgatga atcccctcat 3540
cgaccagtac ttgtactacc tggccagaac acagagtaac ccaggaggca cagctggcaa 3600
tcgggaactg cagttttacc agggcgggcc ttcaactatg gccgaacaag ccaagaattg 3660
gttacctgga ccttgcttcc ggcaacaaag agtctccaaa acgctggatc aaaacaacaa 3720
cagcaacttt gcttggactg gtgccaccaa atatcacctg aacggcagaa actcgttggt 3780
taatcccggc gtcgccatgg caactcacaa ggacgacgag gaccgctttt tcccatccag 3840
cggagtcctg atttttggaa aaactggagc aactaacaaa actacattgg aaaatgtgtt 3900
aatgacaaat gaagaagaaa ttcgtcctac taatcctgta gccacggaag aatacgggat 3960
agtcagcagc aacttacaag cggctaatac tgcagcccag acacaagttg tcaacaacca 4020
gggagcctta cctggcatgg tctggcagaa ccgggacgtg tacctgcagg gtcccatctg 4080
ggccaagatt cctcacacgg atggcaactt tcacccgtct cctttgatgg gcggctttgg 4140
acttaaacat ccgcctcctc agatcctgat caagaacact cccgttcccg ctaatcctcc 4200
ggaggtgttt actcctgcca agtttgcttc gttcatcaca cagtacagca ccggacaagt 4260
cagcgtggaa atcgagtggg agctgcagaa ggaaaacagc aagcgctgga acccggagat 4320
tcagtacacc tccaactttg aaaagcagac tggtgtggac tttgccgttg acagccaggg 4380
tgtttactct gagcctcgcc ctattggcac tcgttacctc acccgtaatc tgtaattgca 4440
tgttaatcaa taaaccggtt gattcgtttc agttgaactt tggtctcctg tgcttcttat 4500
cttatcggtt tccatagcaa ctggttacac attaactgct tgggtgcgct tcacgataag 4560
aacactgacg tcaccgcggt acccctagtg atggagttgg ccactccctc tatgcgcgct 4620
cgctcgctcg gtggggcctg cggaccaaag gtccgcagac ggcagagctc tgctctgccg 4680
gccccaccga gcgagcgagc gcgcatagag ggagtggcca a 4721

<210> 134
<211> 4393
<212> DNA
<213> Artificial
<220>
<223> AAV8
<400> 134
cagagaggga gtggccaact ccatcactag gggtagcgcg aagcgcctcc cacgctgccg 60
cgtcagcgct gacgtaaatt acgtcatagg ggagtggtcc tgtattagct gtcacgtgag 120
tgcttttgcg gcattttgcg acaccacgtg gccatttgag gtatatatgg ccgagtgagc 180
gagcaggatc tccattttga ccgcgaaatt tgaacgagca gcagccatgc cgggcttcta 240
cgagatcgtg atcaaggtgc cgagcgacct ggacgagcac ctgccgggca tttctgactc 300
gtttgtgaac tgggtggccg agaaggaatg ggagctgccc ccggattctg acatggatcg 360
gaatctgatc gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg acttcctggt 420
ccaatggcgc cgcgtgagta aggccccgga ggccctcttc tttgttcagt tcgagaaggg 480
cgagagctac tttcacctgc acgttctggt cgagaccacg ggggtcaagt ccatggtgct 540
aggccgcttc ctgagtcaga ttcgggaaaa gcttggtcca gaccatctac ccgcggggtc 600
gagccccacc ttgcccaact ggttcgcggt gaccaaagac gcggtaatgg cgccggcggg 660
ggggaacaag gtggtggacg agtgctacat ccccaactac ctcctgccca agactcagcc 720
cgagctgcag tgggcgtgga ctaacatgga ggagtatata agcgcgtgct tgaacctggc 780
cgagcgcaaa cggctcgtgg cgcagcacct gacccacgtc agccagacgc aggagcagaa 840
caaggagaat ctgaacccca attctgacgc gcccgtgatc aggtcaaaaa cctccgcgcg 900
ctatatggag ctggtcgggt ggctggtgga ccggggcatc acctccgaga agcagtggat 960
ccaggaggac caggcctcgt acatctcctt caacgccgcc tccaactcgc ggtcccagat 1020
caaggccgcg ctggacaatg ccggcaagat catggcgctg accaaatccg cgcccgacta 1080
cctggtgggg ccctcgctgc ccgcggacat tacccagaac cgcatctacc gcatcctcgc 1140
tctcaacggc tacgaccctg cctacgccgg ctccgtcttt ctcggctggg ctcagaaaaa 1200
gttcgggaaa cgcaacacca tctggctgtt tggacccgcc accaccggca agaccaacat 1260
tgcggaagcc atcgcccacg ccgtgccctt ctacggctgc gtcaactgga ccaatgagaa 1320
ctttcccttc aatgattgcg tcgacaagat ggtgatctgg tgggaggagg gcaagatgac 1380
ggccaaggtc gtggagtccg ccaaggccat tctcggcggc agcaaggtgc gcgtggacca 1440
aaagtgcaag tcgtccgccc agatcgaccc cacccccgtg atcgtcacct ccaacaccaa 1500
catgtgcgcc gtgattgacg ggaacagcac caccttcgag caccagcagc ctctccagga 1560
ccggatgttt aagttcgaac tcacccgccg tctggagcac gactttggca aggtgacaaa 1620
gcaggaagtc aaagagttct tccgctggggc cagtgatcac gtgaccgagg tggcgcatga 1680
gttttacgtc agaaagggcg gagccagcaa aagacccgcc cccgatgacg cggataaaag 1740
cgagcccaag cgggcctgcc cctcagtcgc ggatccatcg acgtcagacg cggaaggac 1800
tccggtggac tttgccgaca ggtaccaaaa caaatgttct cgtcacgcgg gcatgcttca 1860
gatgctgttt ccctgcaaaa cgtgcgagag aatgaatcag aatttcaaca tttgcttcac 1920
aacacggggtc agagactgct cagagtgttt ccccggcgtg tcagaatctc aaccggtcgt 1980
cagaaagagg acgtatcgga aactctgtgc gattcatcat ctgctggggc gggctcccga 2040
gattgcttgc tcggcctgcg atctggtcaa cgtggacctg gatgactgtg tttctgagca 2100
ataaatgact taaaccaggt atggctgccg atggttatct tccagattgg ctcgaggaca 2160
acctctctga gggcattcgc gagtggtggg cgctgaaacc tggagccccg aagcccaaag 2220
ccaaccagca aaagcaggac gacggccggg gtctggtgct tcctggctac aagtacctcg 2280
gacccttcaa cggactcgac aagggggagc ccgtcaacgc ggcggacgca gcggccctcg 2340
agcacgacaa ggcctacgac cagcagctgc aggcgggtga caatccgtac ctgcggtata 2400
accacgccga cgccgagttt caggagcgtc tgcaagaaga tacgtctttt gggggcaacc 2460
tcgggcgagc agtcttccag gccaagaagc gggttctcga acctctcggt ctggttgagg 2520
aaggcgctaa gacggctcct ggaaagaaga gaccggtaga gccatcaccc cagcgttctc 2580
cagactcctc tacgggcatc ggcaagaaag gccaacagcc cgccagaaaa agactcaatt 2640
ttggtcagac tggcgactca gagtcagttc cagaccctca acctctcgga gaacctccag 2700
cagcgccctc tggtgtggga cctaatacaa tggctgcagg cggtggcgca ccaatggcag 2760
acaataacga aggcgccgac ggagtgggta gttcctcggg aaattggcat tgcgattcca 2820
catggctggg cgacagagtc atcaccacca gcacccgaac ctgggccctg cccacctaca 2880
acaaccacct ctacaagcaa atctccaacg ggacatcggg aggagccacc aacgacaaca 2940
cctacttcgg ctacagcacc ccctgggggt attttgactt taacagattc cactgccact 3000
tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagagac 3060
tcagcttcaa gctcttcaac atccaggtca aggaggtcac gcagaatgaa ggcaccaaga 3120
ccatcgccaa taacctcacc agcaccatcc aggtgtttac ggactcggag taccagctgc 3180
cgtacgttct cggctctgcc caccagggct gcctgcctcc gttcccggcg gacgtgttca 3240
tgattcccca gtacggctac ctaacactca acaacggtag tcaggccgtg ggacgctcct 3300
ccttctactg cctggaatac tttccttcgc agatgctgag aaccggcaac aacttccagt 3360
ttacttacac cttcgaggac gtgcctttcc acagcagcta cgcccacagc cagagcttgg 3420
accggctgat gaatcctctg attgaccagt acctgtacta cttgtctcgg actcaaacaa 3480
caggaggcac ggcaaatacg cagactctgg gcttcagcca aggtgggcct aatacaatgg 3540
ccaatcaggc aaagaactgg ctgccaggac cctgttaccg ccaacaacgc gtctcaacga 3600
caaccgggca aaacaacaat agcaactttg cctggactgc tgggaccaaa taccatctga 3660
atggaagaaa ttcattggct aatcctggca tcgctatggc aacacacaaa gacgacgagg 3720
agcgtttttt tcccagtaac gggatcctga tttttggcaa acaaaatgct gccagagaca 3780
atgcggatta cagcgatgtc atgctcacca gcgaggaaga aatcaaaacc actaaccctg 3840
tggctacaga ggaatacggt atcgtggcag ataacttgca gcagcaaaac acggctcctc 3900
aaattggaac tgtcaacagc cagggggcct tacccggtat ggtctggcag aaccgggacg 3960
tgtacctgca gggtcccatc tgggccaaga ttcctcacac ggacggcaac ttccacccgt 4020
ctccgctgat gggcggcttt ggcctgaaac atcctccgcc tcagatcctg atcaagaaca 4080
cgcctgtacc tgcggatcct ccgaccacct tcaaccagtc aaagctgaac tctttcatca 4140
cgcaatacag caccggacag gtcagcgtgg aaattgaatg ggagctgcag aaggaaaaca 4200
gcaagcgctg gaacccccgag atccagtaca cctccaacta ctacaaatct acaagtgtgg 4260
actttgctgt taatacagaa ggcgtgtact ctgaacccg ccccattggc acccgttacc 4320
tcacccgtaa tctgtaattg cctgttaatc aataaaccgg ttgattcgtt tcagttgaac 4380
tttggtctct gcg 4393

<210> 135
<211> 6042
<212> DNA
<213> Artificial
<220>
<223>AAV9
<400> 135
gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgta 60
atagcgaaga ggccccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 120
ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180
agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240
acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300
acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360
agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420
gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540
cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660
gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780
ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840
taacgcgaat tttaacaaaa tattaacgct tacaatttaa atatttgctt atacaatctt 900
cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960
acgattaccg ttcatcgccc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 1020
cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 1080
agtggaattc acgcgtggat ctgaattcaa ttcacgcgtg gtacctctgg tcgttacata 1140
acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 1200
aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 1260
gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 1320
ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 1380
atgggacttt cctacttggc agtacatcta ctcgaggcca cgttctgctt cactctcccc 1440
atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 1500
gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg 1560
cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag 1620
tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg 1680
gcgggagcgg gatcagccac cgcggtggcg gcctagagtc gacgaggaac tgaaaaacca 1740
gaaagttaac tggtaagttt agtctttttg tcttttattt caggtcccgg atccggtggt 1800
ggtgcaaatc aaagaactgc tcctcagtgg atgttgcctt tacttctagg cctgtacgga 1860
agtgttactt ctgctctaaa agctgcggaa ttgtacccgc ggccgatcca ccggtccgga 1920
attcccggga tatcgtcgac ccacgcgtcc gggccccacg ctgcgcaccc gcgggtttgc 1980
tatggcgatg agcagcggcg gcagtggtgg cggcgtcccg gagcaggagg attccgtgct 2040
gttccggcgc ggcacaggcc agagcgatga ttctgacatt tgggatgata cagcactgat 2100
aaaagcatat gataaagctg tggcttcatt taagcatgct ctaaagaatg gtgacatttg 2160
tgaaacttcg ggtaaaccaa aaaccacacc taaaagaaaa cctgctaaga agaataaaag 2220
ccaaaagaag aatactgcag cttccttaca acagtggaaa gttggggaca aatgttctgc 2280
catttggtca gaagacggtt gcatttaccc agctaccatt gcttcaattg attttaagag 2340
agaaacctgt gttgtggttt acactggata tggaaataga gaggagcaaa atctgtccga 2400
tctactttcc ccaatctgtg aagtagctaa taatatagaa cagaatgctc aagagaatga 2460
aaatgaaagc caagtttcaa cagatgaaag tgagaactcc aggtctcctg gaaataaatc 2520
agataacatc aagcccaaat ctgctccatg gaactctttt ctccctccac caccccccat 2580
gccagggcca agactgggac caggaaagcc aggtctaaaa ttcaatggcc caccaccgcc 2640
accgccacca ccaccacccc acttactatc atgctggctg cctccatttc cttctggacc 2700
accaataatt cccccaccac ctcccatatg tccagattct cttgatgatg ctgatgcttt 2760
gggaagtatg ttaatttcat ggtacatgag tggctatcat actggctatt atatgggttt 2820
tagacaaaat caaaaagaag gaaggtgctc acattcctta aattaaggag aaatgctggc 2880
atagagcagc actaaatgac accactaaag aaacgatcag acagatctag aaagcttatc 2940
gataccgtcg actagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 3000
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 3060
ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 3120
gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 3180
ggagagatcg atctgaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 3240
gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg ccccgggcttt gcccgggcgg 3300
cctcagtgag cgagcgagcg cgcagagagg gagtggcccc cccccccccc cccccggcga 3360
ttctcttgtt tgctccagac tctcaggcaa tgacctgata gcctttgtag agacctctca 3420
aaaatagcta ccctctccgg catgaattta tcagctagaa cggttgaata tcatattgat 3480
ggtgatttga ctgtctccgg ccttctcac ccgtttgaat ctttacctac acattactca 3540
ggcattgcat ttaaaatata tgagggttct aaaaattttt atccttgcgt tgaaataaag 3600
gcttctcccg caaaagtatt acagggtcat aatgtttttg gtacaaccga tttagcttta 3660
tgctctgagg ctttattgct taattttgct aattctttgc cttgcctgta tgatttattg 3720
gatgttggaa tcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 3780
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 3840
acccgccaac actatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 3900
agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 3960
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 4020
catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 4080
tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 4140
cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4200
cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4260
tcgccccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4320
tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4380
atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4440
gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 4500
aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 4560
aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 4620
gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 4680
cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4740
atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4800
tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4860
ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4920
ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 4980
ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 5040
tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 5100
tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5160
aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5220
tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5280
tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5340
gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5400
agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5460
tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5520
ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 5580
cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5640
tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 5700
acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5760
gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5820
ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5880
tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5940
attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 6000
cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gc 6042

<210> 136
<211> 4102
<212> DNA
<213> Artificial
<220>
<223>AAV10
<400> 136
atgccgggct tctacgagat cgtgatcaag gtgccgagcg acctggacga gcacctgccg 60
ggcatttctg actcgtttgt gaactgggtg gccgagaagg aatgggagct gccccggat 120
tctgacatgg atcggaatct gatcgagcag gcacccctga ccgtggccga gaagctgcag 180
cgcgacttcc tggtccactg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt 240
cagttcgaga agggcgagtc ctactttcac ctgcacgttc tggtcgagac cacgggggtc 300
aagtccatgg tcctgggccg cttcctgagt cagatcagag acaggctggt gcagaccatc 360
taccgcgggg tagagcccac gctgcccaac tggttcgcgg tgaccaagac gcgaaatggc 420
gccggcgggg ggaacaaggt ggtggacgag tgctacatcc ccaactacct cctgcccaag 480
acgcagcccg agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtctg 540
aacctcgcgg agcgtaaacg gctcgtggcg cagcacctga cccacgtcag ccagacgcag 600
gagcagaaca aggagaatct gaacccgaat tctgacgcgc ccgtgatcag gtcaaaaacc 660
tccgcgcgct acatggagct ggtcgggtgg ctggtggacc ggggcatcac ctccgagaag 720
cagtggatcc aggaggacca ggcctcgtac atctccttca acgccgcctc caactcgcgg 780
tcccagatca aggccgcgct ggacaatgcc ggaaagatca tggcgctgac caaatccgcg 840
cccgactacc tggtaggccc gtccttaccc gcggacatta aggccaaccg catctaccgc 900
atcctggagc tcaacggcta cgaccccgcc tacgccggct ccgtcttcct gggctgggcg 960
cagaaaaagt tcggtaaaag gaatacaatt tggctgttcg ggcccgccac caccggcaag 1020
accaacatcg cggaagccat cgcccacgcc gtgcccttct acggctgcgt caactggacc 1080
aatgagaact ttcccttcaa cgattgcgtc gacaagatgg tgatctggtg ggaggagggc 1140
aagatgaccg ccaaggtcgt ggagtccgcc aaggccattc tgggcggaag caaggtgcgc 1200
gtcgaccaaa agtgcaagtc ctcggcccag atcgacccca cgcccgtgat cgtcacctcc 1260
aacaccaaca tgtgcgccgt gatcgacggg aacagcacca ccttcgagca ccagcagccc 1320
ctgcaggacc gcatgttcaa gttcgagctc acccgccgtc tggagcacga ctttggcaag 1380
gtgaccaagc aggaagtcaa agagttcttc cgctgggctc aggatcacgt gactgaggtg 1440
acgcatgagt tctacgtcag aaagggcgga gccaccaaaa gacccgcccc cagtgacgcg 1500
gatataagcg agcccaagcg ggcctgcccc tcagttgcgg agccatcgac gtcagacgcg 1560
gaagcaccgg tggactttgc ggacaggtac caaaacaaat gttctcgtca cgcgggcatg 1620
cttcagatgc tgtttccctg caagacatgc gagagaatga atcagaattt caacgtctgc 1680
ttcacgcacg gggtcagaga ctgctcagag tgcttccccg gcgcgtcaga atctcaacct 1740
gtcgtcagaa aaaagacgta tcagaaactg tgcgcgattc atcatctgct ggggcgggca 1800
cccgagattg cgtgttcggc ctgcgatctc gtcaacgtgg acttggatga ctgtgtttct 1860
gagcaataaa tgacttaaac caggtatggc tgctgacggt tatcttccag attggctcga 1920
ggacaacctc tctgagggca ttcgcgagtg gtgggacctg aaacctggag cccccaagcc 1980
caaggccaac cagcagaagc aggacgacgg ccggggtctg gtgcttcctg gctacaagta 2040
cctcggaccc ttcaacggac tcgacaaggg ggagcccgtc aacgcggcgg acgcagcggc 2100
cctcgagcac gacaaggcct acgaccagca gctcaaagcg ggtgacaatc cgtacctgcg 2160
gtataaccac gccgacgccg agtttcagga gcgtctgcaa gaagatacgt cttttggggg 2220
caacctcggg cgagcagtct tccaggccaa gaagcgggtt ctcgaacctc tcggtctggt 2280
tgaggaagct gctaagacgg ctcctggaaa gaagagaccg gtagaaccgt cacctcagcg 2340
ttccccgac tcctccacgg gcatcggcaa gaaaggccag cagcccgcta aaaagagact 2400
gaactttggg cagactggcg agtcagagtc agtccccgac cctcaaccaa tcggagaacc 2460
accagcaggc ccctctggtc tgggatctgg tacaatggct gcaggcggtg gcgctccaat 2520
ggcagacaat aacgaaggcg ccgacggagt gggtagttcc tcaggaaatt ggcattgcga 2580
ttccacatgg ctgggcgaca gagtcatcac caccagcacc cgaacctggg ccctgcccac 2640
ctacaacaac cacctctaca agcaaatctc caacgggaca tcgggaggaa gcaccaacga 2700
caacacctac ttcggctaca gcaccccctg ggggtatttt gacttcaaca gattccactg 2760
ccacttctca ccacgtgact ggcagcgact catcaacaac aactggggat tccggccaaa 2820
aagactcagc ttcaagctct tcaacatcca ggtcaaggag gtcacgcaga atgaaggcac 2880
caagaccatc gccaataacc ttaccagcac gattcaggta tttacggact cggaatacca 2940
gctgccgtac gtcctcggct ccgcgcacca gggctgcctg cctccgttcc cggcggatgt 3000
cttcatgatt ccccagtacg gctacctgac actgaacaat ggaagtcaag ccgtaggccg 3060
ttcctccttc tactgcctgg aatattttcc atctcaaatg ctgcgaactg gaaacaattt 3120
tgaattcagc tacaccttcg aggacgtgcc tttccacagc agctacgcac acagccagag 3180
cttggaccga ctgatgaatc ctctcattga ccagtacctg tactacttat ccagaactca 3240
gtccacagga ggaactcaag gtacccagca attgttattt tctcaagctg ggcctgcaaa 3300
catgtcggct caggccaaga actggctgcc tggaccttgc taccggcagc agcgagtctc 3360
cacgacactg tcgcaaaaca acaacagcaa ctttgcttgg actggtgcca ccaaatatca 3420
cctgaacgga agagactctc tggtgaatcc cggtgtcgcc atggcaaccc acaaggacga 3480
cgaggaacgc ttcttcccgt cgagcggagt cctgatgttt ggaaaacagg gtgctggaag 3540
agacaatgtg gactacagca gcgttatgct aacaagcgaa gaagaaatta aaaccactaa 3600
ccctgtagcc acagaacaat acggcgtggt ggctgacaac ttgcagcaag ccaatacagg 3660
gcctattgtg ggaaatgtca acagccaagg agccttacct ggcatggtct ggcagaaccg 3720
agacgtgtac ctgcagggtc ccatctgggc caagattcct cacacggacg gcaactttca 3780
cccgtctcct ctgatgggcg gctttggact taaacacccg cctccacaga tcctgatcaa 3840
gaacacgccg gtacctgcgg atcctccaac aacgttcagc caggcgaaat tggcttcctt 3900
catcacgcag tacagcaccg gacaggtcag cgtggaaatc gagtgggagc tgcagaagga 3960
gaacagcaaa cgctggaacc cagagattca gtacacttca aactactaca aatctacaaa 4020
tgtggacttt gctgtcaata cagagggaac ttattctgag cctcgcccca ttggtactcg 4080
ttatctgaca cgtaatctgt aa 4102

<210> 137
<211> 4087
<212> DNA
<213> Artificial
<220>
<223> AAV11
<400> 137
atgccgggct tctacgagat cgtgatcaag gtgccgagcg acctggacga gcacctgccg 60
ggcatttctg actcgtttgt gaactgggtg gccgagaagg aatgggagct gccccggat 120
tctgacatgg atcggaatct gatcgagcag gcacccctga ccgtggccga gaagctgcag 180
cgcgacttcc tggtccactg gcgccgcgtg agtaaggccc cggaggccct cttctttgtt 240
cagttcgaga agggcgagtc ctacttccac ctccacgttc tcgtcgagac cacgggggtc 300
aagtccatgg tcctgggccg cttcctgagt cagatcagag acaggctggt gcagaccatc 360
taccgcgggg tcgagcccac gctgcccaac tggttcgcgg tgaccaagac gcgaaatggc 420
gccggcgggg ggaacaaggt ggtggacgag tgctacatcc ccaactacct cctgcccaag 480
acccagcccg agctgcagtg ggcgtggact aacatggagg agtatataag cgcgtgtcta 540
aacctcgcgg agcgtaaacg gctcgtggcg cagcacctga cccacgtcag ccagacgcag 600
gagcagaaca aggagaatct gaacccgaat tctgacgcgc ccgtgatcag gtcaaaaacc 660
tccgcgcgct acatggagct ggtcgggtgg ctggtggacc ggggcatcac ctccgagaag 720
cagtggatcc aggaggacca ggcctcgtac atctccttca acgccgcctc caactcgcgg 780
tcccagatca aggccgcgct ggacaatgcc ggaaagatca tggcgctgac caaatccgcg 840
cccgactacc tggtaggccc gtccttaccc gcggacatta aggccaaccg catctaccgc 900
atcctggagc tcaacggcta cgaccccgcc tacgccggct ccgtcttcct gggctgggcg 960
cagaaaaagt tcggtaaacg caacaccatc tggctgtttg ggcccgccac caccggcaag 1020
accaacatcg cggaagccat agcccacgcc gtgcccttct acggctgcgt gaactggacc 1080
aatgagaact ttcccttcaa cgattgcgtc gacaagatgg tgatctggtg ggaggagggc 1140
aagatgaccg ccaaggtcgt ggagtccgcc aaggccattc tgggcggaag caaggtgcgc 1200
gtggaccaaa agtgcaagtc ctcggcccag atcgacccca cgcccgtgat cgtcacctcc 1260
aacaccaaca tgtgcgccgt gatcgacggg aacagcacca ccttcgagca ccagcagccg 1320
ctgcaggacc gcatgttcaa gttcgagctc acccgccgtc tggagcacga ctttggcaag 1380
gtgaccaagc aggaagtcaa agagttcttc cgctgggctc aggatcacgt gactgaggtg 1440
gcgcatgagt tctacgtcag aaagggcgga gccaccaaaa gacccgcccc cagtgacgcg 1500
gatataagcg agcccaagcg ggcctgcccc tcagttccgg agccatcgac gtcagacgcg 1560
gaagcaccgg tggactttgc ggacaggtac caaaacaaat gttctcgtca cgcgggcatg 1620
cttcagatgc tgtttccctg caagacatgc gagagaatga atcagaattt caacgtctgc 1680
ttcacgcacg gggtcagaga ctgctcagag tgcttccccg gcgcgtcaga atctcaaccc 1740
gtcgtcagaa aaaagacgta tcagaaactg tgcgcgattc atcatctgct ggggcgggca 1800
cccgagattg cgtgttcggc ctgcgatctc gtcaacgtgg acttggatga ctgtgtttct 1860
gagcaataaa tgacttaaac caggtatggc tgctgacggt tatcttccag attggctcga 1920
ggacaacctc tctgagggca ttcgcgagtg gtgggacctg aaacctggag ccccgaagcc 1980
caaggccaac cagcagaagc aggacgacgg ccggggtctg gtgcttcctg gctacaagta 2040
cctcggaccc ttcaacggac tcgacaaggg ggagcccgtc aacgcggcgg acgcagcggc 2100
cctcgagcac gacaaggcct acgaccagca gctcaaagcg ggtgacaatc cgtacctgcg 2160
gtataaccac gccgacgccg agtttcagga gcgtctgcaa gaagatacgt cttttggggg 2220
caacctcggg cgagcagtct tccaggccaa gaagagggta ctcgaacctc tgggcctggt 2280
tgaagaaggt gctaaaacgg ctcctggaaa gaagagaccg ttagagtcac cacaagagcc 2340
cgactcctcc tcgggcatcg gcaaaaaagg caaacaacca gccagaaaga ggctcaactt 2400
tgaagaggac actggagccg gagacggacc ccctgaagga tcagatacca gcgccatgtc 2460
ttcagacatt gaaatgcgtg cagcaccggg cggaaatgct gtcgatgcgg gacaaggttc 2520
cgatggagtg ggtaatgcct cgggtgattg gcattgcgat tccacctggt ctgagggcaa 2580
ggtcacaaca acctcgacca gaacctgggt cttgcccacc tacaacaacc acttgtacct 2640
gcgtctcgga acaacatcaa gcagcaacac ctacaacgga ttctccaccc cctggggata 2700
ttttgacttc aacagattcc actgtcactt ctcaccacgt gactggcaaa gactcatcaa 2760
caacaactgg ggactacgac caaaagccat gcgcgttaaa atcttcaata tccaagttaa 2820
ggaggtcaca acgtcgaacg gcgagactac ggtcgctaat aaccttacca gcacggttca 2880
gatatttgcg gactcgtcgt atgagctccc gtacgtgatg gacgctggac aagaggggag 2940
cctgcctcct ttccccaatg acgtgttcat ggtgcctcaa tatggctact gtggcatcgt 3000
gactggcgag aatcagaacc aaacggacag aaacgctttc tactgcctgg agtattttcc 3060
ttcgcaaatg ttgagaactg gcaacaactt tgaaatggct tacaactttg agaaggtgcc 3120
gttccactca atgtatgctc acagccagag cctggacaga ctgatgaatc ccctcctgga 3180
ccagtacctg tggcacttac agtcgactac ctctggagag actctgaatc aaggcaatgc 3240
agcaaccaca tttggaaaaa tcaggagtgg agactttgcc ttttacagaa agaactggct 3300
gcctgggcct tgtgttaaac agcagagatt ctcaaaaact gccagtcaaa attacaagat 3360
tcctgccagc gggggcaacg ctctgttaaa gtatgacacc cactatacct taaacaccg 3420
ctggagcaac atcgcgcccg gacctccaat ggccacagcc ggaccttcgg atggggactt 3480
cagtaacgcc cagcttatat tccctggacc atctgttacc ggaaatacaa caacttcagc 3540
caacaatctg ttgtttacat cagaagaaga aattgctgcc accaacccaa gagacacgga 3600
catgtttggc cagattgctg acaataatca gaatgctaca actgctccca taaccggcaa 3660
cgtgactgct atgggagtgc tgcctggcat ggtgtggcaa aacagagaca tttactacca 3720
agggccaatt tgggccaaga tcccacacgc ggacggacat tttcatcctt caccgctgat 3780
tggtgggttt ggactgaaac acccgcctcc ccagatattc atcaagaaca ctcccgtacc 3840
tgccaatcct gcgacaacct tcactgcagc cagagtggac tctttcatca cacaatacag 3900
caccggccag gtcgctgttc agattgaatg ggaaattgaa aaggaacgct ccaaacgctg 3960
gaatcctgaa gtgcagttta cttcaaacta tgggaaccag tcttctatgt tgtgggctcc 4020
tgatacaact gggaagtata cagagccgcg ggttattggc tctcgttatt tgactaatca 4080
tttgtaa 4087

<210> 138
<211> 4200
<212> DNA
<213> Artificial
<220>
<223>AAV12
<400> 138
ttgcgacagt ttgcgacacc atgtggtcac aagaggtata taaccgcgag tgagccagcg 60
aggagctcca ttttgcccgc gaagtttgaa cgagcagcag ccatgccggg gttctacgag 120
gtggtgatca aggtgcccag cgacctggac gagcacctgc ccggcatttc tgactccttt 180
gtgaactggg tggccgagaa ggaatgggag ttgcccccgg attctgacat ggatcagaat 240
ctgattgagc aggcacccct gaccgtggcc gagaagctgc agcgcgagtt cctggtggaa 300
tggcgccgag tgagtaaatt tctggaggcc aagttttttg tgcagtttga aaagggggac 360
tcgtactttc atttgcatat tctgattgaa attaccggcg tgaaatccat ggtggtgggc 420
cgctacgtga gtcagattag ggataaactg atccagcgca tctaccgcgg ggtcgagccc 480
cagctgccca actggttcgc ggtcacaaag acccgaaatg gcgccggagg cgggaacaag 540
gtggtggacg agtgctacat ccccaactac ctgctcccca aggtccagcc cgagcttcag 600
tgggcgtgga ctaacatgga ggagtatata agcgcctgtt tgaacctcgc ggagcgtaaa 660
cggctcgtgg cgcagcacct gacgcacgtc tcccagaccc aggagggcga caaggagaat 720
ctgaacccga attctgacgc gccggtgatc cggtcaaaaa cctccgccag gtacatggag 780
ctggtcgggt ggctggtgga caagggcatc acgtccgaga agcagtggat ccaggaggac 840
caggcctcgt acatctcctt caacgcggcc tccaactccc ggtcgcagat caaggcggcc 900
ctggacaatg cctccaaaat catgagcctc accaaaacgg ctccggacta tctcatcggg 960
cagcagcccg tgggggacat taccaccaac cggatctaca aaatcctgga actgaacggg 1020
tacgaccccc agtacgccgc ctccgtcttt ctcggctggg cccagaaaaa gtttggaaag 1080
cgcaacacca tctggctgtt tgggcccgcc accaccggca agaccaacat cgcggaagcc 1140
atcgcccacg cggtcccctt ctacggctgc gtcaactgga ccaatgagaa ctttcccttc 1200
aacgactgcg tcgacaaaat ggtgatttgg tgggaggagg gcaagatgac cgccaaggtc 1260
gtagagtccg ccaaggccat tctgggcggc agcaaggtgc gcgtggacca aaaatgcaag 1320
gcctctgcgc agatcgaccc cacccccgtg atcgtcacct ccaacaccaa catgtgcgcc 1380
gtgattgacg ggaacagcac caccttcgag caccagcagc ccctgcagga ccggatgttc 1440
aagtttgaac tcacccgccg cctcgaccac gactttggca aggtcaccaa gcaggaagtc 1500
aaggactttt tccggtgggc ggctgatcac gtgactgacg tggctcatga gttttacgtc 1560
acaaagggtg gagctaagaa aaggcccgcc ccctctgacg aggatataag cgagcccaag 1620
cggccgcgcg tgtcatttgc gcagccggag acgtcagacg cggaagctcc cggagacttc 1680
gccgacaggt accaaaacaa atgttctcgt cacgcgggta tgctgcagat gctctttccc 1740
tgcaagacgt gcgagagaat gaatcagaat tccaacgtct gcttcacgca cggtcagaaa 1800
gattgcgggg agtgctttcc cgggtcagaa tctcaaccgg tttctgtcgt cagaaaaacg 1860
tatcagaaac tgtgcatcct tcatcagctc cggggggcac ccgagatcgc ctgctctgct 1920
tgcgaccaac tcaaccccga tttggacgat tgccaatttg agcaataaat gactgaaatc 1980
aggtatggct gctgacggtt atcttccaga ttggctcgag gacaacctct ctgaaggcat 2040
tcgcgagtgg tgggcgctga aacctggagc tccacaaccc aaggccaacc aacagcatca 2100
ggacaacggc aggggtcttg tgcttcctgg gtacaagtac ctcggaccct tcaacggact 2160
cgacaaggga gagccggtca acgaggcaga cgccgcggcc ctcgagcacg acaaggccta 2220
cgacaagcag ctcgagcagg gggacaaccc gtatctcaag tacaaccacg ccgacgccga 2280
gttccagcag cgcttggcga ccgacacctc ttttgggggc aacctcgggc gagcagtctt 2340
ccaggccaaa aagaggattc tcgagcctct gggtctggtt gaagagggcg ttaaaacggc 2400
tcctggaaag aaacgcccat tagaaaagac tccaaatcgg ccgaccaacc cggactctgg 2460
gaaggccccg gccaagaaaa agcaaaaaga cggcgaacca gccgactctg ctagaaggac 2520
actcgacttt gaagactctg gagcaggaga cggaccccct gagggatcat cttccggaga 2580
aatgtctcat gatgctgaga tgcgtgcggc gccaggcgga aatgctgtcg aggcgggaca 2640
aggtgccgat ggagtgggta atgcctccgg tgattggcat tgcgattcca cctggtcaga 2700
gggccgagtc accaccacca gcacccgaac ctgggtccta cccacgtaca acaaccacct 2760
gtacctgcga atcggaacaa cggccaacag caacacctac aacggattct ccaccccctg 2820
gggatacttt gactttaacc gcttccactg ccacttttcc ccacgcgact ggcagcgact 2880
catcaacaac aactggggac tcaggccgaa atcgatgcgt gttaaaatct tcaacataca 2940
ggtcaaggag gtcacgacgt caaacggcga gactacggtc gctaataacc ttaccagcac 3000
ggttcagatc tttgcggatt cgacgtatga actcccatac gtgatggacg ccggtcagga 3060
ggggagcttt cctccgtttc ccaacgacgt ctttatggtt ccccaatacg gatactgcgg 3120
agttgtcact ggaaaaaacc agaaccagac agacagaaat gccttttact gcctggaata 3180
ctttccatcc caaatgctaa gaactggcaa caattttgaa gtcagttacc aatttgaaaa 3240
agttcctttc cattcaatgt acgcgcacag ccagagcctg gacagaatga tgaatccttt 3300
actggatcag tacctgtggc atctgcaatc gaccactacc ggaaattccc ttaatcaagg 3360
aacagctacc accacgtacg ggaaaattac cactggagac tttgcctact acaggaaaaa 3420
ctggttgcct ggagcctgca ttaaacaaca aaaattttca aagaatgcca atcaaaacta 3480
caagattccc gccagcgggg gagacgccct tttaaagtat gacacgcata ccactctaaa 3540
tgggcgatgg agtaacatgg ctcctggacc tccaatggca accgcaggtg ccggggactc 3600
ggattttagc aacagccagc tgatctttgc cggacccaat ccgagcggta acacgaccac 3660
atcttcaaac aatttgttgt ttacctcaga agaggagatt gccacaacaa acccacgaga 3720
cacggacatg tttggacaga ttgcagataa taatcaaaat gccaccaccg cccctcacat 3780
cgctaacctg gacgctatgg gaattgttcc cggaatggtc tggcaaaaca gagacatcta 3840
ctaccagggc cctatttggg ccaaggtccc tcacacggac ggacactttc acccttcgcc 3900
gctgatggga ggatttggac tgaaacaccc gcctccacag attttcatca aaaacaccccc 3960
cgtacccgcc aatcccaata ctacctttag cgctgcaagg attaattctt ttctgacgca 4020
gtacagcacc ggacaagttg ccgttcagat cgactgggaa attcagaagg agcattccaa 4080
acgctggaat cccgaagttc aatttacttc aaactacggc actcaaaatt ctatgctgtg 4140
ggctcccgac aatgctggca actaccacga actccgggct attgggtccc gtttcctcac 4200

<210> 139
<211> 2205
<212> DNA
<213> Artificial
<220>
<223> AAV2 VP3 only
<400> 139
ctggctgccg atggttatct tccagatgg ctcgaggaca ctctctctga aggaataaga 60
cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120
gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180
aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240
cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360
gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa ggcggctccg 420
ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480
aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540
tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600
aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660
gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780
tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840
tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900
aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960
aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020
caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080
tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140
aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200
cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260
cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320
tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380
cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440
ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500
tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560
ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620
atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680
gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740
accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800
cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860
attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920
caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980
ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040
gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcagtac 2100
acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160
tcagagcctc gccccattgg cacgatac ctgactcgta atctg 2205

<210> 140
<211> 2205
<212> DNA
<213> Artificial
<220>
<223> AAV2 VP2/VP3
<400> 140
ctggctgccg atggttatct tccagatgg ctcgaggaca ctctctctga aggaataaga 60
cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120
gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180
aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240
cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360
gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa gacggctccg 420
ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480
aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540
tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600
aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660
gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780
tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840
tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900
aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960
aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020
caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080
tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140
aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200
cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260
cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320
tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380
cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440
ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500
tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560
ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620
atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680
gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740
accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800
cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860
attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920
caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980
ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040
gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcagtac 2100
acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160
tcagagcctc gccccattgg cacgatac ctgactcgta atctg 2205

<210> 141
<211> 2205
<212> DNA
<213> Artificial
<220>
<223> AAV2 VP1 only
<400> 141
atggctgccg atggttatct tccagatgg ctcgaggaca ctctctctga aggaataaga 60
cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120
gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180
aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240
cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360
gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa ggcggctccg 420
ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480
aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540
tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600
aatacgctgg ctacaggcag tggcgcacca ctggcagaca ataacgaggg cgccgacgga 660
gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780
tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840
tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900
aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960
aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020
caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080
tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140
aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200
cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260
cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320
tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380
cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440
ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500
tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560
ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620
atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680
gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740
accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800
cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860
attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920
caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980
ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040
gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcagtac 2100
acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160
tcagagcctc gccccattgg cacgatac ctgactcgta atctg 2205

<210> 142
<211> 2205
<212> DNA
<213> Artificial
<220>
<223> AAV2 VP1/VP3
<400> 142
atggctgccg atggttatct tccagatgg ctcgaggaca ctctctctga aggaataaga 60
cagtggtgga agctcaaacc tggcccacca ccaccaaagc ccgcagagcg gcataaggac 120
gacagcaggg gtcttgtgct tcctgggtac aagtacctcg gacccttcaa cggactcgac 180
aagggagagc cggtcaacga ggcagacgcc gcggccctcg agcacgacaa agcctacgac 240
cggcagctcg acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt 300
caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcggacgagc agtcttccag 360
gcgaaaaaga gggttcttga acctctgggc ctggttgagg aacctgttaa ggcggctccg 420
ggaaaaaaga ggccggtaga gcactctcct gtggagccag actcctcctc gggaaccgga 480
aaggcgggcc agcagcctgc aagaaaaaga ttgaattttg gtcagactgg agacgcagac 540
tcagtacctg acccccagcc tctcggacag ccaccagcag ccccctctgg tctgggaact 600
aatacgatgg ctacaggcag tggcgcacca atggcagaca ataacgaggg cgccgacgga 660
gtgggtaatt cctcgggaaa ttggcattgc gattccacat ggatgggcga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caaacaaatt 780
tccagccaat caggagcctc gaacgacaat cactactttg gctacagcac cccttggggg 840
tattttgact tcaacagatt ccactgccac ttttcaccac gtgactggca aagactcatc 900
aacaacaact ggggattccg acccaagaga ctcaacttca agctctttaa cattcaagtc 960
aaagaggtca cgcagaatga cggtacgacg acgattgcca ataaccttac cagcacggtt 1020
caggtgttta ctgactcgga gtaccagctc ccgtacgtcc tcggctcggc gcatcaagga 1080
tgcctcccgc cgttcccagc agacgtcttc atggtgccac agtatggata cctcaccctg 1140
aacaacggga gtcaggcagt aggacgctct tcattttact gcctggagta ctttccttct 1200
cagatgctgc gtaccggaaa caactttacc ttcagctaca cttttgagga cgttcctttc 1260
cacagcagct acgctcacag ccagagtctg gaccgtctca tgaatcctct catcgaccag 1320
tacctgtatt acttgagcag aacaaacact ccaagtggaa ccaccacgca gtcaaggctt 1380
cagttttctc aggccggagc gagtgacatt cgggaccagt ctaggaactg gcttcctgga 1440
ccctgttacc gccagcagcg agtatcaaag acatctgcgg ataacaacaa cagtgaatac 1500
tcgtggactg gagctaccaa gtaccacctc aatggcagag actctctggt gaatccgggc 1560
ccggccatgg caagccacaa ggacgatgaa gaaaagtttt ttcctcagag cggggttctc 1620
atctttggga agcaaggctc agagaaaaca aatgtggaca ttgaaaaggt catgattaca 1680
gacgaagagg aaatcaggac aaccaatccc gtggctacgg agcagtatgg ttctgtatct 1740
accaacctcc agagaggcaa cagacaagca gctaccgcag atgtcaacac acaaggcgtt 1800
cttccaggca tggtctggca ggacagagat gtgtaccttc aggggcccat ctgggcaaag 1860
attccacaca cggacggaca ttttcacccc tctcccctca tgggtggatt cggacttaaa 1920
caccctcctc cacagattct catcaagaac accccggtac ctgcgaatcc ttcgaccacc 1980
ttcagtgcgg caaagtttgc ttccttcatc acacagtact ccacgggaca ggtcagcgtg 2040
gagatcgagt gggagctgca gaaggaaaac agcaaacgct ggaatcccga aattcagtac 2100
acttccaact acaacaagtc tgttaatgtg gactttactg tggacactaa tggcgtgtat 2160
tcagagcctc gccccattgg cacgatac ctgactcgta atctg 2205

Claims (83)

An isolated AAV virion having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3, comprising:
Two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome;
at least one of the viral structural proteins present is from a different serotype than other viral structural proteins;
VP1 is derived from only one serotype, VP2 is derived from only one serotype, and VP3 is derived from only one serotype,
The isolated AAV virions. 2. The isolated AAV virion of claim 1, wherein all three viral structural proteins are present. 3. The isolated AAV virion of claim 2, wherein all three viral structural proteins are from different serotypes. 3. The isolated AAV virion of claim 2, wherein only one of the three structural proteins is derived from a different serotype. 5. The isolated AAV virion of claim 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP1. 5. The isolated AAV virion of claim 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP2. 5. The isolated AAV virion of claim 4, wherein the one viral structural protein that is different from the other two viral structural proteins is VP3. A substantially homogeneous population of virions according to any one of claims 1 to 7, which is at least 10 ¹ virions. 9. A substantially homogeneous population of virions according to claim 8, which is at least 10 ⁷ virions. 9. A substantially homogeneous population of virions according to claim 8, which is at least 10 ⁷ to 10 ¹⁵ virions. 9. A substantially homogeneous population of virions according to claim 8, which is at least ¹⁰⁹ virions. 9. A substantially homogeneous population of virions according to claim 8, which is at least 10 ¹⁰ virions. 9. A substantially homogeneous population of virions according to claim 8, which is at least 10 ¹¹ virions. 11. A substantially homogeneous population of virions according to claim 10, which is at least 95% homogeneous. 11. A substantially homogeneous population of virions according to claim 10, which is at least 99% homogeneous. 1. A method of producing adeno-associated virus (AAV) virions, the method comprising:
contacting the cell with the first nucleic acid sequence and the second nucleic acid sequence under conditions for the formation of AAV virions;
AAV virions are formed from at least VP1 and VP3 viral structural proteins;
a first nucleic acid encodes VP1 derived only from a first AAV serotype, but is incapable of expressing VP3;
the second nucleic acid sequence encodes VP3 derived exclusively from a second AAV serotype different from the first AAV serotype, and is further incapable of expressing VP1;
the AAV virion comprises VP1 derived only from the first serotype and VP3 derived only from the second serotype, and
When VP2 is expressed, it is derived from only one serotype,
Said method. the first nucleic acid has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid; 17. The method of claim 16, wherein the VP1 has a mutation in its start codon that prevents translation of VP1 from the RNA to be transcribed. 17. The method of claim 16, wherein VP2 from only one serotype is expressed. 19. The method of claim 18, wherein VP2 is derived from a different serotype than VP1 and a different serotype than VP3. 19. The method of claim 18, wherein VP2 is derived from the same serotype as VP1. 19. The method of claim 18, wherein VP2 is derived from the same serotype as VP3. The first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV. 17. The method of claim 16, wherein: an AAV in which the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV; 17. The method according to claim 16. AAV virions are formed from VP1, VP2, and VP3 capsid proteins;
a viral structural protein is encoded by a first nucleic acid derived exclusively from a first AAV serotype and a second nucleic acid derived exclusively from a second AAV serotype different from the first AAV serotype;
the first nucleic acid has a mutation in the A2 splice acceptor site, and the second nucleic acid has a mutation in the A1 splice acceptor site, and
the polyploid AAV virion comprises VP1 derived only from a first serotype and VP2 and VP3 derived only from a second serotype;
19. The method according to claim 18. An AAV in which the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV 25. The method according to claim 24. an AAV in which the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV; 25. The method according to claim 24. a first nucleic acid sequence in which the viral structural proteins are derived only from a first AAV serotype that is different from a second and third serotype; a second AAV serotype that is different from the first and third AAV serotypes; and a third nucleic acid sequence derived only from a third AAV serotype that is different from the first and second AAV serotypes, and further
the first nucleic acid sequence has a mutation in the start codon of VP2 and VP3 that prevents translation of VP2 and VP3 from RNA transcribed from the first nucleic acid; VP1 and VP3 have mutations in the start codons of VP1 and VP3 that prevent translation of VP1 and VP3 from RNA transcribed from the nucleic acid sequence; and have mutations in the start codons of VP1 and VP2 that prevent translation of VP2,
the AAV virion comprises VP1 derived only from a first serotype, VP2 derived only from a second serotype, and VP3 derived only from a third serotype;
19. The method according to claim 18. An AAV in which the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV 28. The method of claim 27. an AAV in which the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV; 28. The method of claim 27. AAV in which the third AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV 28. The method of claim 27. the first nucleic acid sequence has mutations in the start codons of VP2 and VP3 and mutations in the A2 splice acceptor site that prevent translation of VP2 and VP3 from RNA transcribed from the first nucleic acid sequence, and the second nucleic acid sequence has a mutation in the start codon of VP1 and a mutation in the A1 splice acceptor site that prevents translation of VP1 from RNA transcribed from the second nucleic acid sequence, and the AAV polyploid capsid 19. The method of claim 18, wherein comprises VP1 derived only from the first serotype and VP2 and VP3 derived only from the second serotype. An AAV in which the first AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV 32. The method of claim 31. an AAV in which the second AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or an AAV selected from Table 1 or Table 3, or any chimera of each AAV; 32. The method of claim 31. a viral structural protein is encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and
the start codons for VP2 and VP3 have been mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid sequence;
The capsid protein is encoded by a second nucleic acid derived from only a single AAV serotype, and the second nucleic acid has a mutation in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid. having in it, and
the polyploid AAV capsid comprises VP1 from a first nucleic acid sequence created through DNA shuffling and VP2 and VP3 from only a second serotype;
19. The method according to claim 18. a viral structural protein is encoded in a first nucleic acid sequence created through DNA shuffling of two or more different AAV serotypes, and
the start codons for VP2 and VP3 have been mutated such that VP2 and VP3 cannot be translated from RNA transcribed from the first nucleic acid, and
the A2 splice acceptor site of the first nucleic acid is mutated;
the capsid protein is encoded by a second nucleic acid sequence derived from only a single AAV serotype, and the second nucleic acid is in the start codon of VP1 that prevents translation of VP1 from RNA transcribed from the second nucleic acid. and a mutation in the A1 splice acceptor site, and
the polyploid AAV capsid comprises VP1 from a first nucleic acid created through DNA shuffling and VP2 and VP3 from only a second serotype;
19. The method according to claim 18. AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV 16. The virion according to claim 15. 17. A substantially homogeneous population of virions produced by the method of claim 16. A substantially homogeneous population of virions produced by the method of claim 18. 39. The AAV virion of claim 38, wherein the heterologous gene encodes a protein for treating a disease. If the disease is a lysosomal storage disease, such as mucopolysaccharidosis (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [a-L-iduronidase], Scheie syndrome [a-L-iduronidase], Hurler-Scheie syndrome [a-L-iduronidase], Hunter syndrome [Iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebrosidase) , or a glycogen storage disease (e.g. Pompe disease; lysosomal acid a-glucosidase). An isolated AAV virion according to any one of claims 1 to 7, wherein at least one of the viral structural proteins is a chimeric viral structural protein. 42. The isolated AAV virion of claim 41, wherein the chimeric viral structural protein is derived from an AAV serotype but is different from other viral structural proteins. An isolated AAV virion according to any one of claims 1 to 7, wherein none of the viral structural proteins are chimeric viral structural proteins. 42. The isolated AAV virion of claim 41, wherein there is no serotype overlap between the chimeric viral structural protein and at least one other viral structural protein. A method of modulating transduction using a method according to any one of claims 16-35. 46. The method of claim 45, wherein the method enhances transduction. A method of altering the tropism of AAV virions comprising using a method according to any one of claims 16 to 35. A method of altering the immunogenicity of AAV virions comprising using a method according to any one of claims 16-35. A method of increasing vector genome copy number in a tissue comprising using a method according to any one of claims 16-35. A method for increasing transgene expression comprising using a method according to any one of claims 16-35. an effective amount of a virion according to any one of claims 1 to 7, 36, 43, and 44; substantially a virion according to any one of claims 8 to 15, 37 to 42, and 44; 36. A method of treating a disease comprising administering a homogeneous population or virions produced by the method of any one of claims 16 to 35,
the heterologous gene encodes a protein for treating a disease that is suitable for treatment by gene therapy in a subject having the disease;
Said method. 52. The method of claim 51, wherein the disease is selected from genetic diseases, cancer, immunological diseases, inflammation, autoimmune diseases, and degenerative diseases. 53. The method of any one of claims 51 and 52, wherein multiple administrations are performed. 54. The method of claim 53, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration. administering AAV virions according to any one of claims 1 to 15 and 36 to 44. A method of increasing at least one of transgene, copy number, and transgene expression. An isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion encapsidating an AAV genome, wherein at least one of the viral capsid structural proteins is different from other viral capsid structural proteins; Isolated AAV virions, where the virions contain only each structural protein of the same type. having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, and VP3;
The two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome, and at least one of the other viral structural proteins present is different from the other viral structural proteins, and the virions are the same. containing only each structural protein of the type,
57. The isolated AAV virion of claim 56. 58. The isolated AAV virion of claim 57, wherein all three viral structural proteins are present. 59. The isolated AAV virion of claim 58, further comprising a fourth AAV structural protein. having at least two viral structural proteins from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3;
the two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome, and at least one of the viral structural proteins present is from a different serotype than the other viral structural proteins;
VP1 is derived from only one serotype, VP2 is derived from only one serotype, VP1.5 is derived from only one serotype, and VP3 is derived from only one serotype,
57. The isolated AAV virion of claim 56. 61. The isolated AAV virion of any one of claims 57-60, wherein at least one of the viral structural proteins is a chimeric protein that is different from at least one other viral structural protein. 62. The virion of claim 61, wherein only VP3 is chimeric and VP1 and VP2 are non-chimeric. 62. The virion of claim 61, wherein only VP1 and VP2 are chimeric and only VP3 is non-chimeric. 64. The virion of claim 63, wherein the chimera is composed of subunits from AAV serotypes 2 and 8, and VP3 is derived from AAV serotype 2. 65. An isolated AAV virion according to any one of claims 56 to 64, wherein all viral structural proteins are derived from different serotypes. 65. An isolated AAV virion according to any one of claims 56 to 64, wherein only one of the structural proteins is from a different serotype. 67. A substantially homogeneous population of virions according to any one of claims 56 to 66, which is at least 10 ⁷ virions. 68. The substantially homogeneous population of virions of claim 67, which is at least 10 ⁷ to 10 ¹⁵ virions. 68. The substantially homogeneous population of virions of claim 67, which is at least ¹⁰⁹ virions. 68. The substantially homogeneous population of virions of claim 67, which is at least 10 ¹⁰ virions. 68. The substantially homogeneous population of virions of claim 67, which is at least 10 ¹¹ virions. 72. A substantially homogeneous population of virions according to any one of claims 67-71, which is at least 95% homogeneous. 73. A substantially homogeneous population of virions according to claim 72, which is at least 99% homogeneous. AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV selected from Table 1 or Table 3, and any chimera of each AAV 74. The virion according to any one of claims 56 to 73, wherein the virion is 74. A substantially homogeneous population of virions according to claim 73. 75. AAV virion according to any one of claims 56 to 74, wherein the heterologous gene encodes a protein for treating a disease. If the disease is a lysosomal storage disease, such as mucopolysaccharidosis (e.g. Sly syndrome [-glucuronidase], Hurler syndrome [a-L-iduronidase], Scheie syndrome [a-L-iduronidase], Hurler-Scheie syndrome [a-L-iduronidase], Hunter syndrome [Iduronate sulfatase], Sanfilipo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:a-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [-galactosidase], Maroteau-Ramy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (a-galactosidase), Gaucher disease (glucocerebrosidase) 77. The AAV virion of claim 76, wherein the AAV virion is selected from a glycogen storage disease (e.g., Pompe disease; lysosomal acid a-glucosidase). 78. The isolated AAV virion of any one of claims 56-60 and 66-77, wherein none of the viral structural proteins are chimeric viral structural proteins. 79. The isolated AAV virion of any one of claims 57-78, wherein there is no serotype overlap between the chimeric viral structural protein and at least one other viral structural protein. administering an effective amount of a virion according to any one of claims 56-66, 74, 76-79, or a substantially homogeneous population of virions according to any one of claims 67-73 and 75. A method of treating a disease, the method comprising:
the heterologous gene encodes a protein for treating a disease that is suitable for treatment by gene therapy in a subject having the disease;
Said method. 81. The method of claim 80, wherein the disease is selected from genetic diseases, cancer, immunological diseases, inflammation, autoimmune diseases, and degenerative diseases. 82. The method of any one of claims 80 and 81, wherein multiple administrations are performed. 83. The method of claim 82, wherein different polyploid virions are used to avoid neutralizing antibodies formed in response to pre-administration.

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